Wednesday, March 6, 2019 451a

Symposium: Mapping the Cell by four ankyrin repeats, the linker helical domain (LHD1-9), and the pre-S1 elbow. The N-terminus is followed by six membrane-spanning helices (S1-S6) 2230-Symp and a pore helix connecting to a re-entrant loop that forms the selectivity filter. Simultaneous Cross-Evaluation of Heterogeneous E. coli Datasets via The TRP helix is immediately adjacent to S6, and the C-terminus consists of two Mechanistic Simulation alpha-helices connected by a small linker. TRPC3 transmembrane domain re- Markus Covert. sembles those of other TRP channels, whereas the CPD features major differ- Dept Bioengineering, Stanford Univ, Stanford, CA, USA. ences. A C-terminal domain swap occurs at the center of the CPD, where The extensive heterogeneity of biological data poses challenges to analysis and horizontal helices (HH) transition into a coiled-coil bundle, highlighting a interpretation. Construction of a large-scale mechanistic model of Escherichia unique fold for the TRPC subfamily. Comparison of TRPC3 structures reveals coli enabled us to integrate and cross-evaluate a massive, heterogeneous dataset that the HH can reside in two distinct positions. Electrophysiological analyses based on measurements reported by various labs over decades. We identified in- demonstrate that TRPC3 activity increases by shortening the length of the C-ter- consistencies with functional consequences across the data, including: that the minal loop connecting the HH with the TRP helices; likewise elongating the data describing total output of the ribosomes and RNA polymerases is not suffi- length of the loop has the opposite effect. Our findings demonstrate that the C- cient for a cell to reproduce measured doubling times; that measured metabolic terminal loop impacts channel gating by altering the allosteric coupling between parameters are neither fully compatible with each other nor with overall growth; the cytoplasmic and transmembrane domains. Future functional and structural that essential are absent during the cell cycle - and the cell is robust to experiments are underway to validate whether the upward movement of the this absence. Finally, considering these data as a whole leads to successful pre- HHs corresponds to a positive modulation of the TRPC3 gating cycle. dictions of new experimental outcomes, in this case half-lives. 2234-Plat 2231-Symp Structural Insights into Ligand Modulation of the TRPV2 Channel Towards a Model of the Human Pancreatic Beta Cell Ruth Pumroy1, Amrita Samanta2, Yuhang Liu3, Franklin Pozo2, Raymond C. Stevens. Taylor Hughes1, George R. Dubyak2, Seungil Han3, David T. Lodowski2, Bridge Institute, University of Southern California, Los Angeles, CA, USA. Vera Moiseenkova-Bell1. A key scientific challenge in biology and chemistry is the integration of data 1Department of Pharmacology, University of Pennsylvania, Philadelphia, across the different scales – molecular, cellular, and whole body. In a collabo- PA, USA, 2Case Western Reserve University, Cleveland, OH, USA, 3Pfizer rative open source effort with several different groups across California and Inc, Groton, CT, USA. Shanghai, we are workings towards a multi-scale model of the human pancre- The transient receptor potential (TRP) family of ion channels permits permeation atic beta cell at atomic resolution. Such a model will be useful for advancing the of essential ions, including Naþ and Ca2þ, through the plasma membrane. Mem- field of structure based drug design from the protein scale to the cellular scale bers of the Vanilloid subset of this family (TRPV) can be activated or inhibited by and provide us with better feedback in the understanding and design of new a variety of stimuli, including heat, pH, mechanosensation, and a variety of small medicines to treat type II diabetes. We invite all groups interested in this effort molecules. Agonist activation of TRPV2 plays a role in the inhibition of a variety to join called the Pancreatic Beta Cell Consortium https://dornsife.usc.edu/ of cancers, including glioblastoma multiforme cell proliferation. The mechanism bridge-institute/pancreatic-beta-cell-consortium/. of TRPV2 activity in these cases seems to be related to increasing the uptake of some anticancer therapeutics, like temozolomide and doxorubicin. Here we pre- Symposium: RNA sent structures of TRPV2 structures in apo and ligand-bound forms resolved by cryo-electron microscopy. In this work we have found a novel ligand binding 2232-Symp pocket for TRPV2 which could lead to future drug development. Untangling Messenger RNA Structure with Dead-Box RNA Helicases Elizabeth Tran. 2235-Plat Biochemistry, Purdue University, West Lafayette, IN, USA. Multimerization of Human TRPA1 Cytoplasmic Domains DEAD-box proteins constitute the largest RNA helicase family in eukaryotes, Gilbert Q. Martinez, Sharona E. Gordon. functioning in all aspects of expression. However, the mechanism of ac- Dept Physiol/Biophys, Univ Washington, Seattle, WA, USA. tion in cells for these helicases remains elusive. Over the last several years, we The transient receptor potential Ankyrin-1 (TRPA1) ion channel is modu- have used genetics and classic biochemical techniques to provide insight into lated by myriad noxious stimuli that interact with multiple regions of the the role of the DEAD-box RNA helicase Dbp2 in S. cerevisiae, revealing con- channel, including the cytoplasmic N-terminal Ankyrin repeat domains nections between RNA duplex unwinding and nuclear messenger RNA matu- (ARDs) via covalent modification. The way in which TRPA1 cytoplasmic ration steps. However, the precise enzymatic targets of Dbp2 and the vast domain modification is transmitted across large spatial distances to open majority of the 25-40 DEAD-box RNA helicases in eukaryotes. Using a com- thechannelporehasyettobeelucidated.Thecryo-EMstructureof bination of genome-wide techniques including iCLIP-seq, Structure-seq, and TRPA1 revealed a tetrameric C-terminal coiled-coil (CC) surrounded by RNA seq, we have now characterized the enzymatic targets of Dbp2. This re- the ARDs, an architecture shared with the canonical transient receptor po- veals a role for dynamic changes in RNA structure during transcriptional termi- tential (TRPC) ion channel family. Similarly, structures of the TRP mela- nation. Moreover, we find evidence for regulation of Dbp2 and, consequently, statin (TRPM) ion channel family also showed a C-terminal coiled-coil Dbp2-dependent mRNAs, in response to availability. These findings enclosed by N-terminal cytoplasmic domains. This conserved architecture correlate with changes in metabolic gene expression and provide support for may indicate a common gating mechanism in which modification of cyto- widespread regulation of RNA structure as a mechanism of gene regulation. plasmic domains can transduce conformational changes to open the ion- conducting pore. We developed an in vitro system in which N-terminal ARDs and C-terminal CC domains can be expressed in bacteria and main- Platform: TRP Channels tain the ability to interact. We tested whether temperature, the polyphos- phate compound IP6, and the covalent modifier allyl alters 2233-Plat N- and C-terminal interactions. We found that none of the modifications Structural and Functional Analyses of TRPC3 Reveal Allosteric Gating tested altered ARD-CC interactions. We found that CCs tetramerize in a Modulation by the Cytoplasmic Domain concentration dependent manner, with monomers and trimers observed at Francisco J. Sierra Valdez1, Caleigh M. Azumaya2, Luis O. Romero1, lower concentrations. Although we did not observe ligand- or Terunaga Nakagawa2, Julio F. Cordero-Morales1. temperature-dependent disruption of ARD-CC interactions, our system pro- 1Dept Physiology, Univ Tennessee, Memphis, TN, USA, 2Vanderbilt vides a method for examining the mechanism of oligomerization of TRPA1 University, Nashville, TN, USA. cytoplasmic domains. TRPC3 is associated with neurodegenerative diseases, memory loss, and hyper- tension. In blood vessels, TRPC3 is an essential component of the cellular mech- 2236-Plat anisms by which vasoconstrictors regulate blood pressure. Vasoconstrictors, Approaching to the Molecular Mechanism of the Fast Inactivation of such as angiotensin II, bind to phospholipase C (PLC)-coupled receptors, Calcium Selective TRP Channels increasing the intracellular levels of DAG, which in turn activates TRPC3. Lisandra Flores Aldama1, Kattina Zavala2, Daniel Bustos3, Upon activation, TRPC3 channels induce depolarization increases intracellular Wendy Gonzalez3, Juan Opazo2, Sebastian E. Brauchi1. þ Ca2 and promotes vasoconstriction. Here, we show the full-length and cyto- 1Physiology, Univ Austral de Chile, Valdivia, Chile, 2Faculty of Science, plasmic domain (CPD) cryoEM structures for human TRPC3 in the apo state Universidad Austral de Chile, Valdivia, Chile, 3Universidad de Talca, Talca, at 5.8 and 4.0 A˚ resolution. A TRPC3 subunit consists of an N-terminus formed Chile.

BPJ 9439_9446 452a Wednesday, March 6, 2019

TRPV5 and TRPV6 are inwardly rectifying calcium selective channels, 2239-Plat considered as gatekeepers of epithelial calcium transport and key elements Gain-Of-Function Mutationsin TRPM4 Activation Gate Cause Skin Dis- for calcium homeostasis. Intracellular calcium exert a negative control over ease PSEK the activity of these channels. In mammals, TRPV6 channels show a charac- Huijun Wang1, Zhe Xu2, Bo Hyun Lee3, Simon Vu3, Linghan Hu1, teristic fast calcium-dependent inactivation phase, that is absent in TRPV5 Mingyang Lee1, Dingfang Bu1, Xu Cao1, Samuel Hwang4, Yong Yang1, channels at physiological conditions. It has been evidenced that the intracel- Jie Zheng3, Zhimiao Lin1. lular loop located between the transmembrane segments (TM) S2-S3 and res- 1Dept Dermatology, Peking University First Hospital, , , 2Dept. idues downstream the TM S6 are involved in the mechanism of fast Dermatology, Beijing Children’s Hospital, Beijing, China, 3Dept Physiol inactivation, however the molecular mechanism driving fast inactivation is Memb Biol, Univ Calif Davis, Davis, CA, USA, 4Dept. Dermatology, Univ. not known. In the present study we establish a structural-functional correlation California Davis, Davis, CA, USA. of this process. By means of electrophysiological recordings we identified a Here we report the first cases of mutations in TRPM4 which encodes TRP þ set of conserved residues at the Helix-Loop-Helix (HLH) domain that modu- melastatin 4 (TRPM4), a Ca2 -activated monovalent cation channel, as a lates the inactivation phenotype. Molecular dynamics simulations suggest that cause of an autosomal-dominant form of progressive symmetric erythrokera- the inactivation phenotype can be explained by specific conformational toderma (PSEK). In three separate families with PSEK, we identified two changes induced by calcium coordination to a structural triad formed by the missense mutations (c.3099C>G and c.3119T>C) that produce I1033M and HLH domain, the intracellular connector between TM S2-S3, and the TRP I1040T, both of which are located in the S6 transmembrane domain of helix. TRPM4 protein. The substitutions are expected to directly affect activation gating of TRPM4 according to the cryo-EM structures. Electrophysiological 2237-Plat studies of the mutants showed substantial hyperactivity, as evidenced by pro- Antagonist-Induced Clockwise Rotation in the TRPV1 nounced baseline activity, enhanced sensitivity to intracellular Ca2þ, and an Shoko Fujimura1, Kazuhiro Mio1, Masahiro Kuramochi2, elevated resting membrane potential. In vitro studies revealed enhanced pro- Hiroshi Sekiguchi3, Muneyo Mio1, Tai Kubo1, Yuji C. Sasaki2. 1 liferation in keratinocytes overexpressing either of the mutants. We also de- Operand OIL, National Institute of Advanced Industria, Kashiwa, Japan, tected an upregulationof markers for proliferation and differentiation of 2Graduate School of Frontier Sciences, The Univ. Tokyo, Chiba, Japan, 3 keratinocytes in the affected skin tissues. Our study identified TRPM4as a Japan Synchrotron Radiation Research Institute, Hyogo, Japan. new player in the pathogenesis of skin TRP channelopathies, as well as a po- The TRPV1 is a nonselective cation channel that responds to various signals tential target for treatment of skin hyperkeratotic disorders. including , heat, and low pH condition. The Cryo-EM structures of TRPV1 show twisting of transmembrane helices around the pore axis. To un- derstand the intramolecular dynamics of the TRPV1 associating with the 2240-Plat gating event, we adopted the Diffracted X-ray Tracking (DXT) technique. In TRPM8 Regulates Sexual Reward and Satiety DXT, an individual protein was labelled with gold nanocrystals. Trajectories Yelena Nersesyan1, Ekaterina Gribkova2, Padmamalini Baskaran3, of Laue diffraction spots from nanocrystals attached to the immobilized target Daniel Llano2, Baskaran Thyagarajan4, Eleonora Zakharian1. proteins were investigated as the intramolecular movement of the target pro- 1Dept Cancer Bio/Pharm, University Illinois College of Medicine, Peoria, IL, teins in real time. DXT can monitor the rotational motion of the nanocrystal USA, 2University Illinois, Urbana-Champaign, IL, USA, 3University of at several milliradian scales with two rotational axis views, tilting (q) and Wyoming, Laramie, WY, USA, 4Sch Pharmacy, Univ Wyoming, Laramie, twisting (c) motions. The motion of TRPV1 was evaluated by mean square WY, USA. angular displacement (MSD) curves of the Laue diffraction spots from DXT. Testosterone regulates dimorphic sexual behaviors in all vertebrates. However, The slopes of the MSD curves obtained from TRPV1 were shifted upward the molecular mechanism underlying these behaviors remains inconclusive. in response to capsaicin, which reflects DXT successfully extracted the internal Here, we report that a newly identified rapid testosterone signaling receptor, motion of TRPV1. We then applied a velocity dependent filtering method to TRPM8, regulates dimorphic sexual behaviors. We found that TRPM8/ sampling a biased motion. The filtering method clearly distinguished the male mice exhibit a delayed sexual satiety, increased mounting frequency agonist- and competitive antagonist (AMG9810) -induced intramolecular mo- indiscriminate of sexes, and augmented aggression compared to controls, while tion. The motions under the capsaicin and antagonist were 1 mrad (clockwise TRPM8/ females display increased olfaction-exploratory behaviors. Re- from the Au(111) direction) and 0.5 mrad (counterclockwise) after 3 ms, sponses to the acute testosterone application onto the amygdala were entirely respectively. Such motions were also observed another filtered data. This raises attenuated in TRPM8/ males but remained unchanged in females. Conse- the possibility that there are multiple modes of conformational change in quently, following-mating activation of dopaminergic neurons in the ventral TRPV1. The movement of ligand-evoked conformational changes was slower tegmental area was impaired in TRPM8/ males. Together, these results sug- than the thermal fluctuations. gest that TRPM8 mediates sex-reward mechanism in males and depletion of its function leads to a delayed sexual satiety phenomenon. 2238-Plat The Conformational Wave in Capsaicin Activation of Transient Receptor Potential Vanilloid 1 Ion Channel Platform: Intrinsically Disordered Proteins (IDP) Fan Yang1, Xian Xiao2, Bo Hyun Lee3, Simon Vu3, Wei Yang1, Vladimir Yarov-Yarovoy3, Jie Zheng3. and Aggregates III 1Department of Biophysics, School of Medicine, Zhejiang University, , China, 2Westlake University, Hangzhou, China, 3Department of 2241-Plat Membrane Biology, School of Medicine, University of California, Davis, Tardigrade Intrinsically Disordered Proteins Protect from Davis, CA, USA. Desiccation-Induced Inactivation The capsaicin receptor TRPV1 has been intensively studied by cryo-electron Samantha Piszkiewicz1, Kathryn H. Gunn2, Shannon L. Speer1, microscopy and functional tests. However, though the apo and capsaicin- Owen Warmuth1, Aakash Mehta1, Francis J. Lauzier1, Kenny H. Nguyen1, bound structural models are available, the dynamic process of capsaicin Elizabeth Kuhlman2, Saskia B. Neher2, Gary J. Pielak1,2. 1 activation remains intangible, largely due to the lack of a capsaicin- Dept of Chemistry, The University of North Carolina at Chapel Hill, Chapel 2 induced open structural model and the low occupancy of the transition Hill, NC, USA, Dept of Biochemistry and Biophysics, The University of states. Here we report that reducing temperature toward the freezing point North Carolina at Chapel Hill, Chapel Hill, NC, USA. substantially increased channel closure events even in the presence of Protein-based ‘biologics’ — drugs derived from living organisms — are among saturating capsaicin. We further used a combination of fluorescent unnatural the most effective therapeutic treatments on the market. However, biologics are (fUAA) incorporation, computational modeling, and rate- unstable, have short half-lives and require low temperature storage, making equilibrium linear free-energy relationships analysis (Phi-analysis) to them prohibitively expensive. Although some biologics can be stabilized by derive the fully open capsaicin-bound state model, and reveal how the formulation with excipients, most still require low temperature storage. In channel transits from the apo to the open state. We observed that capsaicin our search for new, more robust excipients, we turned to the tardigrade, a initiates a conformational wave that propagates through the S4-S5 microscopic animal that synthesizes a unique family of intrinsically disordered linker towards the S6 bundle and finally reaching the selectivity filter. proteins (IDPs) that protect its cellular components during desiccation. Encap- Our study provides a temporal mechanism for capsaicin activation of sulating a globular test protein in a tardigrade IDP hydrogel stabilizes the TRPV1. folded state of that protein. These tardigrade IDPs are more effective than

BPJ 9439_9446 Wednesday, March 6, 2019 453a two FDA approved excipients, trehalose and serum albumin, at protecting ism spectroscopy shows no gain of structure upon formation of this large poly- lactate dehydrogenase activity during desiccation. We even observe protection , as in the case of ProTa-H1. of lactate dehydrogenase upon heating desiccated /tardigrade IDP mix- A pattern is emerging that chromatin-remodeling complexes must be kept tures to 95 C. In addition, these IDPs protect the restriction enzyme DpnI from amenable of fine-tuning for optimal regulation. Additionally, both ProTa-H1 desiccation-induced inactivation and lipoprotein lipase against both freezing- and ProTa-NUPR1 undergo liquid-liquid phase transition in the micromolar induced and lyophilization-induced inactivation. Work is underway to quantify concentration range, suggesting that disorder, valency and accessibility of these the protection of against aggregation. In summary, we have demon- complexes might also be important to grant a further level of regulation via strated the ability of these tardigrade IDPs to stabilize a globular protein and phase transition. protect a diverse range of enzymes against desiccation, freezing, and lyophilization. 2244-Plat A Bacterial Biomolecular Condensate Sequesters a Signaling Pathway that 2242-Plat Drives Spatial Regulation of Gene Expression and Asymmetric Cell Division A Novel Molecular Lego Approach to Measure the Marginal Folding Keren Lasker1, Alex von Diezmann2, W.E. Moerner2, Lucy Shapiro1. Cooperativity of Intrinsically Disordered Proteins 1Developmental Biology, Stanford University, Stanford, CA, USA, Suhani Nagpal1, Thinh Luong2, Mourad Sadqi1, Victor Mun˜oz1. 2Chemistry, Stanford University, Stanford, CA, USA. 1Bioengineering, University of California, Merced, Merced, CA, USA, Selective recruitment and concentration of signaling proteins within membrane- 2Chemistry and Chemical Biology, University of California, Merced, less compartments is a ubiquitous mechanism for subcellular organization. We Merced, CA, USA. combined single-molecule tracking and super-resolution microscopy, light- Intrinsically disordered proteins (IDPs) do not fold into a native three- induced spatial mutations, reaction-diffusion modeling, and spatially-resolved dimensional structure, but exist as dynamic conformational ensembles. Their transcriptional profiling to study signal exchange in and out of a <200 nm cyto- flexibility enables IDPs to exhibit sophisticated binding modes, such as binding plasmic microdomain at the cell poles of the asymmetrically dividing bacterium to multiple partners, induced-fit binding, and allosterism. Accordingly, IDPs Caulobacter crescentus. We show that the signaling proteins in a core develop- play key regulatory roles in fundamental biological processes such as transcrip- mental pathway that culminates in the activation of the cell fate transcription fac- tion, cell cycle control and signaling. The key to their functioning is their ability tor CtrA are transiently confined to the polar microdomain. Restricted rates of to fold onto specific structures when bound to their ligand partner(s). The mech- entry into and escape from the microdomain enhance phospho-signaling, leading anistic characterization of IDPs poses a challenge to current methods which to a steep submicron gradient of activated CtrA. Thus, nanoscale protein assem- rely on folding to well-defined structures. blies can modulate signal propagation with fine spatial resolution, and in Caulo- We have devised a novel approach to measure the inherent folding cooperativ- bacter, this modulation serves to prime asymmetric genome readout. ity of an IDP that we term Molecular LEGO. Inspired by the LEGO game in The polar microdomain is defined by self-assembly of the disordered protein which building blocks associate to one another by complementary indentations, PopZ that directly binds signaling proteins in vivo. We show that cytosolic pro- our approach consists on analyzing the conformational properties of fragments teins lacking a microdomain binding partner cannot penetrate the microdomain of an IDP containing each secondary structure element as well as all possible despite being similar in size and charge to proteins that do enter. These combinations of them. Direct comparison of conformational preferences of iso- microdomain-excluded-proteins can be recruited to the microdomain through lated elements, their combinations, and the entire protein, we infer the degree of light-induced dimerization between the excluded protein and PopZ, suggesting energetic coupling between each the various parts. We do this experimentally, that microdomain binding controls entry. We further show that light-induced analyzing conformational properties by circular dichroism and using tempera- recruitment of signaling proteins to the poles of the cell at the wrong time alters ture and TFE as thermodynamic parameters to enhance information, and also the fate of the two daughter cells, leading to loss of asymmetry. Modifying either computationally, by simulating all possible fragments using atomistic Molecu- the charge or composition of the disordered region modulates selectivity proper- lar Dynamics simulations in explicit solvent. Using NCBD as paradigmatic ties, leading to severe phenotypes that are in some cases lethal. Finally, the selec- example of IDP, we observe, although NCBD exhibits an apparently featureless tive properties of PopZ are independent of geometry: In cells with a deformed thermal unfolding profile, the various elements interact with one another, spherical shape, PopZ becomes a droplet that maintains its selectivity properties. biasing the conformational space of the protein in significant ways to populate the native-like partially folded conformations that enable its complex binding 2245-Plat behavior. This approach can generally be applied to any partially disordered Sequence Determinants of Protein Phase Separation of the Intrinsically IDPs thus providing an essential new tool for their analysis. Disordered RGG Domain from LAF-1 Benjamin S. Schuster1, Gregory Dignon2, Craig Jahnke1, 2243-Plat Matthew C. Good1, Daniel A. Hammer1, Jeetain Mittal2. Highly Disordered 10:1 Complex of Two Anti-Apoptotic, Chromatin-Re- 1University of Pennsylvania, Philadelphia, PA, USA, 2Lehigh University, modelling Intrinsically Disordered Proteins Bethlehem, PA, USA. Alessandro Borgia1, Madeleine B. Borgia1, Alain Scaiola2, Robert Best3, Phase separation of intrinsically disordered proteins (IDPs) underlies the for- Benjamin Schuler1. mation of membraneless organelles with prominent biological roles, such as 1Department of Biochemistry, University of Zurich, Zurich, Switzerland, P granules in C. elegans embryos. Determining the sequence-level features 2Department of Biology, Institute of Molecular Biology and Biophysics, responsible for IDP phase separation is important for understanding native bio- ETH, Zurich, Switzerland, 3NIDDK, Laboratory of Chemical Physics, NIH, logical function, protein evolution, and approaches to manipulate phase Bethesda, MD, USA. behavior. However, sequence determinants of IDP condensation are poorly un- The prominence of intrinsically disordered proteins (IDPs) in regulatory roles is derstood because the field lacks a predictive framework for guiding experi- in no small part due to their flexibility, which allows them to interact with many ments. We investigated sequence determinants governing phase separation of partners and be accessible to modifying enzymes. The repertoire of IDPs inter- the RGG domain from the P granule protein LAF-1, using theory and experi- actions has recently been expanded by our discovery and characterization of the ments. We uncovered three features of the LAF-1 RGG domain that mediate first case of an IDP-IDP high-affinity interaction between the linker its phase behavior. First, based on a predictive coarse-grained (CG) model of H1.0 and prothymosin-a (ProTa). Mapping these interactions is an inherently IDPs, we identified an unappreciated region of the RGG domain that is also difficult task due to their transient nature, but thanks to single-molecule FRET highly conserved between species. Experiments revealed that deletion of this (smFRET), NMR and molecular simulations, we demonstrated that disorder of region indeed significantly disrupts phase separation, much more than would the interacting proteins is fully maintained in the complex, where the constitu- have been expected from control deletions of other regions. Second, based ent proteins dynamically interconvert within a broad conformational ensemble on CG molecular dynamics simulations, we predicted amino acid replacements with no preferential binding sites. that may dramatically affect phase behavior. We experimentally determined We have now investigated the interaction between the highly acidic ProTa and that mutating Tyr to Phe, or Arg to Lys, dramatically perturbs RGG phase sep- its basic partner NUPR1: both IDPs are involved in cancer progression, aration; these trends agree with recent findings on other proteins, including apoptosis, and, like H1.0, in chromatin remodeling. Multiple intra- and inter- FUS. Finally, we assessed the effect of sequence charge decoration on phase molecular distances measured with smFRET show a progressive addition of behavior, harnessing a new finding relating single-molecule properties and NUPR1 molecules on a single ProTa chain, which is consistent with results phase separation. Experimentally, we found that sequences with large charge from dual-focus FCS showing a continuous increase in hydrodynamic radius segregation have dramatically enhanced propensity to phase separate compared upon titration of ProTa with NUPR1. Using FRET we were able to determine to wild-type RGG, as expected from simulation and theory. Together, these a 10:1 stoichiometric ratio in saturating conditions, which is highly unusual for studies identify key determinants of RGG phase separation, including proteins of roughly similar sizes. However, strikingly, far-UV circular dichro- conserved residues and charge patterning. These results elucidate physical

BPJ 9439_9446 454a Wednesday, March 6, 2019 principles of IDP condensation and its biological implications, while advancing is completely resistant to liquid-liquid phase separation (LLPS) of the eye lens a computational/experimental hybrid framework for studying phase separation. proteins, also known as cold cataract. Despite this observation, a sub-fraction of the D. mawsoni eye lens proteins was found to undergo LLPS above the body tem- 2246-Plat perature of the toothfish. Six g-crystallins were identified from this fraction and The Ins and Outs of Phase Separation in Nucleolar Biology characterized for their LLPS behavior and general features of stability. No obvious 1 1 1 1 Richard Kriwacki , Diana Mitrea , Mylene Ferrolino , Eric Gibbs , relationship could be discerned between the critical temperatures at which these g- 1 1 2 Aaron H. Phillips , Michele Tolbert , Christopher B. Stanley , crystallins undergo LLPS and the various measures of stability. Additionally, Amanda Nourse1, Paulo L. Onuchic3, Priya R. Banerjee3, Ashok A. Deniz3. 1 2 mutagenesis of predicted surface exposed residues was performed to study their Dept Struct Biol, St Jude Childrens Res Hosp, Memphis, TN, USA, Biology role in the LLPS behavior. Mutagenesis revealed that replacing exposed arginine and Biomedical Sciences Group, Oak Ridge National Laboratory, Oak Ridge, 3 for lysine results in a decrease of the phase transition temperature, while replacing TN, USA, Department of Integrative Structural and Computational Biology, lysine with arginine had the opposite effect, but similar magnitude. These finding The Scripps Research Institute, La Jolla, CA, USA. highlights the importance of arginine in increasing attractive protein-protein inter- The nucleolus is a multi-layered, liquid-like membraneless organelle that medi- actions even when the site specific charge is largely unchanged. Furthermore, we ates the multi-step process of ribosome biogenesis and also coordinates signaling have shown that the components of the buffer solution can further modulate the responses to various cellular stresses. Initiation of ribosomal RNA (rRNA) tran- phase transition temperature of these crystallins. scription during interphase of the cell division cycle triggers phase separation with the nucleolar protein, Fibrillarin, forming the Dense Fibrillar Component region of the nucleolus wherein rRNA is sequentially modified and spliced. Platform: General Protein-Lipid Interactions Mature rRNAs flux outwards from the DFC into the surrounding Granular Component (GC) region wherein they phase separate with another nucleolar pro- 2249-Plat tein, Nucleophosmin (NPM1). Utilizing an intrinsically disordered region (IDR) Understanding the Organization and Dynamics of KRAS4b on a Complex displaying alternating acidic and basic tracts, NPM1 independently phase sepa- 8-Lipid Reconstituted Model Membrane using Microscopy and Spectros- rates with ribosomal proteins (rProteins), sequestering them within the GC and copy Methods enabling their assembly with rRNA to form nascent ribosomal subunits. We seek Rebika Shrestha, Thomas Turbyville. to understand how the molecular interactions that independently drive phase sep- RAS Initiative, Frederick National Laboratory for Cancer Research, aration of NPM1 with rRNA and rProteins also enable the molecular handoffs Frederick, MD, USA. that underlie ribosomal subunit assembly. Our studies employ structural and bio- The enrichment of negatively charged phospholipids, particular phosphatidylser- physical methods that probe molecular interactions at the atomic length scale as ine (PS) and phosphoinositides, in the inner leaflet of plasma membrane (PM) is well as single-molecule, scattering and imaging methods that reveal the struc- one of the underlying principle behind recruitment of numerous intracellualr pro- tural and material properties of liquid-phase condensates on the micron length teins to PM, thus regulating key cellular events. One such protein of interest is scale. Only by bridging these disparate length scales can we understand how KRAS4b, a member of Ras superfamily of small GTPases, which is an oncopro- phase separation contributes to the essential process of ribosome biogenesis. tein implicated in 95% of pancreatic cancer, 45% of colorectal cancer, 35% of We will present our latest findings on the liquid phase organization of the nucle- lung cancer and high extent in other forms of cancer. When Ras is active and olus and how this mediates vectorial ribosomal subunit assembly. bound to the membrane, it binds to its downstream effectors and triggers multiple signal cascading pathways that are essential for cell growth, proliferation and sur- 2247-Plat vival. KRAS4b localizes to PM predominantly by virtue of (i) the electrostatic Sequence Determination of Liquid-Liquid Phase-Separated Assemblies of interaction between sixpositively charged lysine residues located in the hypervar- Engineered Disordered Proteins in Living Cells iable region (C-terminal) of KRAS4b and negatively charged phospholipids in Ming-Tzo (Steven) Wei, Clifford P. Brangwynne. PM and (ii) the hydrophobicity of the farnesyl group, post-translational modifica- Chemical and Biological Engineering, Princeton University, Princeton, NJ, tion in C185 amino acid of KRAS4b, which anchors the protein into the disor- USA. dered lipid domains. In this study, we aim to explore the molecular mechanism There is currently a growing interest in understanding biopolymer phase transi- of KRAS4b interaction with the membrane using recombinant protein on an arti- tions, particularly those involving intrinsically disordered proteins/regions ficial supported lipid bilayer. The biomimetic surface is composed of complex (IDPs/IDRs)and RNA.Ithas been determinedthatintracellularliquid-liquidphase 8-lipids composition that closely resemble the lipid composition of the inner separations underlie the assembly of many non-membranous organelles, including leaflet of biological PM. We use a combination of fluorescence-based microscopy RNA/protein assemblies such as P granules, nucleoli, transcription factors, and and single molecule spectroscopy techniques as well as high resolution scanning stress granules. However, little is known about the physics of these organelles, probe microscopy to study the lateral organization and dynamics of KRAS4b as including their internal molecular organization and feedback between their molec- well as the lipid bilayer. Using a very complex reconstituted model membrane to ular and mesoscale properties. Progress on these questions has been hampered by study KRAS4b, our research provides mechanistic insight into RAS biology and the lack of detailed phase diagrams, which could elucidate how molecular interac- possibly a nobel platform for targeting RAS. tions give rise to emergent droplet properties, particularly condensed-protein con- centrations and their physical characteristics. To answer these questions, we 2250-Plat utilized a novel technique, ultrafast-scanning fluorescence correlation spectros- Structural and Mechanistic Bases of Drp1-Cardiolipin Interactions in copy, to measure the inter-molecular interaction strengths and full binodal of a Mitochondrial Fission 1 1 2 1 disordered protein that induces in-vivo phase transitions. These measurements re- Bin Lu , Mukesh Mahajan , Abhishek Mandal , Nikhil Bharambe , 1 2 1 1 vealed that phase-separated protein droplets have unusually low densities with Rihua Wang , Patrick van der Wel , Matthias Buck , Xin Qi , 1 large void volumes. The data demonstrate how sequence-encoded conformational Rajesh Ramachandran . 1Dept Phys/Biophys, Case Western Reserve Univ, Cleveland, OH, USA, fluctuations of IDRs give rise to low overlap volume fractions for driving phase 2 separations. Furthermore, using the inter-molecular interactions of native non- Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA, USA. membranous organelles, we develop an optogenetic platform for light activation Mitochondria are dynamic organelles that undergo regulated cycles of fission and of IDR-mediated phase transitions in living cells. This powerful technique permits fusion. Emergent data indicate that a direct interaction of the cytosolic, mechano- detailed studies of the mechanisms by which IDR sequences determine intracel- chemical GTPase, dynamin-related protein 1 (Drp1) with mitochondrial outer lular phase separation. Such experiments can elucidate not only physiological membrane-localized cardiolipin (CL) catalyzes mitochondrial fission. However, phase transitions but also their links to pathological aggregates. neither the identity of the amino acid residues nor the mechanism by which Drp1 accomplishes specific CL recognition and subsequent membrane remodel- 2248-Plat ing is known. Here using a comprehensive toolkit of structural, biochemical, bio- Uncovering the Role of Surface Residues and Buffer Composition in physical, and cell biological approaches, we reveal the structure, identity, and Liquid-Liquid Phase Separation of Eye Lens Crystallins from an Antarctic mechanism by which the intrinsically disordered Drp1 variable domain (VD) spe- Toothfish cifically binds and remodels membrane CL. We demonstrate that lipid-packing Jan C. Bierma1, Kyle Roskamp1, Aaron Ledray1, Andor J. Kiss2, defects in CL-containing membranes enable specific Drp1-CL recognition inde- C.-H. Christina Cheng3, Rachel W. Martin1. pendent of CL acyl chain composition or shape. We further show that the Drp1 1UC Irvine, Irvine, CA, USA, 2Miami University, Oxford, OH, USA, VD intercalates into the hydrocarbon core of the membrane to specifically restrict 3University of Illinios at Urbana-Champaign, Urbana, IL, USA. CL motion and induce transient CL non-bilayer topological transitions that facil- The Antarctic toothfish, Dissostichus mawsoni, lives in the Southern Ocean itate membrane constriction. We reveal a disorder-to-order structural transition around Antarctica, requiring adaptations to deal with the challenges of living in of the Drp1 VD upon CL binding, and identify critical VD residues, the substitu- a subfreezing environment. One such adaptation is that the D. mawsoni eye lens tion of which selectively reduces Drp1-CL binding affinity. Remarkably, the

BPJ 9439_9446 Wednesday, March 6, 2019 455a expression of mutant VD in Drp1-null cells induces the formation of hitherto un- 2253-Plat seen, ring-like ‘donut’ mitochondria subsequent to mitochondrial fission. Our data Molecular Simulations Reveal the Dynamics of the Band 3 Anion Trans- reveal that a rapid degradation of pro-fission CL to fusogenic phosphatidic acid porter in a Model Native Red Blood Cell Membrane (PA) at the poles of newly divided mitochondria in mutant VD-expressing cells Dario De Vecchis1,2, Reinhart A. Reithmeier3, Antreas Kalli1,2. enable mitochondrial back-fusion and donut formation. These studies firmly 1School of Medicine, Univ Leeds, Leeds, United Kingdom, 2Astbury Centre establish an indispensable role for Drp1 VD-CL interactions in regulating mito- for Structural Molecular Biology, Leeds, United Kingdom, 3Dept chondrial dynamics and morphology both pre- and post-fission. Biochemistry, Univ Toronto, Toronto, ON, Canada. Band 3, the red blood cell anion exchanger (AE1/SLC4A1), is responsible for the 2251-Plat rapid transport of bicarbonate and chloride across the red blood cell plasma mem- Multiple Stochastic Pathways in Forced Peptide-Lipid Membrane brane, a process necessary for efficient respiration. Human Band 3 is comprised of Detachment a cytosolic domain and a membrane domain that contains 14 transmembrane heli- Milica Utjesanovic1, Tina R. Matin2, Krishna P. Sigdel3, Gavin M. King1, ces. Although structural data are available for both isolated domains, the structure Ioan Kosztin1. of the complete Band 3 remains elusive. This is a limiting factor in the study of 1Dept Phys/Astron, Univ Missouri Columbia, Columbia, MO, USA, 2Dept Band 3 anion transport and of its interactions with cytosolic proteins. By inte- Anesthesiology, Weill Cornell Medicine, New York, NY, USA, 3Dept Phys/ grating molecular modelling and molecular dynamics simulations at the coarse- Astron, California State Polytechnic University, Pomona, CA, USA. grained and all-atom resolutions, we have constructed a model of Band 3 that con- We have used high resolution AFM based dynamic force spectroscopy to inves- sists of both the transmembrane and the cytosolic domains. This model enabled us tigate peptide-lipid membrane interactions by measuring the detachment (last- to identify the orientation of the cytosolic domain relative to the transmembrane rupture) force distribution, P(F), and the corresponding force dependent rupture domain and the role of the linker regions that connect these two domains in their rate, k(F), for two different peptides and lipid bilayers. The measured quantities, interactions. Our model was validated using functional data and molecular simu- which differed considerably for different peptides, lipid-membranes, AFM tips lations. Simulations were performed in complex bilayers that resemble the native (prepared under identical conditions), and retraction speeds of the AFM canti- red cell plasma membrane, containing a full complement of phospholipids, sphin- lever, could not be described in terms of the standard theory, according to which gomyelin and cholesterol. Our results provide novel molecular insights into the in- detachment occurs along a single pathway, corresponding to a diffusive escape teractions of the lipid environment with Band 3. Specific lipids, e.g. cholesterol, process across a free energy barrier. In particular, the prominent retraction speed have been found to localize in the Band 3 dimer interface possibly stabilizing dependence of k(F) was a clear indication that peptide-lipid membrane dissocia- the dimer. Specific lipid head groups were also found to interact with the cytosolic tion occurs stochastically along several detachment pathways. Thereby, we have domain regulating its orientation relative to the transmembrane domain. Moreover, formulated a general theoretical approach for describing P(F) and k(F), by our modeling approach identified key residues involved in anion binding. Under- assuming that peptide detachment from lipid membranes occurs, with certain standing the dynamics and interactions of Band 3 in a model native red cell mem- probability, along a few dominant diffusive pathways. This new method was vali- brane provides new insights into the function of this important human transporter. dated through a consistent interpretation of the experimental data. Furthermore, we have found that for moderate retraction speeds at intermediate force values, 2254-Plat k(F) exhibits catch-bond behavior (i.e. decreasing detachment rate with Huntingtin Aggregation Is Modified in the Presence of a Variety of Lipid increasing force). According to the proposed model this behavior is due to the sto- Membranes chastic mixing of individual detachment pathways which do not convert or cross Maryssa A. Beasley1, Sharon E. Groover1, Justin A. Legleiter1,2. during rupture. To our knowledge, such catch-bond mechanism has not been pro- 1C. Eugene Bennett Department of Chemistry, West Virginia University, posed and demonstrated before for a peptide-lipid interaction. Morgantown, WV, USA, 2Blanchette Rockefeller Neurosciences Institute, Work supported by the Burroughs Wellcome Fund (Career Award at the Scien- Robert C. Byrd Health Sciences Center, West Virginia University, tific Interface), the NSF (CAREER Award #: 1054832), and the MU Research Morgantown, WV, USA. Board. The computation for this work was performed on the HPC infrastructure Huntington’s Disease is a fatal neurodegenerative disorder characterized by an provided by RCSS at the University of Missouri, Columbia MO. The HPC expanded polyglutamine (polyQ) region of the huntingtin protein (htt) that leads equipment for the computational work is supported by NSF CNS-1429294. to the formation of toxic aggregates. The first 17 amino-terminal residues of htt (Nt17) have been shown to promote this oligomer formation, and also comprise 2252-Plat a lipid binding domain. The subcellular localization and interaction of htt contain- The Small Heat Shock Proteins, HSPB1 and HSPB6, Have the Ability to ing expanded polyQ domains with membranous surfaces has been well docu- Get Inserted into Lipid Membranes mented and suggests that these interactions play a role in HD pathogenesis. Antonio De Maio1,2, David M. Cauvi1, Ricardo F. Capone1, Nelson Arispe3, While protein/lipid membrane interactions play an important role in a number Wilbert Boelens4. of amyloid-related diseases, the protein represents only one of the participants. 1Dept. of Surgery, University of California San Diego, La Jolla, CA, USA, For example, the size and charge of the lipid headgroups likely influence the inter- 2Dept. of Neurosciences, University of California San Diego, La Jolla, CA, action between Nt17 and lipid membranes. Any effect on lipid binding would also USA, 3Dept Anat/Phy, Uniform Serv Univ Hlth Sci, Bethesda, MD, USA, affect the aggregation and trafficking of htt. Determining factors that regulate the 4Dept. of Biomolecular Chemistry, Institute for Molecules and Materials, affinity of htt for membranes could not only help understand the normal functions Radboud University Nijmegen, Nijmegen, Netherlands. of htt, but could lead to a better understanding of how to modify protein-lipid Increasing evidence has indicated that heat shock proteins (HSP) escape their cyto- interaction for therapeutic purposes. As a result, we undertook a series of exper- solic environment acting as signaling agents in the extracellular fluid. Since the iments to determine the role of specific lipids in modulating the htt/lipid interac- majority of these proteins lack the information necessary for their export via the tion and resulting aggregation. Htt aggregation in the presence of POPC, DOPC, classical secretory pathways, special attention has been directed at alternative DMPC, POPG, POPE, POPS, and total brain lipid extract were investigated via mechanisms for their release. In this regard, a major obstacle for their secretion thioflavin T aggregation assays and other biochemical assays. In addition, the is the crossing of the plasma membrane. A mechanism that appears equally suit- modulating effect of cholesterol was also explored. able for their release is via exosomes. Indeed, several hsp have been detected within exosomes isolated from various sources. Herewith we report that a member 2255-Plat of the small hsp family, HSPB1 (Hsp27), was found to be associated with exo- Characterization of Phosphatidylinositol Phosphate Binding in Lipid Bila- somes and apparently inserted in the vesicle membrane. In order to investigate yers by Solid-State NMR Spectroscopy the potential interaction of HSPB1 and lipid membranes, pure protein was incu- Jacqueline R. Perodeau, Ashley D. Bernstein, Stefany M. Lazieh, bated with liposomes made of various lipid compositions and insertion was ac- Robert D. Palmere, Andrew J. Nieuwkoop. cessed by high-speed centrifugation. We found that HSPB1 interacts with Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, USA. liposomes made of palmitoyl oleoyl phosphatidylserine (POPS), phosphatidylcho- Phosphatidylinositol phosphates (PIPs) are a seven membered class of lipid that line (POPC) and phosphatidylglycerol (POPG), however, with different character- regulate diverse cell processes in eukaryotic organisms differentiated by the level istics. Another member of the small HSP family, alphaB-crystallin (HSPB5), also of phosphorylation at three sites on the lipid head group. The presence of one or interacts with liposomes, but differently than HSPB1. To address the regions inter- more of these lipids in a membrane serves as a biochemical signal for the local- acting with the membrane, intact proteoliposomes were digested with proteinase K ization and activation of hundreds of intrinsic and peripheral membrane proteins. and the protected domains within the liposomes were detected by mass spectros- Magic-angle-spinning (MAS) solid-state NMR (ssNMR) can provide atomic res- copy. We observed that large parts of HSPB1 and HSPB6 were embedded within olution information about interactions in lipid environments. Direct observation the liposomes. These observations suggest that the interaction with lipid mem- of the lipids and membrane proteins are possible, as well as the interactions be- branes may be part of the mechanisms of export of these proteins. tween them. While ssNMR is capable of investigating all types of lipids, the

BPJ 9439_9446 456a Wednesday, March 6, 2019 existence and importance of up to three additional phosphate groups in PIPs pro- thermodynamic cost imposed by isomerization, and the difference between ATP- vide a distinct advantage for their investigation. We demonstrate sample prepa- and ADP-actin that we calculate exactly matches the values found in experiments. ration and spectroscopic techniques to maximize the sensitivity of 31P spectra of PIP containing liposomes. The location of the PIP binding site on a protein 2258-Plat is important to understand if we seek to detect or modify binding. NMR is Investigations into the Structure and Intermolecular Interface of Human uniquely suited for mapping the binding sites of PIPs to atomic resolution. Mod- Cofilin-2 Assembled on Actin Filaments by Magic Angle Spinning NMR ern ssNMR utilizes very-fast MAS rates (in excess of 100 kHz) and proton detec- Jodi Kraus1, Jenna Yehl1, Elena Kudryashova2, Emil Reisler3, tion to streamline the process of assigning the chemical shifts in moderately sized Dmitri Kudryashov2, Tatyana Polenova1. proteins. PH domains are conserved PIP binding domains found in many different 1Chemisty and Biochemistry, University of Delaware, Newark, DE, USA, proteins. We present progress towards the solid-state assignments of PH domains, 2Chemistry and Biochemistry, The Ohio State University, Columbus, OH, a well characterized class of PIP binding domain. These chemical shifts will be USA, 3Chemisty and Biochemistry, University of California, Los Angeles, used to track the effects of PIP binding on the domain, confirming the binding Los Angeles, CA, USA. site as observed by other techniques. Actin comprises one major component of the eukaryotic cytoskeleton and is one of the most ubiquitous and conserved proteins. The cofilin/ADF family of pro- 2256-Plat teins play an integral role in the regulation of actin polymerization dynamics. Spatial Organization of the Blood Stage Parasitophorous Vacuole of the Specifically, by stimulating severing and depolymerization of actin filaments Malaria Parasite Plasmodium falciparum they potentiate actin recycling in a nucleotide-dependent manner. While actin 1 2 3 4 Matthias Garten , Josh R. Beck , Robyn Roth , Christopher K.E. Bleck , treadmilling is crucial for cell function and is responsible for up to 50% of 1 1 1 John E. Heuser , Tatyana Tenkova-Heuser , Svetlana Glushakova , cellular ATP consumption, there is a lack of atomic-level information regarding Joshua Zimmerberg1, Daniel E. Goldberg3. 1 the structure and dynamics of cofilin/ADF proteins bound to actin filaments. We NICHD/Section on Integrative Biophys, NIH, Bethesda, MD, USA, have previously reported structural characterization of cofilin/actin assemblies 2Department of Biomedical Sciences, Iowa State University, Ames, IA, USA, 3 4 using magic angle spinning (MAS) NMR spectroscopy. Our MAS NMR results Washington University, St. Louis, MO, USA, NIH/NHLBI, Bethesda, MD, for the F-actin binding site agree with the 3.7 A˚ cryo-EM structure of cofilin/ USA. actin complex, with three patches identified on cofilin for F-actin binding. Other The interface of the malaria parasite with its host red blood cell is the parasi- structural changes of cofilin that occur from binding to F-actin will be clarified tophorous vacuole membrane (PVM). The parasite organizes transport of pro- upon the determination of an atomic-resolution structure. Herein, we report in- teins, lipids and small molecules across the PVM. It is unknown how the vestigations into the structure and intermolecular interface of cofilin bound to F- parasite transports lipids across the PVM. Vesicular transport was not reported actin in the ADP and ADP-BeFx nucleotide states. We report 13C and 15N reso- in the PV lumen (the space between the PVM and the parasite plasma mem- nance assignments for cofilin-2 using homo- and heteronuclear MAS NMR brane (PPM)). Curiously, the freely accessible PV lumen is not homogenously experiments acquired at 19.9 T. We also present 13C-13C correlation spectra us- distributed around the parasite indicating regional close apposition of the PVM ing 2-13C and 1,6-13C-labeled cofilin for determination of distance restraints. and PPM. We hence hypothesized that the parasites may establish membrane Paramagnetic relaxation enhancements (PREs) were also used for long-range contact sites for the transport of lipids. distances and the cofilin-actin intermolecular interface. We observe several To explore the protein distribution on the PVM in correlation to the freely peaks disappear and decrease in intensity. Peak intensities are correlated to accessible PV luminal space, we used a parasite line expressing endogenously proximity to unpaired spins, and are used to derive atomic distances. These labeled, PVM resident protein, EXP2-mNeonGreen, and a PV lumen targeted are used in conjunction with our resonance assignments and distance restraints mRuby3. We found that both labels occupy the same PV region, defining from sparse-labeled cofilin to derive the MAS NMR structure of cofilin/F-actin EXP2-rich regions with accessible PV luminal space. We thus hypothesized assemblies, which is currently underway. that EXP2-free regions could be membrane contact sites to facilitate lipid ex- change. Tagging the PPM resident lipid transporter PfNCR1 with GFP, we 2259-Plat found that EXP2-rich and PfNCR1-rich regions anti-correlate to complement Mechanisms for Dendritic Actin Network Formation, Distributed Turn- each other to outline the parasite. over, and Structural Remodeling The data suggests the organization of the PVM and PV lumen in 2 distinct do- Danielle Holz, Aaron Hall, Dimitrios Vavylonis. mains with functions that can be suggested from their resident proteins: EXP2 Physics, Lehigh University, Bethlehem, PA, USA. has been associated with protein export and implicated in nutrient import. It The dendritic network of actin filaments provides the force for lamellipodial pro- may thus define a domain for the exchange of soluble and solubilized constit- trusions, driven by actin filament polymerization and branch generation by the uents and metabolites. PfNCR1 on the other hand may define a domain desig- Arp2/3 complex. Electron microscopy experiments of lamellipodia revealed nated for the exchange of hydrophobic substances. that the network structure of filaments varies with distance to the leading edge. Near the leading edge there is a dense brushwork composed of short filaments. Platform: Actin Structure, Dynamics & Filaments are longer and appear more linear near the center and rear of keratocyte Associated Proteins lamellipodia. Prior modeling of FRAP and single molecule imaging experiments suggested the existence of a diffuse actin oligomer cytoplasmic pool and distrib- 2257-Plat uted turnover of F-actin through the lamellipodium. The precise mechanisms Molecular Dynamics Simulations of G- and F-Actin Explain Aspects of behind network remodeling and the role of the oligomer pool have yet to be deter- Actin Polymerization mined. To answer this question, we created a three-dimensional stochastic model Lauren Jepsen, David Sept. at the filament level that includes rate constants for known mechanisms of poly- Univ Michigan, Ann Arbor, MI, USA. merization, depolymerization, branching, capping, uncapping, severing and de- The proper regulation of actin filament (F-actin) dynamics is key for proper branching. The model reproduces the þ/ 35o orientation pattern when cellular and physiological function. Filament dynamics are regulated through a branching occurs within 10o of the lamellipodial plane as well as the density wide array of actin binding proteins, but the nucleotide state of actin itself is of branches, ends and length distribution near the leading edge for both fibroblast also an important factor. Actin filaments polymerize in a head-to-tail fashion, and keratocyte lamellipodia (that differ in the magnitude of retrograde flow and meaning that the filament is polar and the two filament ends are intrinsically polymerization rates). We show that the simplest implementations of severing, different. The barbed end of the filament polymerizes more quickly, and the debranching, uncapping, and depolymerization from either pointed or barbed pointed end dissociates faster. As of yet, neither a F-actin crystal structure nor ends does not provide mechanisms for network structural remodeling at the level high resolution cryo-EM structures of either filament end are available, limiting observed in electron micrographs. We examine and test several mechanisms that our knowledge of important changes responsible for end-dependent dynamics. include severing and oligomer annealing that may provide an explanation. To explore filament dynamics, we performed extended molecular dynamics sim- ulations of F-actin. We find that the filament’s pointed end is significantly flatter 2260-Plat than the remainder of the filament and is in a conformation not sampled by our G- Different Faces (Phases) of Actin Depolymerizing Factors from Entamoeba actin simulations - this means that incoming monomers would need to undergo Histolytica. significant conformational changes (isomerization) that would both weaken the Pragyan Parimita Rath, Nitesh Kumar, Samudrala Gourinath. binding affinity and slow the rate of polymerization. Conversely, we find that School of Life Sciences, Jawaharlal Nehru University, New Delhi, India. Entamoebahistolytica the barbed end of the filament takes on a conformation nearly identical to that Actin cytoskeleton, the key structural protein present in of the ATP monomer, enhancing ATP G-actin’s ability to polymerize as compared drives the process of phagocytosis, which is an essential requisite for the sur- to ADP G-actin. Applying inverse Boltzmann weighting, we can account for the vival of amoeba. Owing to the importance of actin tread-milling, nature has

BPJ 9439_9446 Wednesday, March 6, 2019 457a recruited many Actin Binding Proteins (ABPs) for its regulation. More than 200 faster translational and rotational dynamics that the water experiences around types of ABPs have been reported, each with a different binding and regulatory hydrophobic residues offsets the structural contribution to the entropy, and mechanism. We have channelled our focus on Actin Depolymerizing Factors overall a negligible change in water entropy is observed in the water layers (ADF) that makes a family of proteins, responsible for actin filament severing immediately surrounding the protein. and this property makes them extremely important during phagocytic cup for- mation. From our in silico analysis of the E. histolytica proteome, we found 10 2263-Plat proteins harbouring the ADF-homology domain. We have deciphered the high Acetylation of Actin K328 Contributes to a Loss in Tropomyosin-Mediated resolution crystal structure of a couple of them and the others are underway. Inhibition of Myosin Binding One of the proteins, called Ehcoactosin, interestingly was shown to tread on William M. Schmidt, D. Brian Foster, Anthony Cammarato. an off beaten track and does not depolymerize or sever the actin filaments. Dept Medicine, Johns Hopkins Univ Sch Med, Baltimore, MD, USA. Although belonging to the ADF family it rather stabilized the filamentous actin Lysine deacetylases (KDAC’s) catalyze the removal of acetyl groups from lysine (F-actin). EhTwinfilin contains two ADF-H domains and we were successful in side chains, while lysine acetylases (KAT’s) perform the reverse reaction. KDAC inhibition was shown to increase lysine acetylation of muscle proteins, and was determining the crystal structure of the N-terminal ADF domain. The unique- 2þ ness of this protein lies in the differential protein activity shown by the two do- associated with enhanced Ca -sensitivity in skinned fibers and increased relax- mains; N-terminal domain stabilized the F-actin whereas the C-terminal ation rate in isolated myofibrils. Interestingly, the KAT enzyme, PCAF, directly in vitro, domain depolymerized it. We are reporting these for the first time. Here we pre- acetylates myosin and actin has been implicated as a potential target of in vitro, sent a comparative study to understand the structural and functional differences PCAF. Therefore, we attempted to acetylate actin with PCAF, to deter- present in the many forms of the same ADF-homology domain proteins and mine its potential effect(s) on muscle function. PCAF underwent autoacetylation their role in phagocytosis. In this study we are reporting the key structural and bound to actin, yet failed to acetylate it. Chemical treatment of actin with ace- and functional differences observed in the mechanistic details of the same tic anhydride, however, increased lysine acetylation roughly 200-fold. Surpris- in vitro m ADF-H domain when present in different proteins. ingly, motility (IVM) of (100 g/ml myosin concentration) and tropomyosin affinity for acetylated F-actin were indistinguishable from unacety- 2261-Plat lated control. However, IVM of acetylated F-actin, performed at low myosin con- m Reconstitution of Dynamic Actin Cables with Tunable Lengths centration (12.5 g/ml) and in the presence of tropomyosin, revealed a significant < Luther W. Pollard, Salvatore L. Alioto, Mikeal V. Garabedian, 63% increase in motile filaments relative to unacetylated control (p 0.0001), Bruce L. Goode. suggesting that actin acetylation reduces tropomyosin-mediated inhibition of Biology, Brandeis University, Waltham, MA, USA. myosin binding. Since K326 and K328 have been reported as the most reactive in vivo The sizes and functions of many actin-based cellular structures, such as micro- lysines on actin, are acetylated , and pseudo-acetylation of both modulates muscle contraction, we assessed the effect of K326Q and K328Q pseudo- villi, stereocilia, filopodia and polarized actin cables, depend critically on the 2þ precise control of actin bundle length and turnover dynamics. Yeast cells offer acetylated actin on Ca -mediated thin filament activation via regulated IVM. 5 an ideal system in which to study principles of cytoskeletal length control, Although reconstituted thin filaments containing K326Q (pCa50 = 6.36 2þ 5 because they contain discrete linear actin cables that extend to well-defined 0.03) did not alter Ca -sensitivity relative to WT (pCa50 = 6.37 0.03), 2þ D lengths (3-7 mm), matching the cell compartment size, despite undergoing high- K328Q induced a significant leftward shift in Ca -sensitivity ( pCa50 = 0.19; < ly rapid turnover. Yeast is also simpler compositionally and more genetically p 0.0001), consistent with a loss in tropomyosin-mediated inhibition. The amenable compared to most animal cells. Over the past 30 years, the factors data indicate that actin K328 acetylation modulates contractile function, which in vivo involved in yeast actin cable formation have been identified, and many of these makes it a potential mechanism for regulating muscle performance . individual parts have been studied for their biochemical activities. Yet it has 2264-Plat remained unclear how this complex group of proteins works in concert to build Leiomodin and Tropomyosin, Binding at the Pointed End of the Thin Fil- a single actin-based structure of a specified length and architecture. Here, we aments used purified components to reconstitute the formation of yeast actin cables Dmitri Tolkatchev1, Garry E. Smith1, John R. Cort2, Gregory L. Helms3, that dynamically turnover and have steady-state lengths similar to those Alla S. Kostyukova1. in vivo observed . Cables are rapidly polymerized at one end by formins immo- 1Voiland School of Chemical Engineering & Bioengineering, Washington bilized on beads, in a profilin-dependent manner, and disassembled at the other State Univ, Pullman, WA, USA, 2Fundamental & Computational Sciences end by the combined actions of Cofilin, Coronin, and AIP1. Cable length is Directorate, Pacific Northwest National Laboratory, Richland, WA, USA, tuned by Tropomyosin, which decorates the sides of actin cables and antago- 3The Center for NMR Spectroscopy, Washington State Univ, Pullman, WA, USA. nizes the disassembly-promoting factors. Capping protein restricts actin poly- Leiomodin is an important actin-binding protein responsible for the thin filament merization to the formin-coated beads. Bundling proteins with different formation. Mutations in leiomodins were demonstrated to cause myopathies. The properties alter the geometry and dynamics of the cable networks. Together, spectrum of leiomodin functions remains a subject of an intensive debate. One of our results offer new mechanistic insights into the sculpting of actin cables the models proposes that leiomodin and its homolog tropomodulin modulate the through orchestrated assembly, turnover, and crosslinking. length of the thin filaments in muscle sarcomeres. The modulation is thought to function through competition between leiomodin and tropomodulin for the 2262-Plat pointed end of the thin filaments in a tropomyosin-dependent manner. We studied Structural, Kinetic, and Thermodynamic Response of Water to Mechani- complexes formed by peptides representing the tropomyosin-binding site of leio- cal Unfolding of Spectrin Repeats modin 2 (Lmod2s1) and the N-terminal coiled coil fragment of tropomyosin. By Sarah J. Moe, Torvin Rajala, Alessandro Cembran. using a combination of NMR and MDS methods, we determined the mode of Dept Chem/Biochem, Univ Minnesota Duluth, Duluth, MN, USA. Lmod2s1 binding to the N-terminus of tropomyosin. The structure places leiomo- Many common biophysical processes involve mechanical forces acting on and din at the pointed end of thin filaments. This finding provides compelling evi- between biomolecules. Among these processes, the mechanical unfolding of dence in favor of the competition hypothesis and has important implications proteins has been extensively studied with both experimental and theoretical for the role of leiomodin in forming thin filaments in muscle sarcomeres. approaches because it allows gaining insight in the mechanism of function of this class of proteins. Among the proteins that have been subject to mechanical Platform: Protein Assemblies/Enzyme Function, unfolding studies, spectrin repeats occupy a prominent place, as spectrins are the building block of many structural proteins that are subject to deformation. Cofactors & Post-translational Modifications II Although the response of spectrin repeats to external forces has been thor- oughly studied, little is currently known about the role that the solvent has in 2265-Plat the unfolding mechanism. In this work, we have investigated with steered mo- Hydroxylation of Type I Collagen: Effects on Fibrillar Structure and lecular dynamics simulations the mechanical unfolding of spectrin repeat 19 of Mechanics human dystrophin. We measured several observables to characterize spatially Alekhya A. Kandoor1, Michele Kirchner2, Vered Wineman-Fisher1, and temporally the response of water to protein unfolding in terms of its struc- Yujia Xu2, Sameer Varma1,3. tural, kinetic, and thermodynamic properties. Our results show that the 1Department of CMMB, University of South Florida, Tampa, FL, USA, response of water to protein unfolding is dictated by many factors, among 2Dept Chemistry, CUNY Hunter Col, New York, NY, USA, 3Department of which the nature of the exposed residues, the curvature of the protein surface, Physics, University of South Florida, Tampa, FL, USA. and the size of the protein chain play a major role. We also have determined Proline hydroxylation contributes to type I collagen assembly by increasing the that the exposure of hydrophobic residues results in a structural ordering of wa- thermal stabilities of constituent triple helices. Results from recent studies on ter around the protein, which should result in lower water entropy. Yet, the rat type I fibrils suggest another potential role of proline hydroxylation – since

BPJ 9439_9446 458a Wednesday, March 6, 2019 proline hydroxyls make up 21% of the hydrogen bonds between triple helices, phase separation of P-body component DCP1a, which is a part of the eukaryotic proline hydroxyls can also be expected to contribute to the structural integrity mRNA decapping complex. This dynamic phase separation occurs within sec- and the mechanical properties of fibrils. Here we investigate this hypothesis by onds, in contrast to stress granules that form over minutes, and is rapidly revers- carrying out comparative molecular dynamics studies of fibrils in the presence ible over the course of minutes. We show that the extent of DCP1a condensation and absence of hydroxylation. We investigate this in both rat and human type I is independent of the osmolyte used, is correlated with the degree of cellular vol- fibrils. The structure of the human type I fibril structure is constructed using ho- ume decrease, and is promoted by the trimerization domain of DCP1a. Other mology modeling, and its hydroxylation sites are determined using MS/MS multimeric proteins with valency of at least three, but not less, exhibit compara- sequencing. Expectedly, we find that in both human and rat fibrils, the presence ble condensation responses to osmotic compression. Together, these observa- of hydroxylation does alter the contact interface between triple-helices. Specif- tions are consistent with predictions from phase-separation theory and suggest ically, hydroxylation affects hydrogen bonding patterns, but in different ways that multimerization domains are programmed to mediate protein condensation in the two collagens, despite the two collagens sharing 94% sequence identity. in response to sudden cellular dehydration. We propose that self-interacting pro- However, in both rat and human fibrils, the altered local interactions have little teins spontaneously assemble to counteract the entropic challenge of cellular effect on macroscopic structural properties, including D-band lengths and gap- compression. These observations reveal an under-appreciated physiological overlap ratios. Additionally, hydroxylation only marginally increases Young’s context in which proteins undergo phase separation and unveil a hidden role moduli, although the effects of hydroxylation are more pronounced under large of conserved multimerization domains in post-transcriptional gene regulation (> 5%) strains. Overall, this study suggests that although local interactions be- and metabolic pathways that may predate modern cell biology. tween triple helices in fibrils are sensitive to hydroxylation and that there does exist some degree of plasticity in interfacial contacts between triple helices, the 2269-Plat hallmark macroscopic properties of fibrils are not governed by hydroxylation. Photonic Platform for Detailed Physical Characterization of Liquid Pro- tein Droplets 2266-Plat Gheorghe Cojoc1, Timon Beck1, Saeed Ahmed1, Titus Franzmann2, Superresolution Imaging of Amyloid Structures over Extended Times us- Paul Muller€ 1, Mirjam Schurmann€ 1, Raimund Schlu€bler1, Kyoohyun Kim1, ing Transient Binding of Single Thioflavin T Molecules Elisabeth Fischer-Friedrich1, Simon Alberti2, Jochen Guck1. 1 2 3 1 2 Kevin Spehar , Tianben Ding , Yuanzi Sun , Niraja Kedia , Jin Lu , 1Biotec, Dresden, Germany, 2MPI-CBG, Dresden, Germany. George R. Nahass3, Matthew D. Lew2, Jan Bieschke1,3. 1 Protein bodies are membrane-less organelles which form intracellularly by Biomedical Engineering, Washington University in St Louis, St Louis, MO, means of a liquid-liquid phase separation. Protein droplet formation can be USA, 2Electrical and Systems Engineering, Washington University in St 3 sometimes reversible,but frequently droplets undergo a phase transition. Louis, St Louis, MO, USA, Institute of Prion Diseases, UCL, , Mounting evidence suggests a direct connection between the phase transition United Kingdom. of protein droplets and diseases. In vitro assays such as droplet fusion assisted Oligomeric amyloid structures are crucial therapeutic targets in Alzheimer’s by optical tweezers,fluorescence recovery after photobleaching or particle and other amyloid diseases. However, these oligomers are too small to be tracking are commonly used to detect and quantify changes in the mechanical resolved by standard light microscopy. We have developed a simple and versa- properties. However, direct measurement of visco-elastic properties and decou- tile tool to image amyloid structures using Thioflavin T without the need for pling of the two is difficult and is based on many assumptions. covalent labeling or immunostaining. Dynamic binding of single dye molecules We present a novel photonic platform for selective droplet delivery into the generates photon bursts that are used for fluorophore localization on a nano- analysis region and their physical characterization. Our method combines a meter scale. Thus, photobleaching cannot degrade image quality, allowing dual-beam laser trap with quantitative phase imaging(QPI). In our in vitro as- for extended observation times. Super-resolution Transient Amyloid Binding says,we used phase separated fused in sarcoma(FUS) protein droplets. The me- (TAB) microscopy promises to directly image native amyloid using standard chanical properties of the FUS droplets can then be inferred from the change of probes and record amyloid dynamics over minutes to days. We imaged amyloid the droplet shape after the optical forces are applied, meanwhile QPI gives us fibrils from multiple polypeptides, oligomeric, and fibrillar structures formed information regarding protein concentration/distribution inside the droplets. b during different stages of amyloid- aggregation, as well as the structural re- Our preliminary results show that the mechanical properties of FUS droplets b modeling of amyloid- fibrils by the compound epi-gallocatechin gallate depend on the salt concentration and pH,but not on the protein concentration (EGCG). in the buffer solution. Higher protein concentration results in more and bigger 2267-Plat droplets,but with similar protein concentration inside. Tuning the power and In Vitro Study of the Effect of Insulin on Amyloid b-Protein Assembly and wavelength of the lasers,we can monitor the dissolving rate and stability of Toxicity the FUS droplet as function of the temperature and for different buffer condi- Kaho Long1, Thomas L. Williams2, Brigita Urbanc1. tions. Moreover, we can track changes, in a decoupled manner, of the viscosity 1Physics, Drexel University, Philadelphia, PA, USA, 2Clarivate Analytics, and elasticity over time. By applying oscillatory mechanical and light mediated Philadelphia, PA, USA. stress on the droplets,we can accelerate the process of phase transition. This in vitro Alzheimer’s disease (AD) is the primary source of dementia among elderly, for photonic platform can be used for detailed screening assays of protein which there is no cure. The leading amyloid hypothesis of AD posits that self- droplet properties and aging. assembly of amyloid b-protein (Ab) into oligomers triggers the AD pathology. Substantial evidence based on longitudinal studies also shows a strong correlation 2270-Plat between AD and type 2 diabetes mellitus, whereby disrupted insulin homeostasis Dissection of Protein Function Within a Bacterial Biomolecular Conden- is associated with an increased risk for AD. Although administration of intranasal sate by In Vitro Reconstitution insulin is explored as a potential therapy against AD, the molecular mechanisms Saumya Saurabh, Lucy Shapiro. of insulin activity in the brain remain unknown. We here investigate the effect of Developmental Biology, Stanford University, Stanford, CA, USA. insulin on Ab42 self-assembly in vitro using Thioflavin T fluorescence spectros- Recent work in micron-sized bacteria has highlighted the presence of membrane- copy and atomic force microscopy. We also examine the effect of insulin on the less microdomains that organize protein signaling complexes. In addition to well ability of Ab42 oligomers to disrupt the integrity of biomimetic lipid vesicles understood ‘‘lock and key’’ protein domains that lie at the core of cell signaling, we using the calcein release assay. Our results demonstrate that insulin attenuates are beginning to understand the role of unstructured protein bimolecular conden- both Ab42 fibril formation and the ability of Ab42 oligomers to disrupt mem- sates in intracellular communication. However, the molecular principles that orga- brane integrity in a concentration dependent manner. Our findings elucidate a nize chemical reactions in these domains are poorly understood. In order to plausible molecular basis for the observed correlations between AD and understand these principles, we have used a combination of in vitro reconstitution type 2 diabetes mellitus and offer a new perspective on therapeutic approaches and single-molecule fluorescence techniques to study one of the two cell pole mi- against AD. crodomains in the Gram negative bacterium, Caulobacter crescentus. Caulobacter divides asymmetrically into a sessile stalked cell and a flagellated swarmer cell. 2268-Plat Much of this asymmetric division is regulated through different signaling path- Multimeric Proteins Reversibly form Condensates Upon Osmotic ways that reside in microdomains at the two cell poles. Specifically, at the stalked Compression pole, two intrinsically disordered proteins, PopZ and SpmX, form a stable bimo- Ameya P. Jalihal. lecular condensate. PopZ recruits the lysozyme homolog SpmX, which further re- Cell and Molecular Biology, University of Michigan, Ann Arbor, MI, USA. cruits the kinase DivJ to establish the stalked cell identity. Reconstitution of this Hyperosmotic stress is known to induce the formation of cytosolic phase- signaling complex on supported lipid bilayers revealed that SpmX and PopZ separated structures called P-bodies in eukaryotes. Using live-cell fluorescence can form phase-separated droplets that sequester DivJ and control its kinase activ- microscopy we observed that hyperosmolarity induces rapid and reversible ity. Further, we have identified molecular determinants of SpmX and PopZ phase

BPJ 9439_9446 Wednesday, March 6, 2019 459a separation that control the material properties and enzyme kinetics in this conden- N-terminal where previous X-ray structures, transmembrane (TM) predicting sate. Our results underscore the relationship between IDR mediated interactions programs, and molecular dynamic (MD) simulations placed it. This is a ma- that govern the physical environment around signaling proteins, and demonstrate jor result that re-frames the nature of the lipid-protein interactions in MscS the applications of disordered scaffolds to modulate biochemical reactions in vivo. and forces us to reexamine years of experimental and computational data and reassess existing mechanistic gating models. In a second major observa- 2271-Plat tion, the structures also reveal that all available MscS crystallographic Effect of Resultant Dipole Moment on Mechanical Stability of Protein- models are in fact, incomplete: Detergent-solubilized MscS is missing an Peptide Complexes anchoring interfacial domain, comprised of the N-terminal end and three 1 2 1 3 Maksim Kouza , Anirban Banerji , Andrzej Kolinski , Irina Buhimschi , additional turns of TM1. In contrast, bilayer-embedded MscS extends >12 Andrzej Kloczkowski2. ˚ 1 2 A towards the outer leaflet of the bilayer when compared to X-ray structures. Faculty of Chemistry, University of Warsaw, Warsaw, Poland, Battelle The N-terminal domain (NTD) forms a novel fold, and we hypothesize, plays Center for Mathematical Medicine, Nationwide Childrens Hospital, 3 a significant role in channel activation. This was further confirmed an alanine Columbus, OH, USA, Center for Perinatal Research, Nationwide Childrens scan of the NTD assessed by cell survival assays together with high-speed Hospital, Columbus, OH, USA. pressure clamp electrophysiology. The present observations reshape our un- Protein-peptide interactions play essential roles in many cellular processes and derstanding of the mechanism of force-from-lipids gating and suggest that their structural characterization is the major focus of current experimental and allosteric interactions between TM segments play a fundamental role in theoretical research. Two decades ago, it was proposed to employ the steered mo- MscS gating. lecular dynamics to assess the strength of protein-peptide interactions1. The idea behind using steered molecular dynamics simulations is that the mechanical sta- 2274-Plat bility can be used as an efficient alternative to computationally highly demanding 2,3 Structures and Simulations of Membrane Adjacent Fragments of estimation of binding affinity and aggregation rate . However, mechanical sta- Protocadherin-15 bility defined as a peak in force-extension profile depends on the choice of the Pedro De-la-Torre1, Yoshie Narui1, Deepanshu Choudhary1, pulling direction. Here we propose an uncommon choice of the pulling direction Raul Araya-Secchi2, Marcos Sotomayor1. along resultant dipole moment vector, which has not been explored in simulations 1Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA, so far. Using explicit solvent all-atom MD simulations, we apply steered molec- 2Structural Biophysics, Section for Neutron and X-ray Science, Niels Bohr ular dynamics technique to probe mechanical resistance of protein-peptide sys- 4 Institute, University of Copenhagen, Copenhagen, Denmark. tem pulled along two different vectors . A novel pulling direction, along the The tip link, a fine filament that connects neighboring stereocilia of inner-ear resultant dipole moment vector, results in stronger forces compared to commonly hair cells, is made of two unique, non-classical cadherins essential for hear- used peptide unbinding along center of masses vector. Our results demonstrate ing. One half of the tip link is protocadherin-15 (PCDH15), which is that resultant dipole moment is one of the factors influencing the mechanical sta- comprised of 11 extracellular cadherin (EC) repeats followed by a mem- bility of protein-peptide complex. brane adjacent domain (MAD12). Here, we report the structures of pig Science 1) H. Grubmuller et al., 271, 997-999 (1996) PCDH15 EC10-12 and mouse PCDH15 EC9-MAD12, which revealed a J Chem Phys 2) M. Kouza et al., 148, 215106 (2018) bent EC9-10 configuration as well as parallel homodimerization mediated Physical 3) M. Kouza, A. Banerji, A. Kolinski, I. Buhimschi, A. Kloczkowski, by both EC11 and a unique MAD12 that adopts a ferredoxin-like fold. Chemistry Chemical Physics , 19, 2990-2999 (2017) Steered molecular dynamics simulations indicate that when force is applied Mole- 4) M. Kouza, A. Banerji, A. Kolinski, I. Buhimschi, A. Kloczkowski, to both ends of the protein, the bent EC9-10 linker fully extends before un- cules 23, 1995 (2018) folding of MAD12 occurs. Simulations also suggest that MAD12 is mechan- 2272-Plat ically weak and may unfold before tip-link rupture. In addition, sequence Coarse-Grained and Atomistic Simulations of the Mature HIV Capsid and analyses and structural modeling predict the existence of similar domains Related Restriction Factors in cadherin-23, protocadherin-24, and the ‘‘giant’’ FAT and CELSR cadher- Alvin Yu1, Barbie K. Ganser-Pornillos2, Owen Pornillos2, Gregory A. Voth1. ins, indicating that some of them may also exhibit MAD-induced parallel 1Chemistry, University of Chicago, Chicago, IL, USA, 2Univ Virginia, dimerization. Together, these results provide key information about the Charlottesville, VA, USA. elastic properties of the tip link and give insight into the architecture of The human immunodeficiency virus (HIV) uses enclosing capsid (CA) proteins the full-length PCDH15 homodimer. to package and release its genetic information into newly infected host cells. CA forms fullerene cones that compartmentalize viral RNA. TRIM5a is an 2275-Plat important antiretroviral restriction factor found in rhesus macaques that blocks Understanding Piezo1’s Relationship with Lipids 1 2 3 4 HIV infection. Recent cryo-electron microscopy analysis demonstrate cyto- Amanda Buyan , Charles D. Cox , Jonathan Barnoud , Boris Martinac , 5 1 plasmic TRIM5a self-assembles into hexagonally patterned nets that encage Siewert-Jan Marrink , Ben Corry . 1Res Sch of Biology, Australian National Univ, Acton, Australia, 2Victor mature capsids, with a symmetry and spacing that matches the underlying 3 CA lattice. In this work, we use atomistic simulations to probe structural and Chang Cardiac Res Inst, Sydney, Australia, University of Groningen, Groningen, Netherlands, 4Dept Molec, Victor Chang Cardiac Res Inst, thermodynamic properties of the HIV capsid, and top-down coarse-grained 5 modeling to understand the mechanisms of how TRIM5a recognizes and as- Darlinghurst, Australia, Dept Biophys Chem/Molecular Dynamics, Univ sembles on CA lattices. Groningen, Groningen, Netherlands. The sensations of touch and hearing, along with many other physiological pro- cesses, require cells to be able to sense and react to mechanical stimuli. One Platform: Mechanosensation way this is done is via membrane embedded mechanically gated channels. These channels can detect forces through deformation of the lipid bilayer, deemed the 2273-Plat ‘‘force-from-lipids’’ principle. Bacterial homologues, such as MscL and MscS, High-Resolution Structures of MscS in a Lipid Bilayer: Reinterpreting exemplify this principle and have been studied for the past couple of decades, and ‘‘Force from Lipids’’ Activation in Mechanosensitive Channels have contributed greatly to our understanding of mechanically gated channels. Bharat Reddy1, Allen Lu1, Navid Bavi1, Allen Hsu2, Mario Borgnia2, However, understanding the underlying molecular force sensing mechanisms, Eduardo Perozo1. and how similar bacterial and eukaryotic mechanosensitive channels are in terms 1Univ Chicago, Chicago, IL, USA, 2GISBL/DIR/NIEHS/NIH, Research of their gating mechanisms, remains an open question. Triangle Park, NC, USA. The recent discovery and structure elucidation of the first eukaryotic mechan- Prokaryotic mechanosensitive (MS) channels are some of the best character- ically gated channels, named the PIEZO family, allows for the mechanisms of ized force-activated ion channels. Given the critical role of lipid-protein in- mechanical gating to be studied in higher organisms. Since their discovery, teractions in defining force through lipid channel gating, this is a question of PIEZO channels have been implicated in many cellular processes, but the paramount importance in the field. We have determined several high- gating mechanism and the role that lipids play in PIEZO’s mechanics remain resolution cryo-EM structures of MscS in nanodisc (ND) environments that elusive. To this end, we are using a combination of electrophysiology and mo- varied the membrane thickness, charge, size, and rigidity. Compared to pre- lecular simulations to understand protein-lipid interactions between Piezo1 and viously solved crystal and low-res EM structures, the key observation from relevant lipids in model mammalian bilayers, and ultimately the role that lipids our MScS ND structures is the unexpected location of the lipid bilayer in have on Piezo1’s activation. We are able to show that piezo has specific inter- relation to MscS TM segments: We find the bulk of the ND-containing lipid actions with a number of membrane components that likely play a role in medi- annulus surrounding the channel to be almost a full bilayer leaflet towards the ating the bilayer-protein interaction.

BPJ 9439_9446 460a Wednesday, March 6, 2019

2276-Plat Piezo1 significantly reduced the nuclear shrinkage under shear stress. Activa- Dietary Fatty Acids Fine-Tune Piezo1 Activity tion of Piezo1 with Yoda1 caused similar nucleus shrinkage without shear Luis O. Romero1, Alejandro Mata-Daboin1, Andrew Massey2, stress. These results demonstrate that Piezo1 channel is accountable for trans- Francisco J. Sierra Valdez3, Chauhan C. Subhash2, Julio F. Cordero-Morales1, mitting the shear force input to nuclei via Ca2þ signaling. To assess the relative Valeria Vasquez1. contributions of Ca2þ and cytoskeleton reorganization on force transduction, 1Physiology, UTHSC, Memphis, TN, USA, 2Pharmaceutical Sciences, we examined F-actin reorganization under shear stress and static conditions, UTHSC, Memphis, TN, USA, 3Science, ITESM, Memphis, TN, USA. and showed that reorganization is not necessary for the nucleus shrinkage. Mechanosensitive ion channels rely on membrane composition to transduce These results emphasize the role of the mechanosensitive channels as primary physical stimuli into electrical signals. The Piezo1 channel is a mechanosen- transducers in force transmission to the nucleus. sitive channel that regulates physiological processes, including vascular architecture and remodeling, cell migration, and erythrocyte volume. Muta- tions in Piezo1 are associated with dehydrated hereditary stomatocytosis, a 2279-Plat hemolytic anemia characterized by increased cation permeability and dehy- Structural Relationship between the Putative Hair Cell Mechanotransduc- drated erythrocytes. While it is widely agreed that Piezo1 is activated by tion Channel TMC1 and TMEM16 Proteins 1 2 membrane tension, the identity of the membrane lipids that modulate Piezo1 Angela Ballesteros Morcillo , Maria Cristina Fenollar-Ferrer , 1 function and the molecular mechanisms by which they do so remain un- Kenton J. Swartz . 1Molecular Physiology and Biophysics Section, NINDS NIH, Bethesda, MD, known. Dietary fatty acids are among the membrane lipid components that 2 regulate ion channel function. The most common fatty acids in membrane USA, NIDCD-NIMH, Bethesda, MD, USA. lipids have 14 to 22 carbon atoms and carry one unsaturation, and as Our senses of touch, hearing, balance, and proprioception depend on mechan- many as six double bonds. These chemical features confer cells the capacity ically gated ion channels, which transduce mechanical energy into electrical to control membrane thickness and fluidity. We use lipidomics and electro- signals that are transmitted to the brain. Previous studies on the mechanisms physiology to demonstrate that Piezo1-mediated mechano-currents are of hearing have elucidated the biophysical properties of the mechanotransduc- modulated by dietary fatty acids in different cell types. We found that tion (MET) channel essential for hearing, yet its molecular identity and struc- margaric acid (MA), a saturated fatty acid, inhibits Piezo1 activation cur- ture remain elusive. The trantasmembrane channel-like 1 protein (TMC1) rents, whereas u-3 and u-6 polyunsaturated fatty acids (PUFAs) modify localizes to the site of the MET channel, interacts with the tip-link responsible its inactivation. Differential scanning calorimetry and atomic force for mechanical gating, and genetic alterations in TMC1 alter MET channel microscopy experiments show that membranes enriched in MA are more properties and cause deafness, supporting the hypothesis that TMC1 forms rigid and display higher bending stiffness than to those containing PUFAs. the MET channel. We generated a model of TMC1 based on X-ray and cryo- Our findings demonstrate that saturated and polyunsaturated fatty acids EM structures of TMEM16 proteins, revealing the presence of a large cavity contained in the plasma membrane modulate the mechanical response of near the protein-lipid interface that also harbors two TMC1 mutations Piezo1. (hTMC1 p.M418K and p.D572N/H) that cause autosomal dominant hearing loss, suggesting that it could function as a permeation pathway. We also find 2277-Plat that hair cells are permeable to 3 kDa dextran labeled with Texas Red, and Enantiomeric Ab Peptides Inhibit the Fluid Shear Stress Response of that dextran permeation requires TMC1/2 proteins and functional MET chan- PIEZO1 nels, supporting the presence of a large permeation pathway and the hypothesis Philip A. Gottlieb1, Mohammed M. Maneshi1, Frederick Sachs1, that TMC1 is a pore forming subunit of the MET channel complex. Susan Z. Hua2. 1Dept Physiol/Biophys, State Univ New York Buffalo, Buffalo, NY, USA, 2Dept Mech & Aerospace Eng, State Univ New York Buffalo, Buffalo, NY, 2280-Plat USA. Cysteine Substitution Reveals the Pore-Forming Region of TMC1 in Hair Using precision fluid shear stress as a stimulus on a cell line overexpressing Cell Sensory Transduction Channels 1 2 1 1 PIEZO1, we show that Ab peptide monomers inhibit PIEZO1 at fM to pM con- Bifeng Pan , Nurunisa Akyuz , Xiao-Ping , Yukako Asai , 1 3 2 1 centrations. The Ab aggregates were much less potent. The effect of Abson Carl Nist-Lund , Kiyoto Kurima , Bruce Derfler , Bence Gyo¨rgy , 2 4 4 PIEZO gating did not involve peptide-protein interactions since the D and L Walrati Limapichat , Sanket Walujkar , Lahiru Wimalasena , b Marcos Sotomayor4, David Corey2, Jeffrey R. Holt1. enantiomers had similar effects. A peptides co-localized with PIEZO1 as 1 b Otolaryngology, Harvard Medical School/Boston Children’s Hospital, seen with a fluorescent derivative of A and a fluorescently tagged PIEZO1. 2 b Boston, MA, USA, Neurobiology, Harvard Medical School, Boston, MA, To better understand the inhibitory effects of A , we examined the effects in 3 4 a wound healing assay. Simply over-expression of PIEZO1 in HEK293 cells USA, Otolaryngology, NIH/NIDCD, Bethesda, MD, USA, The Ohio State increased cell migration velocity 10-fold, and both enantiomeric Ab peptides University, Colubmus, OH, USA. and GsMTx4 inhibited migration. We examined the correlation of PIEZO1 Molecular identification of ion channels that mediate sensory transduction function with the tension in F-actin using a genetically encoded fluorescent has been a major focus in the field of sensory biology for many years, yet, stress probe. Ab peptides increased resting stress in F-actin, correlated with the proteins that form sensory transduction channels required for hearing the Ab block of PIEZO1-mediated Ca2þ influx. Ab inhibition of PIEZO1 in and balance have not been definitively identified. TMC1 and TMC2 have the absence of stereospecific peptide-protein interactions shows that Ab pep- been proposed as possible pore-forming subunits, but the pore region itself tides modulate both cell membrane and cytoskeletal mechanics to control has not been identified. We generated 17 unique cysteine substitutions in PIEZO1-triggered Ca2þ influx. TMC1 and packaged the mutant sequences into AAV2/1 vectors. The vectors were injected into the inner ears of P1 mice that lacked expression of endog- 2278-Plat enous Tmc1 and Tmc2. Injected ears were dissected at P7 and the organ of Piezo1 Mediated Ca2D Signaling Causes Nuclear Shrinkage under Fluid Corti was cultured for an additional 8 to 10 days. We used rapid hair bundle Shear Stress deflections and the whole-cell, tight-seal technique to record sensory trans- Deekshitha Jetta1, Philip A. Gottlieb2, Frederick Sachs2, Susan Z. Hua3. duction currents from 566 inner hair cells. We applied four different cysteine 1Dept of Mech Eng, University at Buffalo, Buffalo, NY, USA, 2Dept Phys modification reagents methanethiosulfonate (MTS) to native mouse sensory and Biophys, University at Buffalo, Buffalo, NY, USA, 3Dept Phys and hair cells. We identified thirteen TMC1 residues that altered the biophysical Biophys/Mech Eng, University at Buffalo, Buffalo, NY, USA. properties of hair cell mechanosensory transduction. Five residues were Nuclei of cells respond to local mechanical cues with changes in their size, rapidly modified by MTS reagents to confer changes in whole-cell current morphology, and motility, to maintain tissue homeostasis and proper functions. amplitudes. Three sites examined in detail, caused changes in single- Previous studies indicated that the external forces are coupled to nuclei through channel current amplitudes. Eleven sites altered calcium selectivity and the surrounding cytoskeleton network linked to the nuclear envelope, but the cysteine substitution at one site blocked current almost entirely. The data molecules that directly interact with outside environments are unclear. Here support a revised TMC1 topology with ten transmembrane domains and iden- we show that the fluid shear stress applied to MDCK cells caused the nucleus tify four TMC1 transmembrane domains that line the permeation pathway of to shrink through a Ca2þ dependent signaling pathway. Inhibiting Ca2þ perme- mechanosensory transduction channels in mammalian auditory and vestibular able mechanosensitive Piezo1 channels, with the specific inhibitor GsMTx4, hair cells. We conclude TMC1 is a major pore-forming subunit of the hair eliminated the nuclear changes. Piezo1 knockdown with miRNA that targets cell sensory transduction channel.

BPJ 9439_9446 Wednesday, March 6, 2019 461a

Symposium: Membrane Organization and and experiments. Molecular dynamics (MD) simulations of an isolated subunit of the pore domain of the Kv1.2 and KcsA channels in a lipid bilayer were Sculpting by Proteins carried out, followed by a Markov State Model (MSM) analysis. The results show that the monomer of these channels can adopt a very wide range of con- 2281-Symp formations while the two transmembrane (TM) helices and the pore helix Reverse Topology Membrane Scission by the ESCRTs retain their secondary structure. This view is supported by NMR spectroscopy James H. Hurley. showing that the TM helices of single monomers of the KcsA channel Molecular and Cell Biology, University of California, Berkeley, Berkeley, embedded in nanodisc or bicelle are partially disordered. A KcsA mutant CA, USA. construct with a disulphide bridge engineered between the two TM helices The narrow membrane necks formed during viral, exosomal and intra- (A29C-A109C) shows an HSQC spectrum with well-dispersed peaks and endosomal budding from membranes, as well as during cytokinesis and limited heterogeneity on NMR time-scale, indicating that the monomer can related processes, have interiors that are contiguous with the cytosol. be locked into a ‘‘native-like’’ conformation by reducing the disorder of the Severing these necks involves action from the opposite face of the membrane 2 TMs helices. The refolding and assembly of the tetrameric channel in lipo- as occurs during the well-characterized formation of coated vesicles. This somes is examined using a gel-based refolding assay and fluorescent energy ‘‘reverse-topology’’ membrane scission is carried out by the endosomal sort- transfer (FRET) experiments. The results suggest that the wild-type monomers ing complex required for transport (ESCRT) proteins, which form filaments, are partially disordered in the bilayer, with a high proclivity to misfold due to flat spirals, tubes and conical funnels that are thought to direct membrane re- stable non-native helical tetramerization, and that re-folding is limited by a modelling and scission. Their assembly, and their disassembly by the slow concentration-independent unimolecular event. We speculate that the ATPase vacuolar protein sorting-associated 4 (VPS4) have been intensively slow step could correspond to the re-orientation of the monomers and the studied, but the mechanism of scission has been elusive. To probe the bio- insertion of the pore helix into a disordered tetrameric assembly of 8 TM physical mechanism of membrane scission, we encapsulated a minimal helices. ESCRT module consisting of ESCRT-III subunits Snf7, Vps24, and Vps2, þ and the AAA ATPase Vps4 such that membrane nanotubes reflecting the 2284-Symp correct topology of scission could be pulled from giant vesicles. Upon Capturing Sequential Steps of Dynamin-Mediated Fission by Cryo-EM ATP release by photo-uncaging, this system was capable of generating forces Jenny E. Hinshaw1, Leopold Kong1, Kem A. Sochacki2, Huaibin Wang1, within the nanotubes in a manner dependent upon Vps4 catalytic activity and Bertram J. Canagarajah1, Andrew D. Kehr1, William J. Rice3, Vps4 coupling to the ESCRT-III proteins. Imaging of scission revealed Snf7 Marie-Paule Strub2, Justin W. Taraska2. and Vps4 puncta within nanotubes whose presence followed ATP release, 1LCMB, NIH, Bethesda, MD, USA, 2NHLBI NIH, Bethesda, MD, USA, correlated with force generation and nanotube constriction, and preceded 3New York Structural Biology Center, Simons Electron Microscopy Center, scission. Implications for the scission mechanism and remaining open ques- New York, NY, USA. tions will be discussed. Dynamin, a large GTPase, mediates membrane fission by assembling around and cleaving the necks of budding vesicles. The molecular details of 2282-Symp dynamin-driven membrane scission, however, remain elusive. Here, we report Membrane Curvature and the ABC Transporter BmrA: A Yin & Yang a 3.75 A˚ resolution cryo-EM structure of the constricted, membrane- Story associated helical polymer of human dynamin-1 in the GMPPCP bound state. Patricia M. Bassereau1, Ajay K. Mahalka1, Su-Jin Paik1, Giovanni Manzi1, Compared to the soluble tetramer, membrane-associated dynamin self- Andrew Callan-Jones2, Daniel Levy1. assembles across the helical rungs via the GTPase domain, and interacts 1PhysicoChimie Curie Lab., Institut Curie, Paris, France, 2Laboratoire with the lipid membrane with its Pleckstrin homology domain. These differ- Matie`re et Syste`mes Complexes, University Paris Diderot, Paris, France. ences translate to a severely bent bundle signaling element (BSE), which con- Matching protein shape to membrane curvature provides a possible targeting nects the GTPase domain with the rest of the protein. The BSE conformation mechanism for membrane proteins. However, how much membrane curvature is asymmetric across the inter-rung GTPase interaction, and is unique can affect function and reciprocally, how protein conforma- compared to all known nucleotide bound states of dynamin. Mutations that tional change modulates its localization has not been investigated. We have disrupt the BSE kink impaired endocytosis but not localization of the protein. studied BmrA, a bacterial homologue of the human ABC (ATP Binding Furthermore, the cryo-EM structure refines the membrane-bound orientation Cassette) transporter P-glycoprotein, to understand the feedback between pro- of the dynamin stalk, which mediates most of the oligomeric interactions tein function, conformation and the physicochemical properties of the sur- that were validated by cell-based assays, and rationalizes previously published rounding membrane including lipid composition and membrane curvature. biochemistry. In addition, the new model also fits well into the 10 A˚ angstrom Upon ATP binding and hydrolysis, BmrA switches conformation between an resolution cryo-EM map of a super-constricted GTP-bound dynamin polymer, apo- and a post-hydrolytic state. The protein was reconstituted in small and gi- confirming that the new interfaces are sufficient to bring the lipid bilayer close ant liposomes (GUVs). We could first demonstrate with small liposomes that to the hemi-fission state. These results reveal molecular snapshots of the bio- different negatively charged and PE lipids stimulate the protein ATPase activ- logically active form of dynamin during the fundamental process of mem- ity. Furthermore, we showed that the activity is reduced by at least two-fold brane constriction. when decreasing the diameter of the liposomes from 150 nm down to 30 nm. Next, from the analysis of fluorescent proteins’ enrichment in nanotubes pulled Symposium: Molecular and Transcriptional from GUVs containing BmrA at surface fraction z 1%, we could demonstrate that the trans-membrane domain of BmrA exhibit 2 contrasted spontaneous cur- Regulation of Cardiac E-C Coupling 1 vature Cp: a high positive Cp in the apo-state, and a negative Cp (1/7.5 nm ) in the post-hydrolytic state. In the apo-state, BmrA sorting kinetics is very slow 2285-Symp but leads to a gradual constriction of the tube at constant membrane tension, Acute Loss of cMyBP-C Induces Auto-Oscillatory Contractions in Per- down to tube radius z 30 nm and an enrichment factor of the order of 30. Even- meabilized Cardiomyocytes: Implications for Reverse E-C Coupling? tually, analysis of protein sorting in the presence of ATP suggests that BmrA Samantha P. Harris. remains a large fraction of its cycle in the post-hydrolytic state. Globally, Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA. this shows that localization of this type of proteins in cell membranes should Mutations in cardiac myosin binding protein-C (cMyBP-C) are a frequent cause strongly depend on their activity, which itself is modulated by membrane of hypertrophic cardiomyopathy (HCM), affecting 1 in 500 and the most com- shape. mon cause of sudden cardiac death in young people. Unlike mutations in myosin, the vast majority of mutations in cMyBP-C are truncation mutations 2283-Symp that lead to reduced cMyBP-C expression (haploinsufficiency). However, the Structural Dynamics of Monomer in a Membrane mechanism(s) by which loss of cMyBP-C affects contraction and how muta- Environment and Tetrameric Assembly tions in sarcomeric proteins ultimately lead to arrhythmias are not well under- Benoit Roux, Kevin Song, Young Hoon Koh, Eduardo Perozo, stood. Here we investigated effects of cMyBP-C loss using a novel ‘‘cut and Tobin R. Sosnick. paste’’ approach in detergent permeabilized myocytes from gene-edited Department of Biochemistry and Molecular Biology, University of Chicago, ‘‘Spy-C’’ mice developed exclusively in our lab. Spy-C mice express Chicago, IL, USA. cMyBP-C with a TEV protease site and a ‘‘SpyTag’’ sequence embedded be- The folding of a potassium channel monomer and its assembly into a tetramer tween domains C7 and C8. Permeabilized Spy-C myocytes were first treated was investigated using a multi-disciplinary approach combining computations with TEV protease to ‘‘cut’’ cMyBP-C and then recombinant C0-C7 domains

BPJ 9447_9453 462a Wednesday, March 6, 2019 were ‘‘pasted’’ back via ‘‘SpyCatcher’’ which covalently binds SpyTag. Re- compromised the gain of E-C coupling. In contrast, we found that the sults showed that similar to genetic knockout of cMyBP-C, acute loss of LCC-RyR signaling efficiency and the E-C coupling gain were increased cMyBP-C after TEV treatment caused an apparent increase in cross-bridge significantly during hibernation in ground squirrels. The structural consoli- 2þ cycling rates (ktr) and reduced Ca sensitivity of tension. Strikingly, there dation of LCC-RyR signaling apparatus was in parallel with increased was also a dramatic increase in auto-oscillatory contractions propagated across expression of JPH2 and caveolin-3 (CAV3), the interaction between which multiple sarcomeres. Oscillations were especially evident at sub-maximal anchors the SR to TTs. Interestingly, the promoters of both JPH2 and CAV3 2þ [Ca ] near the pCa50 of force generation and were sustained for seconds to mi- exhibited binding sites for the serum response factor (SRF), which initiated nutes despite conditions of constant Ca2þ. Oscillations abruptly ceased upon JPH2 and CAV3 transcription by binding the transcription co-factor myocar- ligation of new recombinant cMyBP-C N’-terminal domains via SpyCatcher. din. During hibernation, the expression of myocardin was increased while Results suggest a previously unrecognized role of cMyBP-C to dampen me- that of microRNA-24, which mediates post-transcriptional suppression of chanical activity originating from contracting sarcomeres and raise the JPH2 expression, was decreased. Adenovirus-directed overexpression of intriguing possibility that loss of cMyBP-C through haploinsufficiency could myocardin, but not that of SRF, increased JPH2 and CAV3 expression, elicit mechanical feedback that influences electrical activity through reverse improved LCC-RyR signaling efficiency and enhanced E-C coupling gain E-C coupling in intact cardiomyocytes. This work supported by NIH in ground squirrel cardiomyocytes. The myocardin-mediated conjunct regu- R01HL080367 and AHA IRG 17IRG33411051. lation of JPH2/CAV3 transcription explained the cardiac specificity of JPH2 expression, and revealed a stoichiometry-optimized mechanism for E-C 2286-Symp coupling regulation. This finding not only elucidated the mechanism for cBIN1: From T-Tubule Folds to Dyad Organization, to Microparticles and maintained cardiac contractility in hibernation, but also provides new ideas Clinical Use for studying the ‘‘loss-of-function’’ remodeling in heart diseases, such as Robin Shaw. heart failure. Smidt Heart Institute, Cedars-Sinai Medical Center and UCLA, Los Angeles, CA, USA. Platform: Protein Dynamics and Allostery II BIN1 is a membrane curvature protein which is richly spliced. We have identified a cardiac muscle splice variant (BIN1þ13þ17) now named 2289-Plat cBIN1. In the past decade, it has been found that cBIN1 is responsible for pH-Driven Conformational Reorganization of Proteins: NMR Spectros- trafficking to T-tubules as well as organizing microfolds copy Study with Buried Lys Residues of cardiac T-tubule membrane. The folds affect the local flow of ions and Christos M. Kougentakis1, Ananya Majumdar2, Jamie L. Schlessman3, help arrange cardiac dyads between L-type calcium channels and ryanodine Bertrand Garcia-Moreno1. receptors. In heart failure, cBIN1 is transcriptionally decreased, affecting 1Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA, T-tubule architecture, and negative impacting calcium transients as well 2Biomolecular NMR Center, Johns Hopkins University, Baltimore, MD, as cardiac function. We recently explored the possibility that the cBIN1- USA, 3Chemistry Deptartment, US Naval Academy, Annapolis, MD, USA. microfolds are externally released from cardiomyocytes. Using electron The pH sensitivity of proteins is essential for biological energy transduction microscopy imaging with immunogold labeling, we found that cBIN1 exists and for cellular and physiological homeostasis. The thermodynamic basis of in membrane vesicles about 200 nm in size, which is consistent with the pH effects in proteins is well understood but detailed mechanistic under- size of microparticles (MPs). In mice with cardiac-specific heterozygous standing of how changes in pH drive conformational reorganization in pro- Bin1 deletion, cBIN1-MPs are appropriately reduced in plasma. In human teins is not. This was addressed systematically using NMR spectroscopy to plasma, cBIN1 detection by an enzyme-linked immunosorbent assay characterize pH-dependent conformational changes coupled to the ioniza- (ELISA), is decreased in humans with heart failure, a condition with tion of residues buried in the hydrophobic core of a protein. The study reduced cardiac muscle cBIN1. The clinical utility is significant. A cBIN1 used 25 variants of staphylococcal nuclease with buried Lys residues. The Score (CS) based on plasma cBIN1 levels was studied in patients with heart pKa values of 19 of 25 of these internal Lys residues are depressed relative failure. We find that CS outperforms current biomarkers for the diagnosis of to the normal pKa of Lys in water, some by as much as 5 pH units. This shift heart failure and also can accurately predict future hospitalization in heart in pKa ensures that the ionizable moiety stays neutral at pH values below failure patients. The data indicate that cBIN1 is released from T-tubule 10 when it is buried, consistent with what is expected from the unfavorable membrane in MPs, and that based CS as a blood test can serve as a clinical transfer of a charge from water to a hydrophobic environment. In most biomarker of cardiomyocyte remodeling, aiding in the management of heart cases, ionization of the buried Lys shifts the conformation of the protein failure patients. into a state where the charged Lys can contact water. This study contributes a detailed description of the location, amplitude and time scale of 2287-Symp reorganization coupled to the ionization of buried Lys residues. The struc- Regulation of the RyR2 Calcium Release Channel by SPEG tural details of the conformational response were characterized with back- Xander H.T. Wehrens. bone chemical shift perturbation analysis, titrations of the buried Nz Cardiovascular Research Institute, Baylor College of Medicine, Houston, atom, and in some cases even X-ray crystallography. At least in this protein, TX, USA. the anomalous pKa values of buried groups are governed by the propensity It is well established that altered sarcoplasmic reticulum (SR) Ca handling of the protein to reorganize. These results demonstrate that computational plays a key role in the pathogenesis of heart failure and cardiac arrhythmias. modeling of pH dependent processes in proteins will require prediction Altered post-translational regulation of the (RyR2) is of alternative conformational states and accurate calculation of free en- thought to promote aberrant SR Ca release; however, it remains controversial ergies; this remains a significant challenge even under the most favorable which kinases and phosphatases underlie disease-associated changes. Recent circumstances. studies from our lab have demonstrated that ‘striated muscle preferentially ex- pressed gene’ (SPEG) - a serine/threonine protein kinase - binds to RyR2 and 2290-Plat regulates its activity. In this presentation, the effects of SPEG on RyR2 and SR Rhodopsin Hydration Dynamics Studied by Solid-State Deuterium NMR Ca handling will be discussed. Spectroscopy Nipuna Weerasinghe1, Suchitranga M.D.C. Perera1, Trivikram R. Molugu1, 2288-Symp Andres M. Salinas1, Michael F. Brown1,2. Conjunct Upregulation of Junctophilin-2 and Caveolin-3 Transcription 1Chemistry and Biochemistry, Univ Arizona, Tucson, AZ, USA, Enhanced Excitation-Contraction Coupling Efficiency in Hibernating 2Department of Physics, University of Arizona, Tucson, AZ, USA. Ground Squirrels The physiological properties of proteins strongly depend on the dynamics of Rong-Chang Li, Lei Yang, Yi-Chen Li, Bin Xiang, Li-Peng Wang, water in the hydration shell. Knowledge of hydration water dynamics is vital Xiao-Ting Wang, Jing-Hui Liang, Shi-Qiang Wang. for comprehending protein function [1]. Developing methods for the study Peking University, Beijing, China. of hydration dynamics of membrane proteins such as GPCRs is crucial for Cardiac excitation-contraction (E-C) coupling is controlled by the signaling pharmaceutical applications. We choose rhodopsin as a prototype for our in- between L-type Ca2þ channels (LCCs) in the cell membrane/T-tubules vestigations of GPCRs, where the role of water in its activation mechanism (TTs) and ryanodine receptor (RyR) Ca2þ release channels in the sarco- has been explored using quasi-elastic neutron scattering (QENS) methods plasmic reticulum (SR). In heart failure, decreased expression of [2]. First, we developed methods for generating powdered samples, notable junctophilin-2 (JPH2) decreased the efficiency of LCC-RyR signaling and for exceptionally high protein content while retaining photochemical

BPJ 9447_9453 Wednesday, March 6, 2019 463a functionality. The powdered GPCR samples are suitable for biophysical ex- experimental free energies affirm an accurate and robust approach. Thus, periments to studying the dynamic process of protein activation [3], where alchemical free energy calculations provide a powerful tool for the analysis the protein is hydrated using vapor phase absorption in a controlled manner. of correlated amino acid mutations and their interaction over large distances. We hypothesized that the internal protein dynamics (b-fluctuations) are In fact, the access to accurate mutation free energies and nonadditivities coupled to the solvent shell while large-scale protein domain motion (a-fluc- potentially enables monitoring of the involved interaction pathway throughout tuations) are coupled to the bulk solvent. To gain a comprehensive hold on the entire protein. functional water interactions, we studied rhodopsin hydration shell dy- namics using solid-state 2H-NMR spectroscopy at various hydration levels 2293-Plat and temperatures [4]. From the spectral features at 200 K, we established Towards Comprehensive Control and Design of Targeted Signalling in two dynamically distinct hydration regions. The broad quadrupolar splitting Allosteric Regulation of Protein Activity about 180 kHz is due to bulk water; however, the anisotropic narrow compo- Enrico Guarnera1, Wei-Ven Tee1,2, Zhen Wah Tan1, Igor N. Berezovsky1,2. nent is a result of water molecules dynamically perturbed by rhodopsin. 1Bioinformatics Institute, A*STAR, Singapore, Singapore, 2Department of Additional solid-state 2H-NMR relaxation investigations of molecular dy- Biological Sciences, National University of Singapore, Singapore, Singapore. namics of the rhodopsin-bound water shell are in progress. Our future focus We developed a computational approach combining protein harmonic model is on a comparative study of water dynamics in the light activated state and with statistical mechanical formalism in a perturbation-based approach, which the dark state. [1] M.T. Rao et al. (2009) J.Phys.Chem.B 113, 6994. [2] allows one to evaluate per-residue allosteric free energy as a result of ligand Shresta et al. (2016) J.Phys.Chem.Lett. 7, 4130. [3] S.M.D.C. Perera et al. binding and/or mutations. The model was implemented in the AlloSigMA (2016) J.Phys.Chem.Lett. 7, 4239. [4] T.R. Molugu et al. (2017) Chem.Rev. web-server that provides an interactive framework for estimating allosteric 117, 12087. free energy, can help to find potential latent regulatory exosites and candidates for allosteric mutations. Because of the critical role of global protein dy- 2291-Plat namics in allosteric signaling, we hypothesized reversibility of allosteric Using Histone H1 Derived Peptides to Investigate Binding Affinity and communication, according to which allosteric sites can be detected via the Inter-Domain Dynamics in Human Pin1 perturbation of the functional sites. Validating the ‘‘reversibility hypothesis’’, Dinusha Jinasena1, Jerrano Bowleg1, Robert Simmons1, Yue Zhang2, we also show that, in addition to known allosteric sites, perturbation of func- Steven R. Gwaltney1, Nicholas C. Fitzkee1. tional sites unravels rather extended protein regions, which can host latent 1Department of Chemistry, Mississippi State University, Mississippi State, regulatory exosites. These protein parts that are dynamically coupled with MS, USA, 2National Cancer Institute, National Institutes of Health, functional sites can also be used for inducing and tuning allosteric communi- Frederick, MD, USA. cation. Defining generic characteristic of the allosteric effect of mutation, the Pin1 is an essential Peptidyl-prolyl isomerase (PPIase) that catalyzes cis- modulation range, we build Allosteric Signaling Maps (ASMs) of proteins on trans prolyl isomerization in proteins containing pSer/pThr-Pro motifs. It the basis of residue-by-residue scanning of mutations. ASMs allow one: (i) to has an N-terminal WW domain that targets pSer/Thr-Pro motifs and a C-ter- establish a relationship between mutations allosterically modulating protein minal PPIase domain that catalyzes isomerization. In this study, we report a activity and those that mainly change the protein stability; (ii) to observe a novel series of chimeric peptides that has been designed to optimize binding role of domain/subunit in protein allosteric communication via the distance- across the inter-domain interface of Pin1. The peptide series was designed dependent mode switching in allosteric signaling; (iii) to complement and based on the binding site of the Pin1 and optimal peptide sequences analyzed tune already existing allosteric signaling and to design new elements of in earlier studies. Specifically, we started from a sequence derived from the regulation. human Histone H1.4 sequence (KATGAApTPKKSAKW). Then, we de- signed a series of chimeric peptides based on this H1.4 sequence. NMR ti- 2294-Plat trations were performed for each peptide using both full-length Pin1 as Integration of an Electrostatic Network and Disorder-to-Order Transi- well as the WW domain alone. An analysis of KD values reveals complexity tions in Protein Allostery in the energetics of interaction between the peptide substrate, the PPIase Riya Samanta, Jingheng Wang, Dorothy Beckett, Silvina Matysiak. domain, and the WW domain. 15N relaxation and residual dipolar couplings University of Maryland College Park, College Park, MD, USA. (RDCs) were used to monitor the degree to which peptide binding induced Although allostery-in which action at one site alters the function at another inter-domain interactions. When combined with molecular simulations, our site -is integral to many biological regulatory processes, the molecular mech- results suggest a structural basis for how substrate binding can alter the anism of this phenomenon still eludes our understanding. Transcription inter-domain dynamics. Finally, we investigated whether our chimeric se- repression by E. coli repressor, BirA, is allosterically modulated by 1 1 quences could alter catalysis (kex)using H- H EXSY NMR experiments. activation of dimerization via binding of the small molecule effector bio- Interestingly, no relationship was found between kex and either peptide affin- tinoyl-5’-AMP. Previous studies have shown that disorder-to-order transi- ity or inter-domain interaction, suggesting a lack of allosteric control for this tions in loops on the distant dimerization and effector binding surfaces series of peptides. Thus, while our results suggest that peptide binding can communicate in BirA allostery. We hypothesize that an electrostatic network alter the interaction between the PPIase and WW domains, altering the is key to this communication. In this work, combined experimental and inter-domain interaction by itself does not appear to modulate catalysis in computational methods have been applied to investigate the role of this the PPIase domain. network in BirA allostery. Energy Network Analysis predicts that the elec- trostatic network residues participate in a larger allosteric network, while 2292-Plat ITC measurements support network formation in solution upon bio-5’- Thermodynamic Coupling - Free Energy Calculations of Correlated AMP binding. Thermodynamic measurements reveal the importance of the Amino Acid Mutations network for both effector binding and BirA allostery. Force Distribution Martin Werner, Bert L. de Groot. Analysis combined with Energy Network Analysis show coupling between Max Planck Institute for Biophysical Chemistry, Go¨ttingen, Germany. the electrostatic network and disorder-to-order transitions in BirA. The Interactions between combined amino acid mutations play a key role in pro- experimental and computational studies support an allosteric mechanism in tein engineering affecting properties such as cooperativity and allostery. The which an electrostatic network enables communication, via a global popula- effect of a double mutation on properties like thermostability or binding af- tion shift, between disorder-to-order transitions at two distant functional finity can be significantly different from the sum of effects of the separate sites. single mutations. Such a situation is revealed by a nonadditivity of the cor- responding free energies and indicates the mutations involved to be thermo- 2295-Plat dynamically coupled. While this type of epistatic interaction is expected for Graph Spectral Properties of the Sidechain Networks of Protein Struc- residues in close spatial contact, additivity of the effects of both single amino tures: Implications to Allostery and Structure Comparison acid mutations is widely observed for distant pairs. Indeed, even mutations Saraswathi Vishveshwara1, Anasuya Dighe2, Vasundhara Gadiyaram1. on opposite sides of a protein can exhibit significant nonadditivity in their 1Molec Biophys Unit, Indian Institute of Science, Bangalore, India, 2IISc free energies indicating a correlation of both mutations persisting over large Math Int, Indian Inst Sci, Bangalore, India. distances. The investigation of network metrics such as communities and paths of Molecular dynamics simulations with alchemical amino acid mutations grant communication are shown in literature to yield valuable information on protein access to the free energies and nonadditivities of double mutants via different structure, function and folding. Recently, our laboratory has adopted graph pathways. The comparison of the resulting thermodynamic couplings to spectral approach to study the sidechain interactions in protein structures in

BPJ 9447_9453 464a Wednesday, March 6, 2019 detail (https://doi.org/10.1093/comnet/cnw016; https://doi.org/10.1002/prot. 2298-Plat 25332) by transforming all-atom coordinates into a weighted normalized Lap- Understanding the Biophysics of Protein-Surface Interactions lacian network, since the spectra of a graph is known to capture network fea- Gabriel Ortega1, Martin Kurnik1, Philippe Dauphin Ducharme1, Hui Li2, tures with minimal loss of information. A weighted network represents Netzahualcoyotl Arroyo-Curras3, Bishal Gautam4, Kevin Plaxco1. accurate data and many of the important phenomena such as allostery involving 1Center for Bioengineering, University of California Santa Barbara, Santa long distance communication take place through the global effect of local Barbara, CA, USA, 2Engineering Research Center of Nano-Geomaterials, changes in edge weights. Further, the spectra of normalized Laplacian allows China University of Geosciences, Wuhan, China, 3Pharmacology and a comparison of multiple networks in a rigorous manner. The networks can Molecular Sciences, Johns Hopkins University School of Medicine, be from different proteins or different states of the same protein. The edge Baltimore, MD, USA, 4Chemistry and Biochemistry, University of California weight differences provide one component of the scoring scheme. More impor- Santa Barbara, Santa Barbara, CA, USA. tantly, the clustering of interacting residues is a property uniquely elucidated Despite the biological and biotechnological importance of protein-surface in- from spectral studies and it provides the information on changes at global level teractions, our understanding of how and why they occur is still limited: due to local changes in interactions. What are the thermodynamic consequences of the interaction of proteins Methodology and two applications of the above formalism will be presented: a) with surfaces? Why do proteins generally remain folded and functional on Investigation of the graph spectra of GPCR structures and b) Validation of pro- biological surfaces and cell membranes, but often unfold, adhere to, and tein structure models through network scoring scheme (NSS). As part of (a) The inactivate on artificial surfaces? To respond to these questions we need to clustering of interacting residues in adrenergic receptors-ligand complexes will understand the biophysical origins of protein-surface interactions, however be discussed. Distinct patterns emerge from the apo, agonist, and antagonist the absence of experimental methods to measure the thermodynamics of bound structures. The spectral results are discussed in terms of their possible such interactions has so far precluded quantitative analysis. In response, function such as allostery. As part of (b) A large scale comparison of protein we have developed an approach to measure the free energy of protein- structure models from earlier CASP experiments and from the ensemble of surface interactions, which we have employed to explore the extent to which structures obtained from molecular dynamics simulation will be presented. attachment to a specific, macroscopic surface alters the thermodynamic sta- bility of protein L. We have achieved so by modifying protein L with the 2296-Plat redox reporter methylene blue and then covalently tethering its N-terminus Allostery is Highly Tunable by Amino Acid Substitutions at Long-Range to a gold electrode passivated with a hydroxyl-terminated alkanethiol mono- Rheostat Positions layer. Denaturant-induced unfolding of the surface-attached protein alters the Liskin Swint-Kruse, Aron W. Fenton. ease with which the methylene blue reporter transfers electrons to the surface Dept Biochem Molec Biol, Univ Kansas Med Ctr, Kansas City, KS, USA. of the electrode, and therefore electrochemical techniques, such as square- One mechanism by which organisms evolve and adapt to new niches involves wave voltammetry, allow monitoring the unfolding to extract protein stabil- amino acid changes that ‘‘fine-tune’’ protein function. For several proteins, ity. Comparing the thus obtained stability of the surface-attached protein to we previously showed that one group of nonconserved positions contribute to that of the same protein in bulk solution we find that surface-attachment sta- functional tuning by behaving as evolutionary ‘‘rheostats’’. That is, when a va- bilizes the protein due to excluded volume effects that restrict the conforma- riety of amino acids were substituted at a given rheostat position, functional out- tional entropy of the unfolded state. We have also explored the role of comes sampled a wide range of values. In these prior studies, the rheostat macromolecular crowding, solvent composition and electrostatics to find outcomes could be considered ‘‘short-range’’. For example, DNA binding affin- that their surface biophysics are markedly different from those in bulk solu- ity was tuned by mutating positions near the DNA binding site of LacI homo- tion. We believe that our studies refine our understanding of the biophysics logs. In a second example, properties of allosteric regulation (apparent underlying protein-surface interactions, which may in turn improve the effector binding ‘‘Kix’’ and coupling ‘‘Q’’) were tuned by mutating positions design of protein-surface pairs for protein-incorporating biotechnologies in and near the two distinct allosteric binding sites on pyruvate kinase (PYK). such as protein-based sensors. Here, we used a quantitative score for tunability to show that the same PYK substitutions also exerted ‘‘long-range’’ rheostat effects: Mutations at rheostat 2299-Plat positions in the alanine allosteric site tuned both K and Q for -1,6- ix Tie Up Cytoskeleton to Inhibit Ovarian Cancer Metastasis bisphosphate activation (‘‘FBP’’); mutations at rheostat positions in the Ye Zhang. FBP allosteric site tuned Q for alanine inhibition, but had only modest effect Bioinspired Soft Matter Unit, Okinawa Institute of Science and Technology, on K . For Q ,Q , and K , mutational outcomes sampled the range ix,ala ala FBP ixFBP Kunigami-gun, Japan. from ‘‘better’’ than wild-type to ‘‘dead.’’ The ranges observed from long- Cells modify the migration mechanism in response to their surroundings, range tuning were quantitatively equivalent to those from short-range tuning. which sets the challenge for cancer therapy targeting metastasis through Several amino acid positions contributed to tuning multiple functional parame- signaling pathways. To cope with the diversity and complexity, we have ters. Strikingly, for all mutated positions in this study, the long-range effects on developed a mechanical approach that captures membrane dynamicity to sup- Q tunability were larger than the long-range effects on K . This supports the ix press cancer cell migration and invasion. We designed and synthesized a hypothesis that the functional roles of binding and allostery can be controlled ruthenium (Ru)-complex-based peptidic molecule that selectively initiates by distinct sets of amino acid positions. self-assembly into extracellular nanofibrils on lipid rafts of ovarian cancer cells by reacting with glycosylphosphatidylinositol-anchored placental alka- Platform: Engineering and Detecting Cellular line phosphatase (GPI-anchored PLAP). The growing nanofibrils glue the lipid rafts and chain them into large clusters, leading to reinforced focal adhe- (dys) Faction sion suppressing cell migration. The molecular self-assembly constantly ex- erts mechanical stimuli to raft-associated protein receptors, provoking 2297-Plat opposing cell migration against focal adhesion with enhanced motility. The Hydrogel Engineering with Widefield Patterned Illumination contradictory motions generate a mechanical force transferred through the Aurelien Pasturel1,2, Pierre-Olivier Strale2, Vincent Studer1. 1 2 actin cytoskeleton. When the force increases, the restricted ovarian cancer CNRS UMR 5297, Bordeaux, France, ALVEOLE, Paris, France. cell is ruptured. Hydrogels are the material of choice to emulate the cellular micro-environment thanks to their high hydric content and tunable visco-elastic properties. In field such as cancer research, toxicology or tissue engineering, enabling technolo- 2300-Plat gies to control the structural and biochemical properties of hydrogels are Improvement of Maturation State of Human Induced Pluripotent Stem much sought after. Indeed, structuring and functionnalizing hydrogels could Cell-Derived 3D Cardiac Microtissues by Defined Chemical Factors unlock the design of more advanced organotypic models but existing solutions Chen Yu Huang1, Rebeca Joca2, Chin Siang Ong2, Ijala Wilson2, usually lack the simplicity and flexibility to adapt to the day to day flow of mod- Roald Teuben2, Gordon F. Tomaselli2, Daniel H. Reich1. 1 ern research. Here by harnessing generic chemistry with digitally processed UV Department of Physics and Astronomy, Johns Hopkins University, 2 widefield illumination we achieve additive or subtractive manufacturing and Baltimore, MD, USA, Division of Cardiology, Johns Hopkins Hospital, decoration for common hydrogels such has PEG, Matrigel, Agar or Poly- Baltimore, MD, USA. acrylamide. We grew neurons and cell lines onto chemically and topographi- Recent advances in the understanding and use of pluripotent stem cells have cally complex PEG gels, inside engineered Matrigel structures or within micro- produced major changes in the approach to the diagnosis and treatment of hu- fluidic chambers demonstrating that simple principles may unlock engineering man disease. An obstacle to the use of human pluripotent stem cell-derived car- for hydrogels that lack dedicated chemistry. diomyocytes (hiPSC-CMs) for regenerative medicine, disease modeling and

BPJ 9447_9453 Wednesday, March 6, 2019 465a drug discovery is their immature state relative to the adult myocardium. In this sulted in a low fluorescence, which increased over 4 times in the unfolded research, 3D cardiac microtissues (CMTs) were generated using hiPSC-CMs to state (Caþ2 free) of the protein, resulting in a large change in the fluorescence recapitulate the structural, functional and metabolic properties of normal and signal. The successful in-vitro detection of Caþ2 ions confirms the eligibility diseased adult ventricular myocardium. CMTs were treated with ontologically of the ‘‘folding coupled to binding’’ strategy as the next generation sensor defined biochemical interventions (thyroid hormone, dexamethasone and designing methodology. insulin-like growth factor, TDI) to promote the maturation of hiPSC-CMs. The effects of TDI treatment on both structural and functional (biomechanical, 2303-Plat þ Ca2 handling and electrophysiology) properties at the tissue level were char- pH Sensitive Peptide Functionalized High Stability Polymeric Nanopar- acterized. Also, the molecular correlates of maturation of the hiPSC-CMs in the ticles for Mitochondria Targeted Cancer Drug Delivery CMTs were studied by gene expression, proteomics studies. Our data demon- Palanikumar Loganathan, Mona Kalmouni, Sumaya Al Hosani, strate that TDI treatment improves both the structure and function of CMTs. Mazin M. Magzoub. Structurally, the hiPSC-CMs show improved alignment and longer sarcomere Biology Program Division of Science, New York University Abu Dhabi, Abu length, as shown by immunofluorescence and confocal microscopy. Function- Dhabi, United Arab Emirates. ally, CMTs’ static and dynamic force both increase following TDI treatment, A major focus of cancer nanomedicine is to achieve a high therapeutic index and cardiac electrophysiology assessed by optical mapping showing that the for cancer therapy. Despite the unprecedented growth in the development of CMTs are electrically coupled. TDI-treated CMTs exhibited both chronotropic polymeric self-assembled nanosystems, only a few of them end up in clinics and inotropic response to isoproterenol treatment. The enhanced functional (e.g. Doxil) as a result of (i) poor stability, (ii) drug leakage and (iii) disso- properties that this maturation approach yields have the potential to yield ciation kinetics due to the strong influence of blood components in vivo.The improved hiPSC derived cardiac model systems that can advance both mecha- most common method for improving nanocarrier stability is to tune the drug nistic studies and the development of new therapies for the treatment of cardiac hydrophobicity and polymer matrix miscibility under complex chemical re- diseases. action conditions, that leads to poor drug cytotoxicity and attenuation of therapeutic efficacy. To address these challenges, we have designed a simple and robust poly (lactide-co-glycolide) (PLGA) and bovine serum albumin 2301-Plat (BSA) hybrid nanoparticles to provide high stability and trigger drug release Protein Detection in Blood with Single-Molecule Imaging inside cancer cells. The hybrid nanoparticles were prepared by covalently Shih-Chin Wang, Chih-Ping Mao, Yu-Pin Su, T.C. Wu, Chien-Fu Hung, wrapping BSA over the surface of drug-loaded PLGA nanoparticles using Jie Xiao. simple carbodiimide-chemistry in mild reaction conditions. To increase the Johns Hopkins Univ, Baltimore, MD, USA. stability, the BSA shells were crosslinked. The BSA shells can be dissociated The ability to accurately identify and characterize proteins in blood samples in the high redox microenvironment of the tumor and within the cancer cells. would facilitate disease diagnosis and monitoring. Current blood tests, Subsequently, the drugs loaded into the PLGA core can be released by accel- including ultra-sensitive assays, depend on ensemble measurements which erated degradation (hydrolysis) due to the low pH conditions in the tumor are hampered by background detection errors. Single-molecule imaging ap- microenvironment and lysosomal compartments. The cancer cell targeting proaches, by contrast, directly inspect individual target proteins, offering ability of the hybrid NPs was achieved by coupling of the amino groups much greater reliability than ensemble methods. However, the detection limit of the BSA shell with a pH-sensitive peptide (acidity-triggered rational of single-molecule imaging has thus far been confined to the picomolar (10 12 membrane peptide, ATRAM). The insertion of the ATRAM-BSA-PLGA M) range, and these approaches have not been previously translatable to clin- nanoparticles into cancer cell membranes occurs at the peritumoral pH ical samples. Here we describe Single-Molecule Augmented Capture (6.5). In vitro studies showed highly-efficient pH-dependent uptake and (SMAC), a single-molecule imaging technique to visualize and quantify indi- remarkable cytotoxicity of doxorubicin triphenylphosphine loaded ATRAM- vidual proteins-of-interest down to the sub-femtomolar (<10 15 M) range BSA-PLGA NPs. Our results demonstrate that the novel pH-sensitive pep- with minimal detection errors, even in complex fluids such as blood. We tide-functionalized high stability BSA-PLGA nanoparticles are a highly demonstrate SMAC in a wide variety of applications with human blood sam- promising nanoplatforms. ples, including the analysis of secreted proteins (prostate-specific antigen and anti-p53 autoantibodies), membrane proteins (programmed death-ligand 1), and rare intracellular proteins (mutant p53). SMAC opens the door to the 2304-Plat use of single-molecule imaging in non-invasive disease profiling. This plat- Protein Phase Transition: From Biology Towards New Protein Materials 1 € 1 1 2 form can be adapted to multiplex or high-throughput formats to characterize Miriam Linsenmeier , Andreas Kuffner , Lenka Faltova , Maria Hondele , Karsten Weis2, Paolo Arosio1. heterogeneous biochemical and structural features of circulating proteins-of- 1 interest. Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland, 2Dept Biology, ETH Zurich, Zurich, Switzerland. Many cellular organelles can form via phase separation of proteins and nu- 2302-Plat cleic acids. Yet, the molecular mechanisms that govern the lifetime, the Recombinant Protein Based CaD2Ion Sensor Designing; an In-Vitro Test of composition and the size of these membrane-less compartments remain Folding Coupled to Binding Hypothesis largely elusive. Here, we apply methods based on polymer physics and micro- Abhigyan Sengupta, Mourad Sadqi, Victor Mun˜oz. fluidic technology to analyze the physical chemical processes underlying the Bioengineering, University of California Merced, Merced, CA, USA. phase separation of biological proteins, and we use these lessons to induce Recombinant protein-based sensor designing is a burgeoning topic of interest controlled self-assembly of soluble proteins in biotechnology. In particular, due to its potential in-vivo applications. Proteins are particularly suitable for we focus on the phase separation of the DEAD-box protein ATPase Dhh1, sensor designing as they have high cell viability, negligible cytotoxicity, inter- which is strongly associated with the formation of processing bodies (P- action with the substrate with a large three-dimensional surface and binding bodies) in yeast. We identify the role of ATP and RNA in triggering the with high affinity. But protein-based sensors are scarcely available as engi- nucleation and growth of the protein-rich droplets, as well as in maintaining neering protein as a functional sensor is very challenging. I will describe the protein dense phase in the liquid state. These results reveal molecular the designing of a recombinant protein-based Caþ2 ion sensor using ‘‘folding mechanisms that cells have plausibly developed to accurately control the coupled to binding’’ strategy. The Caþ2 ion binding domain of a natural pro- reversible assembly and the biophysical properties of P-bodies. Moreover, tein is used as a template and site-selective mutations are utilized to destabi- we demonstrate the possibility to mimic these mechanisms and induce similar lize the protein structure and couple its folding-unfolding kinetics with the behaviours in soluble proteins by conjugating low complexity domains to sol- ligand association equilibrium. In the coupled equilibrium, the apo (Caþ2 uble globular regions (Faltova L., Kuffner€ A. et al, ACS Nano, 2018, https:// free) form of the protein folds only in presence of the target analyte and doi.org/10.1021/acsnano.8b04304). We show that these molecular adhesives the structural changes in the protein is exploited as a transducer for the enable? sensitive, controlled self-assembly of these proteins into supramolec- biosensor. We have utilized both FRET and PET-based techniques to estimate ular? architectures via a? multistep process. This multistep pathway involves the ligand-induced changes in protein structure. To probe less than 2 nm dis- an initial liquid-liquid phase transition, which creates protein-rich droplets tance change between folded and unfolded states of the protein, photo-induced that mature into protein aggregates over time. These protein aggregates electron transfer (PET) is exploited as an alternative to FRET. An organic flu- consist of permeable structures that maintain activity and release active solu- orophore is covalently attached to one site of the protein and at another site, a ble proteins. We further demonstrate that this feature, together with the dy- selected amino acid is mutated to tryptophan. In folded state (Caþ2 bound) the namic state of the initial dense liquid phase, allows one to directly intramolecular electron transfer from the tryptophan to the fluorophore re- assemble different globular domains within the same architecture, thereby

BPJ 9447_9453 466a Wednesday, March 6, 2019 enabling the generation of both static multifunctional biomaterials and dy- 2307-Plat namic microscale bioreactors. Enhanced Crossbridge Binding with 2-Deoxy-ATP Results from Increased Electrostatic Interactions between Myosin and Actin in Cardiac Muscle Chen-Ching Yuan1, Joseph D. Powers1, Kimberly J. McCabe2, Platform: Cardiac Muscle Mechanics, Structure, Jason D. Murray3, Morhan Saffie1, Castillo Romi1, Zuzek Carla1, Weikang Ma4, Andrew D. McCulloch5, Thomas C. Irving6, and Regulation II Michael Regnier1. 1Dept BioEngineering, Univ Washington, Seattle, WA, USA, 2UC San 2305-Plat Diego, San Diego, CA, USA, 3Dept Physiol & Biophys, Univ Washington, Stretch-Induced Activation of the Myosin Motors on the Thick Filament in Seattle, WA, USA, 4BioCAT, Argonne, IL, USA, 5Bioengineering, UC San Rat Cardiac Trabeculae Diego, La Jolla, CA, USA, 6Dept Biology, Illinois Inst Tech, Chicago, IL, So-Jin Park-Holohan, Elisabetta Brunello, Thomas Kampourakis, USA. Martin Rees, Malcolm Irving, Luca Fusi. We have demonstrated that 2-deoxy-ATP (dATP) enhances muscle contraction Randall Centre for Cell and Molecular Biophysics, King’s College London, by increasing cross-bridge binding and cycling rates. Our recent molecular dy- London, United Kingdom. namics study (Nowakowski 2017, Protein Sci 26:749-62) suggested that dATP The contractility of cardiac muscle is regulated by thick-filament based increases the positive charge of the actin binding surface of myosin, thus mechanisms in addition to the classical calcium/thin-filament mediated enhanced binding may result from increased electrostatic interactions with actin mechanisms. Here we studied the role of structural changes of the thick in resting heart muscle. To test this hypothesis we employed multiple biophys- filament in length-dependent activation in cardiac muscle. We used fluores- ical approaches. X-ray diffraction analysis comparing dATP vs. ATP was per- cence polarization from bifunctional sulphorhodamine probes on the N- and formed on rat cardiac muscle in physiological (170mM) and low (100mM) C-lobes of the myosin regulatory light chain (RLC) to monitor changes in ionic strength (m) solutions at two sarcomere lengths (SL). In 170mM m relaxing the orientation of the myosin motors induced by increasing sarcomere length solution (pCa9) with dATP, cardiac muscle had a significantly higher I1,1/I1,0 ra- in relaxed and partially calcium-activated demembranated trabeculae. tio, larger M3 spacing (SM3) and lattice spacing (d1,0) at both SL. These results Under relaxing conditions at near physiological temperature and lattice suggest that, compared with ATP, dATP in resting cardiac muscle causes spacing the myosin motors are roughly parallel to the filament axis, consis- myosin to move towards actin filaments, and the space between adjacent crowns tent with the OFF structure of the thick filament. Cooling of the relaxed is similar to the activated myosin conformation (pCa5.2). These effects were trabecula induced a more perpendicular orientation of the myosin motors reduced or eliminated at m=100mM where surface charge is increased (with indicating disruption of the OFF-state. Application of a staircase stretch ATP). This suggests that increased positive charge on the actin binding surface protocol in the sarcomere length range 2.0-2.3 mm(25C, 3% dextran of myosin with dATP may indeed be responsible for enhanced crossbridge bind- T500) at pCa 9.0 also induced a more perpendicular orientation of the ing. Brownian Dynamics simulations predicted the myosin.dADP.Pi structure myosin motors, but despite an increase in passive tension to 30% of has a significantly higher association rate to actin (than myosin.ADP.Pi) over T the maximum active force ( 0), the orientation change was only 4% of a wide range of reaction distances, and this results primarily via an increase in that associated with full calcium activation. Larger stretch-induced orienta- positive (myosin) and negative (actin) contact pairs. Consistent with this, as tion changes were observed at pCa 7 (7%) and at pCa 6.6 (20%), the the m of solutions in the in vitro motility assay was increased, more actin fila- T latter accompanied by an increase in active force of 0.05 0. RLC phos- ments remained on cardiac myosin-coated surfaces with dATP compared with phorylation further increased the stretch-induced orientation changes to ATP. Combined these data suggest that dATP enhances electrostatic interac- 14% and 33% respectively, and the active force response at pCa 6.6 tions that facilitate increased weak myosin-actin binding, leading to increased T increased to 0.2 0. These results indicate that increasing sarcomere length crossbridge binding and contraction in cardiac muscle. in the physiological range at diastolic calcium concentration induces activation of the myosin motors and that this effect is enhanced by RLC 2308-Plat phosphorylation, suggesting that changes in thick filament structure may Basic Amino Acids within the C-Terminal 16 Residues of Troponin T mediate length-dependent activation. Supported by Wellcome Trust and Modulate Calcium Sensitivity and the Distribution of Actin States BHF, UK. Dylan Johnson, Li Zhu, Joseph M. Chalovich. Biochemistry and Molecular Biology, Brody School of Medicine at East 2306-Plat Carolina Univ, Greenville, NC, USA. Cardiomyopathy Mutation at End-End Overlap of Alpha - Tropomyosin We previously presented evidence that the highly conserved C-terminal 14 res- Influences Cooperative Activation and Calcium Sensitivity idues of cardiac troponin T has two regulatory functions. 1) Formation of the 1 2 2 SaiLavanyaa Sundar , Michael J. Rynkiewicz , William Lehman , inactive state (B or 10) does not occur when the last 14 residues are removed þ Jeffrey R. Moore3. from troponin T. 2) Those last 14 residues limit Ca2 activation to 33% of 1University of Massachusetts Lowell, Lowell, MA, USA, 2Dept Physiol/ full activation in solution. Deletion of the last 14 residues leads to doubling þ Biophys, Boston Univ, Boston, MA, USA, 3Dept Biological Sciences, Univ of Ca2 activation by. We recently showed that stepwise deletion of the last Massachusetts Lowell, Lowell, MA, USA. 14 residues of Troponin T changes both regulatory functions in a graded Tropomyosin (Tpm) polymerizes head-to-tail along the long-pitch helix of manner. We proposed that ionic interactions between basic residues within F-actin to form a coiled-coil super-helical uninterrupted cable structure the C-terminal of Troponin T and acidic residues elsewhere in the thin filament that wraps around the complete length of actin filaments. Muscle contraction are important for these functions. We tested this hypothesis by substituting Ala is governed by the ability of Tpm to azimuthally shift between three states for each of 5 Lys and 2 Arg residues within the last 16 residues (to substitute an on F-actin (Blocked, Closed and Open) in response to calcium-binding to additional Lys) of human cardiac Troponin T. We also engineered a C-terminal troponin and acto-myosin cross-bridge formation. It is also known that the Cys residue into both the mutant and wild type troponin T for coupling to þ end-to-end bonds formed between the N- and C- terminus of adjacent probes. In the virtual absence of Ca2 the ATPase rate of actin filaments con- Tpm molecules defines Tpm continuity, plays a critical role in the ability taining that mutant troponin T was slightly elevated with respect to wild type. of Tpm to cooperatively bind to actin and for Tpm conformational switching Also, the fluorescence change characteristic of forming the inactive B state was þ to cooperatively propagate along F-actin. We hypothesize that a missense missing with the mutant troponin T. At saturating Ca2 the ATPase rate was mutation, A277V, in this critical overlap region, associated with dilated car- approximately double that of wild type. Thus the mutant Troponin T behaved diomyopathy, will alter tropomyosin binding, thin filament activation by sta- like Troponin T that lacked the C-terminal 14 residues. We named this mutant bilizing the overlap structure. Here, we used co-sedimentation assays and in HAHA Troponin T (High Alanine-High Activity Troponin T). We plan to use þ vitro motility assays to determine how the mutation alters Tpm binding to HAHA troponin T to explore the mechanism of modulation of Ca2 activation. actin, and its ability to regulate acto-myosin interactions. The A277V Tpm exhibited enhanced actin - Tpm binding affinity and cooperativity, 2309-Plat consistent with overlap stabilization. However, regulated thin filament On the Functional Assessment of Hypertrophic Cardiomyopathy-Causing motility measurements exhibited a decrease in both, cooperativity and Mutations in Human b-Cardiac Myosin and the Role of Myosin Binding Ca2þ sensitivity, when compared to wild-type. The observed effects are Protein-C consistent with a mutation-induced disruption of the blocked-closed equilib- Darshan V. Trivedi1,2,*, Saswata S. Sarkar1,2,*, Arjun S. Adhikari1,2,*, rium, an increased Tpm-Tpm end-end bonding and a decrease in overlap Makenna M. Morck1,2, Kristina B. Kooiker1,3, Daniel Bernstein2,3, stiffness. Kathleen M. Ruppel1,3, James A. Spudich1,2.

BPJ 9447_9453 Wednesday, March 6, 2019 467a

1Dept. of Biochemistry, Stanford Univ, Stanford, CA, USA, 2Stanford variants in desmosomal , however the molecular mechanisms underlying Cardiovascular Institute, Stanford Univ, Stanford, CA, USA, 3Dept. of AC remain unclear. We identified a novel variant (R451G) in the desmoplakin Pediatrics (Cardiology), Stanford Univ, Stanford, CA, USA. (DSP) gene; heart autopsy revealed pronounced fibrofatty scarring and a sig- *Equal Contribution nificant loss of DSP protein at the intercalated disc (ID). Engineered heart tis- Human b-cardiac myosin and cardiac myosin binding protein-C (MyBP-C) sues (EHTs) from induced-pluripotent stem cells generated from a harbor the majority of hypertrophic cardiomyopathy (HCM)-causing point symptomatic R451G-positive family member showed a significant loss of mutations which lead to a hypercontractile heart with systolic and diastolic DSP protein compared to control EHT. Notably, the R451G-variant sits in a defects. Previous studies looking at the effects of HCM mutations on the pocket containing several clinical DSP variants and we show that endogenous force, velocity and ATPase activity of the catalytic domain of human b-car- DSP is cleaved via a calpain-dependent mechanism. From this, we hypothe- diac myosin have not shown clear trends that establish a causal link to hyper- size that mutant DSP is more susceptible to calpain cleavage, resulting in contractility. However, recently, a regulatory, off-state of myosin with its cellular haploinsufficiency. In previous work, we defined a unifying mecha- heads folded back onto its own coiled-coil tail (probably the interacting heads nism for AC-linked variants by assessing the biomolecular properties of motif or IHM described by others) has been experimentally demonstrated to DSP variants. While these variants did not significantly perturb the structure be one of the primary states that yields the very slow turnover of ATP (called of DSP, there were significant changes to stabilizing intramolecular interac- the super relaxed state or SRX) typically observed in muscle fibers. This reg- tions of select variants, which correlated with augmented calpain degradation ulatory off-state is in equilibrium with the on-state of myosin which is avail- due to increased exposure of an auto-inhibited calpain-cleavage site. We have able to interact with actin and generate force. Our model posits that HCM now shown that by changing the molecular environment using site-directed mutations destabilize this SRX state, thus leading to a premature release of mutagenesis, we can ameliorate the calpain sensitivity of clinical variants. myosin heads from the thick filament backbone causing clinically observed Taken together we have identified a unifying mechanism for select AC- hypercontractility. Using IHM as the model for the folded-back state, we linked variants of DSP grounded in increased susceptibility to calpain- have previously presented binding data demonstrating that HCM mutations cleavage leading to decreased functional DSP. DSP protein expression was at the head-tail interface weaken this interaction and possibly destabilize also decreased in end-stage failing human hearts compared to control donor the IHM state. Here we present functional data showing that six separate hearts. The physiological and biochemical impact of this loss is being further HCM mutations located at the myosin head-tail and head-head interfaces of evaluated to define the role DSP haploinsufficiency plays in the fundamental the IHM lead to a significant increase in the number of heads functionally disease mechanism of AC. accessible for interaction with actin. Interestingly, one of the mutations also ablates the binding of myosin with the N-terminal C0C7 fragment of 2312-Plat MyBP-C hinting at a role of MyBP-C in regulating the availability of myosin Determining the In Vivo Role of Microtubule Detyrosination in Healthy heads for contraction of the cardiac muscle. and Diseased Myocardium Christina Yingxian Chen1, Matthew A. Caporizzo1, Kenneth Bedi2, 2310-Plat Michael P. Morley2, Kenneth B. Margulies2, Benjamin L. Prosser1. Development of an Imaging Pipeline to Model and Predict the Integrated 1Dept Physiology, Univ Pennsylvania, Philadelphia, PA, USA, 2Dept Localization of Organelles in hiPSC-Derived Cardiomyocytes Medicine, Univ Pennsylvania, Philadelphia, PA, USA. Melissa Hendershott, Susanne Rafelski. Detyrosinated microtubules provide mechanical resistance that can impede Allen Institute for Cell Science, Seattle, WA, USA. cardiomyocyte motion. Cardiomyocytes from patients with heart failure The Allen Institute for Cell Science is developing an imaging pipeline to study are characterized by a dense, heavily detyrosinated microtubule network changes in cellular organization and gene expression during differentiation of that is associated with increased myocyte stiffness and impaired contractility. human induced pluripotent stem cells (hiPSCs) into cardiomyocytes. We Pharmacological or genetic suppression of detyrosinated microtubules have used CRISPR/Cas9 to generate endogenous fluorescently tagged hiPSC lowers the viscoelasticity and improves contractile function in failing human lines (www.allencell.org), each expressing a monoallelic EGFP-tagged protein myocytes. These data demonstrate that a modified cytoskeletal network im- that localizes to structures including adhesions, actin and microtubule cytoskel- pedes contractile function in cardiomyocytes from failing human hearts and eton, mitochondria, nuclear envelope, desmosomes, endoplasmic reticulum, that targeting detyrosinated microtubules could represent a new inotropic and the Golgi. In addition, we have developed methods for scarless GFP- strategy for improving cardiac function. However, whether an increase in de- tagging of late expressing cardiomyocyte genes, including ACTN2, ssTNNI1, tyrosinated microtubules is sufficient to induce cardiac dysfunction in vivo is MYL2, MYL7 and TTN, to study the organization and morphogenesis of the unknown. To test this, we generated a cardiac-specific tubulin tyrosine ligase contractile apparatus. Furthermore, we have developed an optimized, scalable, knockout (TTL-cKO) mouse model to study the in vivo function of detyro- and robust protocol for differentiation of hiPSCs into cardiomyocytes, proto- sinated microtubules. In TTL-cKO hearts, detyrosinated tubulin increases cols for freezing/thawing cells, and methods for preparing cells for imaging, 4-fold compared to that of the control mice, mimicking similar levels of in- including dyes that label the cell membrane and nucleus. To quantify the crease seen in failing human hearts. Baseline echocardiography shows a sig- live, high-resolution 3D fluorescence images of hiPSC-derived cardiomyo- nificant increase in LV mass/body weight ratio in 3-4 months old TTL-cKO cytes, we developed image-based assays and segmentation algorithms that mice. Interestingly, TTL-cKO male and female hearts appear to undergo enable single-cell analyses of structure localization in differentiated cardio- different types of remodeling at this age. TTL-cKO females show purely myocytes. We have applied this imaging workflow to hundreds of cells for compensatory hypertrophy with increased wall thickness, mild changes in 20 different intracellular structures. These data were used to develop a ma- chamber diameter and no change in LV ejection fraction (LVEF), while chine learning neural net algorithm that can predict the location of cardiomyo- TTL-cKO males demonstrate a dilated remodeling with decreased septal cyte structures from transmitted light images. We are now extending our thickness, increased chamber diameter and a trend of declining LVEF. imaging workflow to incorporate fluorescence in situ hybridization (FISH); Ongoing echocardiography on older mice and mice with transverse aortic this will allow us to combine image data with gene expression profiles on a constriction (TAC) will inform whether remodeling and cardiac function is single-cell level. This imaging pipeline will be scaled up to generate an image perturbed with age and pressure overload induced stress. This will yield database of high-resolution, high-replicate image data of the fluorescently deeper insight on the in vivo functions of detyrosinated microtubules in tagged structures with a goal of generating an integrated image ‘‘state space’’ health and disease. of intracellular reorganization during cardiomyocyte differentiation. Platform: Protein-Nucleic Acid Interactions/ 2311-Plat Defining a Unifying Mechanism for Select Cardiomyopathy-Linked Vari- Chromatin and the Nucleoid II ants of Desmoplakin Heather R. Manring1, Ronald Ng2, Taylor Albertelli3, Trevor Dew1, 2313-Plat Tyler L. Stevens1, Ahmet Kilic4, Paul M.L. Janssen1, Nathan T. Wright3, Shelterin Components Modulate the Phase-Separation Propensity of Telo- Stuart Campbell5, Maegen A. Ackermann1. meres 1Dept Physiol & Cell Biol, Ohio State Univ, Columbus, OH, USA, 2Yale Andrea Soranno, Jeremias Incicco, Paolo De Bona, Eric Tomko, Eric Galburt, Univ, New Haven, CT, USA, 3Dept Chem/Biochem, James Madison Univ, Roberto Galletto. Harrisonburg, VA, USA, 4Ohio State Univ, Columbus, OH, USA, 5Dept Biochemistry and Molecular Biophysics, Washington University School of Physiol & Cell Biol, Yale Univ, New Haven, CT, USA. Medicine, Saint Louis, MO, USA. Arrhythmogenic cardiomyopathy (AC) affects 1 in 2000 Americans every Telomeres are nucleoprotein complexes that provide a mechanism to shelter the year and segregates with sudden cardiac death (SCD). AC is linked to genetic ends of and are essential to maintain stability in

BPJ 9447_9453 468a Wednesday, March 6, 2019

Eukaryotes. In cells, individual telomeres are bound by the six-protein shelterin facilitating the qualitative and quantify describe the non-covalent interac- complex (TRF2-hRap1-Tin2-TRF1-TPP1-Pot1) and form distinct condensed tions inside membrane channels. globules of finite size. Interestingly, incells that maintain their telomeres via an alternative lengthening mechanism, multiple telomeric DNAs and shelterin 2316-Plat proteins coalesce in membrane-less promyelocytic leukemia (PML) nuclear Disordered RNA Chaperones Enhance Nucleic Acid Folding via Local bodies. The mechanism controlling this dual nature of telomere organization Charge Screening 1 2 3 4 is currently not understood. Here, we propose that shelterin proteins modulate Erik D. Holmstrom , Zhaowei Liu , Daniel Nettels , Robert B. Best , 3 the phase-separation propensity of telomeres, leading to either the condensation Benjamin Schuler . 1University of Kansas, Lawrence, KS, USA, 2University of Basel, Basel, of single DNA chains or the coalescence of multiple ones. In particular, we use 3 4 a combination of optical and fluorescence microscopy and single-molecule ex- Switzerland, University of Zurich, Zurich, Switzerland, NIH, Bethesda, periments to quantify the contribution of shelterin components to phase- MD, USA. separation and condensation of single DNA chains. Nucleic acid chaperones are an emergent class of proteins that facilitate for- Our experiments reveal that TRF2 promotes phase-separation of TRF2-DNA mation of natively folded RNAs and DNAs. These chaperones are involved mixtures. Even though the N-terminal basic domain and the dimerization in many nucleic acid-dependent processes, including critical steps in the life domain of TRF2 are sufficient to promote phase-separation, the C-terminal cycles of many viruses. Interestingly, many of these proteins are intrinsically DNA-binding domain imparts specificity for telomeric DNA repeats. More- disordered, raising the fundamental question of how such a chaperone func- over, addition of shelterin components, such as hRap1, provides a fine-tuning tions without a well-defined three-dimensional structure. One such example of the properties of the resulting dense and de-mixed phase. is the nucleocapsid protein of the hepatitis C virus, which catalyzes genome dimerization. Recently, we have uncovered many of the structural and 2314-Plat dynamical aspects of chaperone activity using a variety of smFRET tech- RNA Binding Mode Regulates PKR Activation niques. This positively charged intrinsically disordered protein functions as Stephen J. Hesler, Bushra Husain, Matthew Angeliadis, James L. Cole. a chaperone by acting as a flexible macromolecular counterion that locally Dept Molec Cell Biol, Univ Connecticut, Storrs, CT, USA. screens repulsive electrostatic interactions within the nucleic acid. The re- The antiviral kinase PKR is activated by RNA. The enzyme contains two sulting compaction biases the unfolded nucleic acid towards compact confor- dsRNA-binding domains, dsRBD1 and dsRBD2, and a kinase domain. Co- mations that are primed for folding, thereby increasing the folding rate localization of the kinase domains upon binding of two or more PKRs to a constant. Finally, molecular simulations that treat the nucleic acid and chap- single dsRNA enhances dimerization and subsequent activation. However, erone as a simple polyelectrolytes reproduce the experimental observations multiple lines of evidence indicate that the mode of RNA binding regulates and support the proposed molecular mechanism of these disordered viral PKR activation beyond simply modulating the overall binding affinity. proteins. dsRBD2 binds RNA weakly and engagement of this domain by an RNA is 2317-Plat correlated with enzymatic activation. In order to define the role of dsRBD2 Eukaryotic Transcription Factors Can Track and Control their Target in PKR activation we have systematically mutated residues in this domain Genes using DNA Antennas that typically mediate dsRBD-RNA interactions and probed the consequences Victor Munoz. for PKR activation and overall binding affinity. Several mutations inhibit PKR Bioengineering, Univ Calif Merced, Merced, CA, USA. activation and reduce RNA binding affinity in parallel. However, mutation of Eukaryotic transcription factors (TF) function by binding to short, 6-10 bp outward-facing residues on helix 1 that typically engages the RNA minor DNA recognition sites located near their target genes, which are scattered groove dramatically reduce PKR activation while only slightly decreasing through vast genomes. Such process surmounts enormous specificity, effi- overall RNA binding affinity. Steady-state fluorescence anisotropy analysis ciency and celerity challenges using a molecular mechanism that remains of PKR constructs containing a single tryptophan in either dsRBD1 or poorly understood. Combining biophysical single-molecule fluorescence ex- dsRBD2 indicate that both domains undergo a significant decrease in mobility periments, theory and bioinformatics analysis, we dissected the interplay be- upon RNA binding. These anisotropy changes are not markedly affected by tween the DNA-binding domain of Engrailed, a Drosophila TF, and the the helix 1 mutations, implying that the interaction of dsRBD2 with RNA per- regulatory regions of its target genes. Remarkably, Engrailed binding affinity sists. We propose that instead of interacting with RNA, helix 1 in dsRBD2 is enormously amplified by the DNA regions flanking the recognition site, engages in an intramolecular interaction that regulates kinase activity based which contain long tracts of degenerate recognition-site repeats. Such on RNA binding mode. DNA organization operates as an antenna that attracts TF molecules in a pro- miscuous exchange between myriads of comparatively weaker binding sites. 2315-Plat The antenna ensures a local TF supply, enables gene tracking and fine con- A Single-Molecule Interaction Spectrum for Non-Covalent Interaction In- trol of basal site occupancy. This mechanism illuminates puzzling gene side Membrane Protein Channel expression data, and suggests novel engineering strategies to control gene 1 1 1 2 1 Meng-Yin Li , Yi-Lun Ying , Wei Tong , Yong-Jing Wan , Yi-Tao Long . expression. 1Key Laboratory for Advanced Materials & School of Chemistry and Molecular Engineering, East China University of Science and Technology, 2318-Plat Shanghai, China, 2School of Information Science and Engineering, East Multiple Interaction Modes of the Nucleosomal Histone H3 N-Terminal China University of Science and Technology, Shanghai, China. Tail Revealed by High Precision Single-Molecule FRET Dynamic non-covalent interactions of biomolecules are ubiquitous in almost Kathrin Lehmann1,2, Suren Felekyan2, Ralf Kuhnemuth€ 2, Mykola Dimura2, all biological process. In particular, it underlies the successive transport of Katalin To´th1, Claus A.M. Seidel2. ions or target carriers by membrane protein, controlling many key cellular 1Biophysics of Macromolecules, German Cancer Research Center, functions. However, few experimental approaches can directly define the Heidelberg, Germany, 2Dept Molec Phys Chem, Heinrich Heine Universit€at multiple non-covalent interactions within the channel. Since all the dynamic Dusseldorf,€ Dusseldorf,€ Germany. non-covalent dynamic interaction inside membrane protein have been all re- Nucleosomes, as the basic unit of chromatin, are the key element of chromatin corded but hide in the traditional single-channel recording of ionic current, compaction and gene accessibility. The nucleosomal disassembly process, its here, we present a rational strategy in the combination of experiments, dynamics and the effects of posttranslational modifications have been widely MD simulations and frequency-energy spectra of ionic flow to quantify studied. However, little is known about the actual conformation and dynamics and quality a role of non-covalent interaction for membrane protein channel of the protruding histone tails. Here, we focus on the N-terminal tail of histone in transporting the carriers. We employed wild-type aerolysin transporting of H3 (H3NtT) to augment our recently published model on nucleosome methylcytosine and cytosine as a model to detailed explore the dynamic disassembly. ionic signatures with the non-stationary and non-linear frequency analysis. In this FRET study salt dependent conformational changes of reconstituted Wi- The results demonstrate that The main frequency peak position in dom 601 mononucleosomes with a DNA length of 170 bp, or 210 bp were frequency-energy spectrum represents the main ion mobility inside mem- measured from the perspective of the labeled histone tails by systematic vari- brane protein while its energy suggests the vibration amplitude of the related ation of the DNA labeling positions. Both, ensemble FRET experiments using ions. The sudden spikes in the frequency-energy considered as the fingerprint a Typhoon multimode scanner, and single-molecule experiments using multi- spectra for potential characterization of non-covalent interaction. The dimensional confocal fluorescence spectroscopy with pulsed interleaved exci- frequency-energy spectrum of ions flowing inside membrane channels tation (MFD-PIE) were performed. constituting a Single-Molecule Interaction Spectrum, which bridges the Our results reveal that the highly dynamic H3NtT is located within 10 nm dis- gap in-between traditional ionic current recording and the MD simulations, tance from the nucleosomal dyad axis and the linker DNA featuring multiple

BPJ 9447_9453 Wednesday, March 6, 2019 469a interaction modes. We resolved three DNA associated and one histone core Studies of multi-domain proteins and multi-protein machines necessitate a associated conformations and found that the H3NtTs are following the DNA deeper understanding of dynamic structure and transient conformations. There- motions during the disassembly process. The two major steps of the proposed fore, conventional structure determination methods need to be integrated into NaCl induced nucleosome disassembly process: 1) unwrapping of the weakly dynamic detection approaches. bound DNA side and 2) weakening of the more stable DNA side coincide We developed a hybrid method that integrates x-ray structure information into with the loss of the respective H3NtT:DNA interactions. Those results comple- self-consistent distance networks based on single-molecule Fo¨rster resonance ment our earlier model on the nucleosome disassembly with hitherto unknown energy transfer (FRET) [1,2]. By analyzing time-correlated distance distribu- intermediate steps obtained from the perspective of the labeled histone tails and tions globally, we can separate real protein dynamics and fluctuations from emphasize the pivotal role of the H3NtT for nucleosome integrity. modelling uncertainties, and ultimately generate time-correlated structural en- sembles. On top of that, we reveal correlated small- and large-scale intra-mo- 2319-Plat lecular fluctuations from time-resolved, color- and polarization-sensitive Yeast Pioneering Transcription Factors Rely on Slowed Dissociation Ki- fluorescence measurements. netics to Efficiently Target Nucleosomal Sites We applied our approach to the heat shock protein Hsp90. The chaperone ac- 1 2 1 2 2 Benjamin T. Donovan , Hengye Chen , Caroline Jipa , Chao Yan , Lu Bai , tivates large sets of signal transduction proteins often assisted by co-chaperones Michael G. Poirier1. 1 2 [3]. These conformation- and nucleotide-dependent processes are not yet Physics, Ohio State Univ, Columbus, OH, USA, Penn State Univ, comprehensively understood. Our approach resembled the x-ray structure of University Park, PA, USA. Hsp90’s closed conformation with an RMSD of 2.8 A˚ . Beyond that, we Nucleosomes are key regulators of transcription factor (TF) occupancy by re- resolved the previously unknown dynamic open structure of this multidomain stricting binding and accelerating dissociation rates, while pioneer transcrip- protein. The large-scale fluctuations on the lower millisecond timescale might tion factors (PF) somehow circumvent nucleosome regulation. Here, we be the basis of a general regulation mechanism. investigate the essential S. cerevisiae TF Reb1, which binds nucleosome Finally, I want to show how FRET can be orthogonally integrated into cross- entry-exit sites with high occupancy. We show that Reb1 binds its site within disciplinary platforms such as a microfluidics [4] and others, enabling the nucleosomes, trapping a partially unwrapped state without histone eviction. simultaneous detection of dynamic structure and low-affinity interactions. Reb1 binds nucleosomes and DNA with the same affinity, while surprisingly [1] Hellenkamp B, Wortmann P, Kandzia F, Zacharias M, Hugel T. Multido- the binding and dissociation kinetics are 50-fold slower at nucleosome sites main structure and correlated dynamics determined by self-consistent FRET relative to DNA. We then found similar results for a separate S. cerevisiae networks. Nature Methods 14(2), 174-180 (2017) TF, Cbf1. From these results, we propose that Reb1 and Cbf1 are pioneer fac- [2] Hellenkamp B, Schmid S et al. Precision and accuracy of single-molecule tors that target sites within nucleosome entry-exit regions by decreasing FRET measurements—a multi-laboratory benchmark study Nature Methods their dissociation rates to compensate for reduced nucleosome binding. This 15, 669-676 (2018) allows these PFs to induce nucleosome unwrapping and reside at nucleosome [3] Taipale, M et al. Quantitative Analysis of Hsp90-Client Interactions Re- entry-exit sites for minutes to facilitate the recruitment of transcription veals Principles of Substrate Recognition. Cell 140(5), 987-1001 (2012) coactivators. [4] Hellenkamp B, Thurn J, Stadlmeier M, Hugel T., submitted 2320-Plat 2322-Plat Role of RNA-Binding Activity of Hu in Chromosomal Organization Probing Structural States in Fast Exchanging Proteins by FRET and 1 2 2 2 Kelsey E. Bettridge , Xiaoli Weng , Subhash Verma , Sankar Adhya , Computational Methods 1 Jie Xiao . Hugo Sanabria. 1Biophysics and Biophysical Chemistry, Johns Hopkins Sch Med, Baltimore, 2 Dept Phys/Astro, Clemson University, Clemson, SC, USA. MD, USA, National Cancer Institute, National Institutes of Health, Fo¨rster Resonance Energy Transfer provides distance information between Bethesda, MD, USA. spectroscopic labels that are chemically coupled to the biomolecule of The E. coli chromosome must condense over a thousand fold to fit into the interest. A series of designed positions could provide sparse distance infor- micron-sized cell. This compaction is spatially organized by several factors, mation to describe the topology of the system. Given the fact that the motion including a group of proteins termed nucleoid-associated proteins (NAPs). of the spectroscopic labels is decoupled from the dynamics of the backbone The most highly conserved NAP in eubacteria is HU, comprised of two sub- of biomolecules, we can identify limiting conformational states even when units that can form homo- or heterodimers, which binds to the bacterial these exchange at rates in the microsecond timescales. Complementary in- DNA with no sequence preference. While not lethal, deletion of both subunits formation is provided by independent molecular dynamic information that of HU results in changes to replication, transcription, and chromosomal archi- fill in the missing degrees of freedom of biomolecular motion. Combining tecture. Puzzlingly, the biochemical properties of HU suggest it’s DNA- molecular dynamic simulations and a set of FRET-derived distances, exper- binding activity is particularly poor, in the millimolar range. Recently, it was iments allows one to create structural models of fast exchanging conforma- discovered that HU can bind to various RNAs inside the cell, in particular small tions. We apply this to resolved previous diverging structural models of the non-coding RNAs from a repeat extragenic palindromic (REP) sequence, re- N-terminal PDZ1-PDZ2 tandem of the Postsynaptic density protein of 95 named nucleoid-associated RNAs (naRNAs). Deletion of this REP element kDa (PSD-95). We identified two conformations: an open-like conformation hinders the ability of HU to bridge chromosomal contacts, and this ability with a small contact interface stabilized by salt bridges, and a closed-like can be restored by expression of just one of the naRNAs. This suggests a mech- conformation with a larger contact interface stabilized by surface-exposed anism by which HU binds naRNAs and bridges interactions in the chromosome hydrophobic residues. Both interfaces were independently probed. The through the naRNAs themselves. Here, we investigate the dynamics of HU us- low-energy barrier between conformations allows sub millisecond dy- ing superresolution single molecule tracking. We find that HU has two diffusive namics, which were time-averaged in previous diverging models. previous states corresponding to DNA-bound and freely diffusing, and that HU rapidly diver. Moreover, the small contact interfaces were likely overridden by lat- switches between the two states, confirming prior biochemical data. Addition- tice contacts as crystal structures were rarely sampled in simulations. This ally, HU dynamics and localization are drastically altered in both general RNA hybrid approach can identify transient inter domain interactions, which depletion, and specific naRNA depletion. Finally, our data suggests HU must be are abundant in multidomain proteins yet often obscured by dynamic able to bind both HU and the DNA in order to execute its function. Together, averaging. our data supports a model in which HU mediates chromosomal contacts through naRNAs. 2323-Plat Integrative Dynamic Structural Biology with Fluorescence Spectroscopy Platform: Member Organized Session: Claus A.M. Seidel1, Mykola Dimura2, Hugo Sanabria3, Katherina Hemmen4, Integrative Structural Modeling Using Thomas-Otavio Peulen5, Dmitro Rodnin1, Holger Gohlke6. 1Chemistry, Heinrich Heine University, Duesseldorf, Germany, 2Heinrich Information from Spectroscopic Lables Heine Univ, Dusseldorf,€ Germany, 3Dept Phys/Astro, Clemson University, Clemson, SC, USA, 4Rudolf-Virchow-Zentrum fur€ Experimentelle 2321-Plat Biomedizin, University Wuerzburg, Wuerzburg, Germany, 5Physical Single Molecule FRET - A Multi-Environment Ruler for Determining Chemistry II, Duesseldorf, Germany, 6Inst Pharm Med Chem, Univ Structure and Dynamics Duesseldorf, Duesseldorf, Germany. Bjorn Hellenkamp. Fo¨rster Resonance Energy Transfer (FRET) studies on the ensemble and single- Columbia University, New York, NY, USA. molecule level probe biomolecular structure and dynamics and identify

BPJ 9447_9453 470a Wednesday, March 6, 2019 coexisting conformational states. Integrative structural biology solves bio- measured distances with a modeling approach best suited for the specific sys- molecular structures by combining data from different sources, which indi- tem under investigation. As such, the integrative experimental/computational vidually would be insufficient. Here, we combine FRET experiments with approach described here represents a hybrid method for determining all-atom computer simulations to overcome their sparsity. I will discuss recent meth- models based on experimentally-derived distance measurements. odological developments in integrative structural modelling by satisfying spatial restraints on networks of FRET pairs (hybrid-FRET). We introduce 2326-Plat new procedures for: (i) an automated FRET experiment design tool deter- Dynamic Enzyme: An NMR Study of USP7 mining most informative FRET pairs for structural modeling using prior Irina Bezsonova, Gabrielle Valles, Dmitry M. Korzhnev. structural knowledge (https://github.com/Fluorescence-Tools), (ii) a protocol Molecular Biology and Biophysics, Univ Connecticut Health, Farmington, for efficient FRET-assisted computational structural modelling at multiple CT, USA. scales (http://nmsim.de), (iii) choice of appropriate models for describing Ubiquitin-specific protease 7 (USP7) is a deubiquitinating enzyme (DUB) that the dye behavior and consequences for designing appropriate labels, (iv) plays a pivotal role in multiple oncogenic pathways and therefore is a desir- use of all eight characteristic fluorescence parameters as a vectorial local able target for new anti-cancer therapies. It is a cysteine protease that belongs and dynamic structure information, and (v) a new quantitative quality esti- to the USP family of DUBs. USP7 is unique among other USPs in that its mate for judging the accuracy of determined structures. I will present appli- active site is catalytically incompetent in the apo-state and can rearrange cations simulations and real experiments of our techniques to proteins such into a productive conformation upon substrate binding. Such substrate- as T4 Lysozyme (T4L). We used 33 variants with single-FRET pairs to induced structural rearrangements likely play a role of a ‘‘safety switch’’ resolve three T4L conformers via their characteristic distance sets. Screening that turns the enzyme ‘‘on’’ only when it is engaged with a specific ubiquiti- the known T4L structures, revealed that T4L in solution mainly adopts the nated substrate. Although USP7 has been crystallized in both apo- and known open and closed states in exchange at 4 ms. A newly found minor ubiquitin-bound forms that highlighted the structural differences between state, undisclosed by at present more than 500 crystal and NMR structures the active and inactive protein conformations, these structures represent static of T4L and sampled at 230 ms, may be actively involved in the product snapshots of the enzyme. Whether USP7 samples these conformations in so- release step in catalysis. Importantly, we released together with the PDB lution and the role USP7 conformational dynamics play in its function remain the initial version of the fluorescence (FLR) dictionary extension (https:// unknown. Using the latest developments in Nuclear Magnetic Resonance github.com/ihmwg/FLR-dictionary) on the Integrative/Hybrid Modeling (NMR) spectroscopy we have observed and characterized the conformational (IHM) working group GitHub site. Now, fluorescence-restrained structural dynamics of USP7 in solution. Our data suggest that apo-USP7 has two models can be deposited at PDB-Dev. distinct sites of conformational exchange. Remarkably, they are located in two key functional sites of the enzyme. 2324-Plat 2327-Plat Proteins’ Dynamics, Hydration and Conformational Changes Studied by Specific 13CH3 Labeling and NMR Reveal the Role of Structural Dy- EPR namics to Enzymatic Function Enrica Bordignon. Mioara Larion1, Alexandar Hansen2, Lei Bruschweiler-Li2, Chemistry and Biochemistry, Ruhr University Bochum, Bochum, Germany. Vitali Tugarinov3, Rafael Bruschweiler€ 2, Brian Miller4. Site-directed spin labeling EPR enables detection of conformational changes 1Neuro-Oncology Branch, NIH/NCI, Bethesda, MD, USA, 2Chemistry and in proteins with almost no restriction in the environmental conditions. Key in- Biochemistry, The Ohio State University, Columbus, OH, USA, 3Laboratory formation for structural analysis is provided by changes in the dynamics of of Chemical Physics, NIH/NIDDK, Bethesda, MD, USA, 4Chemistry and spin-labeled sites and by interspin distances between selected pairs of labels. Biochemistry, Florida State University, Tallahassee, FL, USA. Dynamics are detected by continuous wave EPR, interspin distances by pulse To understand the catalytic function of glucokinase, an enzyme that catalyzes dipolar techniques (DEER or PELDOR being the most common). The long- glucose during glycolysis following a kinetic cooperative mechanism, we em- range EPR distance constraints, combined with existing structural data at ployed 13CH3 labeling of methyl groups and NMR. We showed that the origin atomic level for one state of the protein, enable the creation of coarse- of kinetic cooperativity is rooted in intramolecular protein dynamics using kinetic grained models of complex protein rearrangements in their physiological CPMG-NMR data of 17 isoleucine sidechains distributed over all parts of GCK. milieu. Each protein conformational transition due to oligomerization, ligand Residues of glucose-free GCK located in the small domain display a distinct ex- binding, transfer from a water to a membrane environment, etc. is also tightly change behavior involving multiple conformers that are substantially populated coupled to rearrangements in the hydration water surrounding the different (p > 17%) with a kex = 509 5 51 s–1, whereas in the glucose-bound form these protein interfaces. Changes in local hydration dynamics and water accessi- exchange processes are quenched. This exchange process directly competes with bility can be monitored directly and with high precision at physiological tem- the enzymatic turnover rate at physiological glucose concentrations, thereby perature using Overhauser dynamic nuclear polarization (ODNP), which has generating the sigmoidal rate dependence that defines kinetic cooperativity. several advantages with respect to other EPR techniques which provide water accessibility data. We will show examples of structural studies in which the 2328-Plat changes in the protein and its surrounding water environment are observed. Decomposing NMR Ensemble with the Assistance of Single Molecule Examples will be given on ABC transporters, Bcl-2 proteins, and light FRET receptors. Chun Tang. Wuhan Inst Phys/Math, Chinese Academy of Sciences, Wuhan, China. 2325-Plat Proteins are inherently dynamic, and the dynamics allow the protein to fulfill An Integrated Spin-Labeling/Computational-Modeling Approach for specific functions. NMR is exquisitely sensitive to protein dynamics. In partic- Mapping Global Structures of Nucleic Acids ular, paramagnetic relaxation enhancement (PRE) has an distance Peter Z. Qin. dependence, and has been used to visualize sparsely populated protein confor- Dept Chemistry, Univ Southern Calif, Los Angeles, CA, USA. mation(s). However, owing to the averaging of NMR signals over many The technique of site-directed spin labeling (SDSL) provides unique infor- different copies of protein molecules, it can be difficult to resolve the consti- mation on biomolecules by monitoring the behavior of a stable radical tag tuting conformational states that contribute to the observables. Single molecule (i.e., spin label) using electron paramagnetic resonance (EPR) spectroscopy. techniques, on the other hand, can be used to visualize the fluctuation of a single Here we present an approach in which SDSL is integrated with computa- protein molecule over time. Here I will present how the characterization of tional modeling to map conformations of nucleic acids. This approach builds protein ensemble structures can be made easier and more accurate with the upon a validated SDSL tool kit that includes three components: (i) a incorporation of single molecule FRET data. The relative populations of the nucleotide-independent nitroxide probe, designated as R5, which can be effi- inter-converting conformational states can be obtained from single-molecule ciently attached at defined sites within arbitrary nucleic acid sequences; (ii) measurement, and detailed structural information can be characterized by inter-R5 distances in the nanometer range measured via pulsed EPR; and (iii) NMR and other biophysical techniques. Moreover, the calculated ensemble an efficient program, called NASNOX, that computes inter-R5 distances on structures of the protein can be verified by the distance information from single given nucleic acid structures. Following a general framework of data-mining, molecule FRET. Using both bulk and single-molecule techniques, we have our approach uses multiple sets of measured inter-R5 distances to retrieve resolved and visualized the ensemble structures of polyubiquitin and other ‘‘correct’’ all-atom models from a large ensemble of models. The pool of multi-domain/subunit proteins. We envision that such an integrative approach models can be generated independently without relying on the inter-R5 dis- will play an increasingly important role in characterizing the dynamics of bio- tances, thus allowing a large degree of flexibility in integrating the SDSL logical macromolecules.

BPJ 9447_9453 Wednesday, March 6, 2019 471a

Posters 2331-Pos Mutation of Residues in CD Loop and Distal Pocket Impact Protein Stabil- ity of Human Neuroglobin Posters: Protein Structure and Conformation IV Ruipeng Lei1, David Butcher1, Sophie Bernad2, Valerie Derrien2, Jaroslava Miksovska1. 1 2329-Pos Chemistry and Biochemistry, Florida International University, Miami, FL, 2 Neuronal Calcium Sensor Dream Interactions with Insulinotropic Agent USA, Chemistry, University of Paris XI Orsay, Orsay, France. Repaglinide Neuroglobin (Ngb) is a new member of hexa-coordinate vertebrate Heme- Maria D. Santiago, Maria Daniel Santiago, Jaroslava Miksovska. with several proposed functions such as ROS/RNS scavenging, and inhibition of Chemistry and Biochemistry, Florida International University, Miami, FL, USA. hypoxia-induced apoptosis. Nonetheless, the molecular mechanism for most of Downstream Regulatory Element Antagonist Modulator (DREAM) is a its functions is still not clear. Recent studies have shown a link between the struc- neuronal calcium sensor protein which is highly expressed in several areas ture and function of the protein, for example, the CD loop in Ngb is a potential of the central nervous system such as the hippocampus and the neural cortex. binding site for the G-alpha subunit and the distal histidine affects the thermody- It has been related to several physiological processes and linked to patholog- namic profile for gaseous ligands association. In order to characterize the struc- ical conditions such as Alzheimer’s disease. DREAM interacts with multiple ture function relationship in Ngb, we have introduced two mutants, the E53Q in intracellular partners and has a significant role in regulating gene expression, the CD loop region and H64Q in the distal pocket. UV-vis and fluorescence spec- kinetics of potassium channels, calcium homeostasis, and enzymatic activity troscopy together with time of flight-mass spectrometry have been used to deter- of presenilin. Previous studies have shown that the insulinotropic agent repa- mine how the mutations affect the Ngb stability. Our data indicate a distinct glinide targets neuronal calcium sensors by selectively binding to them in a stability profile of the two mutants in both liquid phase and gas phase. In the calcium-dependent manner. It has also been shown that DREAM binds to gas phase, Ngb WT is more stable in neutral pH whereas stability of Ngb mutants small hydrophobic molecules such as 1,8-ANS, CL 88, and NS5806. Here was pH independent. However, both mutants, especially E53Q, are significantly we aim to test the role of repaglinide as a neuronal calcium sensor antagonist less stable in the gas phase than the wild type. In the liquid phase, distal histidine through characterizing its interactions with DREAM and monitoring its replacement leads to a decrease in overall protein stability whereas the stability impact on protein structure and dynamics. Fluorescence studies showed a of E53Q mutant is comparable to that of WT. Nevertheless, the replacement of decrease in the Trp 169 emission upon repaglinide addition, providing a glutamine residue increases the protein solubility during pH induced unfolding 2þ Kd value of 30 mM in the presence of Ca and similar Kd value was deter- studies. Overall, our data provide information on the impact of the CD loop and mined for the apoprotein. Association of repaglinide to DREAM leads to a the distal histidine on protein stability in both gas phase and liquid phase. decrease in the Trp169 lifetime suggesting a dynamic quenching mechanism. 2332-Pos Titration of DREAM-1,8-ANS complex with repaglinide resulted in a Understanding Protein HD Exchange Data using Molecular Dynamics decrease in the emission intensity, pointing towards the binding of repagli- Simulations nide to one of the 1,8-ANS binding sites. Using AutoDock and molecular dy- Dipak B. Sanap1,2, V.V. Hemanth Giri Rao2, Juan R. Perilla1, namics we show that repaglinide binds to a hydrophobic cavity located Shachi Gosavi2. between the N- and C- terminal and the DREAM-repaglinide complex is sta- 1Chemistry and Biochemistry, University of Delaware, Newark, DE, USA, bilized through hydrogen bonds with Lys166 and Arg160. These results sug- 2Simons Centre for the Study of Living Machines, National Centre for gest that several members of the NCS protein family may serve as a target Biological Sciences Tata Institute of Fundamental Research, Bangalore, for repaglinide. India. 2330-Pos Hydrogen deuterium exchange (HDX)coupled with either nuclear magnetic The Effects of Common Disease-Causing Variations on the Structure and resonance (NMR) or mass spectrometry (MS)can be used to probe protein stabil- Stability of TREM2: An In Silico Examination ity and dynamics. Protein backbone amide protons exchange slower if they Hunter B. Dean1,2, Erik D. Roberson3,4, Yuhua Song1. become inaccessible to the solvent protons either through burial in the protein 1Department of Biomedical Engineering, The University of Alabama at structure or through the formation of hydrogen bonds. Protein dynamics further Birmingham, Birmingham, AL, USA, 2Medical Scientist Training Program, modulate this exchange and mapping the rate of amide exchange back onto the The University of Alabama at Birmingham, Birmingham, AL, USA, protein structure can be used to identify mechanisms of conformational transi- 3Department of Neurology, The University of Alabama at Birmingham, tions and of protein folding. In order to gain a theoretical understanding of Birmingham, AL, USA, 4Department of Neurobiology, The University of HDX and in turn, protein dynamics, the rates of HDX have been correlated Alabama at Birmingham, Birmingham, AL, USA. with protein structural parameters like solvent access, atomic depth within the Single nucleotide variations in TREM2 are associated with both a late-onset structure, etc. However, individually, these parameters have patchy correlations form of Alzheimer’s disease (AD) and a severe, early-onset demyelinating de- with experimental data. Here, we computationally examine the simpler problem mentia, which can present in isolation as a variant of frontotemporal dementia of differentiating between those amides whose dynamics are visible through (FTD) or with cystic bone lipomas in a condition called Nasu-Hakola disease HDX experiments and those which exchange so fast that they have already (NHD). Published X-ray crystallography models of the TREM2 Ig domain exchanged on the time scale of the experiment. We perform short atomistic mo- have suggested that homozygous NHD- or FTD-causing mutations are grossly lecular dynamics (MD)simulations of three representative proteins, CBTX, inactivating while variations associated with increased risk of AD in hetero- ACBP and BPTI, calculate several previously proposed structural parameters zygous patients are centered on a prominent surface patch of positively (solventaccessible surface area, atomic depth, population of open states) from charged residues. This surface patch, which is not present in other members these simulations and then compare them with experimental data. We also of the TREM family, has been proposed as the primary binding site for construct a new parameter based on interatomic forces. We find that no individ- various polyanionic ligands. Importantly, AD-associated variations in ual parameter explains the experimental HDX data completely. However, com- TREM2 appear to alter the electrostatics or gross structure of this surface binations of parameters give better agreement. We then prescribe a specific patch, but do not cause larger structural changes in published crystal struc- combination which gives near perfect correlation with experimental amide pro- tures. Because crystal structures for many rare variants involved in disease tection patterns across all three proteins. We conclude that the mechanism of remain unpublished, the precise effects of these variants on binding site struc- HDX includes several physical processes and diverse structural parameters are ture and dynamic motion remain unclear. Here we perform molecular dy- required to capture the combined effect of all these steps. namics using AMBER14 to study the effects of mutations associated with AD, NHD, and FTD on stability of TREM2. Preliminary results show 2333-Pos increased conformational flexibility in FTD and NHD variants, which is un- The Microphthalmia-Associated Transcription Factor Associates with changed in AD variants. Consistent with proposed losses of structural stability Multiple Domains of CBP/P300, Including the E1A Binding Face of TAZ2 from previous reports, this increased flexibility is present near the putative Kathleen Vergunst, Alexandra Brown, Makenzie Branch, ligand-binding region and could directly contribute to lost or weakened bind- David N. Langelaan. ing between TREM2 and putative ligands, such as the AD-associated proteins Biochemistry & Molecular Biology, Dalhousie University, Halifax, NS, APOE and amyloid-b. Thus, further work toward thorough understanding of Canada. conformation and dynamical motion in this region of TREM2 remains critical Transcription factors control gene expression and coordinate fundamental pro- to our understanding of the role of TREM2 in AD and other neurodegenera- cesses such as cell growth and differentiation. The microphthalmia-associated tive diseases. transcription factor (MITF) is a melanocyte-specific protein essential for

BPJ 9454_9455 472a Wednesday, March 6, 2019 melanocyte development and differentiation. MITF has also been identified as a simulations to explore the structural dynamics of the three different VP40 inter- lineage-specific oncoprotein in melanoma, and knockdown of MITF function face structures. Normal mode investigations and analysis of our MD simula- results in the activation of cellular senescence. MITF contains N-terminal tions show that the CTD-CTD structural interface is the most flexible of the and C-terminal activation domains as well as a central basic helix-loop-helix three different interface. This indicates that the high flexibility of the CTD- DNA binding motif. Both the N-terminal and C-terminal activation domains CTD interface may be essential for the supple bending of the Ebola virus, directly interact with the homologous histone acetyltransferases CBP/p300 and possibly the other filoviruses. The flexibility of this interface may also pre- and are required for MITF-dependent transcriptional activation. We have sent a target for molecular interventions to disrupt the functioning of the Ebola used a combination of pull-down experiments, NMR spectroscopy, biophysical virus. studies, and functional transactivation assays to show that the N-terminal acti- vation domain of MITF is intrinsically disordered in solution and directly inter- 2336-Pos acts with the TAZ2 domain of CBP/p300 with high affinity. NMR-based Linking the Sequence, Anti-Tumor Function, and Shared Structural Fea- titrations indicate that MITF also interacts with the KIX and TAZ1 domains tures of Class Ib Hydrophobins of CBP/p300, raising the possibility of co-operative interactions between these Calem Kenward, David N. Langelaan. domains. Competition experiments indicate that E1A is able to displace MITF Biochemistry & Molecular Biology, Dalhousie University, Halifax, NS, from TAZ2. Experiments aimed at determining the functional effect of block- Canada. ing the MITF:TAZ2 interaction are ongoing. These results provide insight as to Hydrophobins are low molecular weight (5-20 kDa) self-assembling proteins how MITF may control gene expression in melanoma and support a model in secreted by fungi that accumulate at hydrophobic-hydrophilic interfaces and which MITF interacts with multiple domains of CBP/p300 to activate transcrip- are extremely surface-active. Hydrophobins may undergo structural rear- tion of MITF-target genes. rangement and oligomerize to form rodlets, which are an insoluble func- tional amyloid that coats fungal spores to act as a water repellent, 2334-Pos facilitate dispersal into the air, and prevent immune recognition. Due to their Evolution of Stability/Flexibility Relationships in Beta-Lactamase biochemical properties hydrophobins are a target a wide range of potential John Patterson1, Matthew C.B. Tsilimigras1, Dennis R. Livesay2, commercial applications. Hydrohphobins might also find use as a cancer Donald J. Jacobs3. treatment as they have also been shown to have anti-tumor properties, 1Bioinformatics and Genomics, UNC Charlotte, Charlotte, NC, USA, 2Dept although the precise mechanism of this action is unclear. To better under- Chem, Wichita State University, Witchita, KS, USA, 3Dept Phys, Univ North stand which sequence characteristics determine hydrophobin properties, we Carolina Charlotte, Charlotte, NC, USA. are characterizing the structure and properties of class IB hydrophobins We have curated over 100 structures of class A beta-lactamase proteins, a fam- from various fungal sources. Target proteins from Serpula lacrymans ily of enzymes that is responsible for a considerable percentage of multidrug (SL1), Wallemia ichthyophaga (WI1), and Phanerochaete carnosa (PC1) antibiotic resistance in bacteria. Our dataset includes three ancestral reconstruc- were chosen for study due to their different sequence composition. We ex- tions of pre-hominid beta-lactamase structures unperturbed by modern day se- pressed uniformly 13C/15N-labeled protein in E. coli and then purified it to lective pressures on antibiotic resistance. Having ancient to extant protein homogeneity using Ni2þ affinity and RP-HPLC and determined the high- structures represented, we sweep over the feature space governing thermody- resolution structure using NMR spectroscopy. We discovered that each hy- namic stability to identify underlying cooperativity motifs that are conserved drophobin contains shared structural features despite their dissimilar over the evolution of this protein family. Across all proteins within our dataset, sequence compositions. The core feature is a four strand anti-parallel b-sheet mechanical cooperativity is analyzed by a minimum Distance Constraint Model that is connected by three loop sequences (L1-L3). In all hydrophobins the in a similar fashion to previous work [1] to obtain quantitative stability/flexi- b-sheet folds to form a b-barrel-like structure, however the first loop is disor- bility relationships (QSFR). We create an innovative thermodynamic landscape dered in WI1 while it is an a-helix in SL1 and PC1. Spectroscopic amyloid that describes the variance in thermodynamic stability across this family of formation assays indicate that these hydrophobins have differing propen- proteins based upon a model parameter space that is relevant to enzyme activ- sities to form rodlets and are using AFM to characterize rodlet morphology. ity. Using clustering and machine learning techniques, we identify relevant We are also examining which sequence and structure characteristics of hy- chemo-physical constraints that each beta-lactamase structure places on func- drophobins influence their anti-tumour activity. Overall, this work will pro- tion and mechanical cooperativity over a diverse range of thermodynamic con- vide a fundamental correlation between the structure, function, and anti- ditions. Under appropriate stability conditions for functionality, we aim to tumour properties of hydrophobins. elucidate why antibiotic resistance differs between members in this class, despite all members sharing similar functional sites, global dynamics, and over- 2337-Pos all structural similarity. We also take into consideration the known pharmaco- Measuring Ionic Strength Changes using Fluorescence Lifetime and Time- logical metadasta for each of these proteins to better understand the binding Resolved Anisotropy 1 1 1 1 1 mechanisms between these enzymes and their various substrates. Our bio- Robert Miller , Cody Aplin , Anh Cong , Christin Libal , Rowan Simonet , 1 2 1 informatics approach is designed to give insight into selective pressure and Emma Kauffman , Margaret Gurumani , Ryan Leighton , 1 1 3 possible hidden mechanisms behind substrate selectivity in modern day beta- Alexander Naughton , Jessica Marshik , Arnold J. Boersma , 1 1 lactamases, while revealing the relevant evolutionary factors that guide ligand Ahmed A. Heikal , Erin D. Sheets . 1Dept Chem & Biochem, Univ Minnesota Duluth, Duluth, MN, USA, 2Dept selectivity to change. 3 [1] Verma, Jacobs and Livesay, PLOS Computational Biology 2013, 9(7) Chem & Biochem, College of St Scholastica, Duluth, MN, USA, DW1- e1003155. Leibniz Institute for Interactive Materials, Aachen, Germany. Intracellular ionic strength affects many factors such as cell volume, cata- 2335-Pos lytic activities, and molecular interactions. A family of hetero-FRET sensors Dynamics of the Protein Interfaces of the Ebola Virus VP40 Structural has been designed to detect changes in ionic strength in vivo and in vitro. Matrix Filament These proteins consist of a donor (mCerulean) and an acceptor (mCitrine) Elumalai Pavadai, Nisha Bhattarai, Prem P. Chapagain, that are connected via a single flexible polypeptide hinge with a basic helix Bernard S. Gerstman. and an acidic helix. The basic helices are enriched with either arginine or Physics, Florida International University, Miami, FL, USA. lysine, and the acidic helices are enriched with either glutamate or aspartate. We used molecular dynamics (MD) computations to investigate the dynamics The FRET sensors (RE, RD, and KE) are designed to have maximal energy of the structural matrix of the Ebola virus. The Ebola protein VP40 is one ofs- transfer at low ionic strength and to decrease monotonically as electrostatic even proteins that are encoded by the Ebola virus genome. VP40 plays crucial screening of the charged helices occurs with increasing ionic strength. In this roles in viral assembly and budding at the plasma membrane of the infected study, we investigate the effects that different linker structures have on the cells. VP40 is a transformer protein that exists in different conformational energy transfer efficiencies as measured using fluorescence lifetime and and oligomeric states. One of VP40’s roles is to form the long, filamentous time-resolved anisotropy. We find that RE consistently has the greatest structural matrix for the virus. The VP40 monomer is composed of two do- energy transfer across all salt concentrations and RD has the lowest, with mains: NTD and CTD. The basic building block of VP40 filaments is a KE having intermediate sensitivity. This family of proteins has been VP40 dimer that oligomerizes into a hexamer. The hexamers then join end- characterized as a function of potassium chloride and other ions in the Hof- to-end to form a long filament. The filaments contain three different interfaces: meister series. Our solution studies indicate that these proteins are useful monomer-to-monomer NTD-NTD to form a dimer, central dimer-to-dimer sensors of biologically relevant ionic strengths. Importantly they have poten- NTD-NTD oligomerization to form a hexamer, and end-to-end hexamer tial for dynamically mapping ionic strength changes in response to biological CTD-CTD to build the filament. We performedexplicit-solvent, all-atom MD stimuli.

BPJ 9454_9455 Wednesday, March 6, 2019 473a

2338-Pos in conformation and corresponding energetics of dynamin oligomers will Advances in the Structural and Biochemical Determination of Several help shed light on how dynamin helical oligomers are able to induce membrane Dynamin-Like GTPases scission. Andrew D. Kehr1, Shuxia Meng2, Matthew F. Martin1, David Chan2, Jenny E. Hinshaw1. 2341-Pos 1NIDDK, NIH, Bethesda, MD, USA, 2Caltech, Pasadena, CA, USA. The Development of Intrinsically Fluorescent Unnatural Amino Acids for Dynamins are a class of GTPase enzymes responsible for the fusion, fission, In Vivo Incorporation into Proteins and vesiculation of lipid membranes throughout the cell. The dynamin-like Chloe M. Jones1, Itthipol Sungwienwong2, E. James Petersson2. protein Optic Atrophy 1 (Opa1) is responsible for the fusion of the mito- 1Biochemistry and Molecular Biophysics, University of Pennsylvania, chondrial inner membrane and plays a role in maintaining cristae shape. Philadelphia, PA, USA, 2Chemistry, University of Pennsylvania, Atlastin mediates fusion of homotypic three-way junctions in the endo- Philadelphia, PA, USA. plasmic reticulum (ER). Mutations in these, and other dynamin-like proteins, The amino acid acridonylalanine (Acd) can be a valuable tool for studying pro- can lead to neuropathies including Dominant Optic Atrophy, Hereditary tein dynamics. On its own, Acd’s long fluorescence lifetime of 15 ns has been Spastic Paraplegia, and Charcot-Marie-Tooth, among others. Currently, beneficial for polarization experiments to reveal protein binding dynamics in structural and biochemical data is limiting for Opa1. In addition, crystal biological pathways. Acd can also reveal structural dynamics as part of a Fo¨r- structures of ATL with nucleotide do not entirely explain the GTPase cycle. ster resonance energy transfer (FRET) or photoinduced electron transfer (eT) We have developed a protocol for expressing and purifying biologically probe pair. We have reported successful, selective genetic incorporation of relevant and biochemically active shortened isoforms of Opa1 (Opa1GG) Acd by an aminoacyl tRNA synthetase. This allows us to site-specifically label in sufficient quantity to begin crystallography and biochemical assays. a protein of interest expressed in E. coli with minimal perturbation. While Acd Furthermore, we have optimized sample prep and begun reconstructions is valuable in itself, we are developing on a series of Acd derivatives that can of aproteolytically processed short form, Opa1S, by cryo-EM on a lipid sur- shift Acd’s fluorogenic properties while still being ribosomally permitted for face and are examining the role nucleotide plays in structural rearrange- genetic incorporation. We have shown that we can red shift Acd’s absorption ments. In addition, to verify work performed by x-ray crystallography, we and emission spectra and modulate it’s brightness. One derivative has been have begun probing the GTPase cycle of Atlastin using single particle shown be an enhanced FRET acceptor in a peptide cleavage assay and can cryo-EM methods. be activated by the aminoacyl tRNA synthetase for genetic incorporation. Our long-term goal is to develop FRET pairs that can be completely genetically 2339-Pos encoded to enable monitoring of protein processes that cannot be accessed with Evolution of Transient Helicity and Disorder in Late Embryogenesis other labeling methods, such as real-time monitoring of co-translational protein Abundant Protein COR15A folding. Oluwakemi Sowemimo1, Wade Borcherds1, Patrick Knox-Brown2, Tobias Rindfleisch2, Anja Thalhammer2, Gary Daughdrill1. 2342-Pos 1Cell Biology Microbiology and Molecular Biology, University of South Applying Hydrogen Exchange Mass Spectrometry Coupled with Numeri- Florida, Tampa, FL, USA, 2Physical Biochemistry, University of Potsdam, cal Simulations to Investigate Toxic Misfolding of b2-Microglobulin Potsdam, Germany. Angelika Hirsch1, John Strahan2, Amy Wagaman3, Sheila Jaswal1. Cold regulated protein 15A (COR15A) is a nuclear encoded, intrinsically 1Biochemistry and Biophysics, Amherst College, Amherst, MA, USA, disordered protein that is found in Arabidopsis thaliana. It belongs to the 2Chemistry, Amherst College, Amherst, MA, USA, 3Mathematics and Late Embryogenesis Abundant (LEA) family of proteins and is responsible Statistics, Amherst College, Amherst, MA, USA. for increased freezing tolerance in plants. COR15A is intrinsically disordered b2 microglobulin (b2M) is normally part of the major histocompatibility com- in dilute solutions and adopts a helical structure upon dehydration or in the plex 1 which sits on the surface of human cells and presents peptides for the presence of co-solutes such as TFE and ethylene glycol. This helical structure surveillance of the immune system. In patients with dialysis-related amyloid- is thought to be important for protecting plants from dehydration induced by osis (DRA), however, this normally soluble protein aggregates into plaques freezing. Multiple protein sequence alignments revealed the presence of that deposit into joint spaces. Although there are slight differences in the several conserved glycine residues that we hypothesize keeps COR15A plasma conditions under dialysis (elevated copper and urea levels), these from becoming helical in dilute solutions. Using AGADIR, the change in appear not to significantly affect the global stability of the protein. Therefore, helical content of COR15A when these conserved glycine residues were sub-global events must underlie the transformation of b2M into amyloid pla- mutated to alanine residues was predicted. Based on the predictions, glycine ques. Hydrogen exchange mass spectrometry (HXMS) experiments probing to alanine mutants were made at position 68, and 54,68,81, and 84. Labeled these events reveal that b2M populates a partially unfolded intermediate under samples of wildtype COR15A and mutant proteins were purified and NMR slightly destabilizing conditions of 20% Acetonitrile (AcN). Here we apply nu- experiments were performed to examine any structural changes induced by merical simulations to fit HXMS data for b2M at amyloid-inducing concentra- the mutations. To test the effects of dehydration on the structure of tions of copper and at progressively lower concentrations of AcN, including COR15A, trifluoroethanol, an alcohol based co solvent that is proposed to 0 denaturants. The denaturant- dependent results will help determine to what induce/stabilize helical structure in peptides was added to the NMR samples, extent this intermediate is populated under more native conditions. The dena- and the results of the experiment showed an increase in helical content, turant dependence of the extracted rate constants for the transitions between the compared to the samples without TFE. To test the functional differences be- native, intermediate, and unfolded conformations of b2M will provide insight tween wild type and the mutants, liposome leakage assays were performed. into how their structural features differ. Furthermore, this work will illuminate The results from these assays suggest the more helical mutants may augment the denaturing properties of AcN, which has not been extensively investigated membrane stability. as a denaturant, but holds promise for use in HXMS studies of protein folding and dynamics because of its volatility. 2340-Pos Molecular Dynamics Studies of Dynamin Oligomers in Solution 2343-Pos Dalia Hassan, Frank X. Vazquez. Studying Mutations in Geobacillus kaustophilus Tils to Probe Changes in Chemistry, St. John’s University, Jamaica, NY, USA. Structure and Mobility using Molecular Dynamic (MD) Simulations Dynamin is an enzyme that forms large protein oligomers on membrane vesicle Ferdiemar C. Guinto, Rebecca W. Alexander. necks and causes membrane scission in the final stage of endocytosis. Several Chemistry, Wake Forest University, Winston-Salem, NC, USA. models for dynamin induced membrane scission have been proposed, but there tRNA isoleucine lysidine synthetase (TilS) is a nucleotide modifying enzyme are still several unanswered questions about the molecular mechanism of dyna- that contributes to translational accuracy. TilS catalyzes formation of lysidine min induced membrane fission. Understanding how the various domains of dy- at the C34 wobble position of tRNAIle2, which alters recognition and specificity namin move with respect to each other could help shed light on the mechanism during protein biosynthesis at the ribosome. This base modification is essential of dynamin induced membrane fission. To this end, we simulated dynamin for decoding the minor isoleucine AUA codon in prokaryotes. The opportu- monomers, dimers, and tetramers in solution to understand how the energetics nistic human pathogen Burkholderia cenocepacia evolved TilS mutations in of the protein motions are affected by oligomerization. The missing loops from response to nutrient depletion. These mutations exhibit a loss of catalytic func- available crystal structures for the human dynamin 1 monomer and dimer were tion while retaining binding to the TilS Substrate tRNAIle2. Using molecular modelled and then relaxed. Afterwards, monomers and oligomers were equili- modeling and MD simulations, mutations have been evaluated in the context brated in solution to observe the conformational changes that dynamin un- of the 3.65 A˚ Geobacillus kaustiphilus TilS:tRNAIle2 crystal structure. The re- dergoes as it forms naturally occurring oligomers. Understanding the changes sulting changes in structure, dynamics, or intramolecular communication can

BPJ 9454_9455 474a Wednesday, March 6, 2019 be used to evaluate the rationale for these loss-of-function, gain-of-fitness 2346-Pos mutations. Interaction of the Curli Accessory Proteins CsgE and CsgF with the Hu- man Islet Amyloid Polypeptide 2344-Pos Osmar Meza-Barajas, Isamar Aranda, Ashwag Binmahfooz, Differentiating Structural Changes of Glycoproteins in Solution using Sajith A. Jayasinghe. Small Angle Scattering Analysis Chemistry and Biochemistry, California State University, San Marcos, CA, Taylor N. Segally1,2, Luis A. Palacio2,3, Jason Kim2,4, USA. Christopher B. Stanley5, Soenke Seifert6, Horia I. Petrache3. Gram-negative bacteria, such as E.coli and Salmonella, contain proteinaceous, 1Department of Psychology, Indiana Univ. Purdue Univ. Indianapolis, 2 hair-like, cell surface filaments known as curli. Curli serve to facilitate cell-cell Indianapolis, IN, USA, Innovation and Research Division, Health & Science interactions and are essential for host cell colonization. Curli assembly involves Innovations, Inc., Indianapolis, IN, USA, 3Department of Physics, Indiana 4 six proteins, CsgA, CsgB, CsgC, CsgE, CsgF, and CsgG. CsgE and CsgF are Univ. Purdue Univ. Indianapolis, Indianapolis, IN, USA, Department of thought to act as chaperones to help prevent the premature aggregation of Chemistry & Chemical Biology, Indiana Univ. Purdue Univ. Indianapolis, CsgA and/or CsgB, and to help transport these proteins, through the outer- Indianapolis, IN, USA, 5Biology and Soft Matter Division, Oak Ridge 6 membrane protein CsgG, to the cell surface where they assemble to form curli. National Laboratory, Oak Ridge, TN, USA, Chemical and Materials We have observed that both CsgE and CsgF are able to inhibit the aggregation Science, X-ray Science Division, APS, Argonne National Laboratory, of CsgA, as well as human Islet Amyloid Polypeptide (hIAPP), an amyloido- Lemont, IL, USA. genic polypeptide that is unrelated to curli. We are investigating the nature Alpha 1-Antitrypsin (A1AT) and Fibrinogen are plasma glycoproteins with of the CsgE/CsgF interaction with hIAPP using a series of single cysteine mu- different, but specific biological functions. A1AT (which forms part of the ser- tants of the two proteins and fluorescence spectroscopy. Fluorescence quench- pin family) has been shown to have protective roles of lung cells against ing studies indicate that the C-terminal regions of CsgE/CsgF may be involved emphysema, a disease characterized by lung tissue destruction, while fibrin- in interacting with hIAPP. ogen is a major factor in the blood clotting process. While biologically distinct, the two proteins present similarities in biophysical studies due to their size and 2347-Pos polymerization tendency. Previous approximations, using radius of gyration Comparative Investigation of Native-State Dynamics in Trypsin Func- analysis (shape independent), have shown that there is a phase transition for tional Variants by Hydrogen Exchange Mass Spectrometry A1AT under osmotic stress. The goals of this study are to better understand: Kimberly Burnett, Maxum Paul, Katie Ventre, Abel Samanez, the stability of A1AT’s native conformation when its environment deviates Sheila Jaswal. from physiological conditions; and the effects on fibrin’s final structure as Amherst College, Amherst, MA, USA. fibrinogen undergoes polymerization under different conditions. Environ- Trypsin is the mammalian homolog of the bacterial alpha-lytic protease (aLP). mental variables include osmotic stress, crowding, and presence of other mol- Although aLP has evolved extreme longevity through dampening dynamics on ecules (e.g. lipids, catalytic enzyme thrombin, or cholesterol) found in plasma. the local and the global scales, trypsin’s function over its lifetime is regulated The method used to assess the stability of the native conformation was to through a series of catalytic cleavage events. Trypsin must, therefore, possess observe changes in the structural envelope of the protein. Static and dynamic enough local flexibility to be accommodated into the protease active site. In addi- small angle x-ray scattering (SAXS) and small angle neutron scattering tion, these cleaved trypsin variants are virtually identical structurally, despite (SANS) experiments of the proteins in solution were conducted for this pur- different functional activities. This suggests that trypsin’s functional variants pose. Scattering data was fitted using various shape dependent models in SAS- must also differ in dynamics. Here we report results from applying hydrogen ex- View. Pair distance functions (PDF) were used to validate any changes change mass spectrometry (HXMS) to compare protein dynamics ranging from identified by the model fits. Multi-variable phase diagrams were constructed individual residue fluctuations to global unfolding among the variants. In addi- from the fitting results in order to quantify structural and conformational tion, we leverage the mass differences to measure HXMS for sets of trypsin var- changes of the proteins. iants simultaneously. Preliminary results suggest that the precursor and mature variants of trypsin possess significant global stability with distinct differences 2345-Pos in their sub-global dynamics. In addition, the singly cleaved trypsin variant main- Structural Investigations into the Serum Endonuclease Dnase1L3, as it tains partial enzymatic activity and remarkable stability. Quantitative investiga- Relates to Systemic Lupus Erythematous tion of both local and global events across these variants will provide insight into Jon J. McCord1, Faraz Harsini1, Peter Keyel2, Roger B. Sutton1. 1 2 the coupling of dynamics with function in trypsin. Furthermore, comparison with CPMB, Texas Tech Univ HSC, Lubbock, TX, USA, Biological Sciences, aLP will shed light on how dynamics are harnessed for the different functional Texas Tech Univ, Lubbock, TX, USA. needs of the structural homologs trypsin and alpha-lytic protease. Systemic Lupus Erythematosus (SLE) afflicts at least 5 million people world- wide. SLE is an autoimmunological disease characterized by multisystem 2348-Pos inflammation. SLE has poly and monogenic etiologies. SLE is characterized Structure and Function of Human Vitronectin, a Key Mediator of Host- by anti-dsDNA antibodies and anti-neutrophil cytoplasmic antibodies. Dna- Pathogen Interactions se1L3 is primarily expressed by myeloid cells in the blood, and dendritic Kyungsoo Shin, L. Miya Fujimoto, Luz M. Meneghini, Chandan Singh, and macrophage cells in the liver and spleen as an excreted endonuclease. Yong Yao, Ye Tian, Francesca M. Marassi. Dnase1L3 is a serum endonuclease in the Dnase1 family, that cleaves phos- Sanford Burnham Prebys Medical Discovery Institute, San Diego, CA, USA. phodiester bonds in dsDNA. As contrasted with Dnase1, Dnase1L3 is more Yersinia pestis is the gram-negative bacterium responsible for plague. It re- active in apoptotic microparticle degradation and, has more activity in chro- mains prevalent to this day and given its potential to be weaponized, its mech- matin DNA laddering. Undigested apoptotic microparticles and chromatin anism of infection must be elucidated to develop medical countermeasures. cause anti-dsDNA antibodies that give rise to lupus symptoms. Loss of func- Vitronectin (Vtn) is a 459-residue multidomain, multifunctional human serum tion mutations (such as R206C and a murine mutation T89I) in Dnase1L3 are protein that regulates many physiological responses, including blood clotting associated with pediatric onset lupus in humans. Dnase1L3 is not inhibited by and complement-mediated immune response. We recently identified Vtn as a actin and shows higher degradation activity than Dnase1 against DNA-protein binding partner of the Y. pestis outer membrane protein, attachment invasion complexes, this makes Dnase1L3 an important immunological enzyme, as locus (Ail). Strikingly, many other bacterial species have been shown to recruit well as a valuable biotechnical tool. The C-terminus helix present in Dna- Vtn at the bacterial surface to escape membrane attach complex formation, and se1L3 homology models, likely explains some of the function of Dnase1l3. promote bacterial survival and growth. Thus, the interaction between Ail and The C- terminal helix represents a substantial difference in secondary struc- Vtn appears to be critical for Y. pestis pathogenesis. Despite its importance, ture from Dnase1. Here we explore the relation of the C-terminal helix to structural characterization of Vtn is limited to the N-terminal 51 residues, vesicle interactions, through lipid vesicle co-sedimentation assays. While which fold as a somatomedin B domain. To rectify this issue, we have recom- the structure for Dnase1 is known, the structure for Dnase1L3 is still un- binantly generated a series of polypeptides spanning the sequence of Vtn, using known. Dnase1 has been proven ineffective as an enzyme therapy for SLE a bacterial expression system. One of these constructs encompasses C-terminal but, combined with Dnase1L3 effectiveness may increase. To investigate residues 134-458, which share homology with hemopexin (HPX) but remain the potential for Dnase1L3 to be utilized in enzyme therapy research, we first structurally and functionally uncharacterized. We are using NMR spectros- need to understand the structural basis that underlies Dnase1L3’s immunolog- copy, in combination with other biophysical and biochemical approaches, to ical disorder preventing activity. This is accomplished through expression, pu- characterize the protein structure, oligomerization state, and folding kinetics. rification and, crystallization of Dnase1L3. Towards the end of structural The Vtn generated polypeptides are also used to probe for Ail-binding sites analysis through X-ray crystallography. by means of biochemical and biophysical assays. Our results provide the first

BPJ 9454_9455 Wednesday, March 6, 2019 475a insights into the structure and function of this key human protein, Vtn, and its recombinant aciniform spider silk proteins, their potential as drug carriers, and molecular interactions with bacterial pathogens. the role of these self-assembled species in the initial stages of fibre formation. The aciniform spidroin protein (AcSp1) from Argiope trifasciata consists of 2349-Pos R14 repeat units of 200 amino acids (‘‘W’’, from wrapping silk) flanked by Single Molecule Imaging of DNA Structure: CLIC Microscopy Powers a non-repetitive N- and C-terminal domains. Recombinant proteins consisting Mechanistic Insights for Drug Development of a single repeat (W1) spontaneously self-assemble into spherical micelle-like Francis Stabile1, Cynthia Shaheen1, Shane Scott1, Daniel Berard1, nanoparticles in potassium phosphate buffer. Insoluble W1 nanoparticles also 2 3 1 David Levens , Craig Benham , Sabrina Leslie . form in highly concentrated phosphate buffer. Features of conformational tran- 1McGill University, Montreal, QC, Canada, 2National Cancer Institute, sitions associated with nanoparticle formation from monomeric W1 are delin- Bethesda, MD, USA, 3Genome Center and Applied Mathematics, California, eated here using tryptophan mutants of W1 through intrinsic tryptophan CA, USA. fluorescence and 19F NMR spectroscopy. Nanoparticle properties are correlated Ideally, a single-molecule platform would be simple to implement (in terms of to these features using electron microscopy, dynamic light scattering, and hardware, method, and analysis) and mimic cell-like conditions. This inspired Raman spectroscopy. Ultimately, reproducible and specific production of these us to develop Convex Lens-induced Confinement (CLiC) microscopy, to enable nanoparticles will assist in the design and optimization of aciniform silk-based direct visualization, manipulation, and quantification of biomolecules at the materials for biomedical and other materials applications. single molecule level. CLiC overcomes the limitations of existing techniques like TIRF (total internal reflection fluorescence), confocal microscopy, and op- 2352-Pos tical/magnetic trapping. By mechanically confining molecules to nanoscale 77Se-NMR Probes the Protein Environment of Selenomethionine wells (and other features), CLiC enables long observation times (from seconds Shiping Xu, Maggie Chen, Mike Boeri, Sharon Rozovsky. to hours) of untethered and freely diffusing biomolecules. Looking at hundreds Chemistry and Biochemistry Department, University of Delaware, Newark, of copies of molecules (or more) at once, it provides high statistics and good DE, USA. signal-to-noise, enabling dissection of complex processes. By enabling reagent 77Se NMR is highly sensitive to its chemical environment and can report on exchange and control over confinement geometry, CLiC mimics crowded con- the interactions and degrees of freedom available for selenium-containing ditions in cells. amino acids in biological systems. Particularly, selenomethionine can be In this work, we use CLiC imaging to investigate the role of DNA structure used as a probe for methionine environment, an amino acid which is often in mediating the binding activity of small molecules (oligonucleotide found in protein-protein interfaces and in ligand binding sites. Yet, for these probes, proteins) to target sites on DNA, building on our recent publication larger and more complex biological systems, reliable data interpretation that (Scott et. al., Nucleic Acids Research, 2018). We study the impact of tem- systematically connects variations of system variables to changes in the perature and DNA supercoiling upon the binding affinity and kinetics of NMR spectra is still lacking. To build a biological 77Se magnetic resonance small molecules to individual, untethered DNA plasmids. By interpreting databank, we thus constructed a library of GB1 variants with a single seleno- our microscopy results with supporting simulations, we investigate the methionine at different locations within the protein. The structure of five of role of specific higher-order structures (such as ZDNA) in actively medi- such GB1 variants was solved to 1.2 A˚ , allowing the conformation of the se- ating site-unwinding; for instance, we establish that ZDNA is prevalent at lenomethionine to be unambiguously determined. We recorded the isotropic 77 low temperature, but suppressed at high temperature. Further, we explore Se chemical shift for six GB1 variants along with their T1 relaxation times how oligonucleotide sequence, solution salinity, crowding agents, and other and line width. For four of the GB1 variants we were able to determine the cofactors (such as oligo-binding proteins) impact the interaction kinetics and chemical shielding tensor of SeM by 77Se solid-state NMR. Finally, we affinities. Looking ahead, we extend our CLiC nucleic acid assay to interro- combine solution and solid-state NMR with the structural information to gate and quantify the binding of modified DNA to RNA targets, a funda- arrive at general recommendations for the use of selenomethionine as NMR mental parameter in understanding the mechanisms and efficacies of probe in biological systems. nucleic acid therapeutics. 2353-Pos 2350-Pos Investigate the Existence of Domain Swapped Dimer in iLPB Family Temperature Dependence of the Protein-Chromophore Hydrogen Bond Nona Ehyaei1, Zahra Assar-Nossoni2, James H. Geiger1, Babak Borhan1. Dynamics in the Far-Red Fluorescent Proteins mNeptune1, mNeptune2.5 1Michigan State University, East Lansing, MI, USA, 2Cayman Chemical and mCardinal2 Company, East Lansing, MI, USA. Chandra Dhakal, Prem Chapagain, Xuewen Wang. Intracellular lipid binding proteins (iLBPs) are responsible for transport of Physics, Florida International University, Miami, FL, USA. different insoluble hydrophobic molecules in cytosol. Structures of members Low temperature experiments on mneptune1, mNeptune2.5 and mcardinal2 of this family include ten stranded beta barrel and two alpha helices which show a reduced stokes shift compared to room temperature. This suggests are like a cap for binding pocket of these proteins. Recently, we discovered a that the increased flexibility of the chromophore environment at higher temper- domain swapped dimer for Human cellular retinol binding protein II (hCRBPII) atures along with its ability to reorganize after excitation is related to the larger which is a member of iLBP. In Domain swapping two or more monomers ex- stokes shift. We used molecular dynamics (MD) simulations to investigate the change an identical part of their structures and form dimer or higher order olig- dynamics of the hydrogen bonds formed in the chromophore regions of the far- omers. The swapped region in hCRBPII is three beta strands with two alpha red fluorescent proteins mNeptune1, mneptune2.5 and mcardinal2. We helixes. Existence of domain swapping for this protein may have physiological explored the protein-chromophore hydrogen bond pattern at various tempera- relevant and may have huge effect on the folding pathway for iLBPs.1 We suc- tures and correlate the hydrogen bond dynamics to the experimentally observed cessfully expressed hCRPBII by Hela Cells and we got closer to find the size of Stoke’s shifts. hCRBPII in mammalian expression by using size exclusion chromatography and western blotting technique. The discovery of domain swapping in hCRBPII 2351-Pos also led us to research domain swapping in other types of iLBPs, such as human Investigating Recombinant Aciniform Silk Nanoparticles as Potential fatty acid binding proteins. We studied domain swapping in fatty acid binding Drug Carriers and as Intermediates in Silk Fibrillogenesis protein 5 (FABP5). FABP5 is responsible for endocannabinoid Stefan A. Warkentin, Jan K. Rainey. (AEA) transport in cytocol.2 We recently found the structure of domain swap- Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, ped dimer for FABP5 binds to palmitic acid. Our eventual aim is to predict Canada. domain swapping from amino acid sequences in iLBPs. Spider silks form biomaterials including fibres, films, and nanospheres with excellent mechanical properties and biocompatibility. As such, silk materials 2354-Pos have been investigated as a component of biomedical textiles for grafts and im- Exploring the Effect of Preorganization on Binding Affinity in Cyclized plants, as a scaffold for tissue regeneration, and as a drug delivery vehicle. Silk Peptidomimetics fibres generally form in vivo in a process where soluble proteins are forced Allison Terry1, Vincent Voelz2. through a narrowing spinning duct, giving rise to shear force. One theory of 1Physics, Temple University, Philadelphia, PA, USA, 2Chemistry, Temple fibre formation is that silk proteins are stored in micellar form, with these mi- University, Philadelphia, PA, USA. celles elongating while being forced together in the spinning duct, undergoing Peptidomimetics are a class of molecules able to bind and disrupt protein- morphological and conformational transitions as shear forces are applied. Silk protein interactions (PPIs) by mimicking complementary structure of a protein nanoparticles can be self-assembled, with concomitant or post-assembly incor- binder, making them potential candi-dates for drug molecules. Cyclization is a poration of a variety of drug-like molecules into their interior. Here, we focus common strategy intended to increase the binding affinity of peptidomimetics on the mechanism of formation of previously uncharacterized nanoparticles by and therefore produce better drug molecules. A simple conformational

BPJ 9454_9455 476a Wednesday, March 6, 2019 selection model predicts that increased preorganization leads to increased bind- amide PTM. In order to better utilize this modification for biophysical and ing affinity. To explore this idea, we constructed all-atom molecular topologies bioengineering purposes, a thioamide-specific probe is being developed to and performed molecular dynamics simulations of cyclized peptidomimetics to identify unknown thioamide-containing proteins. test if their relative preorganization shows a correlation to binding affinity. Our simulations of stapled peptide binders of PUMA suggest this indeed is the case, 2358-Pos at least for peptide staples which do not interfere with the binding interface. If Exploring the Space of Antimicrobial Peptides Guided by a Deep Learning further all-atom studies show similar trends, we may conclude that peptide pre- Model Manpriya Dua1, Amarda Shehu2. organization is an efficient way to screen potential drug molecules, circumvent- 1 2 ing the computational expense of receptor-ligand dissociation studies. Computer Science, George Mason University, Fairfax, VA, USA, Dept Compu Sci, George Mason Univ, Fairfax, VA, USA. 2355-Pos Resistance antibiotics among bacteria is on the rise. Research on finding ther- Expression and Purification of Complement Proteins for Protein Interac- apeutics against antibiotic-resistant bacteria is currently active. One promising tion Studies direction involves building over known antimicrobial peptides that constitute Matthew Gehm1, Veronica Singh2, Julia R. Koeppe2. the host’s innate immunity. There are now growing databases of antimicrobial 1Biological Sciences, SUNY Oswego, Oswego, NY, USA, 2Dept Chemistry, peptides discovered on several species, but such peptides do not currently pre- State Univ New York Oswego, Oswego, NY, USA. sent suitable therapeutic template for several reasons, including toxicity at the Complement is a system of plasma proteins that can be activated directly by concentrations needed to effectively kill bacteria. Given these constraints, it is pathogens or indirectly by pathogen-bound , leading to a cascade of of great interest to design novel antimicrobial peptides. Many laboratories that reactions that occurs on the surface of pathogens and generates active compo- take on this task proceed via mutagenesis, introducing mutations in a system- nents with various effector functions. Our lab is interested in gaining a atic manner over a small family of known antimicrobial peptides. Due to its la- molecular-level understanding of the protein interactions that regulate comple- bor, the process is limited to small families and yields small libraries. In our ment. We began our study looking at regulatory interactions that occur between laboratory, we have built a body of work that focuses on learning the relation- the blood clotting cascade and complement, with particular focus on comple- ship between sequence and antimicrobial activity. We have done so via ma- ment component 3 (C3) and thrombomodulin, which is a suspected regulator. chine learning models. Our models have gotten better over the years and We are now expanding our study of complement regulation to include comple- have recently culminated in a deep learning model that currently outperforms ment factor H (CFH), which is a known regulator of C3. CFH is a 150-kDa many known models. Given a peptide sequence, the model assigns an antimi- plasma glycoprotein made up of 20 globular complement control protein crobial activity score to a sequence that can be used to make a determination on (CCP) modules, each made up of nearly 60 amino acids, where domains 1-4 the presence or absence of activity. In this work we show how such a model can maintain regulatory activity. We have successfully expressed the full length be used in a generative setting to find novel antimicrobial sequences. Specif- CFH in Pichia pastoris and purified the protein for use in interaction studies ically, we couple the model with explorative algorithms that explore the with C3 and thrombomodulin. Work to create constructs expressing different sequence space of a template peptide. The model is used to score generated se- domains of CFH are ongoing. To investigate complement activation, our focus quences and guide the exploration towards regions of the sequence space likely has been on complement component 2 (C2), a multi-domain serine protease, to contain active sequences. Visualization and analysis of the explored which forms the C3 convertase activation complex when bound to complement sequence-activity landscape show regions of antimicrobial activity that present component 4 (C4). We are expressing C2 in E. coli and purifying it for use in rich data for further evaluation and refinement in the wet laboratory. activation of C3 (both C3 and C4 are already available in the lab). Future ex- periments will include the study of various protein complexes by surface plas- 2359-Pos mon resonance (SPR) and hydrogen-deuterium exchange mass spectrometry Simulating the Folding States of Lattice Proteins within an Oscillatory (HDXMS). Environment Austin H. Cheng, Cory J. Kim, Amy Y. Wang, Xuanye Zhu, Qizhang Jia, 2356-Pos Kateri H. DuBay. pH-Dependent Properties of Ionizable Residues in the Hydrophobic Inte- University of Virginia, Charlottesville, VA, USA. rior of a Protein Proteins are macromolecules composed of long chains of amino acids that play a Ankita Sarkar1, Adrian E. Roitberg2. vital role in the function of life, ranging from tissue structure and intracellular 1Physics, Univ Florida, Gainesville, FL, USA, 2Chemistry, Univ Florida, transport to enzymatic catalysis. Key to the function of proteins is their conforma- Gainesville, FL, USA. tional structure, and many proteins fold to a unique conformation called the native Internal ionizable residues in proteins have shifted apparent pKa values with state. Protein folding under static conditions has been extensively studied through respect their intrinsic pKa in bulk water. To understand the atomistic mecha- experiment and simulation, but complicating factors exist in the cell, such as the nism responsible for this shift in pKa, we study the pH-dependent conforma- presence of crowded agents, chemical gradients and oscillations, and supramo- tional changes in mutants of Staphylococcal Nuclease (SNase) having single lecular assemblies that influence folding. In our work, we investigate how oscil- internal ionizable residues. We used pH Replica Exchange Molecular Dy- lations in the interactions between amino acids can influence the distribution of namics (pH-REMD) for our calculations. We present thermodynamic models folded states occupied by a small protein. Our study employs a small, 3 dimen- to find the ‘conformation-specific pKa values’ of the internal ionizable residues sional lattice protein and uses Monte Carlo algorithms to simulate folding. and explain their association with the observed ‘apparent pKa’. Further, we study the pH-dependent structural and thermodynamic properties of ionizable 2360-Pos residue pairs within the hydrophobic interior of SNase. Studying Complex Biomolecular Dynamics by Single-Molecule Three-Color FRET 2357-Pos Anders Barth1,2, Claus A.M. Seidel2, Don C. Lamb1. Thioamide Effects on Protein Structure 1Department of Chemistry, Ludwig Maximilians University, Munich, Kristen E. Fiore1, D. Miklos Szantai-Kis2, E. James Petersson1. Germany, 2Institute for Molecular Physical Chemistry, Heinrich-Heine- 1Chemistry, University of Pennsylvania, Philadelphia, PA, USA, Universit€at, Dusseldorf,€ Germany. 2Biochemistry and Molecular Biophysics, University of Pennsylvania, Single-molecule Fo¨rster resonance energy transfer (FRET) is a powerful tool to Philadelphia, PA, USA. study conformational dynamics of biomolecules. Using solution-based single- The thioamide is a naturally-occurring single atom substitution on the peptide pair FRET by burst analysis, conformational heterogeneities and fluctuations of backbone. The exchange of oxygen to sulfur alters the chemical character, fluorescently labeled proteins or nucleic acids can be studied by monitoring a sin- thereby expanding functionality of the bond. This allows for its use as a min- gle distance at a time. Three-color FRET offers the possibility to monitor three imal biophysical probe. Thioamides quench fluorescence in a distance- distances simultaneously, allowing one to address complex and coordinated mo- dependent manner, and therefore they can be used for studying protein folding tions within single molecules. We have recently developed a three-color photon and conformational changes. To expand upon its potential, the Petersson lab distribution analysis (3C-PDA) to extract quantitative information about the has studied the effects of thioamide incorporation into prevalent secondary three-dimensional distribution of interdye distances from three-color FRET ex- structure motifs. Based on thermodynamic data, perturbation or stabilization periments. This method allows us to characterize the conformational space of bio- appears dependent upon the location of the bond and the surrounding molecules in unprecedented detail and, through the co-occurrence of distance, hydrogen-bonding network. In order to confirm these findings, high- address concerted conformational changes. Going beyond the static picture, resolution structures of thioamide-containing proteins are needed. In addition, single-molecule FRET has proven a well-suited tool to resolve conformational the best way to understand the functionally of the thioamide is to study its dynamics on the timescale of nanoseconds to seconds. I will show how the exist- occurrence in nature. To date only a single protein is known to contain a thio- ing toolbox to study dynamics in two-color FRET experiments, consisting of

BPJ 9454_9455 Wednesday, March 6, 2019 477a qualitative indicators of dynamics and the quantitative methodsof filtered fluores- gene CISD1 and has been implicated to play still undefined role(s) in type-2 cence correlation spectroscopy (fFCS) and dynamic photon distribution analysis diabetes and Parkinson’s Disease. Like all NEET proteins, the mNT monomer (PDA), can be extended and applied to three-color FRET experiments. Specif- possesses a CDGSH domain with three cysteine and one histidine residue coor- ically, in three-color pulsed interleaved excitation experiments (PIE) with multi- dinating a [2Fe-2S] cluster. Removal of this cluster by treatment with acidic parameter fluorescence detection (MFD), the multidimensional information of conditions induces mNT unfolding. While there are numerous crystal structures color, lifetime and anisotropy offers superior contrast to separate different confor- in the Protein Data Bank, little is known about holo mNT dynamics and stabil- mational states. This increases the robustness of the analysis and the sensitivity to ity or the unfolding of apo mNT. Here we apply hydrogen exchange mass spec- minor conformational changes that are otherwise not detectable in two-color trometry (HXMS) to holo and apo mNT to detect dynamics ranging from local FRET. breathing to global unfolding under native-state conditions. With mild shifts in pH and temperature, our N-HXMS approach can detect partially unfolded in- 2361-Pos termediates, extract the free energy of unfolding and/or the rate constants of un- Structural Studies of the Fc Region of Murine Immunoglobulin G Anti- folding and folding for the native state and intermediates. In addition, we can bodies using Single Molecule FRET simultaneously monitor holo and apo states of mNT due to their differences in Cathrine A. Southern, Jenna Henning, Kirsten Kochan. mass. By systematically comparing their HXMS behavior, we will gain insight Department of Chemistry and Biochemistry, DePaul University, Chicago, IL, into the connection between mNT dynamics and function. USA. The glycosylation of antibodies is known to influence their ability to instigate an immune response. The biantennary sugars present in the crystallizable frag- Posters: Protein-Small Molecule Interactions ment (Fc) region of antibodies are thought to stabilize the conformation of the Fc region so it can successfully bind to its receptors, thereby initiating an im- 2364-Pos mune response. Prior studies have suggested that sugar removal leads to an in- The Effects of Ligand Structure on Protein-Multimodal Ligand Interac- crease in the flexibility of the Fc region. Other results show a collapse of the Fc tions Camille Bilodeau1, Edmond Y. Lau2, Steve Cramer1, Shekhar Garde3. region upon sugar removal. Either of these conformational alterations would 1 impact receptor binding, thereby explaining the decreased immune response Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY, USA, 2Lawrence Livermore National Laboratory, Livermore, CA, observed upon sugar removal. To examine the structure of the Fc region of mu- 3 rine immunoglobulin G (IgG) antibodies, click chemistry was used to attach USA, Dean’s Office, Rensselaer Polytech Inst, Troy, NY, USA. dye molecules to azide-modified sugars in the Fc region. In addition, the Many important biophysical phenomena are driven by the structuring of water enzyme EndoS was used to cleave the majority of the sugars from the Fc region. around solutes. Solutes perturb the structure (i.e., the packing and orientational This enzyme leaves behind a single N-acetylglucosamine moiety, which was organization) of water molecules in the vicinity - the extent of the perturbation modified with an azide group and then reacted via click chemistry with dye depending on the nature of solute and strength of solute-water interactions - re- molecules. These dye-labeled glycosylated and deglycosylated fully intact sulting in solute-solute interactions that are strongly mediated by water. IgG antibodies were then examined using single molecule FRET to observe Although the role of water in mediating solute-solute interactions has been the structural impact of sugar removal. well characterized for simple systems, such as small ions or simple hydropho- bic solutes, most biological systems are larger and more complex molecules 2362-Pos that contain regions of mixed charge, hydrophobicity, hydrogen bonding, and Binding Free Energy Analysis of Programmed Cell Death Protein PD1 to other modes of interaction. Here, we focus on the role of water in governing its Ligand PD-L1 the behavior of small, flexible molecules containing multiple modes of interac- Peter C. Pan1, Alireza Tafazzol2, Xianwei Zhang3, Yong Duan2. tion (e.g., charge, hydrophobicity, and hydrogen bonding interactions) and how 1Department of Neurology, Univ Calif Davis, Davis, CA, USA, 2Genome this behavior impacts their interactions with proteins. In particular, we study a Center, Univ Calif Davis, Davis, CA, USA, 3Biophysics Program, UC Davis, set of multimodal chromatographic ligands which are commonly used for pro- Davis, CA, USA. tein separation applications. We characterize the conformational and hydration Immune checkpoint inhibitors targeting programmed cell death protein recep- preferences of each ligand in water including water density fluctuations in the tor PD1 and the ligand PDL1 represent a step forward for treatment of previ- solvation shell. By decomposing ligand contributions intramolecular versus ously difficult-to-treat cancers such as non-small cell lung cancer, metastatic water-mediated parts, we quantify the role of water in ligand conformational melanoma, and renal cell carcinoma. However, the same degree of benefit equilibria. We also perform molecular simulations of multimodal ligands in has unfortunately not been seen in glioblastoma, the most common and dead- aqueous solutions with different proteins. We find that regardless of which re- liest of glial tumors with a median patient survival between 10 to 20 months. gion of the protein surface the ligand binds to, its conformational preferences in Despite promising pre-clinical data, anti-PD1 monoclonal antibody nivolumab- free solution are maintained upon binding. Finally, we identify ligand design failed to reach its primary efficacy endpoint in a recent phase III randomized characteristics that lead to differences in the overall strength of protein- clinical trial, CheckMate-143. One possible reason for failure in glioblastoma ligand interactions. This work provides a basis for characterizing the role is poor diffusion of large monoclonal antibodies across the blood-brain barrier. that water plays in governing protein-ligand interactions as well as serves to Detailed characterization of the PD1-PDL1 complex may aid in development guide the design of new multimodal ligands for protein separations. of a small molecule inhibitor that would overcome the size limitation. We investigated the binding free energies involved in PD1-PDL1 complex forma- 2365-Pos tion with a molecular dynamics approach and identified the specific amino acid Peptide Assisted Supramolecular Polymerization of the Anionic Porphyrin sites important for PD1-PDL1 complex formation. Five simulations of 500 ns Meso-Tetra(4-Sulfonatophenyl)Porphine Eric Kohn1,2, David Shirly1, Christopher H. Fry3, Gregory A. Caputo1,4. each were performed for the binding domains of wild-type (PDB: 4ZQK) and 1 high-affinity mutant (PDB: 5IUS) PD1-PDL1 complexes in an explicit-water Chemistry and Biochemistry, Rowan University, Glassboro, NJ, USA, 2Bantivoglio Honors Concentration, Rowan University, Glassboro, NJ, USA, environment with AMBER force fields. Binding free energies estimated by 3 4 Molecular Mechanics Generalized Born Surface Area and entropy calculated Argonne National Labs, Argonne, IL, USA, Molecular and Cellular by normal mode analysis confirmed high-affinity PD1 mutant receptor had a Biosciences, Rowan University, Glassboro, NJ, USA. stronger binding free energy to PD-L1 than wild-type PD1 receptor. Among The photoelectronic properties of porphyrins have been harnessed by nature to residues of the high-affinity PD1 mutant receptor, I132,I134,T78,E70,H68, power such widely known processes as photosynthesis. When excited by light, and E136contribute the most free energy difference between bound and free many porphyrins release electrons; however, without an efficient or directed states and are implicated as the most important residues for stable complex for- method to transport them, this energy is typically limited to facilitating local mation. We suggest that ability of PD1 to bind stably to PD-L1 would be most enzymatic reactions. We investigated a series of peptides containing partial and complete porphyrin binding regions (PBRs) for their ability to promote vulnerable to interference at these sites. 2 meso-tetra(4-sulfonatophenyl)porphine (TPPS4 -) to form electrically conduc- 2363-Pos tive structures known as J-aggregates. While aggregation has previously been Determining Native-State Dynamics of Mitoneet using Hydrogen Ex- shown to occur in the absence of peptide, this requires very acidic conditions change Mass Spectrometry (pH < 1) and the resultant aggregates are too small and irregular to effectively Namita Khajanchi1, Rebeca Mena2, Mary Konkle2, Sheila Jaswal1. conduct electrons. Our approach uses amphiphilic peptides containing com- 1 2 Amherst College, Amherst, MA, USA, Ball State University, Muncie, IN, plete PBRs (Lys3) as well as partial (Lys1 or Lys2) regions located at the peptide USA. termini. Our goal is to induce the concurrent binding of TPPS to the incomplete The protein mitoNEET (mNT) is a protein first described in 2004 that binds the domains of two peptides, forming a bridge between them. Binding was assessed 2 type- 2 diabetes drug pioglitazone. The homodimeric protein is encoded by the in aqueous solution by monitoring the soret and Q-bands of TPPS4 -) across

BPJ 9454_9455 478a Wednesday, March 6, 2019 various peptide concentrations and pH levels. The absorbance transitions are 2368-Pos sensitive to the formation of the electronically conductive J-aggregate, allow- Integration of Text Mining and Binary QSAR Models for Novel Anti- ing for rapid spectroscopic identification. Fluorescence spectroscopy, circular Hypertensive Antagonist Scaffolds dichroism, and dynamic light scattering were also used to assess J-aggregate Serdar Durdagi1, Ismail Erol1, Berna Dogan1, Taha Berkay Sen2. formation. The peptides were shown to induce the formation of J-aggregates 1Department of Biophysics, Bahcesehir University, School of Medicine, up to solution pH 3.0, but maintains the ability to bind porphyrin at neutral Istanbul, Turkey, 2Bahcesehir University School of Medicine, Istanbul, pH. Notably, the peptides were shown to adopt an unstructured conformation Turkey. at low pH but become somewhat helical at neutral pH. Studies of mixed peptide AT1 antagonists is the most recent drug class of molecules against hypertension binding stoichiometry indicate formation of complementary heterooligomers and they mediate their actions through blocking detrimental effects of angio- that retain their porphyrin-binding capabilities and produce an additional bind- tensin II (A-II).(1) In this study, text mining approaches and binary QSAR ing site at their junction. These results have implications for the design of pho- modeling techniques were integrated to propose novel AT1 antagonist scaf- toelectronically active biomaterials. folds. For this aim, around 212000 small molecules were retrieved from Specs-SC database. Their 2D structural information were converted to IUPAC 2366-Pos .name format as text file. Then common pharmacophore groups as finger prints Optimization of the Site-Identification by Ligand Competitive Saturation were searched within this database. Therapeutic activity prediction of selected (SILCS) as an Accurate and Reliable Technique in Lead Optimization molecules using 25-common disease QSAR models as well as pharmacokinetic Vincent D. Ustach1, Sirish Kauhik Lakkaraju2, Sunhwan Jo2, Wenbo Yu1, and toxicity profiles (with 26-different toxicity QSAR models) were studied Fang-Yu Lin1, Wenjuan Jiang1, Alexander D. MacKerell1. 1 with MetaCore/MetaDrug approach. Binary QSAR models were constructed Computer Assisted Drug Design Center, Department of Pharmaceutical based on manually curated database of molecular interactions, molecular path- Sciences, University of Maryland Baltimore, Baltimore, MD, USA, 2 ways, gene-disease associations, chemical metabolism and toxicity informa- SilcsBio, LLC, Baltimore, MD, USA. tion. Particular therapeutic activity and toxic effect predictions are based on Chemical fragment cosolvent sampling techniques has become a versatile tool the ChemTree ability to correlate structural descriptors using recursive parti- in protein-ligand binding prediction. Site Identification by Ligand Competitive tioning algorithm. Selected hit molecules were then docked to the AT1 mono- Saturation (SILCS) is one such method that maps the hotspots of chemical frag- mer and dimer target structures and results were compared with known positive ments on a protein as free energy fields called FragMaps. Drug candidates are controls. Molecular Dynamics (MD) simulations were also performed for simulated via Monte Carlo techniques in the field of the FragMaps (SILCS- selected hits at the binding pockets of monomer and dimer targets to investigate MC) in order to predict their binding conformation and affinity for the target their detailed structural and dynamical analysis beyond docking studies. As a protein. Many protocols of SILCS-MC were considered and tested in order result, a new scaffold is proposed for new generation AT1 antagonists. to optimize the predictive capability of SILCS-MC. The tested parameters (1). Durdagi, S. et al. Integration of Multi-scale Molecular Modeling Ap- included the classification of ligand atom SILCS types, the scaling of the Frag- proaches with Experiments for the in silico Guided Design and Discovery of Maps, the calculation of cosolvent probe concentration, the expansion of probe Novel hERG-Neutral Antihypertensive Oxazalone and Imidazolone Deriva- types to include halogens and ethers, and the details of initial ligand placement tives and Analysis of Their Potential Restrictive Effects on Cell Proliferation. and attempted Monte Carlo moves in SILCS-MC. A set of 7 protein targets and European Journal of Medicinal Chemistry 145, 273-290, 2018. 552 ligands with broad chemical variability were used to train the model to maximize correct relative binding affinity, Pearlman’s Predictive Index, and 2369-Pos Pearson’s correlation coefficient. Across the protein-ligand sets, the relative af- The Two Faces of Bitter Sugars: Insights from Multiscale Simulations finities of the ligands was predicted correctly an average of 69.1% of the time. Fabrizio Fierro1,2, Alejandro Giorgetti3,1, Paolo Carloni1,2, The results support SILCS-MC as a powerful and inexpensive tool to support Wolfgang Meyerhof4, Mercedes Alfonso Prieto1,5. lead optimization in drug discovery. 1INM-9/IAS-5 Computational Biomedicine, Forschungszentrum Juelich, Juelich, Germany, 2RWTH Aachen University, Aachen, Germany, 2367-Pos 3University of Verona, Verona, Italy, 4Saarland University, Homburg, The Weak Enzymatic Activity of Truncated Lecithin Retinol Acyltransfer- Germany, 5Cecile and Oskar Vogt Institute for Brain Research, Heinrich ase (LRAT) Mutants cannot be Explained by their Affinity for All-Trans Heine University Duesseldorf, Duesseldorf, Germany. Retinol Bitter sugars are molecules present in food that can have either health benefits Sarah Roy1,2, Ana Coutinho3, Line Cantin1, Marie-Eve Gauthier1,2, (such as salicin, long used as analgesic and antipyretic) or toxic effects (such as Manuel Prieto3, Stephane M. Gagne2, Christian Salesse1. the cyanogenic glycosides in bitter almonds and apricot seeds). These bitter 1 Departement d’Ophtalmologie et d’ORL-CCF, FacultedeMedecine, sugars are detected by TAS2R16, a human bitter receptor expressed not 2 Universite Laval, Quebec, QC, Canada, Departement de Biochimie, only in the tongue but also in the brain. Although TAS2R16 is highly special- Microbiologie et Bio-informatique, Faculte des Sciences et de Genie, ized in detecting bitter b-glucopyranosides, its ligands display a large diversity 3 Universite Laval, Quebec, QC, Canada, Instituto Superior Tecnico, of aglycons, varying in size and hydrophobicity. This poses the question of how Universidade Lisboa, Lisbon, Portugal. the binding cavity of TAS2R16 is capable to adapt to such a broad range of li- Lecithin retinol acyltransferase (LRAT) is responsible for transforming all- gands. By means of multiscale molecular mechanics/coarse grained molecular trans retinol (ROH) into all-trans retinyl ester. LRAT participates in two enzy- dynamics simulations, here we show that bitter sugars interact with the receptor matic reactions. First, it hydrolyzes a phospholipid, resulting in the acylation of through a previously unrecognized dual binding mode. Such mechanism may LRAT (PLA1 activity); then, it transfers this acyl group to ROH, producing an offer a seamless way to fit different aglycons inside the binding cavity, while all-trans retinyl ester and deacylated LRAT (acyltranferase activity). Mutations maintaining the sugar bound, similar to the strategy used by several found in LRAT are responsible for severe degenerative eye diseases. These mu- carbohydrate-binding . Our prediction is fully consistent with mutagen- tations cause an almost complete loss of activity in a truncated LRAT without esis data and rationalize a wealth of structure-activity relationship data. Consid- its hydrophobic N- and C-terminal segments (tLRAT). However, nuclear mag- ering the multiple physiological and pathological roles of TAS2R16, the netic resonance data shows that these mutations have no effect on the global molecular information obtained in this work might help to design new mole- folding of tLRAT. Electrophoresis data suggests that tLRAT mutants have cules, either food additives or therapeutic drugs. PLA1 activity but no acyltransferase activity. This absence of activity could be explained by a deficiency in their binding of ROH. The objective was 2370-Pos thus to compare the binding of ROH to tLRAT and its mutants by titrating Structural Bases for Chemical and Mechanical Gating in the Piezo1 the increase in fluorescence upon ROH binding. An important increase in the Channel fluorescence intensity of ROH was observed in presence of tLRAT. The fitting Wesley M. Botello-Smith1, Han Zhang1, Alper D. Ozkan2, Wenjuan Jiang1, of the curve of the fluorescence intensity as a function of ROH concentration Christine N. Pham1, Yun Luo1, Jerome J. Lacroix2. using a mathematical model allowed to calculate the dissociation constant of 1College of Pharmacy, Western U of Health Sciences, Pomona, CA, USA, the binding of ROH to tLRAT and its natural mutants. A value of 34 5 12 2Graduate College of Biomedical Sciences, Western U of Health Sciences, nM was obtained for native tLRAT. Astonishingly, only the natural mutant Pomona, CA, USA. P173L-tLRAT showed a dissociation constant whose value (250 5 70 nM) Mechanosensitive Piezo channels possess a propeller-like structure with a cen- differed significantly from that of native tLRAT. These results suggest that tral cation-selective pore and three putative force-sensing blades encompassing all tLRAT mutants bind ROH as efficiently as native tLRAT, except for mutant several helical bundles called Piezo repeats. How membrane stretch and chem- P173L-tLRAT. The efficiency of ROH binding to LRAT is therefore not ical agonists modulate Piezo channels is unknown. Here, using all-atom molec- responsible for the large decrease in activity of this enzyme. ular dynamic stimulations and experimental assays, we localize the binding site

BPJ 9454_9455 Wednesday, March 6, 2019 479a of Yoda1, a small molecule Piezo1 activator, and identify stretch-induced 2373-Pos conformational rearrangements of the blades. Yoda1 binding uncouples two Structure Dynamics Guides the Enhancement of Ligand Affinity for adjacent Piezo repeats, facilitating stretch-induced blade motions, and alters MdmX long-range residue-residue contacts between pore and blades, as evidenced Zhengding Su, Yongqi Huang, Xiyao Cheng. by Yoda1-induced channel activation of a mechanically-insensitive mutant. Department of Biological and Food Engineering, Hubei University of In addition, cation-selective fenestrations allow potassium, not chloride, ions Technology, Wuhan, China. to enter a pore vestibule. Our work reveals the structural bases of cationic selec- Mdm2 and MdmX are highly homologous and Mdm2 inhibitors exhibit weak af- tivity, chemical modulation and mechanical sensing in a mammalian Piezo finity for MdmX. The structural mechanism underlying how Mdm2/MdmX channel. distinguish ligand binding affinity remains elusive. Guided by NMR study, we have identified three flexible regions on N-MdmX that affected inhibitor- 2371-Pos binding affinity. Furthermore, using disulfide staple, we found that the Phe19- Molecular Insight into the Agonist Properties of the Multimodal Antide- binding site on MdmX was one of the most flexible regions weakening nutlin- pressant Vortioxetine in Human 5-HT3A Receptors 3a binding. On the other hand, we continued to pursue high-resolution structure Lucy Kate Ladefoged1, Lachlan Munro2, Anders S. Kristensen2, of Mdm2 in complex with ntutin-3a. An X-ray crystallographic structure of Birgit Schiøtt3. ˚ 1 Mdm2/nutlin-3a at 1.10 A identified that His72 had two conformations, a bound Interdisciplinary Nanoscience Center, Aarhus University, Aarhus C, state and an unbound state. Molecular dynamics simulation also revealed that Denmark, 2Department of Drug Design and Pharmacology, University of 3 His72 in MdmX existed two states. Thus, our work started to search fragments Copenhagen, Copenhagen, Denmark, Department of Chemistry, Aarhus that could rigidify Phe19-binding site. We designed a fusion protein model University, Aarhus C, Denmark. that p5315-29 was fused with MdmX through a flexible linker, i.e., p5315-29- Human (h) 5-HT3A receptors are serotonin receptors belonging to the cys- MdmX, for fragment screening. By use of the intrinsic fluorescence signal of loop family that are important for physiological processes such as digestion p5315-29 to monitor high-throughput screening, we found that the imidazoindole and emesis as well as cognition and pain. Currently, the mechanism underly- was a bona fide scaffold for template-based design of MdmX inhibitors. Next the ing agonist and antagonist responses by the receptor remain largely elusive. p5315-29-MdmX fusion protein was modified to leave Phe19-binding site Vortioxetine is a multimodal antidepressant with high affinity for human unoccupied, i.e., p5323-29-MdmX. By screening a fragment library, a small frag- 5-HT3A receptors. Vortioxetine has a distinct activity profile which includes ment, CPD-01, could bind to Phe19-binding site in an allosteric mode that an initial brief partial agonist effect that is followed by insurmountable enhanced the binding of p5323-29 to MdmX. Furthermore, CPD-01 was conju- inhibition in human receptors. The bioactive binding mode of vortioxetine gated with p5323-29 to synthesize p53 peptide analogues with different linkers. in h5-HT3A receptors has been determined, and vortioxetine has a unique These peptide analogues exhibited enhanced binding affinity for MdmX, binding mode not similar to other known ligands. However, how vortioxetine compared with p5323-29. Then, CPD-01 was integrated into nutlin-3a to replace binding is coupled to receptor activation remains elusive. To address this, we Phe19-binding-site-pharmacophore. The new nutlin analogue exhibited high- applied an interdisciplinary approach consisting of mutagenesis experiments, affinity binding MdmX and Mdm2 as well. Taken together, our work provided structure/activity relationships, and in silico docking calculations to determine a promising strategy to design inhibitors of MdmX/Mdm2. This strategy should the molecular basis for vortioxetine’s distinct activity profile. Using mutant be applicable for lead optimization in drug discovery. receptors the activity profile of vortioxetine was established, and it was found that vortioxetine’s agonist activity can be eliminated through a single point 2374-Pos mutation, V202I. Vortioxetine analogs that retain agonist activity in mutant Characterizing the Direct Influence of a Small Molecule on a RAS-Related receptors were determined, and the underlying molecular interactions were Protein Interaction elucidated. Understanding how vortioxetine achieves its distinct activity Djamali Muhoza, Alix Montoya-Beltrand, Emilio Duverna, Paul D. Adams. profile and invokes an agonist response can aid in the greater understanding Dept Chem/Biochem, Univ Arkansas, Fayetteville, AR, USA. of h5-HT3A receptors on the whole, and the methodology presented here Ras-related Protein-Protein Interactions (PPIs) are critical to events involved can be applied to other ligands. Ultimately, knowledge of how the receptors in cellular proliferation, inhibition of cell death, and cell transformation. It is function can aid in the future development of improved pharmacotherapies therefore important to characterize molecular features of Ras protein interac- for emesis and digestion, and novel treatment options for pain and cognition tions that dictate their function as ‘timing switches’ in signal transduction improvement. pathways. This is an important step towards targeting the inhibition of cell transformation as well as controlling abnormal signaling activity of tumor- 2372-Pos causing Ras variants. Cell division cycle 42 (Cdc42) is a Ras-related protein, Energetics of Nucleotides Translocation through HIV-1 CA Hexamer and previous studies from this laboratory have outlined differences in confor- Chaoyi Xu1, Robert A. Dick2, Marc C. Johnson3, Volker M. Vogt2, mational dynamics in an important region that interacts with various effectors, Juan R. Perilla1. known as the Switch 1 region. These differences resulted in an increase rate of 1Department of Chemistry and Biochemistry, Univ Delaware, Newark, DE, 2 3 GTP hydrolysis in the presence of an effector protein that inhibits GTP hydro- USA, Cornell Univ, Ithaca, NY, USA, University of Missouri, Columbia, lysis when bound to wild type Cdc42. These findings suggest that induced MO, USA. conformational changes in key effector-binding regions of Ras-related pro- The HIV-1 capsid is a protein shell of the virus genome and other viral pro- teins may serve to alter effector protein interactions. As such, the targeting teins. Viral reverse transcription is initiated inside the capsid and nucleoside of small molecules towards this Switch 1 region of Cdc42 may facilitate triphosphates (NTPs) are synthesized into cDNA using viral RNA as a tem- changes in local conformation as well as dynamics without affecting the over- plate, which requires NTPs translocation from cytoplasm into the capsid. all stability of the protein. We are currently using biochemical and biophysical Recently, it was reported that the center pore of HIV-1 capsid hexamer is a approaches to characterize the influence of two small molecule targets on the potential nucleotide channel to fuel cDNA synthesis and the R18 ring in interaction between Cdc42 and an effector protein that inhibits Cdc42- the pore plays an essential role in recruiting nucleotides. However, the molec- stimulated GTP hydrolysis. Preliminary results on theses protein interaction ular mechanism of nucleotide translocate through the capsid hexamer is still are presented. unclear. Here, we designed and performed a series of all-atom molecular dy- namic simulations sampling over 100 microseconds to study the translocation 2375-Pos of the nucleoside triphosphates (NTPs) and inositol hexakisphosphate (IP6) Inhibition of Zinc-Mediated Amyloid Beta Aggregation and Cytotoxicity through the central cavity of a CA hexamer. The latter was recently found by Alpha Helix Mimetics to be a cellular cofactor of HIV-1 Gag assembly and maturation. The Maria C. Vogel1, Sunil Kumar2, Debabrata Maity2, Mazin M. Magzoub1, potential-of-mean-forces (PMFs) of the nucleoside triphosphates (NTPs) and Andrew D. Hamilton2. inositol hexakisphosphate (IP6) translocation were calculated by employing 1Biology, New York University Abu Dhabi, Abu Dhabi, United Arab Hamiltonian replica exchange (HREX)/Umbrella sampling (US) method. Emirates, 2Chemistry, New York University, New York, NY, USA. The single nucleotide translocation results suggested that nucleotides binding Amyloid proteins misfold from their natural, functional conformations and inside channel is energetically favorable and thus a passive translocation of aggregate into insoluble, highly structured beta-sheet-rich fibers. These pro- single nucleotide seems unlikely. On the other hand, a translocation pathway teins are associated with several diseases, including Huntington’s disease, involving two dATPs or a dATP and an IP6 molecule was found in the 2D- Parkinson’s disease, type II diabetes (T2DM), and Alzheimer’s disease (AD). PMF results. Together, a cooperative translocation mechanism for nucleotide T2DM and AD share common amyloid pathologies, wherein islet amyloid translocation through HIV-1 CA hexamer was proposed and tested experimen- polypeptide (IAPP) and amyloid beta (Ab), respectively, form toxic intermedi- tally. ate oligomers, some of which sample a-helical conformations, during their

BPJ 9454_9455 480a Wednesday, March 6, 2019 transition to fibrils. Our approach takes advantage of these intermediates to NANP repeats (NPNA3) for avid binding. For 3 out of the 4 mAbs tested, the het- target these peptides in a conformation- and chemical property-specific erologous repeat peptide binding was similar to that of homologous NANP repeat manner. We designed a library of small molecule a-helical mimetics, specif- peptide NPNA3, if NVDP residues were upstream of the NANP residues, but was ically oligoquinolines and oligopyrrolamides, that present surface functional- drastically decreased (>100 fold higher AppKd) when the order was reversed. ities in a well-defined order to mimic the residues at position i, i þ3/þ4, and Remarkably, although the N-JP was not included in the RTS,S vaccine, the iþ7, of the helical surface. The rationale is to stabilize these peptides, thereby mAbs tested recognized this peptide (1-150 nM apparent Kd ). These data are preventing their oligomerization and fibrillation, by matching the scaffold side- partly consistent with the ability of previously reported RTS,S vaccine-induced chains to complementary residues in the helical peptide domain. Previously we mAbs to recognize NVDP repeat-containing epitopes. Most importantly, our re- showed that the oligoquinoline 5, with alternating negatively-charged and hy- sults reveal the additional specificity of some of the RTS,S-elicited mAbs to the drophobic side-chains, is a potent antagonist of Ab aggregation. Here we have N-JP that was previously observed for the potent protective mAbs isolated from investigated whether 5 maintains inhibitory potential in the presence of zinc, a attenuated Pf sporozoite vaccinees. Selection of CSP-specific mAbs optimal in metal ion that has been reported to associate with Ab and affect its conforma- epitope targeting and affinity will advance preventative and therapeutic antibody tion, aggregation, and toxicity. Other work on oligopyrrolamides demonstrated development and immunogen design. their antagonistic effects on IAPP aggregation and toxicity. Due to the similar- ity between IAPP and Ab, oligopyrrolamides were also screened against Ab. 2378-Pos Our results show that ADH-120 is a potent inhibitor of Ab aggregation but Systematic Biophysical Insights into the Interaction of Anti MERS-CoV only in the absence of zinc. Biophysical assays were used to compare the Drug Ribavirin with Major Transport Protein in Human Serum: activity of 5 and ADH-120 against Ab, both in the presence and absence of In-Vitro Studies and Implications in Diabetes and Uremia zinc, as well as to determine trends in Ab inhibition within the oligopyrrola- Fahad Almutairi, Mohammad Rehan Ajmal. mide library to elucidate structure-activity relationship of the scaffold. Biochemistry, University of Tabuk, Tabuk, Saudi Arabia. The effect of glycosylation in the interaction of antiviral drug ribavirin is stud- 2376-Pos ied in detail with major transport protein in blood, human serum albumin Modifying Zinc Fingers: Targeting the Inflammatory Zinc Finger Protein, (HSA) under in vitro disease mimetic conditions. Ribavirin is an antiviral Tristetraprolin, with Exogenous Gold Complexes and Determining a Role drug used in treatment of Middle East Respiratory Syndrome Coronavirus for H2S in Modifying Trisetrapolin Endogenously (MERS-CoV) infection in Tabuk region, kingdom of Saudi Arabia. In present Kiwon Ok1, Wenjing Li1, Geoffrey D. Shimberg1, Sharon Batelu2, study interaction mechanism for binding of ribavirin with HSA has been eluci- Mike Lange3, Ivana Ivanovic-Burmazovic3, Dr. Timothy Stemmler2, dated. We explored the binding interaction under physiological environment Maureen A. Kane1, Milos R. Filipovic4, Sarah L. Michel1. for alterations in their binding characteristics in co-morbid disease environ- 1Department of Pharmaceutical Sciences, University of Maryland School of ments namely uremia and diabetes. Binding affinity for drug was found to Pharmacy, Baltimore, MD, USA, 2Department of Pharmaceutical Sciences, decrease with glycosylation also there are conformational changes pertaining Wayne State University School of Pharmacy, Detroit, MI, USA, 3Department to binding of drug to target protein. To evaluate the drug binding defects we of Chemistry and Pharmacy, University of Erlangen-Nurnberg,€ Erlangen, employed combination of biophysical and bioinformatics techniques. Fluores- Germany, 4Institut de Biochimie et Genetique Cellulaires - UMR 5095, cence and UV-visible spectroscopy has been used to elucidate the parameters of University of Bordeaux, Bordeaux, France. the drug complexation in presence of urea and glucose. Binding of ribavirin to Tristetraprolin (TTP) is a non-classical Zinc Finger protein (ZF), that contains human serum albumin has been found to decrease both in uremia and diabetic two Cys3His zinc binding domains, and which regulates the inflammatory mimetic conditions. Molecular topology of protein has also been studied by response at the mRNA level. TTP is a potential target for exogenous anti- dynamic light scattering under different conditions. Ligand binding is an inflammatory gold complexes and the endogenous signaling molecule, H2S. important phenomenon; it essentially controls drug pharmacokinetics. Treat- To understand how TTP is targeted by gold complexes, the interactions ment outcome in MERS-CoV infection have been strongly correlated to dia- between the model gold complex, [Au(III)(terpy)Cl]Cl2 (Auterpy) and TTP betes and chronic kidney disease. This study is an attempt to examine were investigated using a combination of optical spectroscopy and native elec- binding refashioning under these conditions and will help to understand the trospray ionization mass spectrometry (ESI-MS) as well as X-ray absorption changes in drug behavior, this will help in dosage design; appropriate for study (XAS). We discovered that gold exchanges with zinc bound to TTP form- achieving optimum therapeutic treatment outcome. 3þ ing a series of Aux-TTP-2D complexes, with reduction of the gold from Au to Au1þ. These protein is then functionally inactive (no RNA binding). When the 2379-Pos same experiments were performed with TTP bound to RNA, the Zn-TTP/RNA Perturbing Lipopolysaccharide Biosynthesis through Inhibition of Hepto- complex is not disrupted by Auterpy providing potential evidence possible syltransferase I Jozafina Milicaj. mechanism for its anti-inflammatory properties. To understand how H2S, a naturally occurring signaling molecule, targets Zn-TTP, its reactivity was Chemistry, Wesleyan University, Middletown, CT, USA. determined using a combination of cryo-ESI-MS, fluorescence and electron Gram-negative bacteria utilize a variety of extracellular polymeric substances (EPS) as cell wall protectants; a means conserved amongst all gram-negative paramagnetic resonance (EPR) spectroscopies. We discovered the H2S oxidizes the cysteine residues of Zn-TTP via a mechanism that involves atmospheric bacteria. Lipopolysaccharides (LPS) are the EPS in highest abundance on the oxygen, a persulfide intermediate and a radical reaction. The results of both cell surface with highly hydrophobic o-antigen chains that perturb antimicro- studies will be presented in the context of TTP’s biological role. bial penetration as well as contributing greatly to species specific virulence. Biosynthesis of the LPS core region begins with a glycosylation reaction cata- 2377-Pos lyzed by Heptosyltransferase I (Hep I). After extensive work to characterize Breadth of Human Monoclonal Antibodies Isolated from RTS,S/AS01 Hep I, inhibitor discovery began with the computational and experimental char- Vaccinees Binding to Plasmodium falciparum Circumsporozoite Protein acterization of a library of compounds from the National Cancer Institute. Antigens Numerous compounds were potent in silico binders to Hep I, and these mole- S. Moses Dennison, Milite Abraha, Richard H.C. Huntwork, Kan Li, cules are being evaluated for their ability to inhibit Hep I via kinetic and cell Dustin L. Mauldin, S. Munir Alam, Georgia D. Tomaras. based assays that test for disruption of bacterial growth and LPS biosynthesis. Duke Human Vaccine Inst, Duke Univ, Durham, NC, USA. Additionally, compounds identified by our collaborators at the University of Recently, the structural basis of epitope recognition by vaccine-elicited human Minnesota, have been tested as inhibitors of Hep I using the same approach. monoclonal antibodies (mAbs) specific for the Plasmodium falciparum (Pf) It has been demonstrated that many of these tested compounds have bound circumsporozoite protein (CSP) repeat region (containing NANP repeats inter- to Hep I and hindered product formation in ways that require further investiga- spersed with NVDP repeats) and the preceding N-terminal junction was detailed. tion with methods including but not limited to: isothermal titration calorimetry, Here we examined the interactions of mAbs isolated from RTS,S/AS01 (RTS,S) circular dichroism, and fluorescence spectroscopy. We are in the process of vaccinees with CSP antigens using Biolayer Interferometry and Surface Plasmon testing all of these prospective inhibitors via a growth challenge assay where Resonance. Optimal epitope recognition of mAbs was examined for the NANP Hep I containing and lacking cell lines will exhibit a different growth pattern repeats, NANP and NVDP residues containing heterologous repeats, and the in the presence of an inhibitor. Futhermore, total lipid extractions separated N-terminal junction peptide (N-JP) dual specificity, which was reported previ- through gel electrophoresis show truncation in the LPS of a Hep I containing ously for a potent protective antibody against malaria. Here we show that cell line in the presence of Hep I inhibitors. These cells should then have mAbs that avidly (10-50 nM apparent Kd [AppKd]) bound to 1-1.5 NANP repeat increased susceptibility to antimicrobials; therefore, as a combined therapy containing peptide (NPNA2) also bound to all the other repeat peptides tested. In the inhibitor would make traditional antimicrobials effective treatments for contrast, mAbs that bound weakly (AppKd R 1mM) to NPNA2, required two resistant bacterial strains.

BPJ 9454_9455 Wednesday, March 6, 2019 481a

2380-Pos consideration. A biochemical luminescence assay of cyclin A2 function was Control of Protein Self-Assembly with Water-Soluble Porphyrins used for experimental verification and identified three nanomolar inhibitors Tyler J. Brittain1, Samuel D. Fontaine1, Coleman Swaim2, of cyclin A2. These novel cyclin A2 inhibitors are the first reported cyclin Daniel R. Marzolf3, Oleksandr Kokhan3. A2 inhibitors that do not target the cyclin A2 - CDK 2 protein-protein interface. 1Chemistry, James Madison University, Harrisonburg, VA, USA, 2James Madison Univ, Harrisonburg, VA, USA, 3Dept Chem & Biochem, James 2383-Pos Madison Univ, Harrisonburg, VA, USA. Exploring the Toxicity of Small Molecule Metabolite 3-Hydroxy-3-Methyl- Interactions between charged porphyrins and complimentary or similarly glutaryl-Coenzyme a (HMG-CoA) in the Pathogenic Bacterium Entero- charged proteins provide important models systems for studies of electron coccus Faecalis transfer processes, artificial photosynthesis, and control of protein-protein in- Gillian M. Barth. teractions. Typically, the experimental results are analyzed and discussed Biol Sciences, Purdue Univ, West Lafayette, IN, USA. assuming that the proteins exist in a monodisperse state. Here, we explored Antibiotic-resistant Enterococcus spp. is considered a serious global health haz- interaction of wild-type and 12 mutants of PpcA, a 3-heme c-type cytochrome ard, causing infections of the bloodstream, urinary tract, and surgical sites. (cyt) from Geobacter sulfurreducenswith several anionic water-soluble deriv- Enterococcus spp. is one of the Gram-positive pathogens that uses the atives of tetraphenylporphyrin. Combined small- and wide-angle X-ray scat- conserved mevalonate pathway to produce isopentenyl diphosphate (IPP). tering experiments revealed formation of multimers with a wide range of The substrate of HMG-CoA Reductase and an intermediate of the pathway, complex sizes. Thermodynamic interaction parameters and complex binding HMG-CoA is thought to cause cell toxicity at accumulated levels in these stoichiometries were established with isothermal calorimetry. All-atom mo- mevalonate-dependent pathogens. Initial evidence was produced by Kizer et. lecular dynamics simulations revealed quick complex formation with binding al. (2008), who introduced HMG-CoA production into E. coli and saw inhibi- sites well matching the areas identified in our experimental work. The ob- tion of essential fatty acid enzyme Malonyl-CoA Acyltransferase (MCAT). tained results demonstrate that multimerization of solution-state proteins by They concluded that an abnormally high level of endogenous HMG-CoA is large water-soluble ligands can be tuned to control shape and size of the toxic due to MCAT inhibition by HMG-CoA. Inhibition of HMG-CoA Reduc- formed complexes. Molecular level mapping of the binding sites allows us tase, which implies accumulation of HMG-CoA, has proven to be toxic in E. to build a theory explaining the size of the formed complexes and provides faecalis despite mevalonate supplementation. Under native conditions, opportunities for targeted design and assembly of multi-subunit protein MCAT uses malonyl-CoA and Acyl Carrier Protein (ACP) to produce complexes. malonyl-ACP. Given the striking similarity of HMG-CoA to Malonyl-CoA, it is proposed that HMG-CoA is able to inhibit Malonyl-CoA binding. 2381-Pos HMG-CoA inhibition of MCAT is being explored via crystallographic and Biophysical Characterization of Binding Interactions of Ppar with THC fluorometric studies. Diffraction data from E. faecalis MCAT crystals has Iulia Bodnariuc, Margaret Renaud-Young, Justin L. MacCallum. been collected to 2.5 Angstroms, and co-crystallization of MCAT with Chemistry, University of Calgary, Calgary, AB, Canada. HMG-CoA and Malonyl-CoA is underway. Kinetic analysis with thiol- Peroxisome proliferator-activated receptors (PPAR) is a nuclear receptor with reactive dye 7-Diethylamino-3-(4’-Maleimidylphenyl)-4-Methylcoumarin three isoforms a, g, and d that are involved in the regulation of lipid metabolism, (CPM) has begun as well. Elucidating the mechanism of toxicity caused by inflammatory processes, and cell differentiation. Each isoform contains a DNA high endogenous levels of HMG-CoA in bacteria will aid in understanding binding domain, ligand binding domain (LBD), and two activation function do- the downstream effects of HMG-CoA Reductase inhibitors, assisting pursuit mains. Upon ligand binding, PPARs heterodimerize with retinoid X receptor to of a double target antibiotic with two different mechanisms. bind DNA and modulate transcription of target genes. Ligands corresponding to each isoform are unique despite similar secondary protein structure. Recent ev- 2384-Pos idence shows activate PPARa and PPARg. However, few studies Mapping the Binding Trajectory of a Suicide Inhibitor in Human Indole- have been done on PPARd. This implies that synthetic cannabinoids, such as amine 2,3 Dioxygenase 1 D9- (THC), may act by mediating PPAR-related pathways Khoa N. Pham, Syun-Ru Yeh. Physiology & Biophycics, Albert Einstein College of Medicine, Bronx, NY, unrelated to canonical CB1 and CB2 receptor interactions. Due to the specific ligand profiles and target genes of each PPAR isoform, understand- USA. ing each PPAR isoforms’ binding with THC would shed light on the role PPAR Human indoleamine 2,3-dioxygenase 1 (hIDO1) is a small heme-containing plays in cannabinoid activated pathways. Here we use microscale thermophore- enzyme that is an important cancer immune therapeutic target. Several sis, isothermal titration calorimetry, and fluorescence to quantify the binding af- hIDO1 inhibitors have entered clinical trials, among which BMS-986205 finities of PPAR LBD isoforms with THC. (BMS) stands out as the only suicide inhibitor. Here, we crystalized the hIDO1-BMS complex and identified three snapshots along the inhibitor binding 2382-Pos trajectory. BMS first binds in a surface cleft near the active site in an extended Identification of Novel Cyclin A2 Binding Site and Nanomolar Inhibitors conformation. The initial binding leads to partial protein unfolding that triggers Stephanie Kim1, Michele Alves2, Patrick Gygli2, Jose Otero2, heme release and subsequent large-scale movement of the inhibitor to the Steffen Lindert1. vacant heme binding site, where it adopts a high-energy kinked conformation. 1Department of Chemistry and Biochemistry, The Ohio State University, Finally the protein reaches its energy-minimum state with the inhibitor relaxed Columbus, OH, USA, 2Departments of Neuroscience, Pathology, to a bent conformation. The data offers new insights into structure-based design Neuropathology, The Ohio State University, Columbus, OH, USA. of hIDO1-selective inhibitors. It has been shown that dysfunction in tight regulation of DNA repair pathways can induce various syndromes, increase the risk of cancer, and trigger neurolog- 2385-Pos ical defect. However, there are cases where inhibition of DNA repair process is Structure-Guided Development of Dual Inhibitors of EGFR and JNK to necessary. For example, after treating cancer cells with radiotherapy, it is Treat GBM and NSCLC essential to inhibit DNA repair function of the radiation-induced cancer cells Haikui Yang, Ying Jiang, Ruohong Yan, Tingting Zhang, Jiajie Zhang. for successful apoptosis. Therefore, the key is to target a protein that is respon- Guangdong Provincial Key Laboratory of New Drug Screening, School of sible for controlling the rapid and proper resolution of DNA strand breaks. The Pharmaceutical Sciences, Southern Medical University, Guangzhou, China. recent discovery of CCNA2 (cyclin A2), the main mammalian S-phase cyclin, Abnormal epidermal growth factor receptor (EGFR) signaling is widespread in as a regulator of neuronal genomic stability and DNA repair positioned cyclin cancer, making the EGFR as an important target for therapy. The third- A2 as a potential therapeutic target for DNA repair dysfunction and as a radi- generation EGFR small-molecule inhibitors are at the forefront clinically for ation sensitizer. Thus, identifying small molecule regulators of cyclin A2 activ- treatment of patients with non-small cell lung cancer (NSCLC). However, ac- ity carries significant potential to regulate diverse cellular processes in both quired resistance limits the effectiveness. The c-Jun N-terminal kinase (JNK) ageing/neurodegeneration and in cancer. No functional modulators of cyclin signaling axis mediates primary resistance to EGFR inhibition in glioblastoma A2 are known to date. Here, we identified a potential allosteric cyclin A2 ligand (GBM) and NSCLC. Therefore, it’s a new approach of using a dual EGFR and binding pocket based on high-resolution structural data. Molecular dynamics JNK inhibition to treat EGFR-expressing GBM and NSCLC is a new approach. simulations were used to generate diverse binding pocket conformations for On the basis of an iterative approach of docking, two series of trisubstituted application of the relaxed complex scheme. We then used structure-based 1,2,4-triazoles and thiazoles were designed and synthesized. Some compounds virtual screening to find potential cyclin A2 inhibitors. Based on a consensus demonstrated inhibitory activity for EGFR and JNK kinases with IC50 values in score of docked poses from Glide and AutoDock Vina, we identified about the low nanomolar range. This work may lead to design and optimize the potent 60 promising hit compounds, where all PAINS scaffolds were removed from EGFR and JNK inhibitors in the future.

BPJ 9454_9455 482a Wednesday, March 6, 2019

2386-Pos might be one of the mechanisms underlying TopoIIa inhibition. The obtained Tight Binding of Natural Polyphenols to the Intrinsically Disordered theoretical information is useful as rational guide for further development of Mammalian High Mobility Group Protein At-Hook 2 new anticancer agents containing naphthoquinone moiety against TopoIIa. Linjia Su1,2, Jeremy Chambers2,3, Fenfei Leng1,2. 1Department of Chemistry and Biochemistry, Florida International 2389-Pos University, Miami, FL, USA, 2Biomolecular Sciences Institute, Florida Development of Small Molecule Inhibitors Targeting Clostridium Difficile International University, Miami, FL, USA, 3Department of Environmental Binary Toxin using the Site-Identification by Ligand Competitive Satura- Health Sciences, Florida International University, Miami, FL, USA. tion (SILCS) Method The mammalian high mobility group protein AT-hook 2 (HMGA2) is an intrin- Wenbo Yu1,2, Edvin Pozharskiy2,3, Kristen Varney2,3, David J. Weber2,3, sically disordered protein (IDP) that specifically recognizes the minor-groove Alexander D. MacKerell1,2. 1Dept Pharm Sci, Univ Maryland, Baltimore, MD, USA, 2Inst Biosci of AT-rich DNA sequence. It works as a multifunctional DNA-binding protein 3 and plays important roles in tumorigenesis and adipogenesis. Previous studies Biotechnol Res, Rockville, MD, USA, Dept Biochem/Molec Biol, Univ showed that HMGA2 is a potential therapeutic target of anticancer and anti- Maryland, Baltimore, MD, USA. obesity drugs by inhibiting its DNA-binding activities. In this study, we found Clostridium Difficile (C. diff) infection (CDI) is a commonly diagnosed infec- that several natural polyphenols, such as aurintricarboxylic acid, gallic acid, el- tion especially among in-hospital patients due to the imbalance between protec- lagic acid, and ()-epigallocatechin gallate (EGCG) tightly bind to HMGA2 tive microbiota and C. diff caused by long term antibiotic treatments. According and, as a result, strongly inhibit HMGA2 binding to AT-rich DNA sequences. to the Center for Disease Control (CDC), more than half million CDI cases are Since these polyphenols can inhibit adipogenesis in vitro and in animal models, reported annually in the United States and the number of patients with hypervir- our results suggest that their anti-obesity activities may stem from their inhibi- ulent strains of CDI increased significantly over the last decade. Hypervirulent tion of HMGA2-DNA interactions. Because polyphenols are naturally occur- strains of C. diff express binary toxin CDTa/CDTb in which CDTa plays cata- ring chemical compounds enriched in vegetables and fruits, our discovery lytic role while CDTb helps with transportation of CDTa. CDTa contains a cat- strongly support that vegetables and fruits are natural remedies to treat obesity. alytic domain and a CDTb binding domain. Targeting the CDTb binding domain of CDTa might be a promising way to interrupt the formation of the binary toxin 2387-Pos and thus inhibit its biological activity in CDI. In this study, we applied our Improved Modeling of Halogenated Ligand-Protein Interactions using the fragment-based approach based in explicit solvent all-atom molecular dynamics Drude Polarizable Force Field and Additive CHARMM36/CHARMM simulations (SILCS: Site Identification by Ligand Competitive Saturation) to General Force Field (CGenFF) explore small molecule inhibitors targeting the CDTb binding domain of Fang-Yu Lin. CDTa. SILCS includes both protein flexibility and desolvation considerations Dept Pharma Sci, Univ Maryland, Baltimore, MD, USA. when explore protein surface and can quantitatively describe the binding of The treatment of halogens by a force field is of particular importance to drug small molecules to protein accurately. The SILCS FragMaps were then used design as halogen atoms are able to participate in nonbonding interactions with to direct docking of a ligand obtained from an NMR chemical shift perturbation proteins via halogen bond (XB) or halogen-hydrogen bond donor (X-HBD) inter- screen using the SILCS-MC approach. The resulting docked orientation helped actions. In this work, we improved the additive CHARMM36/CHARMM to interpret its binding mode of the ligand, which was subsequently verified by General force field (CGenFF) and Drude polarizable force field (FFs) to more X-ray crystallography. Based on this confirmed small molecule binder, ligand accurately reproduce XB and X-HBD interactions with proteins. Both FFs optimization directed by SILCS is ongoing to enhance its binding and related were optimized using atom pair-specific Lennard-Jones (LJ) parameters by tar- tests are underway toward the development of drug candidates targeting C. diff. geting QM interaction energy profiles between chloro- or bromobenzene model 2390-Pos compounds and those representative of protein functional groups. With the opti- Diffusion-Influenced Reversible Ligand Binding to Two Inequivalent Sites mized FFs, the halogenated-ligand binding geometries from MD simulations Irina V. Gopich, Attila Szabo. were in good overall agreement with experimental crystal data, showing an NIDDK NIH, Bethesda, MD, USA. improvement over the non-optimized models and indicating the ability of the We have recently presented a general theory for a complex network of coupled FFs to treat both the XB and X-HBD interactions. In addition, systematic analyses association-dissociation reactions. The main result of the theory is a set of non- of the ligand-protein interactions that take into account the contribution of elec- Markovian diffusion-influenced rate equations, which involve time-dependent tronic polarizability show that the explicit treatment of polarization has differen- irreversible association fluxes instead of rate constants. The association flux is tial effects on the interactions. Interestingly, the electronic self-polarization defined as a probability density that an isolated pair irreversibly associates via energy can make both favorable and unfavorable contributions to ligand binding. one reaction channel having started out with the dissociation product of the Dipole moment analyses of the ligands suggest that the treatment of the polariza- same or another channel. The time integral of the association flux is the capture tion in the Drude model can capture local variations in the environment of the li- (also called splitting or commitment) probability, i.e., the probability that a pair gands, and the water molecules in the vicinity of the ligands, that are not of reactants, just formed by dissociation of one reaction channel, irreversibly accounted by the mean-field approximation in the additive model. Accordingly, associates via another (or the same) channel rather than diffusing apart the present work demonstrates the utility of the improved additive CHARMM36/ (escape). Here we apply this theory to reversible diffusion-influenced ligand CGenFF and polarizable Drude FFs for in silico studies of halogenated-ligand- binding to a macromolecule with two active sites. We find that the influence protein interactions as well as the role that polarization can play in ligand binding. of diffusion cannot be described by simply modifying the rate constants that appear in ordinary chemical kinetics. Diffusion actually modifies the chemical 2388-Pos kinetics scheme by inducing a direct transition between the two bound states. Molecular Recognition of Naphthoquinone-Containing Compounds The physical reason for this modification is that a ligand that has dissociated Against Human DNA Topoisomerase II ATPase Domain: A Molecular from one site can directly rebind to the other site rather than diffuse away in Modeling Study the bulk. This process would never occur if diffusion were fast, hence it is Panupong Mahalapbutr. not included in the ordinary chemical kinetics. As a result of the direct transi- Biochemistry, Chulalongkorn University, Pathum Wan, Thailand. tion between the two bound states, the kinetics of ligand binding can be accel- Several quinone-based metabolites of anticancer drugs and naturally occurring erated or slowed down depending on the initial condition. quinone-containing compounds have been characterized as potent inhibitors to- ward topoisomerase IIa (TopoIIa), an essential enzyme involved in maintaining 2391-Pos genomic integrity during DNA replication and mitotic division. Mansonone G Edema Factor Of Bacillus Anthracis Interacting with its Inhibitors (MG), a naphthoquinone-containing compound extracted from the heartwood Ire`ne Pitard1, Catherine Simenel1, Damien Monet1, Christophe Thomas2, of Mansonia gagei, exhibits various biological activities including antitumor po- Peggy Suzanne3, Arnaud Blondel1, Jacques Bellalou2, Patrick Dallemagne3, tential. In the present study, MG and its semi-synthetic derivatives were selected Inaki Guijarro1, Daniel Ladant2, Pierre Goossens2, Therese E. Malliavin1. to study the preferential binding site and dynamics behavior as well as to predict 1Inst Pasteur, Unite Bioinfo Struct, Paris, France, 2Institut Pasteur, Paris, the inhibitory activity against TopoIIa using molecular modeling approaches. France, 3Centre d’etudes et de recherche sur le medicament de Normandie, The molecular docking results revealed that the entire series of MG preferentially Caen, France. target to the ATPase domain. Among all studied MGs, the ester derivative MG14 Edema factor (EF), a toxin of Bacillus anthracis, is activated by calmodulin containing C-10 length exhibited the highest binding affinity against TopoIIa and (CaM). The EF/CAM interaction induces the appearance of a site catalyzing greater than that of the ATP-competitive inhibitor salvicine as well as 1,4-benzo- the production of cyclic AMP (cAMP), through the folding of a disordered quinone. Interestingly, the MG14 binding could induce the closed form of the turn switch. Previous studies (Laine, 2010) have shown that thiophen ureidoacids region (residues 145-151) inside the ATP-bindingpocket, implyingthatthis event inhibit the cAMP production, but their binding site is not precisely known.

BPJ 9454_9455 Wednesday, March 6, 2019 483a

These inhibitors were discovered by targeting in silico the switch, and, interest- gates and the formation of amyloid-like fibers as the surfactant concentration ingly, similar compounds were shown to target the switch region of bacterial increased, whereas circular dichroism (CD) showed that BSA second structure RNAP (Sahner, 2013; Fruth, 2014). Beside, interactions between adefovir com- changes from a-helix to b-sheet. Transmission electron microscopy (TEM) pound and EF have been extensively studied (Shen, 2004; Cesnek, 2018). An permitted us to obtain images of fibers and aggregates in the micrometers scale. X-ray crystallographic structure of the EF/adefovir complex displays the com- Further, small angle x-ray scattering (SAXS) measurements provided informa- pound bound into the catalytic site in the presence of a Rubidium ion. The tion about the protein’s quaternary structure as a function of surfactant concentra- disordered switch observed in the inactive EF and the partial knowledge of tion and a more detailed analysis allowed us to suggest a pathway of fibrillation the interactions between the protein and these inhibitors open a wide range process. Lastly, we performed molecular dynamics (MD) simulations to obtain an of possibilities for the interactions. In the present work, we use various exper- all atom structure of BSA at pH 3.7 to study in the molecular level the different imental approaches, biochemical assays and NMR, as well as molecular effects of SDS or SPFO in the BSA conformation and we also used the MD struc- modeling to investigate these interactions. tures to modeling the SAXS experimental curve. Cesnek et al. Analogues as Potent and Selective Inhibitors of Adenylate Cy- clases from Bordetella pertussis and Bacillus anthracis. ChemMedChem 2018. 2394-Pos Fruth et al. Binding mode characterization of novel RNA polymerase inhibitors Nuclear Magnetic Resonance at the Interface: Identifying Preferred Bind- using a combined biochemical and NMR approach. ACS Chem Biol 2014. ing Regions in Multimodal Cation Exchange Chromatography using Func- Laine et al. Use of allostery to identify inhibitors of calmodulin induced acti- tionalized Nanoparticles 1 1 2 vation of Bacillus anthracis edema factor. Proc Natl Acad Sci USA 2010. Ronak B. Gudhka , Camille L. Bilodeau , Scott A. McCallum , 3 3 1 Sahner et al. Novel small molecule inhibitors targeting the ‘‘switch region’’ of Mark A. Mccoy , David J. Roush , Steven M. Cramer . 1Chemical and Biological Engineering, Rensselaer Polytechnic Institute, bacterial RNAP: structure-based optimization of a virtual screening hit. Eur J 2 3 Med Chem 2013. Troy, NY, USA, Rensselaer Polytechnic Institute, Troy, NY, USA, Process Shen et al. Selective inhibition of anthrax edema factor by adefovir, a drug for Development and Engineering, Merck & Co., Inc., Kenilworth, NJ, USA. chronic hepatitis B virus infection.Proc Natl Acad Sci USA 2004. The Fc domain is an important component of complex molecules such as monoclonal antibodies (mAb) based biologics as well as fusion proteins and 2392-Pos is highly conserved across a given class of mAbs. Recent work in our lab Study on the Mechanism of Anti C-Met Activity of Boc-Protected Amino has shown the importance of specific domains towards binding for mAbs to Groups of Bithiazolophanes by using SILCS multimodal (MM) cation exchange chromatographic systems. In this collabo- Tatsuya Takimoto1, Ozge Yoluk2, Sunhwan Jo3, rative work, we employ a combination of Nuclear Magnetic Resonance Alexander D. MacKerell Jr.2, Hideaki Sasaki1. (NMR) spectroscopy and Umbrella sampling simulations to develop a funda- 1Faculty of Pharm. Sci., Gakuin Univ., Kobe, Japan, 2Dept. Pharm. mental understanding of how multimodal ligands and surfaces interact with Sci., Univ. of Maryland, Baltimore, Baltimore, MD, USA, 3SilcsBio, LLC, the Fc domain. We performed NMR titration experiments with isotopically Baltimore, MD, USA. labeled Fc and 15 nm diameter gold nanoparticles (Au NPs) functionalized We have synthesized some bithiazolophane derivatives and analyzed inhibitory with Self Assembled Monolayers (SAMs) presenting different MM ligands at activity against protein kinases, including c-Mets. Only 2,15-Bis(tert-butoxy- different ligand densities. The results showed the interface of CH2 and CH3 do- carbonyl)-2,15-diaza[3.3](2,2’)(4,4’-bithiazolophane) (BOCBTP) showed mains and the hinge region to be preferred binding regions for interaction of Fc anti-c-Met activity (IC50 = 603 nm) among the bithiazolophane analogs. to MM ligand surfaces. These flexible regions on the protein surface which are When we changed the tert-butoxy groups in this compound into iso-butoxy rich in positive and aliphatic residues are important for the binding of Fc to MM groups, the anti-c-Met activity of this compound diminished. To understand ligand surfaces. Further, to shed light on the binding mechanisms, we per- why only BOCBTP showed anti-c-Met activity, we applied a computational formed umbrella sampling with the Fc held at key orientations above a SAM simulation method: Site-Identification by Ligand Competitive Saturation surface presenting MM ligands at relevant ligand densities similar to those (SILCS) and c-Mets X-ray crystal structures with bound ligands. Characteriza- tested experimentally. These simulations suggested that as the Fc was brought tion and identification of the binding site of our compounds was performed us- close to the surface, the residues near the hinge region first made contact with ing docking simulation in SILCS. the surface followed by a tighter binding facilitated by interaction of His, Val, Based on X-ray crystal structures one main active site in c-Mets was identified, Leu and Ile residues at the interface of the CH2 and CH3 domains. This com- which can accept two types of ligands, bent or long straight ligands. Both these bined experimental and simulations approach enabled us to develop a molecu- ligands have the following interactions; hydrophobic interaction, hydrogen lar level understanding of the binding for complex biological products in MM bond and p-p stacking. As SILCS simulation also showed the hydrophobic CEX systems. and some hydrophilic areas in the same site, the results confirmed the active site of c-Met. Our compounds were also placed at a possible alternative binding 2395-Pos site identified by SILCS. When our compounds were docked into the site, only How does Glycosylation Affect Drug Binding on Influenza? The Roles one side chain placed there and basic skeleton was out of the pocket. Although of Electrostatics and Sterics Examined through Brownian Dynamics BOCBTP also placed at same position, BOC groups of BOCBTP located near as- Simulations partic acid residues. If two BOC groups will be cleaved with protons of aspartic Christian Seitz, Lorenzo Casalino, Gary Huber, Robert Konecny, acid, the steric shape of BOCBTP would change into a flat shape. As the flat shape Yu-Ming Huang, Rommie Amaro, J. Andrew McCammon. of BOCBTP derivatives can enter into the active site deeply, it is hypothesized Chemistry & Biochemistry, UC-San Diego, San Diego, CA, USA. that this is the reason only BOCBTP showed anti-c-Mets activity. Influenza is one of the most well-known viruses, yet much remains to be discovered. Glycans, the ubiquitous polysaccharides that decorate the surface 2393-Pos of influenza membrane proteins, play a complex part in influenza’s life func- Unveiling the Role of Surfactants on Amyloid-Like Protein Self-Assem- tions. They are known to modulate influenza’s transmissibility, virulence, elec- bling trostatics, sterics, cooperativity, drug binding, dynamics, antibody response, Gustavo Scanavachi1, Yanis Ricardo Espinosa2, Juan Ruso3, Rosangela Itri1. and immune evasion, amongst other aspects, but their exact role is not always 1Institute of Physics, University of Sa˜o Paulo, Sa˜o Paulo, Brazil, 2Institute of well-understood. Additionally, experimental influenza work is challenging to Physics of Liquids and Biological Systems, National University of La Plata, deconvolute due to complex and overlapping interactions, and due to influ- La Plata, Argentina, 3Soft Matter and Molecular Biophysics Group, enza’s mutability. These difficulties can be bypassed by creating these systems University of Santiago de Compostela, Santiago de Compostela, Spain. computationally. To examine how glycan and influenza strain differences Some proteins can undergo structural changes that may trigger an aggregation affect the binding kinetics of Tamiflu and Relenza, we created in silico systems process where they self-assembly into highly ordered aggregates called amyloid of neuraminidase from avian influenza (H5N1 A/Vietnam/1203/2004) and fibers. In vivo, these amyloid fibers are related to more than 25 different diseases, swine flu (H1N1 A/California/04/2009) with experimentally-derived, biologi- some of them are lethal as Creutzfeldt-Jakob disease and others can lead a person cally-relevant glycoprofiles. We then benchmarked association rates of the to incapacities, as diabetes type II, Alzheimer’s and Parkinson’s diseases. With drugs to the active site of neuraminidase with Brownian dynamics, a type of the aim of understanding the conditions and mechanisms by which proteins simulation evaluating the electrostatics and sterics of a system. Next, we modu- form amyloid fibers, we mixed bovine serum albumin (BSA) at pH 3.7 with so- lated this association rate by removing glycans, to deconvolute the role of elec- dium dodecyl sulfate (SDS) and sodium perfluorooctanoate (SPFO) to induce the trostatics and sterics in drug association to neuraminidase, and how this varies amyloid fibers formation. BSA conformational changes were followed in order to across strain and glycoprofile. These results increase our understanding of how suggest a possible pathway of aggregation. Dynamic Light Scattering (DLS) and the viral glycoprofile influences drug association, and has implications for vac- Thioflavin T fluorescence data revealed, respectively, the presence of large aggre- cine optimization.

BPJ 9454_9455 484a Wednesday, March 6, 2019

Posters: Protein Dynamics and Allostery II 2398-Pos Effect of Plasmin Cleavage on the Dynamics of the Protease Domain of the 2396-Pos Urokinase-Type Plasminogen Activator (uPA) Mechanism of Hsp104 Function Potentiation Studied by Hydrogen- Constanza Torres-Paris, Yueyi Chen, Elizabeth A. Komives. Deuterium Exchange Detected by Mass Spectrometry (HX-Ms) Department of Chemistry and Biochemistry, University of California San Xiang Ye, Jiabei Lin, Leland C. Mayne, James Shorter, S. Walter Englander. Diego, San Diego, CA, USA. Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, The uPA is a serine protease that catalyzes the cleavage of the inactive USA. zymogen plasminogen into the active protease plasmin. This protease can The unfoldase/disaggregase function of Hsp104 is achieved by ATP binding also cleave uPA into a more active two-chain form, which has the two chains and hydrolysis. Some single-site mutations of Hsp104 enhance its innate linked by a disulfide bond. Recent studies in our lab show that the heavy chain capability of aggregate dissolution and suppression of proteotoxicity of of uPA, that catalyzes the protease activity, is more dynamic than expected. On amyloid-forming proteins. Hsp104 function potentiation is frequently asso- the other hand, the light chain of uPA includes a linker region of 27 residues, a b ciated with faster ATP turnover. Illustration of the mechanism of potentia- with part of the protease domain, that can bind the integrin v 5. It is known tion requires a better understanding of the Hsp104 ATPase cycle e.g. that plasmin cleavage generates a new N-terminus that orders the active site, the identity of the rate limiting step. ADP release has been identified as rat- however, how plasmin cleavage affects the dynamics of the protease domain ing limiting for several protein systems with ATPase activity. Measuring of uPA has not been studied. We carried out hydrogen deuterium exchange ADP release is however challenging. In particular for Hsp104, there are at coupled to mass spectrometry (HDXMS) to determine the effect of plasmin least two obstacles i) it has two different AAA (ATPases Associated with cleavage on the dynamics of the protease domain of uPA containing the linker diverse cellular Activities) nucleotide binding domains (NBD) both are region. We found that the light chain was dramatically more dynamic after a b capable of ATP binding and hydrolysis making just measuring ADP binding plasmin cleavage, possibly exposing the binding site for v 5. Interestingly, affinity ambiguous as a result of averaging between the two NBDs. regions far away from the plasmin cleavage site were also affected, such as Moreover, its steady-state ATP hydrolysis rate is in the range of 1s1 the 140s, 170s, 180s and 220s loops. These loops were more protected from rendering the commonly applied methods of kinetic measurements difficult amide exchange after plasmin cleavage, suggesting that they are more ordered to implement. or less dynamic. These findings are consistent with the model by which the Hydrogen-deuterium exchange probes conformational dynamics of protein protease is transitioning between two states: an active state with the C-terminal b main-chain amides. By taking advantage of the substantially different HX rates -barrel and the active site ordered, and an inactive state with a disordered b of Walker A motif (the most critical nucleotide binding structure element) with C-terminal -barrel and a distorted active site. Therefore, our results suggest or without ADP bound, we apply a fast HX labeling strategy as a non- that plasmin cleavage favors the ordered active conformation over the disor- perturbing and label-free way to measure the kinetics of ADP binding and dered inactive one. release in a NBD-resolved fashion. We’ve found that the ADP off rate of 2399-Pos NBD1 matches perfectly with the steady-state ATP hydrolysis rate which Kinetic and Structural Comparison of HINT Enzymes: The Role of Distant mainly takes place in NBD1. We’ve also found that, of two of the most poten- Dynamics on Catalysis tiated Hsp104 variants, ie. I187F and A503S, their elevated ATPase turnover Alex Strom. can be entirely attributed to faster ADP off from NBD1. In addition, I187F ap- Medicinal Chemistry, University of Minnesota, Minneapolis, MN, USA. pears to hydrolyze ATP more cooperatively due to a strengthened protomer HINT1, or Histidine Triad Nucleotide Binding Protein 1, is a cytosolic interface. enzyme and is therapeutically notable due to its utility for activating antiviral prodrugs and as a target for modulating neuropathic pain and opioid tolerance. HINT1 is a member of the HIT enzyme superfamily and has nucleotide acyl- 2397-Pos phosphate hydrolase as well as nucleotide phosphoramidase activity. Addi- Binding Interface of Gapdh to the Au Rich Elements from TNF-Alpha tionally, neither the natural substrate nor the precise mechanistic cellular func- mRNA Revealed by Hydrogen Deuterium Exchange Coupled with Mass tion are known for this enzyme or the homologous mitochondrially localized Spectrometry isozyme HINT2. Recently, preliminary CPMG-NMR dynamics experiments 1 2 1 1 Daniel J. Deredge , Michael White , Anh Tran , Patrick Wintrode , in our lab with WT human HINT1 revealed several clusters of highly dynamic Elsa Garcin3. 1 2 residues, oscillating in the micro to millisecond time regime. Interestingly, Sch Pharmacy, Univ Maryland, Baltimore, MD, USA, Department of there is a lack of evidence for HINT1 dynamics from the over 30 crystal struc- Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN, 3 tures of human HINT1 in the PDB given that only one enzyme conformation USA, Department of Chemistry and Biochemistry, Univ Maryland has ever being captured. Several mutant HINT1 enzymes were made to test Baltimore County, Baltimore, MD, USA. the importance of these residues that were found to be dynamic by NMR as Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous pro- they pertain to the enzyme’s kinetic rates and structure. We have found that tein primarily known for its glycolytic function. More recent studies have mutations at residues Q62 and E100 have a significant effect on the steady revealed GAPDH as a multifaceted, multifunctional protein involved in state and pre-steady state rates of HINT1 while not significantly altering the various cellular processes including iron homeostasis, heme transport, protein’s secondary structure by CD. We hypothesize that these dynamic res- apoptosis, tumerogenesis, oxidative stress response, chromatin structure idues which lay 13A˚ away from the catalytic H112 in the enzyme’s active site among many others. As part of its multifunctional purpose, GAPDH has through a chain of conserved waters are an unappreciated part of enzyme ki- been shown to bind to various cellular RNAs and has been classified as a netic mechanism, until now. Ongoing research aims to delineate if these dy- non-canonical RNA binding protein. In particular, it has been shown to namic residues that reside on the protein’s exterior not only act to regulate the bind AU-rich element of mRNA transcripts and consequently affecting protein’s internal catalytic function, or if they also play a part in HINT1’s role translation. However, the detailed structural characterization of the RNA as a regulator of opioid tolerance through known interactions with the NMDA bound complex has remained elusive. The RNA binding site remains un- receptor. known thereby restricting potential therapeutic strategies. Our previous studies established that GAPDH binds the AU rich element of TNF-a 2400-Pos mRNA transcript via a two-step model. In addition, we have shown that a Characterizing HP1-Driven Chromatin Compaction using Nuclear Mag- T229K mutant of GAPDH displayed altered RNA and NADþ binding prop- netic Resonance Spectroscopy erties, potentially signaling to a linkage between RNA binding and the Bryce E. Ackermann, Galia T. Debelouchina. cofactor. Hydrogen deuterium exchange coupled with mass spectrometry Department of Chemistry and Biochemistry, University of California San (HDX-MS) revealed that the T229K mutant induced conformational changes Diego, La Jolla, CA, USA. into distal grooves along the P-axis of the . In this study, Eukaryotes package their DNA by wrapping it around a histone protein core we have mapped the binding interfaces of three GAPDH constructs display- to form a structure known as the nucleosome. The nucleosomes form large ing varying NADþ binding properties with the AU rich element from polymers called chromatin which separate into two distinct compartments TNF-a mRNA using HDX-MS. A largely common pattern of localized pro- in the nucleus. In constitutive heterochromatin compartments, chromatin dis- tection from exchange pointed to the grooves along the P-axis as the RNA plays dense compaction and dampened gene expression. Heterochromatin binding interfaces, as hypothesized. Additionally, some variations in HDX protein 1 (HP1) is a multivalent chromatin binding protein enriched at these protection point to potential conformational basis for the linkage with heterochromatin sites. It is known that HP1 is capable of compacting single NADþ binding. chromatin strands and cross-linking separate strands. Here we map the

BPJ 9456_9458 Wednesday, March 6, 2019 485a structural and biophysical properties of HP1 in vitro as it interacts with differ- need for new approaches to interpret mechanisms of dynamic systems entially modified semi-synthetic chromatin. Nuclear magnetic resonance through mutagenesis. spectroscopy provides the selectivity to study HP1 in dense chromatin net- works while resolving residue-specific biophysical changes. These results 2403-Pos provide insight into the function of HP1 in near-physiological chromatin An Allosteric Signaling Governs the CRISPR-Cas9 Function environments. Giulia Palermo1, Clarisse Gravina Ricci2, Ivan Rivalta3, Victor S. Batista4, James A. McCammon5. 2401-Pos 1Department of Bioengineering, Univ Calif Riverside, Riverside, CA, USA, Dynamics Mediate Substrate Recognition and Remote Communication in 2Dept Pharmacology, Univ Calif San Diego, San Diego, CA, USA, 3ENS a Peptide-Bond Forming NRPS Cyclization Domain LYON, Lyon, France, 4Dept Chemistry, Yale Univ, New Haven, CT, USA, Subrata H. Mishra1, Aswani K. Kancherla1, Santrupti Nerli2, 5Chemistry & Biochemistry, Univ Calif San Diego, La Jolla, CA, USA. Nikolaos Sgourakis3, Daniel Dowling4, Dominique P. Frueh1. CRISPR-Cas9 is a bacterial adaptive immune system that recently emerged as 1Biophysics and Biophysical Chemistry, Johns Hopkins University School of the centerpiece of a transformative genome editing technology. This system is Medicine, Baltimore, MD, USA, 2Department of Computer Science, based on the single Cas9 protein, which can be programmed with single guide University of California Santa Cruz, Santa Cruz, CA, USA, 3Dept Chemistry RNAs to site-specifically target and cleave any desired DNA sequence bearing and Biochemistry, University of California Santa Cruz, Santa Cruz, CA, a short recognition region, known as protospacer adjacent motif. Here, all-atom USA, 4Chemistry, University of Massachusetts Boston, Boston, MA, USA. molecular dynamics simulations reveal the important role of allostery in Nonribosomal peptide synthetases (NRPSs) are large multidomain enzymatic enabling regulation and function in CRISPR-Cas9. We disclose an allosteric systems that synthesize complex secondary metabolites from simple substrates mechanism in which the DNA recognition activates the catalytic function at like amino acids. The products often have invaluable pharmacological appli- distal sites of the CRISPR-Cas9 complex. In this mechanism, the binding of cations such as antibiotics and immunosuppressants. To assemble the prod- a short recognition sequence acts as an ‘‘allosteric effector’’, establishing a ucts, condensation or cyclization (Cy) domains catalyse the formation of a tight communication network (and increased correlated motions) between the peptide bond between two substrates each tethered to phosphopantetheinyl catalytic domains. Analysing the communication pathways within the system arms of an upstream donor or a downstream acceptor carrier protein (CP) allowed the identification of residues responsible for the information relay. domain. Cy domains also catalyze cyclodehydration of serine, threonine, or Mutating two of these residues (K775A and R905A) was shown to decrease un- cysteine sidechains to produce heterocycles. For enzyme function, donor selective cleavages of partially complementary DNAs, opening new avenues and acceptor CPs dock on opposite sides of a 40 A˚ channel harboring the for modulating the activity of CRISPR-Cas9 systems. Overall, these findings active site. The molecular determinants for substrate recognition, substrate contribute in clarifying the mechanistic function of this unique genome- introduction to the buried active site, and catalysis itself have proven difficult editing tool, while also offering key insights for future efforts aimed at to determine. Using NMR relaxation dispersion, we demonstrate that the improving its selectivity. channel connecting the remote CP binding sites of the yersiniabactin synthe- tase cyclization domain Cy1 is subject to ms-ms dynamics, suggesting allo- 2404-Pos steric communication between the sites through a network of dynamic Positive and Negative Substrate Interference Supported by Coinciding residues. By parsing transient distances provided by NOESY spectra, we Enzyme Residues determined a structural ensemble representing the conformational landscape Magnus Wolf-Watz, Per Rogne, Elisabet Sauer-Eriksson, Uwe Sauer, of Cy1. We then demonstrate that the network of dynamic residues responds Christian Hedberg. specifically to the presence of substrate when Cy1 is presented to its partner Chemistry Department, Umea University, Umea˚, Sweden. donor CP. We find that the response remodels the entire active site channel, Enzymes bridges the time-scale gap between chemistry and biology and are thus confirming allostery. These studies throw light on how dynamics in as such essential for cellular viability. A fundamental property of enzymatic Cy1 can simultaneously mediate substrate recognition and allosteric commu- function inside the complex and crowded cellular milieu is recognition of nication between partner CPs. correct over in-correct substrates. This is particularly challenging when chemically related substrates such as ATP and GTP should be discriminated. 2402-Pos Specificity can, in principle, be accomplished with positive interference with Molecular Responses of Mutagenesis in Nonribosomal Peptide Synthetase the correct substrate and negative interference with in-correct substrates. Cyclization Domains Here we present the atomic mechanism for ATP versus GTP selectivity of Kenneth Marincin1, Aswani Kancherla1, Subrata H. Mishra1, the small enzyme Adenylate kinase. The finding was enabled through a com- Daniel Dowling2, Dominique P. Frueh1. bination of protein NMR spectroscopy, x-ray crystallography and synthetic 1Dept Biophys/Biophysical Chem, Johns Hopkins Sch Med, Baltimore, MD, organic chemistry. We discovered that while ATP activates the enzyme by USA, 2Chemistry, University of Massachusetts Boston, Boston, MA, USA. triggering a massive conformational change, the enzyme is arrested in an Nonribosomal peptide synthetases (NRPSs) implement a modular architec- inactive conformation in its complex with GTP. Unexpectedly, we found ture to assemble a series of substrates into secondary metabolites, often that a set of residues that are vital for ATP dependent activation also are with medicinal properties. EngineeringofhybridNRPSsystemshasbeen responsible for stabilizing the inactive GTP-complex. Hence, this set of res- an attractive approach in production of new pharmaceuticals due to their idues has evolved to accomplish positive interference with ATP and at the modular organization. Unfortunately, understanding the molecular mecha- same time negative interference with GTP. From a structure-function anal- nisms of NRPSs is challenging and artificial NRPSs are often beset by ysis of a key hydrogen bond formed between ATP and the enzyme, we low product yields. Here, we focus on the cyclization domain from the discovered that this single hydrogen bond is essential for substrate selec- yersiniabactin synthetase, which condenses and cyclizes salicylate and tivity. This particular aspect of ATP recognition is conserved throughout cysteine (each tethered to donor and acceptor carrier proteins), to form a the entire family of eukaryotic protein kinases thus highlighting its precursor for the virulence factor yersiniabactin. Notably, enzymatic importance. mechanisms inferred from mutagenesis interpreted through traditional Rogne et. al, (2018), PNAS, 115, 3012-3017 biochemical and structural approaches are routinely revisited. Previous studies by Dowling, Schmeing, and coworkers have identified mutations 2405-Pos in the active site of the cyclization domain leading to incorrect product The Placement of Vibrational Probe Labeled Substrates to the Phospho- formation and hence thought to contribute to catalysis through acid-base pantetheine Arm of the E.Coli Acyl Carrier Protein for Site Specific mechanisms and/or substrate positioning. However, our lab has shown Vibrational Spectroscopy that the native cyclization domain encompasses large-scale dynamics, Joie Ling1, Eliana V. von Krusenstiern2, Bashkim Kokona3, departing from the accepted rigid model of this domain. Employing Louise Charkoudian2, Casey H. Londergan1. NMR, a technique sensitive to both structural and dynamic properties of a 1Chemistry, Haverford Coll, Haverford, PA, USA, 2Haverford Coll, molecule, we revisited these mutations and observed a global molecular Haverford, PA, USA, 3Dept Biology, Haverford Coll, Haverford, PA, USA. remodeling of the domain rather than expected local effects. In an effort The study of acyl carrier proteins (ACP) can provide unique insights into bac- to determine how these mutations impact domain communication, we terial bio-synthetic pathways due to their integral role of ferrying intermediate probed the molecular response of one mutant to its substrate-loaded donor substrates to desired enzymes’ active sites. Understanding bacterial biosyn- partner using in situ biochemistry with an NMR readout. Our results reveal thesis can broaden our abilities to synthesis unnatural bioproducts using the that mutagenesis has global rather than local effects and emphasizes the cell’s machinery. e.Coli’s fatty acid ACP is a model for understand the broad

BPJ 9456_9458 486a Wednesday, March 6, 2019 family of carrier proteins. One centrally important but poorly understood of the single chitin chain from crystal surface is the rate-limiting step of feature of ACPs is how they protect and present their growing substrates movement with time constant of 17 ms, achieved by binding free energy via ‘‘chain flipping’’. Substrates are placed at the end of a phosphopante- at the product binding site of SmChiA. Our results demonstrate that theine (Ppant) arm of holo ACPs and depending on the carbon chain length, SmChiA operates as a burnt-bridge Brownian ratchet wherein the Brownian the arm can sequester itself in a hydrophobic cavity preventing degradation. motion along the single chitin chain is rectified forward by substrate-assisted Vibrational (infrared and Raman) spectroscopy is an extremely attractive catalysis. ways to study this process due to the short time scale on which they operate. Vibrational probes of particular interest include isonitriles (which have a very 2408-Pos strong, unique IR peak at 2150 cm-1), azides, and alkynes. Recent work Observing Histone H2A.Z Exchange at the Single-Molecule Level points to a modified SFP ligation reaction as a highly effective and versatile Matthew F. Poyton1, Ashlee Feng2, Anand Ranjan2, Qin Lei2, Sheng Liu2, method of attaching probe-labeled CoA derivatives to apo ACPs: the chal- Carl Wu2, Taekjip Ha1. lenge is to synthesize the probe-labeled CoA’s, which we have attempted us- 1Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, ing transamidation chemistry. Another, convergent strategy that we have Baltimore, MD, USA, 2Biology, Johns Hopkins University, Baltimore, MD, adopted is to incorporate probes within the hydrophobic cavity of the ACP USA. itself via the use of unnatural amino acids. Results from both strategies Genetic information is stored as chromatin, a well-organized assembly of will be presented. Coupled with the placement of vibrational probes at the DNA and proteins that must be dynamically packed and unpacked to regulate end of the Ppant arm, vibrational spectroscopic experiments can offer a gene expression. One way of modulating chromatin packing is through ex- well rounded image of ACP’s dynamic nature as it interacts with itself and change of canonical histone proteins in nucleosomes with histone variants, its catalytic partners. such as the replacement of histone H2A with histone H2A.Z. H2A.Z is a non-canonical histone variant that is highly conserved across all species. A special class of nucleosome remodelers insert H2A.Z-H2B dimer into nucle- 2406-Pos osomes via an ATP-dependent process. Despite its importance, our High-Speed Atomic Force Microscopy Shows Conformational Dynamics of D molecular-level understanding of enzyme-mediated histone exchange is Ca2 /Calmodulin-Dependent Protein Kinase II 1,2 3,4 poor. To fill this knowledge gap, we have developed single molecule assays Mikihiro Shibata , Hideji Murakoshi . to monitor the interaction of SWR1, the 1 MDa protein complex responsible 1WPI-NanoLSI, Kanazawa Univ, Kanazawa, Japan, 2Institute for Frontier 3 for histone exchange in S. cerevisiae, with nucleosomes and histone dimers in Science Initiative, Kanazawa Univ., Kanazawa, Japan, Supportive Center real time. By strategically placing fluorophores on individual histone proteins for Brain Research, National Institute for Physiological Sciences, Okazaki, and nucleosomal DNA we can observe histone eviction in real time on single Japan, 4Department of Physiological Sciences, Sokendai, Okazaki, Japan. þ nucleosomes, as well as disruption in the DNA wrapped around the histone Ca2 /calmodulin-dependent protein kinase II (CaMKII) is a downstream þ octamer. effector of Ca2 signaling in eukaryotic cells. An elevation in intracellular Ca2þ leads to binding of Ca2þ to calmodulin (CaM), which binds to and ac- 2409-Pos tivates CaMKII. Upon activation, CaMKII has the ability of autophosphor- Basis of Specificity in Ets-1 DNA Binding Domain to Variable DNA Se- ylation, then CaMKII phosphorylates numerous target proteins and is quences involved in many cellular functions, including synaptic plasticity. Previous Kenneth Huang, Suela Xhani, Amanda V. Albrecht, Gregory M.K. Poon. EM micrographs show that CaMKII forms a 12-meric holoenzyme that þ Chemistry, Georgia State University, Atlanta, GA, USA. could respond to the frequency of the activating signal of Ca2 .However, Transcription factor Ets-1 is a member of the ETS family, characterized by a the relationship between structural dynamics of CaMKII oligomers and its conserved winged helix-turn-helix DNA binding domain that specifically rec- function is still unknown. Here we used high-speed atomic force microscopy ognizes 5’-GGA(A/T)-3’. Nonspecific sites are dictated by the lack of this (HS-AFM) to directly visualize conformational dynamics of CaMKII þ consensus, while high/low affinity binding arise from discrepancies in se- oligomers during Ca2 /CaM binding and phosphorylation. HS-AFM is a quences flanking the consensus. Affinities for consensus bearing sequences powerful technique to visualize conformational changes of proteins at a are dispersed, but nonetheless possess biologically relevant binding. Struc- single molecular level in action under near physiological conditions tures of Ets-1/high affinity complexes are available, but corresponding struc- [1, 2]. At a basal state, HS-AFM images of CaMKII showed a highly fluc- tures for low affinity are lacking. To understand the molecular basis of how tuated structure of kinase domains, indicating that a fluctuation of kinase do- þ Ets-1 discriminates between DNA sequences, we performed molecular dy- mains generates a space to access Ca2 /CaM to the binding site of CaMKII. þ namics simulations of Ets-1 unbound, and bound to nonspecific, low, or Upon binding of Ca2 /CaM, neighbor kinase domains contact each other, þ high affinity DNA. Ets-1 binding to DNA results in Ets-1 becoming less dy- and leads to generate more space to bind additional Ca2 /CaM. These namic relative to unbound in a rank order correspondent with affinity, and in- conformational dynamics probably causes a positive cooperativity of þ duces bending in the DNA in a trend inverse to affinity. Ets-1/nonspecific Ca2 /CaM binding. In the phosphorylated state, kinase domains in CaMKII complexes exhibit a generalized reduction in DNA contacts, behaving similar form a disordered structure, suggesting that a disordered structure phosphor- to unbound Ets-1. Contrarily, Ets-1/ nonspecific complexes display the largest ylates other subunits within a CaMKII oligomer as a non-specific manner. degree bending, suggesting a prelude to localized searching. High and low af- Thus, a sequential HS-AFM images shows importance of structural dy- finity bound Ets-1 form similar complexes, showing significant deviation at namics of kinase domains in CaMKII oligomers, such as fluctuation, confor- three residues- Q336, E343, and R378. Q336 contacts the DNA backbone mational dynamics and a transition to a disordered structure. [1] M. Shibata in high and low affinity; E343 and R378 form a salt bridge aiding in the hy- et al. Nat. Commun. 8, 1430 (2017). M. Shibata et al. Sci. Rep.8,8262 drophobic packing of a-helix H1. These interactions are maintained in Ets-1/ (2018). high affinity DNA; in low affinity, Q336/DNA weakens significantly, and the E343-R378 salt bridge is severed. Loss of the salt bridge enables R378 to con- 2407-Pos tact DNA by distorting the terminus of a-helix H2, thereby exposing the Processive Chitinase is Burnt-Bridge Brownian Motor Operated by Fast hydrophobic core of helix H1 to water. Mutations (Q336L and E343L) in Catalysis after Peeling Rail from Crystalline Chitin high and low affinity perturbed binding affinity; this suggests a mechanism Akihiko Nakamura1, Kei-ichi Okazaki1, Tadaomi Furuta2, Minoru Sakurai2, distal to the DNA interface that differentiates Ets-1 binding to high/low affin- Ryota Iino1. ity DNA sequences. 1Institute for Molecular Science, Okazaki, Japan, 2Tokyo Institute of Technology, Yokohama, Japan. 2410-Pos Processive chitinase is a linear molecular motor which moves on the surface Anomalous Non-Gaussian Viscoelastic and Age-Dependent Dynamics of of crystalline chitin driven by processive hydrolysis of single chitin chain. Histone-Like H-NS Proteins in Live Here, we analyze the mechanism underlying unidirectional movement of Asmaa Sadoon, Yong Wang. Serratia marcescens chitinase A (SmChiA) using high-precision single- Department of Physics, University of Arkansas, Fayetteville, AR, USA. molecule imaging, X-ray crystallography, and all-atom molecular dynamics We report our measurements of the dynamics of H-NS proteins, which interact simulation. SmChiA shows fast unidirectional movement of 50 nm s1 with both proteins and DNA simultaneously, in live E. coli bacteria. The dy- with 1-nm forward and backward steps, consistent with the length of reac- namics turn out to differ significantly from other molecules reported previ- tion product chitobiose. Analysis of the kinetic isotope effect reveals fast ously. A new power-law distribution was observed for the diffusion substrate-assisted catalysis with time constant of 3 ms. Decrystallisation coefficients of individual H-NS proteins. In addition, we observed a new

BPJ 9456_9458 Wednesday, March 6, 2019 487a distribution of displacements, which does not follow the Gaussian, Cauchy, or 2413-Pos Laplace distributions, but the Pearson Type VII distribution. Furthermore, we Utilizing Empirical Data and Structural Dynamics Prediction to Optimize experimentally measured, for the first time, the time/frequency dependence Rational Design of Therapeutic Phospholamban Mutations to Tune of the complex modulus of the bacterial cytoplasm, which deviates from the SERCA Function viscoelasticity of homogeneous protein solutions and shows a glass-liquid tran- Kim N. Ha1, Hannah M. Johnson1, Ariana Schneiderhan1, Daniel Weber2, sition. Lastly, we observed that the dynamics of H-NS protein is cell-length/ Joseph Roith3, Gianluigi Veglia2. cell-age dependent. The findings are expected to fundamentally change the cur- 1Chemistry and Biochemistry, St Catherine University, St Paul, MN, USA, rent views on bacterial cytoplasm and diffusional dynamics of molecules in 2Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, bacteria. Minneapolis, MN, USA, 3Mathematics, St Catherine University, St Paul, MN, USA. 2þ 2411-Pos The interplay between the sarco(endo)plasmic reticulum Ca -ATPase (SERCA) and its endogenous cardiac inhibitor phospholamban (PLN) lends Investigation of Conformational Dynamics Involved in Genome Editing 2þ Events by CRISPR-Cpf1 a critical role in Ca signaling in cardiac myocytes. Dysfunctions in their in- Chun Chan, Xiaolin Cheng. teractions can lead to reduced cardiac contractility, and a number of naturally- Medicinal Chem & Pharm, The Ohio State Univ, Columbus, OH, USA. occurring PLN mutations have been linked to hereditary cardiomyopathy. CRISPR (clustered regularly interspaced short palindromic repeats) and Targeting PLN is a promising avenue for treating cardomyopathies, and in CRISPR-associated (Cas) proteins are part of the adaptive immune system of this study, we seek to improve the design of therapeutic PLN mutants by bacteria and archaea. CRISPRs are stretches of DNA with two distinct charac- creating correlations between NMR spectroscopy data characterizing the teristics: the presence of nucleotide repeats and spacers, which serve as a mem- structural dynamics of PLN, SERCA functional data collected from coupled ory bank, enabling bacteria to recognize the invading viruses. The Cas proteins enzyme assays, and structure prediction data garnered from the programs Dis- are enzymes that cut foreign DNA guided by the information encoded in EMBL and GlobPlot. We have placed particular focus on domain Ib of PLN CRISPRs. In 2012, it has been demonstrated that bacterial CRISPR-Cas9 can due to its relevance in regulating PLN’s inhibitory potency and mitigating be transformed into a simple, programmable genome-editing tool. Cas9 re- allosteric signaling through the juxtamembrane region of the protein. The ul- quires two RNA molecules: a CRISPR RNA (or ‘‘crRNA’’) and another timate goal of these studies is to better inform rational structure-based design tracrRNA (or ‘‘trans-activating crRNA’’) to make its cut on a target DNA of therapeutic PLN species that could be used to treat heart failure. The proj- with a 20-nucleotide stretch complementary to the crRNA. Recently, Cpf1 pro- ect is a working collaboration between St. Catherine University, the Univer- teins have been discovered to show comparable genome-editing capability to sity of Minnesota, and the Minnesota NMR Center, and provides a model of Cas9. Cpf1 functions through a single crRNA without an additional tracrRNA. conducting collaborative undergraduate research in partnership between a pri- Cpf1 is an RNA-guided endonuclease of a class II CRISPR/Cas system, which marily undergraduate institution, a major research institution, and an instru- is a smaller and simpler than Cas9, and overcomes some of the CRISPR/Cas9 ment center. system limitations. We are interested in rationally modifying the CRISPR-Cpf1 system to 2414-Pos improve its genome editing efficiency. The target efficiency and specificity Substrate Driven Allostery in a Mitochondrial Enzyme of the CRISPR technology are influenced by multiple factors. Our specific Amit Kumar, D. Estrada Fernando. objective is to first perform large-scale molecular dynamics (MD) simulations Biochemistry, University at Buffalo New York, Buffalo, NY, USA. to elucidate various conformational states involved in the Cpf1 catalysis The mitochondrial cytochrome P450 24A1 (CYP24A1) is responsible for (especially those upon the binding of crRNA and DNA) and the interconver- side chain hydroxylation of vitamin D, the initial step in deactivation of sion rates between different states. We further aim to use these MD simula- the bioactive hormone. Mutations in CYP24A1 lead to incomplete clear- tions to uncover the molecular mechanisms underlying the higher genome ance of active vitamin D and correlate with idiopathic infantile hypercalce- editing efficiency of certain crRNAs. We will systematically explore how mia, or elevated calcium in blood serum, and can lead to loss of appetite, the incorporation of chemically and structurally modified nucleotides in nausea, vomiting, and failure to thrive in newborns. In order to mediate crRNAs may affect the genome editing efficiency and off-target effects and the vitamin-D hydroxylation step, CYP24A1 requires transfer of two elec- to establish comprehensive structure-activity relationships of crRNA analogs. trons from the iron-sulfur cluster protein Adrenodoxin (Adx). In non- mitochondrial P450 enzymes, conformational selection and long-range 2412-Pos allostery have been demonstrated to occur in connection to substrate bind- Determining the Internal Allosteric Architecture of DHFR with Total ing and redox partner recognition. However, such long-range allostery has Saturation Mutagenesis not been demonstrated for a mitochondrial P450 enzyme, which relies on an James W. McCormick1, Samuel Thompson2, Kimberly A. Reynolds1. electron donor distinct from enzymes in the endoplasmic reticulum. In this 1The Green Center for Systems Biology, UT Southwestern, Dallas, TX, USA, work we report a combined approach of site-directed mutagenesis, protein 2Department of Bioengineering and Therapeutic Sciences, University of nuclear magnetic resonance spectroscopy (NMR), and chemical cross- California, San Francisco, CA, USA. linking to evaluate the CYP24A1-adrenodoxin complex from rat (a carbon Dihydrofolate reductase (DHFR) is a model enzyme for understanding the 24 hydroxylase). As expected, the interaction was primarily driven by the connection between long range conformational change and catalysis. In putative recognition domain of Adx (helix-3). However, the NMR data from titrations with CYP24A1 bound to substrate (1,25-dihydroxyvitamin prior work, we showed that DHFR contains a co-evolving network of amino a acids, termed a sector, which coincides with positions undergoing milli- D) versus the analog (1 -hydroxyvitamin D) indicates that modulation of second conformational exchange associated with catalysis. Moreover, we the redox-binding surface occurs in response to ligand binding. These find- found that inserting a light sensing domain from plants (LOV2) at sector ings provide the first demonstrated evidence in support of functional long- connected surfaces led to weak allosteric regulation in about 40% of posi- range and substrate driven allostery in a mitochondrial P450 enzyme and tions, while it never lead to allosteric regulation at non-sector connected sur- provide further evidence of substrate driven allostery for P450 enzymes faces. This suggests that sector connected edges are hotspots for engineering in general. regulation, and that sector positions might provide the basis of allosteric communication within DHFR. To test this, we constructed a saturation 2415-Pos mutagenesis library of DHFR, in the context of two LOV2 fusions: one in Salt Bridges in Ubiquitin Determine the Protein Conformational Flexi- which LOV2 is connected at a sector position and provides 2foldregula- bility tion of DHFR activity by light, and one in which LOV2 is not sector- Shrabasti Bhattacharya, Nidhi Acharya, Sri Rama Koti Ainavarapu. connected and does not regulate DHFR activity. We then measured the Department of Chemical Sciences, Tata Institute of Fundamental Research, DHFR activity of every library member in the light and dark, using a Mumbai, India. next-generation sequencing based assay in which DHFR activity is coupled Ubiquitin family proteins are involved in a wide variety of functions, ranging to growth rate. From these data, we determine the coupling of each mutation from proteasomal degradation to signal transduction. Apart from their unique to allostery in DHFR. This global map of allosteric contributions inside the b-grasp fold, with 5 b-strands wrapping around an a-helix, a unique feature enzyme provides insight into how allostery can be evolutionarily optimized, of them is a conserved salt bridge connecting the a-helix to a loop between and allows the evaluation of computational methods to model allosteric the b3 and b4 strands. Here, we investigated the role of this conserved salt pathways inside the protein. bridge in determining the protein conformational flexibility using ubiquitin

BPJ 9456_9458 488a Wednesday, March 6, 2019 as a model system and utilizing time-resolved fluorescence anisotropy and a = 88.2, b = 108.9, g = 111.9, belonging to the P1 space group, which was near-UV circular dichroism (CD). Therefore, we inserted a Trp (L43W) at determined both before and after undergoing THz spectroscopy from x-ray the core near to the conserved salt bridge (K27-D52), as a reporter of pro- data. Tri- N-acetyl-D-glucosamine (3NAG) inhibitor bound triclinic crystals tein’s microenvironment. CD and fluorescence anisotropy showed that ubiq- are similarly grown with the adding of an equal molar amount of 3NAG in- uitin’s core is conformationally rigid in the temperature range 5-55 0C. hibitor to the protein solution before setting up the crystallization well. The Disrupting the salt bridge by a double mutation (K27A/D52L) increased protonated, deuterated and inhibitor bound triclinic crystals were measured the conformational flexibility of the protein core at 5 0C, which further using a newly developed technique: ideal polarization varying anisotropic increased with temperature up to 55 0C. Interestingly, SUMO2 lacking Terahertz microscopy (ideal PV-ATM). We compare the relative frequency such a conserved salt bridge, has recently been shown to have similar flexi- shifts of the measured protonated and deuterated protein vibrations to the bility in this temperature range.1 These results suggest that the core flexibility calculated shifts, to make a tentative assignment with the calculated vibra- of the salt bridge (K27A/D52L) disrupted ubiquitin resembles that of tions. We then test our tentative assignments by comparing to the measured SUMO2. Furthermore, a more severe salt bridge mutation (K27M/D52L) of changes with inhibitor binding. ubiquitin, replacing Lys with Met to mimic SUMO2, showed that the pro- [1] K. A. Niessen, M. Y. Xu, A. Paciaroni, A. Orecchini, E. H. Snell, and A. G. tein’s core is highly flexible even at 5 0C and devoid of any tertiary structure. Markelz, Biophys. J. 112, 933 (2017). Our studies on ubiquitin and SUMO2 suggest that the conserved salt bridge [2] S. Mahajan and Y.-H. Sanejouand, Arch. Biochem. Biophys. 567,59 of ubiquitin family proteins plays an important role in determining protein (2015). conformational flexibility. 1. Shrabasti Bhattacharya and Sri Rama Koti Ainavarapu. Mechanical Softening of a Small Ubiquitin-Related Modifier Protein Due to Temper- 2418-Pos ature Induced Flexibility at the Core. J. Phys. Chem. B (2018), 122, Dynamic and Structural Allosteric Events between the D/E Linker and p9128-9136. N-Domain of Cardiac Troponin C Reveal a Novel Mechanism for Cardiac Muscle Regulation Mayra A. Marques1, Guilherme A.P. de Oliveira1, Adolfo H. Moraes2, 2416-Pos Maicom Landim-Vieira3, Karissa D. Jones3, Elio A. Cino4, P. Bryant Chase5, a-Catenin Structure and Nanoscale Dynamics in Solution and in Complex Jerson L. Silva1, Jose R. Pinto3. with F-Actin 1Medical Biochemistry Institute Leopoldo de Meis, Federal University of Rio Iain Nicholl1, David Callaway2, Zimei Bu2. de Janeiro, , Brazil, 2Institute of Exact Sciences, Chemistry 1Department of Biomedical Science and Physiology, Faculty of Science and Department, Federal University of Minas Gerais, Minas Gerais, Brazil, Engineering, University of Wolverhampton, Wolverhampton, United 3Department of Biomedical Sciences, College of Medicine, Florida State Kingdom, 2Department of Chemistry and Biochemistry, City College of New University, Tallahassee, FL, USA, 4Department of Biochemistry and York, CUNY, New York, NY, USA. , Federal University of Minas Gerais, Minas Gerais, Brazil, As a core component of the adherens junction, a-catenin stabilizes the cad- 5Department of Biological Sciences, College of Medicine, Florida State herin/catenin complexes to the actin cytoskeleton for the mechanical University, Tallahassee, FL, USA. coupling of cell-cell adhesion. a-catenin also modulates actin dynamics, Troponin complex exhibits allosteric networks modulating both the dynamics cell polarity, and cell-migration functions that are independent of the adhe- and the structural signaling along the myofilament. The mutation C84Y in rens junction. We have determined the solution structures of the a-catenin cardiac troponin C (cTnC) has been reported in a proband with hypertrophic monomer and dimer using in-line size-exclusion chromatography small- cardiomyopathy (HCM). Despite the relevance of HCM disease, little is angle X-ray scattering, as well as the structure of a-catenin dimer in com- known concerning the function of cTnC D/E linker where the residue 84 is plex to F-actin filament using selective deuteration and contrast-matching located and its consequential allosteric phenomena governing Ca2þ-binding small angle neutron scattering. We further present the first observation, to affinity. Our goal is to understand the structural and allosteric effects of our knowledge, of the nanoscale dynamics of a-catenin by neutron spin- this D/E linker mutation in cardiac muscle regulation. Ca2þ-titrations re- echo spectroscopy, which explicitly reveals the mobile regions of a-catenin vealed that C84Y exhibits enhanced Ca2þ-binding affinity and distinct that are crucial for binding to F-actin. In solution, the a-catenin monomer conformational changes compared to WT. However, the overall dimensional is more expanded than either protomer shown in the crystal structure values and molecular envelopes generated by SAXS remained similar. Using dimer, with the vinculin-binding M fragment and the actin-binding domain circular-dichroism, C84Y revealed significantly lower thermostability being able to adopt different configurations. The a-catenin dimer in solution compared to WT. NMR data and molecular dynamic simulations showed is also significantly more expanded than the dimer crystal structure, with altered binding of TnI switch peptide in WT and C84Y. Distinct allosteric fewer interdomain and intersubunit contacts than the crystal structure. events were shown upon TnI inhibitory peptide binding, suggesting a possible When in complex to F-actin, the a-catenin dimer has an even more open mechanism of contractile dysregulation. The largest chemical shifts were and extended conformation than in solution, with the actin-binding observed in N-Helix residues and at the end of D-helix and D/E linker in domain further separated from the main body of the dimer. The a-cate- C84Y. Temperature-coefficients analysis indicated different temperature- nin-assembled F-actin bundle develops into an ordered filament packing dependent conformational changes between WT and C84Y. Moreover, arrangement at increasing a-catenin/F-actin molar ratios. Together, the different time-scale exchanging rates existed between C84Y and WT. To structural and dynamic studies reveal that a-catenin possesses dynamic mo- further evaluate the role of this mutation in an intact system, knock-in lecular conformations that prime this protein to function as a mechanosensor mice bearing the cTnC-C84Y mutation (KI-TnC-C84Y) were generated. protein. The KI-TnC-C84Y mice displayed HCM measured by echocardiography and histopathology. Permeabilized cardiac muscle preparations from KI- TnC-C84Y exhibited increased myofilament Ca2þ-sensitivity and slower 2417-Pos kTR compared to WT. Altogether, the cTnC allosteric events captured in Deuteration and Inhibitor Binding Dependence of Protein Collective our study bring new insights to the understanding of muscle contraction Vibrations and communication within this intricate machinery. Yanting Deng, Jeffrey Mckinney, Andrea Markelz. Physics, State Univ of New York-Buffalo, Buffalo, NY, USA. Recently long range intramolecular protein structural vibrations have been 2419-Pos isolated by their directionality using anisotropic terahertz microscopy [1]. Dynamical Comparison between and Reveals the These vibrations possibly contribute to conformational change [2]. Calcula- Effect of the Quaternary Structure of Hemoglobin on its Subunits’ tions of the anisotropic collective vibrational spectra using semi-empirical Dynamics force fields along with limited accuracy water models have only qualitative Rotem Aharoni, Dror Tobi. agreement with these measurements. To improve the assignments we measure Molecular Biology Computer Science, Ariel University, Ariel, Israel. changes in the intramolecular vibrations with deuteration and inhibitor bind- Myoglobin (Mb) and Hemoglobin (Hb) are globular hemeproteins, when ing for chicken egg white lysozyme (CEWL) using triclinic crystals which the former is a monomer and the latter a heterotetramer comprised of provide absolute protein alignment and compare these measurements to four symmetrically arranged as a dimer of dimers (a1b1anda2b2). Mb normal mode ensemble analysis (NMEA). Triclinic lysozyme nitrate crystals has a higher affinity for oxygen than Hb, so it can bind oxygen near the were grown from a D2O solution. Similar unit cell parameters were observed capillary and then transport it to the mitochondria. However, Mb lacks ˚ ˚ ˚ for crystals grown in D2O as for H2O, i.e. a = 28.5A, b = 32.7A, c = 35.1A, allosteric and cooperative function in contrast to hemoglobin’s globin

BPJ 9456_9458 Wednesday, March 6, 2019 489a chains. There is a high structural similarity between Myoglobin and the a 2422-Pos and b subunits of Hemoglobin. The functional difference between the two The Effect of Crystal Contact Forces on the Protein Global Motions proteins arise due to the quaternary structure of Hemoglobin. The effect of Jeffrey A. McKinney, Yanting Deng, Deepu George, Andrea Markelz. the quaternary structure of Hemoglobin on the intrinsic dynamics of its Physics, University at Buffalo, Buffalo, NY, USA. subunits is explored by dynamical comparison of the two proteins. Aniso- Protein crystals are widely used for structural determination using X-ray crystal- tropic Network Model modes of motion were calculated for Hemoglobin lography and solid state NMR. X-ray free electron lasers have enabled direct and Myoglobin. Dynamical comparison between the proteins was per- dynamical structural movies of photo or chemically initiated biochemical reac- formed using global and local Anisotropic Network Model mode align- tions. However the perturbation of protein dynamics by the crystal contact forces ment algorithms based on the algorithms of Smith–Waterman and has been persistently under debate. If crystal contact forces are sufficiently Needleman-Wunsch for sequence comparison. The results indicate that strong, the time resolved X-ray diffraction measurements will not accurately the quaternary structure of Hemoglobin substantially alters the intrinsic reflect the in vivo biological dynamics. Here we probe the effect of crystal con- dynamics of its subunits, an effect that may contribute to the functional tact forces on protein structural dynamics by directly measuring the collective difference between the two proteins. Local dynamics similarity between intramolecular vibrations as a function of crystal symmetry for chicken egg the proteins is still observed at the major exit route of the ligand. white lysozyme (CEWL). These vibrations extend throughout the protein, involving coherent motion of entire domains. While the collective vibrational density of states is a broad continuous spectrum peaked near 100 cm1, 2420-Pos (3 THz), polarization dependent terahertz absorption isolates vibrations with Micro-Second X-Ray Single Molecule Dynamics of Allosteric Twisting displacements primarily along the polarization direction [1]. Triclinic, mono- Motions in Hemoglobin clinic, tetragonal and orthorhombic CEWL crystals are grown according to pre- 1,2 2,3 1 Yuji C. Sasaki , Masahiro Kuramochi , Yuu Okamura , vious published protocols. X-ray measurements verify their crystal structure and Hiroshi Sekiguchi4, Naoki Yamamoto5, Naoya Shibayama5. 1 2 quality before and after terahertz measurements. The anisotropic terahertz Dept Adv Matl Sci, Univ Tokyo, Kashiwa City, Japan, AIST- Univ Tokyo absorbance is measured using our new technique: ideal polarization varying Opeland-Oil, Kashiwa, Japan, 3Univ Tokyo, Kashiwa, Japan, 4Dept Res, 5 anisotropic THz microscopy (IPV-ATM). This near field THz microscopy Japan Synchrotron Rad Res Inst, Sayo, Japan, Jichi Medical Univ, method allows rapid determination of the anisotropic THz absorption. By start- Shimotsuke, Japan. ing from measurements of the lowest symmetry, triclinic crystals, which have a Allosteric structural changes underlie the biological functions of many pro- single protein per unit cell and absolute alignment of the protein molecule array, teins. The best studied is the tense (T) to relaxed (R) state transition in hemo- we can predict the spectra for higher symmetry groups, neglecting crystal con- globin (Hb), which allows for efficient oxygen transport. These two end tact perturbations. We compare our predicted spectra to the measurements of the structures have been solved by crystallography, but little is known about monoclinic, orthorhombic and tetragonal crystals, attaining a measure of the the transition pathway and motions between them. Here we use Diffracted impact of the intermolecular crystal forces on the intramolecular vibrations. X-ray Tracking (DXT) to monitor single molecule allosteric dynamics in [1]G. Acbas, K. A. Niessen, E. H. Snell, and A. G. Markelz, Nat. Comm.(2014) Hb. DXT allows us to monitor the protein motions at high time resolution https://doi.org/10.1038/ncomms4076. by tracking diffracted spots from a gold-nanocrystal attached to the protein. X-ray from SPring-8 BL40XU with pink beam was used for the DXT mea- 2423-Pos surements. Data from two rotational directions of the protein molecule, those A Ligand-Binding Site in the Glua3 AMPA Receptor N-Terminal Domain concerning tilting angle q and twisting angle c, are detected. Hb trout III ex- Observed in Druggability Simulations and X-Ray Crystallography tracted from rainbow trout was used as the sample, which exhibits a remark- Ji Young Lee1, James Krieger1, Beatriz Herguedas2, Javier Garcı´a-Nafrı´a2, able Root effect. It is expected that at pH 8 Hb trout III would exist as an Anindita Dutta1, Saher A. Shaikh2, Ingo H. Greger2, Ivet Bahar1. ensemble of the R states with a high oxygen affinity, while at pH 6 it would 1Computational and Systems Biology, University of Pittsburgh, Pittsburgh, exist as the T state with a low affinity for oxygen, which is one-thousandth of PA, USA, 2MRC Laboratory of Molecular Biology, Cambridge, United that of the R state. These unique properties allow us to select conformational Kingdom. populations of Hb by simply adjusting the pH values of the solvents used. Ionotropic glutamate receptors (iGluRs) play a fundamental role in the central Basic time resolution of the system was 100ms/frame. However, we have nervous system, and their dysfunction has been implicated in many neurobio- confirmed that we can observe much faster motions in a solvent containing logical diseases, rendering iGluRs potential drug targets. We performed a sys- 30% glycerol at low temperature, where molecular motions of Hb are mark- tematic study of the druggability of iGluR sub-families using molecular edly decelerated. As a result of the DXT observations, we succeeded to dynamics simulations in the presence of drug-like probe molecules. We demon- clearly detect motions in different twisting direction at a specific moving strated the applicability of our method by identifying known agonist and modu- speed (5-10mard/ms). lator sites on AMPA and NMDA receptors. Our simulations produced domain closure of the ligand-binding domain, which is critical to channel opening in response to agonist. For the N-terminal domain (NTD), we also identified a 2421-Pos highly probable drug-binding site at the NTD dimer interface of AMPAR sub- Diffracted X-Ray Blinking from Nanocrystal on Protein used as Internal type GluA3. This is attributed to its unique conformational flexibility that we Motion Probe explored further via X-ray crystallography revealing three new crystal struc- Hiroshi Sekiguchi1, Masahiro Kuramochi2, Yuji C. Sasaki3. tures in different states. Our study permits the determination of pharmacophoric 1Dept Res & Utilization Div, Japan Synchrotron Rad Res Inst, Sayo, Japan, features towards novel iGluR modulators. 2Univ Tokyo, Kashiwa, Japan, 3Dept Adv Matl Sci, Univ Tokyo, Kashiwa City, Japan. 2424-Pos Diffracted X-ray Tracking (DXT) is one of techniques for investigating intra- Molecular Analysis of Dengue NS3 Helicase Function molecule dynamics of proteins at single molecule level [1-3]. A nanocrystal Kelly E. Du Pont1, Russell B. Davidson1, Brian J. Geiss2, immobilized on the target protein is used as motion probe, and the trajectory Martin McCullagh1. of its diffracted spot is investigated with broad-band X-ray. The radiation dam- 1Chemistry, Colorado State University, Fort Collins, CO, USA, age of X-ray for the sample are serious problem in this method. Here, we ad- 2Microbiology, Immunology, and Pathology, Colorado State University, Fort dressed the issue by recording diffraction intensity fluctuations with Collins, CO, USA. monochromatic X-ray. The transition of X-ray diffraction intensity, diffracted The unwinding of double-stranded RNA intermediates is a critical component X-ray blinking, from moving single nanocrystal were observed to cycle in and for the replication of flavivirus RNA genomes. This function is achieved by the out of the Bragg condition, and the internal motions of a protein molecule C-terminal helicase domain of nonstructural protein 3 (NS3). As a member of labelled with nanocrystals could be extracted from the time-trajectory of this the superfamily 2 (SF2) helicases, NS3 is known to require the binding and hy- diffraction intensity. We succeeded in monitoring rotational motions’ en- drolysis of ATP/NTP to translocate along and unwind double-stranded nucleo- hancements of an individual acetylcholine-binding protein (AChBP) interact- tides. However, the mechanism of energy transduction between the ATP and ing with acetylcholine (ACh) by analyzing auto-correlation of diffraction RNA binding pockets is not well understood. Previous molecular dynamics X-ray intensity from gold nanocrystal on the AChBP using even laboratory simulations published by our group have identified Motif V as a potential X-ray sources. ‘‘communication hub’’ for this energy transduction pathway. In order to inves- [1] Y.C. Sasaki et al., Phys. Rev. E 62:3843 (2000) tigate the role of Motif V in this process, a study combining molecular dy- [2] H. Sekiguchi et al, PLoS ONE 8:e64176 (2013) namics, biochemistry and virology has been employed. Recombinant NS3 [3] H. Sekiguchi et al, Scientific Reports 4:6384 (2014) with mutations in Motif V were expressed and purified from E. coli

BPJ 9456_9458 490a Wednesday, March 6, 2019 andbiochemically assessed for their NS3 ATPase and helicase activity as well The diffusion coefficient D thus provides an effective way to infer the energy as RNA binding affinity. The effects of each mutant on viral genome replication roughness, of which the quantification has been a long pursuit in protein in cell culture were also tested. Additionally, simulations of each mutant were research field. Here, with the great successes of extending energy landscape used to probe structural changes within NS3 caused by each mutation and to intrinsically disordered proteins (IDPs), we employed structure-based improve our model of energy transduction within NS3. These data help define models (SBMs) to quantify the binding diffusion coefficient D of a prototype the linkage between ATP hydrolysis and helicase activity within NS3 and pro- IDP during its binding-coupled-folding process. By modulating the degree of vide insight into the biophysical mechanisms for ATPase driven NS3 helicase conformational disorder in isolated IDP, we found a complex trend of diffusion function. relying on conformational disorder during IDP’s binding that initial binding is accelerated by ‘‘fly-casting’’ mechanism through conformational flexibility 2425-Pos while the late binding is accelerated by the emergence of multiple pathways Investigating the Role of the Auxiliary Nucleotide Binding Sites in the thanks to the stiffness of chain. In the presence of electrostatic interactions, Recbcd DNA Helicase the binding diffusion of IDP is facilitated by the steering ionic interactions in Sivasubramanyan Mangapuram Venkata, Rani Zananiri, Vera Gaydar, the encounter complex but hindered by the non-native salt bridges during the Oded Kleifeld, Ariel Kaplan, Arnon Henn. formation of the final binding complex. With the quantified D, we are able to Biology, Technion, Haifa, Israel. portray the energy landscape of IDP’s binding with great details on the hierar- RecBCD is highly processive and very fast DNA helicase-nuclease, responsible chical roughness. for the initiation of double-stranded break repair in E. coli. By employing a combination of equilibrium and time-resolved unwinding and binding experi- 2428-Pos ments, both ensemble and single-molecule, we previously demonstrated the ex- Long-Range Interactions Mediated by the Disordered NFkB Transcription istence of auxiliary binding sites in RecBCD, where ATP binds with lower Activation Domain affinity and with distinct chemical interactions as compared to the known cat- Dominic Narang, Wei Chen, Allen Po, Elizabeth A. Komives. alytic sites. We showed that RecBCD achieves its fast unwinding rate by uti- Dept Chem & Biochem, Univ Calif San Diego, La Jolla, CA, USA. lizing the auxiliary binding sites to increase the flux of ATP to its catalytic The Nuclear factor kappa B (NFkB) family of transcription factors regulates sites located in RecB and RecD subunits. Equilibrium dialysis indicated the genes involved in the immune response, cell growth and development. The number of nucleotide-binding sites to be at least four, and their likely location activity of NFkB proteins is mediated by its binding to specific DNA se- in the RecC subunit. In this current work, we provide further support for the quences (kB sites) and is tightly regulated by a family of proteins known functional role of the auxiliary sites, by abolishing the nucleotide-binding sites as the inhibitor proteins (IkBs). All the in vitro studies carried out to under- in RecC. A list of possible nucleotide-binding sites was obtained by using a stand the dynamics of DNA binding and stripping of DNA by IkBa have combination of UV-crosslinking and mass spectrometry, as well as molecular been done with the truncated form of the RelA-p50 (NFkB) which lacks docking studies. Based on these predictions, several mutations in RecC were the transactivation domain (TAD). In vivo experimentssuggest that both carefully designed, producing unique RecBCmutD. We have successfully binding of NFkBtokB site and the interactions of TAD with other co- cloned and purified RecBCmutD and shown that RecBCmutD has DNA- transcription factors are critical for the activation of gene transcription. activated ATPase activity. Furthermore, an equilibrium nucleotide binding TAD is predicted to be disordered. We have now been able to produce the assay suggests that the RecBCmutD does not display the same binding isotherm full-length RelA with the TAD and we have shown that it exchanges as the WT RecBCD. Based on these observations, we propose that the auxiliary 100% of its amides within 30 sec except for two short Linear Motifs (SLIMs) binding sites play an integrated role in the reaction mechanism of RecBCD. that only exchange 75%. We observe that full-length NFkB with the TAD binds more tightly to DNA than the truncated protein suggesting that the 2426-Pos TAD affects the DNA binding. This observation is intriguing since the Allostery & Dynamics in Nuclear Hormone Receptor Transactivation TAD is approximately 230 residues apart from the DNA binding domain. David Lohry, Taylor Stevens, Mark Remec Pavlin, Balananda D.K. Putcha, Stopped-flow fluorescence kinetics experiments show that the presence of Tongye Shen, Elias J. Fernandez. TAD does not affect the DNA binding rate constant, but it affects the disso- Biochemistry & Cellular and Molecular Biology, University of Tennessee, ciation rate constant. This suggests that the TAD stabilizes the DNA binding Knoxville, TN, USA. either by interacting with DNA binding domain by direct contacts or alloste- Nuclear hormone receptors (NR) are ligand-mediated transcription factors that rically. HDXMS experiments reveal decreases in exchange in parts of the relay cellular signals through distinct multiprotein assemblies. Hormonal li- DNA-binding domains and the dimerization domains that are most consistent gands produce structural changes within NRs that determine the composition with an allosteric effect. Overall, our results provide new insights into the of the interacting proteins. NRs are characteristically modular proteins. At role of Intrinsically disordered TAD in DNA binding. the N terminus is an intrinsically disordered (IDP) N-terminal domain (NTD) followed by a DNA-binding domain (DBD). The ligand-binding domain 2429-Pos (LBD) at the C terminus contains a dimerization interface that associates Control of Cellular Networks by Structural Disorder with the retinoid X receptor (RXR). Agonist binding results in conformational Nikolay V. Dokholyan1, Onur Dagliyan2, Klaus M. Hahn3. changes associated with a transcriptionally active state where the LBD rear- 1Pharmacology, Penn State College of Medicine, Hershey, PA, USA, ranges to create a docking site for coactivator proteins such as the steroid recep- 2Department of Neurobiology, Harvard University, Boston, MA, USA, 3Dept tor coactivator 1 (SRC1). With a combination of biophysical and cell-based Pharmacology, Univ North Carolina Chapel Hill, Chapel Hill, NC, USA. assays, we are proposing models for allostery within NRs that are designed We developed allosteric control of structural disorder-to-order transition in to address the following questions: (i) What role does the NTD play in the ac- proteins using light or small ligand thereby regulating their functions. We tivity of these receptors? (ii) What role do ligands play in the stoichiometric also develop computational tools, based on graph theory, to rapidly map allo- assembly of the NR:coregulator complex? (iii) How do discrete sites regulate steric connections within proteins. These optogenetic or chemogenetic tools the activity of these receptors? (iv) How do the DBD and LBD communicate allow direct interrogation of cellular networks, protein-protein interactions, to regulate coactivator and DNA recognition? and the roles of individual proteins in cellular life. We demonstrate the util- ity of the chemogenetic and optogenetic approaches to protein regulation in a 2427-Pos number of applications relevant to cellular motility. We demonstrate trans- Binding-Coupled-Folding of Intrinsically Disordered Protein Exhibits a ferability and broad applicability of our approach to diverse families of Hierarchical Energy Landscape proteins. Xiakun Chu1, Jin Wang1,2. 1Department of Chemistry, State University of New York at Stony Brook, 2430-Pos Stony Brook, NY, USA, 2State Key Laboratory of Electroanalytical Structure and Dynamics of Intrinsically Disordered and Unfolded Pro- Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of teins: Investigations using Small-Angle Scattering and Neutron Spin- Sciences, Changchun, China. Echo Spectroscopy Energy landscape theory has successfully predicted that the high-dimensional Felix Ameseder1, Laura R. Stingaciu2, Aurel Radulescu3, Olaf Holderer3, protein folding process can be enormously reduced into a diffusion along Peter Falus4, Michael Monkenbusch1, Ralf Biehl1, Dieter Richter1, limited number of reaction coordinates and the dynamics can be subsequently Andreas M. Stadler1. described by the Kramers reaction rate theory. Therefore the kinetics of protein 1ICS-1 & JCNS-1, Forschungszentrum Julich€ GmbH, Julich,€ Germany, 2Oak folding is basically controlled by the thermodynamic free energy barrier and Ridge Natl Lab, Oak Ridge, TN, USA, 3Julich€ Centre for Neutron Science at pre-factor. Such critical pre-factor directly reflects the local diffusive rate MLZ, Forschungszentrum Julich€ GmbH, Julich,€ Germany, 4Institut Laue- that inherently encodes the magnitude of roughness on the energy landscape. Langevin, Grenoble, France.

BPJ 9456_9458 Wednesday, March 6, 2019 491a

A characteristic property of intrinsically disordered and unfolded proteins is induce an aggregation prone conformation and another that stabilize IAPP at their high molecular flexibility, which enables the exploration of a large confor- the membrane interface. Our studies provide new insight into the membrane mational space. We present neutron scattering experiments on the structure and interaction of IAPP that will improve the understanding of the process of am- dynamics of the intrinsically disordered myelin basic protein (MBP) and the yloid aggregation in a physiologically relevant context. chemically denatured bovine serum albumin (BSA) in solution. Small-angle neutron scattering (SANS) experiments allowed us to gain information of struc- 2433-Pos tural aspects of MBP and denatured BSA as response to denaturant conditions. Inhibition of A-Synuclein Amyloid Fibril Elongation by Blocking Fibril Using neutron spin-echo spectroscopy (NSE), we were able to investigate col- Ends 1 1 2 lective motions of the protein chain up to several hundred nanoseconds on the Volodymyr V. Shvadchak , Kseniia Afitska , Anna Fucikova , 1 nanometre length-scale. NSE results showed a high flexibility of the unfolded Dmytro A. Yushchenko . 1Chemical Biology, Institute of Organic Chemistry and Biochemistry of the proteins. Internal motions of the intrinsically disordered MBP and denatured 2 BSA were described using normal mode analysis and concepts derived from CAS, Prague, Czech Republic, Faculty of Mathematics and Physics, Charles polymer theory. The contribution of residue-solvent friction was accounted University, Prague, Czech Republic. a a for using the Zimm model including internal friction. Motions of MBP are Misfolding of the protein -synuclein ( Syn) into amyloid fibrils plays a cen- well described by collective normal modes, while dynamics of denatured tral role in the development of Parkinson’s disease, a neurodegenerative disor- BSA shows polymer-like properties. Disulphide bonds forming loops of amino der that has no cure at present. The majority of existing approaches for a acids of the peptide backbone have a major impact on internal dynamics of de- inhibition of Syn fibril formation are based on stabilizing the native disor- natured BSA that can be interpreted with a reduced set of Zimm modes. dered monomeric form of the protein or destabilizing the fibrillized misfolded form. They require high concentrations of inhibitor and therefore cannot be easily used for therapies. We decided to use a different approach and to target Posters: Intrinsically Disordered Proteins (IDP) fibril ends, reactive species present at much lower concentrations. We designed a chimeric protein consisting of aSyn and a bulky globular protein. aSyn moi- and Aggregates III ety ensures selective binding of the inhibitor to aSyn fibril end while globular protein creates a steric hindrance and prevents binding of aSyn monomers to 2431-Pos z the end capped by the inhibitor. This approach permits inhibition of fibril for- Understanding the Kinetic Roles of 14-3-3 during Tau Filament mation at inhibitor concentrations much lower than the concentration of mono- Formation meric aSyn (IC50 is 850 nM). We studied kinetic mechanism of the inhibition Junwen Xiong, Meng Gao, Yongqi Huang. and found that the inhibitor efficiency is limited mostly by its slow binding to Hubei University of Technology, Wuhan, China. z the fibril end [1] and it is therefore the most promising direction for the The microtubule associated protein tau and 14-3-3 protein are two proteins further improvements. Our work demonstrates that blocking of aSyn fibril highly expressed in the central nervous system. Tau protein promotes microtu- ends is an effective approach to inhibit fibril growth and provides insights bule assembly and aberrant accumulation of tau and formation of amyloid fi- for development of effective inhibitors of aSyn aggregation. [1] V V Shvad- brils are pathological hallmarks of neurodegenerative tauopathies including chak, K Afitska, D A Yushchenko, Angew Chem Int Ed Engl, 2018, 57, 5690. Alzheimer’s disease. Extensive studies have suggested that polyanionic in- ducers, such as heparin, interact with tau directly and induce specific conforma- 2434-Pos tional changes within tau, accelerating the amyloidogenesis process. 14-3-3z Comparing Early Stages of Amyloid-Beta Aggregation in Different Mem- proteins form homodimers and bind their targets through specific pSer/pThr branous Environments motifs. It has been found that tau protein contains two 14-3-3z-binding motifs, Abhilash Sahoo1, Hongcheng Xu1, Silvina Matysiak2. i.e., pSer214 and pSer324, and tau protein interacts with 14-3-3z protein 1Biophysics Program, Institute for Physical Science and Technology, directly. Furthermore, 14-3-3z is able to induce tau aggregation, although it’s University of Maryland, College Park, MD, USA, 2Fischell Department of still controversial whether this process depends on tau phosphorylation. Bioengineering, University of Maryland, College Park, MD, USA. Here, we investigated the kinetic roles of 14-3-3z during tau filament forma- Aggregation of misfolded Amyloid-beta (Abeta) peptides into highly structured tion. We used the K18 constructs of tau that consists of the four microtubule oligomeric forms, particularly on lipid membranes is suspected as the primary repeats and the pSer324 14-3-3z-binding motifs. Our results showed that event in Alzheimer’s pathogenesis. Coarse grained molecular dynamics (CG- 14-3-3z induced tau K18 aggregation at a rate much larger than that from MD) simulations can provide detailed structural and dynamical characteriza- full-length tau. Kinetics analyses showed that the lag time and rate of aggrega- tion of membrane-induced amyloid-beta oligomers. To investigate the differen- tion were dependent on the stoichiometry of tau to 14-3-3z. Although phos- tial effects of anionic and zwitterionic lipids on aggregation of Abeta central phorylation at Ser324 increased the binding affinity of tau K18 to 14-3-3z, hydrophobic core (residues 16-22: K-L-V-F-F-A-E) in presence of lipid bila- phosphorylation only showed marginal effect on 14-3-3z mediated tau aggre- yers, we employed CG-MD simulations of model lipid bilayers with solvated gation. Our results suggest that the formation of the 2:1 complex of tau mono- monomeric peptides. Similar to experimental observations, the fraction of or- mer and 14-3-3z dimer plays an important role in the 14-3-3z mediated tau dered beta sheet rich fibrils is higher on anionic - 1-palmitoyl-2-oleoyl-sn-glyc- aggregation process. ero-3-phospho-L-serine (POPS) bilayer as compared to zwitterionic - 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. On the other 2432-Pos hand, the total rate of aggregation (both ordered and disordered), is higher for Membrane Interactions of IAPP POPC. We also evaluate other lipid induced morphological features of peptide- 1,2 3 Mikkel H. Christensen , Birgit Schiøtt . aggregates and provide mechanistic explanations derived from lipid-peptide 1Department of Chemistry, INANO, Aarhus University, Aarhus, Denmark, 2 3 interaction for our observations. This work highlights the importance of lipid Sino-Danish Center, Beijing, China, Department of Chemistry, Aarhus headgroup in introducing kinetic variations and structural diversity on University, Aarhus C, Denmark. Amyloid-beta 16-22 peptide aggregation. Islet amyloid polypeptide (IAPP, or amylin) is co-secreted from the pancreatic beta cells together with insulin to regulate the glucose metabolism. In patients 2435-Pos with type II diabetes mellitus, IAPP aggregates to form amyloid fibrils, a high Understanding and Preventing Aggregation in Alpha-Synuclein concentration of which are tightly correlated with a loss of beta cells, thus lead- Lisa J. Lapidus. ing to dysregulation of glucose metabolism and hyperglycemia. Membrane Dept Phys/Astron, Michigan State Univ, East Lansing, MI, USA. induced aggregation and membrane damage has been observed in vitro and it The protein alpha-synuclein is intrinsically disordered, and its aggregation is has been found that lipids such as anionic lipids, cholesterol and ganglioside involved in Parkinson’s disease. There has been many efforts to discover lipids have a large impact on the propensity of IAPP to form fibrils and damage why this protein is so prone to aggregation while other IDPs are not and to lipid bilayers. Using a combination of atomistic and coarse-grained molecular find the aggregation-prone precursor structures. Our hypothesis is that aggrega- dynamics simulations we have investigated the membrane bound state of IAPP tion is kinetically controlled by the reconfiguration rate of the protein compared with various membrane compositions with combinations of cholesterol, zwit- to the biomolecular diffusion rate. The measured intramolecular diffusion con- terionic-, anionic- and ganglioside lipids. We have identified specific lipid stant of this protein is highly correlated with aggregation conditions such as binding modes on IAPP that affect the structure and dynamics of membrane temperature, pH or mutation, with faster diffusion corresponding to slower ag- bound peptide, which provide detailed information about which parts of the gregation. Furthermore, the measured reconfiguration depends on how it is peptides are important for lipid interaction. In the presence of ganglioside measured; using long-range or close range probes produces diffusion constants lipids, IAPPs accumulate in areas with a high content of ganglioside lipids that differ by an order of magnitude. Recent MD simulations have revealed the and we identified two binding modes of membrane associated IAPP, one that molecular basis for these observations: close range probes are most sensitive to

BPJ 9456_9458 492a Wednesday, March 6, 2019 conformations that also have long residence times. Such conformations could moment. Recently we showed that bulky molecules binding to the ends of be the elusive aggregation-prone precursors. Finally we present ongoing aSyn fibrils can efficiently slowdown fibril growth and developed the first ratio- work to find small molecules that bind to the protein, raise the reconfiguration nally designed inhibitor working on this principle [1]. The constructed fusion rate and prevent aggregation and possibly disease. protein, consisting of a bulky protein moiety attached to the C-terminus of aSyn molecule, has IC50 of 850 nM. Nevertheless, its inhibitor efficiency 2436-Pos can be further improved by increasing steric hindrance and affinity to the fibril Terminal Capping of Amyloidogenic Tau Fragments Modulates their end. We studied the structure-activity relationship in a series of inhibitors Fibrillation Propensity focusing on the structure, size, and position of the bulky group. We prepared 1 1 2 3 Shruti Arya , Pritam Ganguly , Sarah L. Claud , Andrea Arsiccio , 12 protein-based inhibitors varying: a) sequence of aSyn moiety to improve 2 1 1 Kristi Lazar Cantrell , Joan Emma Shea , Michael T. Bowers . binding of inhibitor to the fibril end; b) structure of bulky moieties; c) attach- 1Department of Chemistry and Biochemistry, University of California Santa a 2 ment position of the bulky group to Syn to improve steric hindrance. Their Barbara, Santa Barbara, CA, USA, Department of Chemistry, Westmont inhibition activities were tested in a kinetic ThT-based assay and compared College, Santa Barbara, CA, USA, 3Department of Applied Science and by IC50 values. Decreasing the electrostatic repulsion between the fibril end Technology, Politecnico di Torino, Torino, Italy. and inhibitor molecule by deleting aSyn C-terminus or neutralizing its charge The aberrant folding of proteins leading to the formation of characteristic cross- significantly improved inhibitor efficiency (IC =500 nM and 700 nM, respec- b 50 -sheet rich amyloid structures is implicated in the pathogenesis of a variety of tively). Changing the bulky group structure and positioning it at the N-terminus debilitating diseases. In many cases, often depending upon amino acid compo- allowed us to prepare an inhibitor with IC50 340 nM, that is the most active sition, only a small segment of a large protein participates in amyloid core for- aSyn fibril growth inhibitor to the best of our knowledge. Our structure- mation and is in fact capable of self-assembling into amyloid, independent of activity relationship studies provide background for rational design of the rest of the protein. Therefore, such peptide fragments serve as useful model protein-based inhibitors of aSyn fibrillization targeting ends of fibrils and hav- systems for protein aggregation studies. An important factor that has often been ing potential to reach low nanomolar IC50. [1] V V Shvadchak, K Afitska, D A underestimated while using peptides to mimic full length protein, is the charge Yushchenko, Angew Chem Int Ed Engl, 2018, 57, 5690. on the peptide termini. We have investigated the effect of the termini on aggre- gation of amyloidogenic peptides from microtubule associated intrinsically 2439-Pos disordered protein Tau, implicated in Alzheimer’s disease (AD) and Tauopa- Oligomer Cross-Propagation between Wild-Type and Mutant Amyloid-b thies. Unlike other amyloidogenic proteins, Tau is highly soluble and does Implicate Conformational Strains in AD Phenotypes not readily aggregate without anionic co-factors, such as heparin. Our experi- Morgan Malone, Dexter N. Dean, Vijay Rangachari. ments show that modification of the termini can drastically modulate the fibril- Chemistry and Biochemistry, The University of Southern Mississippi, lation of hexapeptide from repeat 3 (R3)-paired helical filament 6 (PHF6) and Hattiesburg, MS, USA. full length R3 of Tau, both with and without heparin. We found that even Nearly ten percent of Alzheimer disease (AD) cases are the result of familial without heparin, PHF6 and R3 peptides that were capped at both the termini mutations. While mutations that lie outside the Ab sequence in APP lead to readily formed amyloid fibrils. In presence of heparin, only PHF6 with free elevation in Ab levels, those that lie within the Ab sequence increase the termini and PHF6 with free N-terminus were unable to form fibrils. Our molec- rate of Ab aggregation and cause the cerebral amyloid angiopathy (CAA) ular dynamics simulations on PHF6 capping variants agree well with our exper- phenotype. Several mutations of the Ab peptide involved in AD have been iments and provide insights into how terminal capping affects the self-assembly identified. Recently, we reported that Large fatty-acid-derived oligomers process. In addition to the effect of terminal capping on Tau fibrillation, we are (LFAOs) of wild-type Ab42, propagate their mesoscopic structure and cause also studying cross-interactions between peptides associated with AD and CAA preferentially in mice. Here, we demonstrate that wild-type LFAOs Type-2-diabetes, in order to understand the underlying link between these high- cross-propagate oligomer formation of Arctic mutant (E22G) of Ab42 as ly prevalent diseases. I will discuss some of our recent results from these well as wild-type Ab40. Seeding of LFAOs to Ab42 E22G monomers gener- studies. ates oligomers with size distributions similar to that of wild-type monomer 2437-Pos seeded with LFAOs. Similarly, soluble oligomers of E22G (ArcOs) formed b Observation of Structural Growth of Fibrils of Amylin Protein in the presence of fatty acid interface, when seeded upon wild-type A 42 b Suparna Khatun1, Shikha Kumari2, Agneyo Ganguly3, Nisha Pawar1, monomers, generated cross-propagated oligomers. While A 40 is the predom- b Amar Nath Gupta1. inant species present physiologically, A 42 exhibits faster aggregation, and b 1Department of Physics, IIT Kharagpur, Kharagpur, India, 2School of Bio higher proportion of A 42 is associated with increased incidence of AD. Our Science, IIT Kharagpur, Kharagpur, India, 3Department of Biotechnology, results demonstrate a fibril-free, oligomer-to-oligomer cross propagation be- b IIT Kharagpur, Kharagpur, India. tween different isoforms of A leading to enhancement in CAA phenotype. The misfolding and aggregation of human amylin are known be involved in the These results also implicate a wider mechanism involving cross-propagation pathogenesis of type II diabetes disease. Understanding how the conversion among other amyloid proteins leading to the clinically observed interplay be- into an infectious form takes place is essential for understanding the disease tween many neurodegenerative diseases. and developing new targets for therapies, but the conversion mechanism re- mains still unclear. Light scattering is the most sensitive method in detecting 2440-Pos even small amounts of aggregates in macromolecular solutions. We have Sumoylation of the Nt17 Domain of Huntingtin Influences Aggregation measured the scattered intensity from amylin solution under conditions and Binding to Lipid Membranes 1 1,2 affecting conversion e.g. pH, temperature, aging and the interaction parameter Faezeh Sedighi , Justin A. Legleiter . 1The C. Eugene Bennett Department of Chemistry, West Virginia University, second virial coefficient (A2). The nucleation-dependent growth of fibrils was 2 measured by the shape factor which has suggested a rod like elongation of fi- Morgantown, WV, USA, Blanchette Rockefeller Neurosciences Institute, brils as well as stacking of individual fibril structure perpendicular to the fibre Robert C. Byrd Health Sciences Center, West Virginia University, axis held by hydrogen bonding which is supported by analytical and high res- Morgantown, WV, USA. olution TEM image. Observed fibrils are composed of several protofilaments Huntington’s disease (HD) is a member of a group of neurodegenerative disor- ders caused by an expansion of polyglutamine (polyQ) domains in specific pro- with multi-stranded structures. A2 has shown an interesting switch from repul- sive to attractive interactions between protein molecules as function of time. teins. The expanded polyQ domains appear to destabilize protein structure Initially the aggregation leads with repulsive interactions and forms protofila- leading to the formation of distinct aggregates such as oligomers, protofibrils, ments. The hydrophobic and liquid crystalline interactions among protofila- annular aggregates, and fibrils. Beyond the polyQ domain, flanking sequences ments stable the system by forming multi-stranded fibrils, thus switching the also heavily influence aggregation. In particular, the first N-terminal seventeen A favours the growth of fibrils. amino acids (Nt17), which are directly adjacent to the polyQ domain, self- 2 associates to form oligomeric intermediates associated with htt aggregation. 2438-Pos The Nt17 domain is also involved in lipid binding. Within Nt17, there are three Structural Optimization of a-Synuclein Fibril Growth Inhibitors lysine residues (K6, K9, and K15) that can be SUMOylated, and SUMOylation Kseniia Afitska, Volodymyr V. Shvadchak, Dmytro A. Yushchenko. modifies htt’s accumulation and toxicity within cell in a variety of HD models. Chemical Biology, Institute of Organic Chemistry and Biochemistry of the To determine the impact of SUMOylation on htt aggregation, ex situ atomic CAS, Prague, Czech Republic. force microscopy (AFM) experiment were performed, and SUMOylation Aggregation of alpha-synuclein (aSyn) is involved in the development of Par- inhibited the formation of htt fibrils. Using in situ AFM experiments, kinson’s disease, a severe neurodegenerative disorder that has no cure at the we demonstrate that SUMOylation greatly reduced the extent of htt

BPJ 9456_9458 Wednesday, March 6, 2019 493a aggregation and accumulation of model lipid bilayers comprised of total binding behavior. The results of in vitro experiments comparing the amyloid brain lipid extract. This reduced htt/lipid interaction was further demon- formation kinetics of acetylated (Ac-aSyn) and the more widely studied strated using a polydiacetylene (PDA) lipid binding assay. Collectively, non-acetylated a-synuclein (NH3-aSyn) have been contradictory. However, these observations suggest that the SUMOylation of Nt17 strongly influ- these assays largely rely on the extrinsic dye thioflavin T (ThT), which dis- ences htt aggregation and its interactionwithlipidmembraneswithpoten- plays a dramatic increase in quantum yield upon binding to the cross-b sheet tial implications for htt toxicity. structure in amyloids. We find that the ThT response is approximately tenfold lower in Ac-aSyn, and that Ac-aSyn aggregates more slowly than NH3-aSyn 2441-Pos at physiological pH. Intrinsic fluorescence of Trp mutants confirms that ThT The Presence of Mitochondria Influences Huntingtin Aggregation emission accurately reports on the kinetics of amyloid formation for both Adewale Adegbuyiro1, Justin A. Legleiter1,2. forms of the protein. Seeding experiments indicate that the ThT fluorescence 1Dept of Chemistry, West Virginia Univ, Morgantown, WV, USA, intensity is determined by the final fibrillar structure templated from the seeds 2Blanchette Rockefeller Neurosciences Institute, West Virginia Univ, rather than the presence or absence of the N-terminal acetyl group. Structural Morgantown, WV, USA. differences of the de novo and seeded fibrils are corroborated by Raman spec- Huntington’s disease (HD) is the most common genetic neurodegenerative troscopy, limited proteolysis experiments, and transmission electron micro- disease. It is caused by the abnormal expansion of a polyglutamine tract scopy. These results indicate that N-terminal acetylation has important near the N-terminus of the huntingtin (htt) protein. This expansion directly consequences for the aggregation propensity and amyloid structure of aSyn, leads to htt aggregation into insoluble b-sheet rich fibrils. Mutant htt localizes and that nonacetylated aSyn is not a substitute for the biological form of the and interacts with membranous surfaces associated with a variety of organ- protein. elles. Mitochondria are of interest as they have been observed to be morpho- logically altered and dysfunctional in a variety of HD models. Therefore, understanding htt aggregation in the presence of mitochondria could provide 2444-Pos Investigation of the Various Structures of Alpha-Synuclein and their Inter- valuable insights into a variety of toxic mechanisms associated with HD. The actions with Small Molecules aggregation of a htt-exon1 protein with an expanded polyglutamine was monitored in the presence and absence of isolated mitochondria. Atomic John Ferrie, Sam G. Giannakoulias, E. James Petersson. Force Microscopy (AFM) was used to evaluate the morphology of heteroge- Chemistry Department, University of Pennsylvania, Philadelphia, PA, USA. neous aggregate populations as a function of time. In addition, filter-trap Intrinsically disordered proteins (IDPs) and proteins with disordered regions assay was performed to validate observations from the AFM experiments. (IDRs) make up an ever-expanding class of protein which lack consistent secondary structure resulting in their propensity for populating a myriad The presence of mitochondria inhibited htt fibrilization and promoted the for- of conformational states. These proteins have been recognized for their mation of oligomers. Oligomers formed in the presence of mitochondria were smaller than those formed in the absence of mitochondria. Collectively, these essential role in many processes through their ability to undergo conforma- observations suggest that mitochondrial surfaces strongly alter htt tional rearrangement, often adopting a more canonically ordered state. How- aggregation. ever, since these proteins lack a consistent compacted structure, they are often highly predisposed to aggregation, resulting in their implication in several neurodegenerative diseases. Of these, we have focused on studying 2442-Pos a-synuclein (aS) whose aggregation has long been linked to Parkinson’s Post Translational Modification of Ab Influences Aggregation in the Pres- disease pathogenesis. Our previous efforts have focused on utilizing Forster ence and Absence of Lipids Resonance Energy Transfer (FRET) based experiments to obtain informa- Albert W. Pilkington, Justin A. Legleiter. tion about the disordered and fibrillar forms of aS. Furthermore, pairing Dept Chemistry, West Virginia Univ, Morgantown, WV, USA. of these measurements with Monte-Carlo based modeling approaches has A pathological hallmark of Alzheimer’s disease (AD), a late onset neurode- allowed us to develop two modules within the PyRosetta-platform within generative disease, is the development of neuritic amyloid plaques, predomi- the Rosetta Modeling Suite which are capable of 1) predicting the structure nantly comprised of the b-amyloid (Ab) peptide. Ab aggregation is associated of a protein, whether ordered or disordered, and 2) accurately positing struc- with a variety of nanoscale, toxic aggregate species ranging from small olig- tural ensembles of the disordered state of IDPs and IDRs from primary omers to fibrils. Ab directly interacts with lipid membranes, which can heavi- sequence. Recent efforts have focused on expanding our computational ly influence the aggregation process and thus a variety of toxic mechanisms. and experimental efforts to monitor the effects of small molecules on the Here, we utilize post translational modifications to understand the role of spe- disordered state of aS. Ultimately we intend to use the previously described cific amino acids on Ab -membrane interactions and self-assembly. Utilizing 40 computational methods, along with others currently under development, to Thioflavin T (ThT), atomic force microcopy (AFM), circular dichroism (CD), inform our selection of small molecules which can either bind selectively and polydiacetylene (PDA) lipid binding assays, we investigated the impact of to aS fibrils, for further development as probes, or interact with the disor- oxidation and acetylation on aggregation kinetics, aggregate morphologies, dered state of aS, with the intention of reducing the protein’s aggregation and secondary structure of Ab in the presence or absence of a model mem- 40 propensity. brane system, total brain lipid extract (TBLE). In the absence of lipids, oxida- tive environments that resulted in the the oxidation of M35 decreased the extent of fibril formation and eventually promoted a distinct fibril 2445-Pos morphology. In the presence of TBLE, oxidation increased aggregation rela- Investigating C99 in Amyloid Formation using Molecular Dynamics: From tive to Ab40 and reduced the ability of Ab to bind membranes. In the absence Simple to Complex Neuronal Models of lipid, acetylation of K16 had minimal impact on aggregation, but K28 acet- Jenny Pin-Chia Hsu, Birgit Schiøtt. ylation completely inhibited fibril formation. In the presence of membranes, Department of Chemistry, Aarhus University, Aarhus C, Denmark. acetylation of K16 modulated peptide-membrane interactions, but further Cell membranes consist of a chemically diverse set of lipids that have been acetylation of K28 had no observable effect. Collectively, our data suggests shown to play a key role in numerous cellular process including defence, intra- that K28 plays an important role in fibril formation, oxidation of M35 alters cellular transport, signal transduction and membrane fusion. Additionally, indi- the structural transitions associated with fibril formation, and K16 facilitates vidual lipids have recently been found to be of great importance for protein Ab-membrane interactions. regulation, and changes in molecular composition of the lipid environment has been associated with development of diseases such as Alzheimer’s disease 2443-Pos (AD). Impact of N-Terminal Acetylation on a-Synuclein Amyloid Amyloid peptides aggregate on the surface of neuronal membranes and form Formation amyloid fibrils that are the cytotoxic hallmarks of neurodegenerative diseases Matthew D. Watson, Jennifer C. Lee. causing loss of neurons. Hypothesis relating these neurological disorder dis- National Heart Lung and Blood Institute, National Institutes of Health, eases to the membrane surface charge, hydrophobicity, and the presence of Bethesda, MD, USA. raft domains have been published; however, no clear mechanism has yet The accumulation of a-synuclein (aSyn) as b-sheet-rich amyloid in Lewy been established. bodies is one of the hallmarks of Parkinson’s disease. Recent data have demon- C99 is the 99 amino acid transmembrane carboxyl-terminal domain of the am- strated that aSyn is constitutively N-terminally acetylated in mammalian cells, yloid precursor protein (APP), which is the immediate precursor of the amyloid a co-translational modification that has been shown to increase a-helical pro- b (Ab) and cleaved by g-secretase to form Ab.Ab is been found to associate pensity in the N-terminal region of the protein and modulate membrane- with AD. The structure of C99 is composed of highly mobile intra- and extra-

BPJ 9456_9458 494a Wednesday, March 6, 2019 cellular loops and a-helical transmembrane domains. The transmembrane 2448-Pos domain of C99 has been studied extensively with different lipid models, but b-Synuclein Ameliorates a-Synuclein Toxicity by Modulating Fibril Shed- studies of the full-length structure are limited. To this end, we constructed ding and Seeding Processes the full-length C99 structure using homology modelling technique and to Xue Yang, Jonathan K. Williams, Jean Baum. understand the impact of individual lipids on the structure and function of Chemistry & Chemical Biology, Rutgers University, Piscataway, NJ, USA. full-length of C99, we employ molecular dynamics simulations to study Alpha-synuclein (aS) fibrils have been recognized as the main component that C99-lipid interplay at atomistic resolution in multiple membrane compositions, make up Lewy bodies, which are a hallmark for Parkinson’s disease (PD). The ranging from simple toward highly realistic neuronal membranes. This study misfolding and aggregation of aS is implicated as a causative agent of PD, sheds light on how individual lipids in different membrane compositions however the correlation between aS aggregation and the ultimate progression may interact with the peptides, regulate their dynamics and hence, physiolog- of PD is not yet clear. Oligomers, short proto-fibrils and mature amyloid fibrils ical function. of aS have all been shown to be cytotoxic, and these aS species are also able to spread from neuron-to-neuron and induce endogenous aS aggregation. The in- hibition of aS fibril formation and reduction of the toxic species produced is 2446-Pos an active target for therapeutic intervention. A homologous protein, beta- Interplay between Two Isoforms of the Functional Amyloid Pmel17 Repeat synuclein (bS), has been found to delay aS fibril formation in vitro and reduce Domain the formation of Lewy bodies in vivo. However, the influence of bS on the cyto- Dexter N. Dean, Jennifer C. Lee. toxicity of aS aggregate species is unknown. Here, we show that co-incubation National Heart, Lung, and Blood Institute, National Institutes of Health, of aS and bS monomers forms an amyloid fibril which appears to have the same Bethesda, MD, USA. fibril core structure as the aS fibril, but exhibits a reduced cytotoxicity, reduced The pre-melanosomal protein (Pmel17, also known as SILV and gp100) seeding ability, and greater thermostability. In addition, as a dynamic equilib- forms functional amyloid fibrils within melanocytes, which promote melano- rium is reached, oligomers and proto-fibrils that shed from mature co-incubated genesis by serving as a scaffold for melanin polymerization. Of the various fibrils show reduced toxicity relative to oligomers shed from mature aS fibrils. Pmel17 domains, the repeat (RPT) region (10 imperfect repeats of 13 amino b in These results suggest that S directly modulates the fibril shedding and seeding acids each) has been shown to form amyloid aggregates when expressed processes to act as a neuroprotective modifier of aS toxicity. vitro. Biochemical studies using MNT-1 melanoma cells have identified mul- tiple isoforms of Pmel17 which can be expressed independently, two of 2449-Pos which arise from alternative splicing within the RPT domain. Interestingly, Alpha-Synuclein Modulates Stimulated Exocytosis and Binds to the shorter isoform of RPT (RPTS) readily forms amyloid fibrils despite lack- Mitochondria ing residues which were identified to constitute the amyloid core of the Meraj Ramezani1, Marcus Wilkes1, Tapojyoti Das2, David Holowka1, longer RPT (RPTL). To understand this seemingly contradictory observation, David Eliezer2, Barbara Baird1. we have carried out a detailed kinetics study of RPTS aggregation, revealing 1Cornell Univ, Ithaca, NY, USA, 2Weil Cornell Medicine, New York, NY, a bi-phasic behavior involving the formation of a distinct intermediate. USA. Based on Thioflavin-T and intrinsic tryptophan fluorescence, RPTS forms fi- Alpha-synuclein (a-syn) is the predominate marker of Parkinson’s disease brils more rapidly than RPTL. Testing our hypothesis that there is a potential in vivo (PD), a prevailing neurodegenerative disorder that impairs motor skills. Dupli- interplay between the two isoforms , cross-seeding experiments have cation or triplication of the a-syn gene, as well as familial mutations, lead to been performed. We find that RPTS can cross-seed and nucleate RPTL aggre- early onset PD. Elevated levels of a-syn are observed in many patients who gation, but RPTL cannot cross-seed RPTS. Morphological differences were develop sporadic forms of PD, further pointing to the critical need to under- also visualized by transmission electron microscopy. Overall, this work stand the pivotal role of this protein. A-syn is a 140-amino acid, presynaptic yields mechanistic insight into Pmel17 amyloid formation and suggests protein found predominantly in neurons, but also in other cells. A prominent that RPTS may serve as a ‘‘nucleator’’ of RPTL amyloid formation during role for a-syn in regulating synaptic vesicle trafficking has been implicated melanogenesis, similar to what has been detailed for other functional amy- in several studies. Increased levels of a-syn are thought to interact with mito- loid systems. chondria in familial PD, and this interaction may be involved in sporadic PD. We are examining the influence of human a-syn on stimulated exocytic 2447-Pos trafficking of recycling endosomal vesicles in RBL-2H3 and PC-12 cells, uti- Structural Evaluation of Aromatic Residues in a-Syn and their Role in lizing VAMP8-pHluorin as a reporter probe. A-syn expressed ectopically at Glycan Binding and Cellular Uptake low levels inhibits stimulated vesicle exocytosis, whereas at higher expression Jonathan M. Musila, Elizabeth Rhoades. levels does not inhibit and somewhat enhances exocytosis. Several mutants of Chemistry, University of Pennsylvania, Philadelphia, PA, USA. a-syn were compared to wildtype, and results can be related to binding to lipo- a-synuclein (a-syn) is a cytosolic protein abundant in presynaptic terminals somes and micelles, mediated by N-terminal helices 1 and 2, as measured by of neurons, whose native role remains unknown. Point mutations to a-syn NMR spectroscopy. We are also examining a-syn interactions with the mito- and gene multiplication have been linked to early onset Parkinson’s disease chondria in the presence or absence of mitochondrial stress. We find that a- (PD), which is characterized by intracellular deposits of aggregated a-syn. In syn binds weakly to mitochondria under normal conditions, and treatments the cytoplasm, a-syn is intrinsically disordered which becomes partially causing mitochondrial stress significantly increase a-syn binding. High- alpha-helical upon binding to cellular membranes. Transient helical structure resolution imaging provides some details about these binding interactions. is observed in its N-terminus in solution, which is enhanced by acetylation of We find a correlation between a-syn variants that inhibit vesicle exocytosis the N-terminal methionine. Among the PTMs identified for a-syn, N-termi- and those that bind to the mitochondria. Collectively our results suggest that nal acetylation is the only modification ubiquitous to a-syn. Prior work from the membrane binding properties of a-syn tend to stabilize membrane- our lab established N-terminally acetylated a-syn binds to complex N-linked membrane interactions, and pathological alterations may lead to dysregulation glycans and that cellular uptake of both monomer and fibrillar forms of of membrane trafficking and mitochondrial function. a-syn were impacted by this modification. Aromatic residues are important for binding to complex N-linked glycans; here, we targeted these residues to 2450-Pos understand how they impact glycan binding and cellular uptake of a-syn. Alpha-Synuclein Disrupts Inter-Membrane Interactions The mutants selected were: Phe4 (F4A), Tyr39 (Y39A and Y39F), and Peter J. Chung1, Qingteng Zhang2, Hyeondo Luke Hwang1, His50 (H50A and H50Q), and double mutants F4A, Y39A and F4A, Alessandra Leong1, Eric Dufresne2, Suresh Narayan2, Erin J. Adams3, Y39F, as the F4A, Y39A variant has been shown to be defective in mem- Ka Yee C. Lee1. brane binding. Lastly, a variant with N-terminal asparagine, N1, was used 1Chemistry/James Franck Inst., Univ Chicago, Chicago, IL, USA, 2X-Ray to probe the role of transient helical structure in glycan binding. Using Science Division, Argonne National Laboratory, Lemont, IL, USA, SH-SY5Y cells as a model system, we quantified cellular uptake of these 3Biochemistry and Molecular Biology, Univ Chicago, Chicago, IL, USA. mutants. Glycan sensitivity was determined by pretreatment of the cells a-Synuclein is an abundant protein found in the presynaptic terminal of with specific endoglycosidases, prior to incubation with a-syn. Biophysical neuronal axons but its physiological function remains unclear, even as the characterization performed includes quantifying binding to isolated glycans dysfunction of a-Synuclein is fundamentally linked to Parkinson’s disease. and membrane binding trends. The results will provide insight into how tran- While a-Synuclein is accepted to peripherally bind to the membrane of synap- sient structure in a-syn is linked to glycan binding and identifies specific res- tic vesicles (organelles that sequester neurotransmitters), there is increasing ev- idues important to this interaction, with the goal of relating aberrant idence that a-Synuclein can subsequently regulate the size and architecture of behavior to PD. synaptic vesicle ‘‘pools’’ through an unknown mechanism. Here, we present

BPJ 9456_9458 Wednesday, March 6, 2019 495a strong evidence that a-Synuclein (at biologically relevant concentrations) dis- tions of C55-dimers, we are supplementing these observations with enhanced rupts inter-membrane interactions between synaptic vesicle-mimics at physi- sampling methods. ological salt and temperature conditions through a combination of synchrotron small-angle X-ray scattering and X-ray photon correlation spec- 2453-Pos troscopy (XPCS). In designing synaptic vesicle-mimics with membranes a-Synuclein Binds Extracellular Complex N-Linked Glycans encapsulating a silica nanoparticle core, not only do we enforce membrane Melissa Birol1, Slawomir P. Wojcik2, Andrew D. Miranker2, curvature and charge at the physiological limit necessary for a-Synuclein Elizabeth Rhoades1. binding, but the high scattering cross-section of silica nanoparticles makes 1Univ Pennsylvania, Philadelphia, PA, USA, 2Yale University, New Haven, feasible the direct measurement of synaptic vesicle-mimic diffusivity via CT, USA. state-of-the-art XPCS. With increasing addition of a-Synuclein, our synaptic Fibrillar deposits of a-Synuclein (aS) are the hallmark of Parkinson’s dis- vesicle-mimics transition from a sub-diffusive, weakly-correlated state (with ease. While significant efforts have focused on deciphering pathological transient attractions most likely occurring due to charge fluctuations) to the mechanisms of aS, comparatively little is understood of its native function. expected Brownian diffusion of non-interacting colloidal particles. This tran- Recent evidence suggests that disease may propagate through cell-to-cell sition is highly indicative of a-Synuclein sterically-stabilizing synaptic transmission of toxic forms of aS. Relevant to this mechanism, our work re- vesicle-mimics, most likely due its projecting, highly-charged, unstructured ports on the discovery that aS binds extracellular N-linked glycans. We find C-terminal domain. In unveiling a novel, biophysical function for the C-termi- that aS binds to proteoliposomes derived from neuronal-lineage cells and that nal domain of a-Synuclein, we will demonstrate the effect of phosphorylation removal of N-linked glycans both decreases binding and alters the conforma- of a-Synuclein at S129, the post-translation modification most associated with tion of bound aS. Cellular internalization of both monomer and fibrillar aSis Parkinson’s, and discuss how a-Synuclein may affect the formation of synap- specific to neuronal-lineage cells is reduced dramatically upon cleavage of tic vesicle pools. extracellular N-linked glycans. Cleavage of extracellular glycans from pri- mary neurons reduces the uptake of aSby>10 fold. Importantly, all of 2451-Pos our results are specific to N-terminally acetylated aS, the physiologically Alpha Synuclein Increases Membrane Binding with Rising Lateral relevant form of the protein. Our results suggest that interactions with extra- Tension cellular glycans may play an important role in cellular uptake of aS relevant Jaclyn Ann Robustelli, Zheng Shi, Tobias Baumgart. both to normal proteostasis as well as to transmission of pathogenic aS Chemistry, University of Pennsylvania, Philadelphia, PA, USA. species. The amphipathic helix (AH) motif is found in many membrane proteins, and it is crucial for membrane curvature sensing and curvature generation. a-Synu- 2454-Pos clein, an intrinsically disordered protein (in solution) of 14kD, forms a uniquely Targeting Soluble Amyloid Oligomers in Alzheimer’s Disease through long AH when bound to membranes. In our previous work, we showed that Disordered Prion Peptides a-synuclein significantly expands the membrane proportionally to synuclein’s Zachary A. Levine. membrane binding density. In general, molecules that increase membrane area Dept. of Molecular Biophysics & Biochemistry, Yale University, New upon binding are expected to adjust their binding density in response to lateral Haven, CT, USA. membrane tension changes. Using model membranes and micropipette aspira- Cognitive decline in Alzheimer’s Disease (AD) and many other age-related tion, we observed that a-synuclein functions as a mechanosensory protein by dementias have long been associated with the presence of insoluble amyloid reversibly changing its membrane binding density in response to membrane plaques that disrupt normal synaptic functioning. However, recent studies tension changes. Our observations reveal a general mechanism by which have revealed that synapse impairment from AD is much more potently asso- different types of peripheral membrane proteins might change their membrane ciated with soluble amyloid-b oligomers (abo), rather than from insoluble fi- binding during processes such as endo- and exocytosis that involve membrane brils. Soluble oligomers are able to adopt heterogeneous structures that are tension changes. irregular and promiscuously bind to membrane proteins, thereby dysregulating downstream amyloid assemblies such as tau. Multiple screening methods have 2452-Pos identified cellular prion protein (PrPc) as a putative target of abo, and subse- The Effect of Amyloid Precursor Protein Dimerization on its Conforma- quent studies have confirmed a pathophysiological pathway in AD involving tion and Cleavage abo-PrPc binding. However, ab monomers and insoluble fibrils do not bind to Jacob B. Usadi1, Karl Freed2, Esmael Haddadian3. PrPc, thus the disordered binding domain of PrPc can be used to isolate abo 1Physical Sciences Collegiate Division, University of Chicago, Chicago, IL, assemblies. In order to exploit these interactions, we designed biomimetic USA, 2Department of Chemistry and James Frank Institute, Univ Chicago, PrPc peptides that complex soluble abo in replica-exchange molecular dy- Chicago, IL, USA, 3Biological Sciences Collegiate Division, Univ Chicago, namics simulations. While peptides are unlikely to act as AD therapeutics, Chicago, IL, USA. they can help inform the types of side-chain interactions that drive soluble Alzheimer’s disease (AD) is an important, increasingly prevalent neurological amyloid formation for use in next-generation therapeutics that cross the disorder. Aggregation of Amyloid b (Ab) is important in etiology of AD. Ab blood-brain barrier. Similarly, targeting abo through disordered PrPc peptides arises from a specific cut of the Amyloid precursor protein (APP). The C99 pep- can enable the tracking of soluble oligomers through fluorescent tagging dur- tide part of APP is the intermediary that contains the toxic Ab1-40 and Ab1-42 ing ab fibril formation. To the extent possible, results are compared to solid- peptides (among other products). A potentially significant feature of C99 is state NMR spectroscopy of abo-PrPc complexes, where heterogeneous struc- its dimerization behavior that may affect its recognition by the g-secretase tures are identified. Taken together, this study highlights how small disordered enzyme for cleavage. To understand the mechanisms and effects of dimeriza- peptides modulate pathological protein behaviors in AD and other degenera- tion, we simulate the C55 part of C99 peptide (the intermembrane part) at tive diseases where soluble amyloid oligomers, rather than insoluble fibrils, monomeric and dimeric levels using POPC and DMPC lipids. To accelerate drive cell toxicity. the membrane contribution to the conformational changes of C55 without the loss of atomistic detail, we also used the novel high mobility mimetic mem- 2455-Pos brane (HMMM) models of POPC and DMPC. Our large-scale molecular dy- Tau Amyloid Aggregates: The Choice of Pathways Makes the Difference namics simulations showed that at the monomeric level the structure Yann Fichou, Songi Han. displayed dependence on the membrane lipid composition. While the trans- Chemistry and Biochemistry, UC Santa Barbara, Santa Barbara, CA, USA. membrane helix remained stable, the G37/38 kink observed experimentally The intrinsically disordered tau protein is known to aggregate into amyloid did not manifest under different lipid compositions. The extracellular part (res- filaments involved in many neurodegenerative diseases, including Alz- idues 1-23) develops b-conformation and the juxta-membrane helix disap- heimer’s disease (AD) and frontotemporal dementia (FTD). Different types peared rather quickly. At the dimeric level, we built four conformations: we of aggregates, called strains, are thought to be involved in distinct pathologies clustered our monomer simulations and chose the representatives of the largest and to faithfully propagate by converting naı¨ve monomers, recalling a prion- clusters as the basis for two initial dimer configurations. For the other two we like behavior. Yet, there is no structural basis for this seed-assisted conver- used structures from Sanders’ experimentally determined C99 structure (PDB: sion and for the selection of well-defined strain. Here, we combined electron 2LP1). We observed that when the N-terminal residues (50-55) of the mono- paramagnetic resonance spectroscopy (EPR) with various biochemical tech- mers are in close proximity, the dimer stabilizes. Similarly, interaction of ex- niques to show the links between aggregation pathways and some key prop- tramembrane residues (1-23) help stabilize the dimer, especially with the erties of mature filaments such as stability and structure. Polyanions, such as presence of b-strands in this region. To better explore the possible conforma- heparin and RNA, have been widely used to trigger tau aggregation and

BPJ 9456_9458 496a Wednesday, March 6, 2019 create filaments used as models for disease-relevant strains. We show that tions. The resulting misfolded proteins aggregate into various polymorphic these model aggregates spontaneously depolymerize and release monomers structures due to alterations in the chemical environment. The deposition when their cofactors are removed. We demonstrate that the cross-b sheet as- of such aggregates on model surfaces and lipid bilayers can cause morpholog- semblies formed in vitro with polyanion addition is unstable at room temper- ical and mechanical changes impacting neuronal structure and function. To ature, counter to common assumptions. We argue that tau filaments formed in understand how various regions of the prion protein aggregate and interact brains might be supported by cofactors, which may be critical in the patho- with model surfaces, we selected a series of synthetic prion protein (PrP) pep- logical propagation of tau aggregates and strain differentiation. Furthermore, tides to study including PrP57-64, PrP89-106, PrP106-126, and PrP127-147. we show that the presence of mice-extracted seeds in the aggregation assay These peptides were chosen due to their sequences spanning various regions makes a selection toward well-defined structured aggregates, as opposed to of interest of the cellular prion protein. We hypothesize each prion peptide heterogeneous filaments formed without seeds. We conjecture that the will have distinct amyloid-forming morphologies and aggregation kinetics, convergence toward a given tau strain, which determine the biological impact facilitating unique peptide-surface interactions that vary with surfaces of of the aggregates, is a combination of three factors: an amyloid template differing chemistry. To test this hypothesis, we observed prion peptide aggre- (seed), a positively charged cofactor, and the tau protein conformation gation formed in free solution, and on mica under near physiological condi- ensemble, which is modulated by modifications such as mutation and tions by in situ tapping mode atomic force microscopy and Thioflavin-T phosphorylation. fluorescence kinetic aggregation assays. To further determine how lipid sur- face composition influences aggregation, we studied protein-lipid surface in- 2456-Pos teractions by means of a colorimetric, biosensing lipid/polydiacetylene Multiscale Investigation of Monomeric Alpha-Synuclein Structure and vesicle-binding assay. These studies will help to understand the pathological Aggregation mechanisms and physical properties of model surfaces, and how they Daisy Alvarado, Frank X. Vazquez. contribute to modulating amyloidogenic aggregation associated with prion Chemistry, St.John’s University, Jamaica, NY, USA. encephalopathies. Large aggregation of the protein a;-synuclein, known as Lewy bodies are the major hallmark of Parkinson’s disease. The fact that the truncation of the C-ter- 2459-Pos minal region leads to enhanced aggregation suggests that the proline-rich Metal-Promoted Alpha-Synuclein Modifications Steer the Aggregate domain may serve a protective role against toxic aggregation. We believe Conformation this may occur through molecular interactions between the C-terminal and Heather R. Lucas, Dinendra L. Abeyawardhane, Ricardo D. Ferna´ndez, the NAC domains that may act to destabilize b-rich structural motifs. Our cen- Denver R. Heitger, Ashley K. Forney, Cody J. Murgas, Alyson Curry. tral goal is to determine whether the removal of the C-terminal proline-rich Dept of Chemistry, Virginia Commonwealth Univ., Richmond, VA, USA. domain leads to changes in the a;-synuclein folding landscape that makes The intrinsically disordered protein, alpha-synuclein (aS), can adopt structural the protein monomer more prone to aggregation. To study the folding land- folds that have been connected to both functional and/or disease-relevant bio- scape of the intrinsically disordered region we used temperature replica ex- logical assemblies depending on the environmental contributors. Most notably, change molecular dynamics to simulate the system without any structural aSaggregates to form cross b-sheet fibrillar strands that make up the principal biases. We started with a total of six domain deletion variants in a fully component of Lewy bodies, the insoluble proteinaceous deposits prevalent in extended configuration. These simulations will show whether the removal of Parkinson’s disease (PD) inflicted brain tissue. An imbalance of metal ions the of the C-terminal region leads to increased sampling of aggregation has been thought to stimulate fibrillar aggregation due to local variations in prone-rich structural motifs. the concentration of brain biometals, namely copper and iron, with aging and I PD. We recently reported that Cu /O2 induces the formation of intermolecular 2457-Pos Y39-Y39 dityrosine crosslinks during the fibrillization process and also C-termi- Biophysical Insights into How Lipid Membranes Modulate Huntingtin nal intramolecular YY crosslinks, based on high-resolution mass spectrometry Aggregation Associated with Huntington’s Disease and immunoblotting analyses. Long-distance H50 interactions with the N-ter- Justin Legleiter. minal CuI site are required for dimerization, indicating a key role for metals Dept Chemistry, West Virginia Univ, Morgantown, WV, USA. in modulating the protein conformation. In the absence of this critical residue, The accumulation of nanoscale protein aggregates is a hallmark of many sys- such as in the PD-relevant H50Q genetic variant, only intramolecular YY mod- temic and neurodegenerative disorders, including Alzheimer’s disease (AD), ifications were observed that resulted in a dramatic increase in the aggregation Huntington’s disease (HD), and Parkinson’s disease (PD). Disease-associated kinetics based on a fluorescence assay performed with thioflavin-T, an indicator II protein aggregation results in b-sheet-rich fibrils. However, a variety of glob- of parallel b-sheet structure. Conversely, Fe /O2 promotes the formation of an ular protein aggregates, such as oligomers, are also associated with these dis- oligomer-locked conformation that does not elongate into fibrils, which may eases and may represent the most potent toxic species. Expansion of CAG have pathological relevance due to the elevated levels of iron in the PD brain. triplet repeats encoding polyglutamine (polyQ) are responsible for at least Spectral deconvolution of this iron-induced structure has also hinted at a func- ten disorders associated with polyQ induced protein aggregation. One of the tional role for iron coordination under controlled conditions. In this presenta- aggregating proteins that has been extensively studied is exon1 of the hunting- tion, recent structural homology studies will be discussed that suggest iron as tin (htt) protein, which is directly implicated in HD and contains a prototype a potential modulator of protein-protein hetero-interactions. Through this expanded polyQ domain. While mutant htt is detected predominantly in micro- work, new roles for metals in protein dynamics have been demonstrated, which scopic inclusion bodies in the cytoplasm and nucleus, it is also associated with will contribute to a mechanistic understanding of PD and/or neuronal many types of membranous organelles, including mitochondria, endoplasmic biochemistry. reticulum, tubulovesicles, endosomes, lysosomes and synaptic vesicles. Using a combination of ex situ and in situ AFM with other biochemical techniques, we 2460-Pos are obtaining a detailed understanding of how htt interacts with lipid mem- Characterization of the Early Stages of Tau Aggregation in the Presence of branes with a focus in determining the role of polyQ length, flanking sequences Polyphosphates adjacent to the polyQ domain, the lipid composition of the membrane, and how Sanjula P. Wickramasinghe1, Hope E. Merens2, Justine Lempart3, small molecules/peptides can interfere with this process. We are able to quan- Ursala Jakob3, Elizabeth Rhoades2. tify the relative number of aggregate types formed as a function of time and also 1Biochemistry and Molecular Biophysics Graduate Group, University of gain insight into their interactions with each other and surfaces along the aggre- Pennsylvania, Philadelphia, PA, USA, 2Chemistry, University of gation pathway. Pennsylvania, Philadelphia, PA, USA, 3Biological Chemistry, University of Michigan, Ann Arbor, MI, USA. 2458-Pos Tau is a microtubule associated protein implicated in the pathology of Alz- Investigating the Effects of Model Surfaces on Synthetic Prion Peptide heimer’s disease and other neurodegenerative disorders through its aggregation Aggregation and deposition as neurofibrillary tangles. However, the triggers of tau aggrega- Elizabeth A. Yates, Catherine M. Yip. tion are not well characterized or understood. Tau is both an intrinsically disor- Chemistry, United States Naval Acad, Annapolis, MD, USA. dered protein and it aggregates very slowly in vitro, making it challenging to Protein-misfolding diseases such as prion-related encephalopathies are recog- study the mechanism of aggregation. Using single molecule fluorescence tech- nized by forming proteinaceous fibrillar aggregate deposits classified as am- niques we investigated the early stages of tau aggregation in the presence of the yloids. Many protein misfolding diseases are related to protein aggregation physiologically relevant aggregation inducer, polyphosphate (polyP). We find and deposition as a result of protein rearrangement to non-native conforma- evidence that polyP accelerates tau aggregation through two different

BPJ 9456_9458 Wednesday, March 6, 2019 497a mechanisms: (1) conformational changes of monomer tau; and (2) intermolec- 2463-Pos ular cross-linking of multiple tau through multiple polyP binding sites. PolyP Probing Bama’s Role in the Assembly of Trimeric Autotransporter binds to both the proline rich and microtubule binding regions, but the proline Adhesins rich region is required for changes of conformation. The magnitude of the David Ryoo, Karl Lundquist, James C. Gumbart. conformational shift and the aggregation of tau are dependent on the chain Georgia Institute of Technology, Atlanta, GA, USA. length of the polyP polymer, with longer polyP polymers causing greater Outer membrane proteins (OMPs) are transmembrane beta barrels in gram- conformational change and more rapid aggregation. PolyP also competes negative bacteria, mitochondria, and chloroplasts. The primary complex with tubulin, tau’s native binding partner, providing further insight into responsible for the assembly of OMPs in bacteria has been identified as the possible triggers for the early stages of tau aggregation. multiprotein beta-barrel assembly machinery (BAM). Its central member, BamA, is the centerpiece for models describing the catalysis of OMP assembly. These models, including the budding and assisted model, fail to account for 2461-Pos OMPs with unique topologies such as trimeric autotransporter adhesins Neurotoxic HIV-Tat Autocleaves and Forms Novel Amyloid-Like Fibrillar (TAAs). TAAs possess three protein chains, each with four C-terminal beta Structures strands and an N-terminal passenger domain that assembles into a 12 stranded Alina L. Popescu Hategan1, Edward L. Mertz2, Joseph Steiner1, transmembrane beta-barrel and a trimeric virulence factor. The current model Elena Karnaukhova3, Lisa Henderson1, Jeff Kowalak1, for TAA secretion assumes hairpin translocation occurs after beta-barrel forma- Emilios K. Dimitriadis4, Avindra Nath1. tion. However, its plausibility has yet to be demonstrated. In order to address 1Dept Infections Nervous Sys, NINDS NIH, Bethesda, MD, USA, 2Section this, we have performed a series of MD simulations in order to test the plausi- on Physical Biochemistry, NICHD NIH, Bethesda, MD, USA, 3CBER, FDA, bility of intermediate assembly states of the well-characterized TAA, YadA. Silver Spring, MD, USA, 4Scanning Probe Microscopy Unit, NIBIB NIH, Bethesda, MD, USA. 2464-Pos HIV-Tat protein is released extracellularly in large amounts from HIV-infected Developing a Single-Molecule Platform to Understand Outer Membrane cells and causes activation of lymphocytes, glial cells and neurotoxicity, even Protein Biogenesis under antiretroviral therapy. Our atomic force microscopy (AFM) and mass Megan Mitchell, Marcelo Sousa. spectrometry data showed that HIV-Tat can produce spontaneously shorter Biochemistry, Colorado University Boulder, Boulder, CO, USA. peptides, some of which are consistent with the protein’s predicted inteins. The outer membrane (OM) of Gram-negative bacteria is essential for growth We found that the HIV-Tat 32-62 piece is neurotoxic by itself. AFM showed, and contributes to their ability to survive even our most powerful antimicrobial at 32 micrograms/ml, formation of micron-long HIV-Tat 32-62 fibrillar struc- attacks. Unique ß-barrel outer membrane proteins (OMPs) reside in the OM, tures. These fibrils present branching and have a persistence length in the 80-90 with hydrophilic residues on their interior and hydrophobic residues interacting nm range. With increasing concentration, the fibrils thicken and their branching with the asymmetric lipid bilayer. These amphipathic proteins are transported angles decrease. With further increasing concentration, large irregularly shaped to the OM in an unfolded state from the cytoplasm and must be folded into the aggregates form in solution and, long fibers and sheet-like aggregates both ex- OM by the ß-Barrel Assembly Machine (BAM) complex. The mechanism of tending over tens of microns form at air-liquid interface. Birefringence micro- how the BAM complex folds and inserts OMPs has remained elusive. The scopy, FTIR and polarized Raman microspectroscopies of the aggregates BAM complex is able to fold and insert OMPs into the OM without the use suggest that the peptide has highly oriented side chains and backbone and con- of the universal cellular energy currency, as there is no ATP in the bacterial tains 45% beta-sheet, consistent with the amyloid fiber structure. This is periplasm. We aim to obtain experimental mechanistic data using single mole- different than the full-length HIV-Tat, which is alpha-helical in bound state cule Fo¨rster Resonance Energy Transfer (smFRET) in an in vitro system of sup- and in aggregates. Unlike other amyloids, the peptide aggregates have a distinct ported bilayers via total internal reflectance microscopy. We have confirmed mechanism of irreversibility. Raman measurements showed that the free cyste- the deposition of lipid vesicles forming uniform bilayers via Fluorescence Re- ines in aggregates form in time S-S bonds, which make the aggregates insol- covery After Photobleaching (FRAP). Using orthogonal labeling strategies, we uble. The replacement of Cys34, Phe38 and Ile45 with Ala in the peptide have labeled BamA, the central component of the BAM complex, with the chain led to reduced aggregation, no fibril formation and lower neurotoxicity. organic dyes Cy3 and Cy5 to observe its dynamics via FRET. This establishes We identified microns-long Tat 32-62 fibers in cells expressing the peptide or the basis for monitoring conformational changes in the BAM complex on a sin- the full-length Tat. Our model of the thinnest fibril involves a beta sheet back- gle molecule level. bone of the fibril to which small aggregates of various sizes are bound. These results suggest that HIV-Tat autocleavage makes possible the aggregation of the 32-62 fragment which leads to neurotoxicity through an amyloid type of 2465-Pos mechanism. Dimeric FkpA Acts as an Anti-Aggregase on a Native Unfolded Membrane Protein Client Michaela A. Roskopf, Dagan C. Marx, Ashlee M. Plummer, Posters: Membrane Protein Folding Quenton R. Bubb, Karen G. Fleming. TC Jenkins Dept Biophysics, Johns Hopkins Univ, Baltimore, MD, USA. 2462-Pos Unfolded outer membrane proteins (uOMPs) transit through the aqueous peri- Bilayer Depth Dependence of Hydrophobic Amino Acid Transfer Free plasmic compartment of E. coli before reaching their native location in the bac- Energies terial outer membrane. The periplasm of E. coli contains a chaperone network Dagan C. Marx, Karen G. Fleming. that suppresses aggregation of nascent membrane protein polypeptides. FkpA, TC Jenkins Dept Biophysics, Johns Hopkins Univ, Baltimore, MD, USA. a heat shock periplasmic peptidyl-prolyl isomerase, functions as one of these Over 15,000 disease causing mutations have been identified for membrane pro- chaperones. The periplasm does not contain external energy sources (i.e. teins in humans, the majority of which are thought to disrupt folding or traf- ATP), which means that any chaperone activities of FkpA are accomplished us- ficking. At its core, membrane protein folding involves the transfer of amino ing the free energy change available from complex formation. The crystal acid side chains from an polar, aqueous solvent into a lipid bilayer. Previously, structure of FkpA is dimeric, which is thought to be the active form of the pro- we measured the transfer free energies of side chains to the nonpolar center of a tein. To better understand the function of FkpA, we measured the equilibrium lipid bilayer in two membrane proteins (beta-barrels OmpLA and PagP). How- dissociation constant (KDIM) for FkpA dimerization. We found that FkpA has a ever, the polarity and solvation state of the lipid bilayer is heterogeneous and KDIM in the low micromolar range, approximately equal to estimates of expres- contingent upon the position across the bilayer normal. Thus, it is imperative sion level in vivo. Additionally, we showed that the dimerization constant is to measure the bilayer position dependence of these energies for all twenty relatively insensitive to changes in temperature and salt concentration. Using naturally occurring amino acids to accurately describe membrane protein the population distribution expected for FkpA, we determined the affinity of folding. Presently, we have measured the transfer free energies for all hydro- dimeric FkpA for a model OMP (OmpA171). Surprisingly, this interaction is phobic amino acids at six different depths in the membrane using OmpLA as weak compared to previous estimates of FkpA-client affinities. Using this in- a protein scaffold. We find, surprisingly, that hydrophobic residues energeti- formation, we assessed the chaperone activity of dimeric FkpA in vitro by cally prefer to be about halfway between the bilayer center and the lipid measuring the efficiency and rate of OmpA171 folding into large unilamellar head groups. Using these results we have calculated a depth dependent function vesicles in the presence and absence of FkpA. Our results demonstrate FkpA describing the nonpolar solvation parameter in the membrane. These results increases the efficiency of OmpA171 folding while also decreasing the apparent allow us to begin to better predict the consequences of mutations and confor- folding rate. Further analysis revealed that the increase in OmpA171 folding is mational changes on membrane protein stabilities and structure in a depth accompanied by a decrease in the previously described, lipid-mediated, aggre- dependent manner. gated, so-called ‘‘elusive’’ state of OmpA171. These findings increase our

BPJ 9456_9458 498a Wednesday, March 6, 2019 understanding of OMP biogenesis and provides insight into the mechanisms lipid modulation of the insertion transition. Our results indicate that the core that E. coli employs to counteract heat-induced stress in the periplasm. hydrophobic helix a6 inserts into the bilayer, without adopting a transmem- brane orientation. This insertion disrupts the packing of Bcl-xL and releases 2466-Pos the regulatory N-term BH4 domain (a1) from the rest of the protein structure. Variable Consequences of Membrane Targeting Motifs for Genetically Our data demonstrate that both thermodynamic and kinetic properties of the Encoded Voltage Indicators insertion and refolding transition of Bcl-xL are modulated by lipid composi- 1 1 1 2 Sungmoo Lee , Bok Eum Kang , Minyoo Kim , Yoon-Kyu Song , tion. We hypothesize that conformational rearrangements associated with the 1 Bradley J. Baker . bilayer insertion of Bcl-xL result in its switching to a so-called non-canonical 1Center for Functional Connectomics, Korea Institute of Science and 2 mode of apoptotic inhibition. Presented results indicate that the alteration in the Technolog, Seoul, Republic of Korea, Graduate School of Convergence lipid composition before and during apoptosis can serve as an additional factor Science and Technology, Seoul National University, Seoul, Republic of regulating permeabilization of the mitochondrial outer membrane. Korea. Golgi and ER export signals have widely been used to improve membrane traf- 2469-Pos ficking of optical bio-sensors. Previously, we reported a linker modified version Role of Dynamic Hydrogen-Bond Networks in Protein Allostery of a genetically encoded voltage indicator engineered to image action potential Konstantina Karathanou, Michalis Lazaratos, Malte Siemers, spikes of neurons. Optimization of membrane expression was expected to Ana-Nicoleta Bondar. improve the signal to noise ratio by minimizing unwanted none voltage depen- Dept Physics, Freie Univ Berlin, Berlin, Germany. dent fluorescence. Accordingly, either Golgi or ER trafficking motif, or both Motor and transporter proteins couple protein conformational dynamics to were introduced into Bongwoori-R3 and affected its voltage sensing properties chemical reactions such as binding and cleavage of a nucleotide, or proton D such as the kinetics, responsive voltage range and F/F signal size. The ER transfer. Hydrogen bond networks are particularly important here, because motif improved the fluorescence response but it also caused increased time con- they can mediate coupling between remote regions of the protein. These net- stants and a shifted V1/2 toward hyperpolarization. Introducing spacers between works can be rather dynamic, and involve water molecules that visit transiently the fluorescent protein and the ER targeting motif recovered the response speed the interior of the protein. Algorithms inspired from graph theory allow us to while maintaining the improved fluorescence signal size. This research was analyze efficiently the dynamics of hydrogen bond networks in complex protein supported by the National Institute of Neurological Disorders and Stroke of environments and at lipid membrane interfaces. Computations on a set of pro- the National Institutes of Health under the award number U01NS099691. teins whose functioning requires allosteric coupling between a chemical reac- 2467-Pos tion, protein and water dynamics, suggest mechanisms by which dynamic Free and Chaperone Bound Unfolded States of Outer Membrane Proteins hydrogen bond networks shape protein conformational dynamics. Neharika Chamachi1, Georg Krainer2, Andreas Hartmann1, Research was supported in part by the German Research Foundation (DFG) 1 Collaborative Research Center SFB 1078 Protonation Dynamics in Protein Michael Schlierf . € 1B CUBE - Center for Molecular Bioengineering, TU Dresden, Dresden, Function (Project C4), by the Freie Universitat Berlin within the Excellence Germany, 2TU Dresden & TU Kaiserslautern, Dresden, Germany. Initiative of the German Research Foundation, and by computing time from Structural and dynamic investigations of unfolded proteins are important for the North-German Supercomputing Alliance, HLRN. understanding protein-folding mechanisms as well as the interactions of unfolded polypeptide chains with chaperones. In the case of bacterial outer- 2470-Pos membrane proteins (OMPs), unfolded-state properties are of particular physio- Tuning the Stability of Membrane Protein Dimerization by Changing the logical relevance, because they remain unfolded for extended periods of time Lipid Solvent 1 2 2 2 during their biogenesis and rely on interactions with periplasmic chaperones Rahul Chadda , Alejandro Gil Ley , Kacie Griffith , Lauren E. Hughes , 2 2 2 to prevent aggregation and support correct folding. Here, we study the Ana Castro , Kacey Mersch , Venkatramanan Krishnamani , 3 4 5 unfolded-state properties of outer-membrane proteins. Using a combination Elizabeth G. Kelley , Susana Marujo-Teixeira , Jose Faraldo-Go´mez , 1,2 of ensemble and single-molecule spectroscopy techniques including single- Janice L. Robertson . 1Biochemistry and Molecular Biophysics, Washington University, St. Louis, molecule FRET, we find that under strongly denaturing conditions and in the 2 absence of chaperones, OMPs adopt conformationally heterogeneous unfolded MO, USA, Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA, USA, 3Center for Neutron Research, National Institute for Standards states that lack the fast chain reconfiguration motions expected for an unstruc- 4 tured, fully unfolded chain. Interestingly, when complexed with periplasmic and Technology, Gaithersburg, MD, USA, Chemical and Biomolecular Engineering, Center for Neutron Science, University of Delaware, Newark, chaperones seventeen kilodalton protein (Skp) and survival factor A (SurA), 5 we observe for the protein OmpLA adopts an expanded, yet squished confor- DE, USA, Theoretical Molecular Biophysics Laboratory, National Heart, mational ensemble, in which the overall broad distribution of unfolded states Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA. is preserved. Differently sized OMPs like OmpX (8 b-strands) and OmpF In cell membranes, proteins encounter a unique problem where the solvent is a (16 b-strands) allow a broader perspective on chaperone enabled conformations mixture of diverse chemical composition. The question arises, how do of OMPs. These findings indicate that periplasmic chaperones Skp and SurA changes in lipid composition affect a simple reaction such as protein associ- ation? Previously, we experimentally measured the equilibrium dimerization prestructure OMPs for their next binding partner (e.g., the b-barrel assembly þ machinery (BAM)) to facilitate insertion and proper folding into the outer reaction of the CLC-ec1 Cl /H antiporter in a synthetic mimic of the E. coli membrane. membrane - 2:1 POPE/POPG comprised of C16:0/C18:1 acyl chains (Chadda et al., eLife 2016). In this condition, the free energy of association 2468-Pos is 11 kcal/mole, one of the strongest membrane protein complexes studied Lipid Modulation of the Activator-Independent Membrane Insertion and so far. Examination of the dimerization interface shows that the monomer in- Refolding of the Apoptotic Inhibitor Bcl-xL troduces a region of hydrophobic mismatch 10 A˚ shorter than the surround- Victor Vasquez Montes, Alexey S. Ladokhin. ing membrane. Coarse-grained molecular dynamics (MD) simulations show Dept Biochemistry, Univ Kansas Med Ctr, Kansas City, KS, USA. that C16:0/C18:1 lipids adopt constrained configurations at the dimerization Bcl-xL is a member of the Bcl-2 family of apoptotic regulators, responsible for interface that introduce an energetic penalty in the dissociated state. Based inhibiting the permeabilization of the mitochondrial outer membrane (MOMP), on this, we hypothesized that short chain lipids, e.g. 2:1 DLPE/DLPG with and a promising anti-cancer target. Bcl-xL exists in the following conforma- C12:0 acyl chains, could solvate the shorter interface without penalty, and tions, each believed to play a role in the inhibition of apoptosis: (a) a soluble thus stabilize the dissociated state. Chloride transport function was measured folded conformation, (b) a membrane-anchored (by its C-terminal a8 helix) in the DL membranes demonstrating that the protein is folded. On the other form, which retains the same fold as in solution and (c) refolded membrane- hand, dimerization stability is shifted, with a gradual increase in monomeric inserted conformations, for which no structural data are available. The population for DL < 1%, and a complete shift to monomers at higher DL. recruitment, anchoring and insertion of Bcl-xL into the mitochondrial outer Small-angle neutron scattering experiments were carried out on the mixed membrane is currently believed to require Bcl-xl interaction and activation DL/PO membranes, showing a reduction in bilayer thickness from 0 - 40% by an effector protein, such as tBid. Here we demonstrate that under DL that correlates strongly with the changes in dimerization. MD simulations physiologically-relevant conditions Bcl-xL can be targeted to and inserted of mixed PO/DL membranes show preferential accumulation of short-chain into membranes in the absence of any protein activators. We employed various lipids along the dimerization interface. Altogether, these results indicate techniques of fluorescence spectroscopy (e.g., FRET and Distribution Analysis that short-chain lipids modulate CLC dimer stability by local hydrophobic of depth-dependent quenching with lipid-attached Doxyl and Tempo probes) to matching, with preferential solvation capable of tuning the reaction across a characterize structural features of bilayer-inserted conformation of Bcl-xL and wide-range of compositions.

BPJ 9456_9458 Wednesday, March 6, 2019 499a

Posters: DNA Structure and Dynamics II By adjusting the optimization parameters, we can evaluate the effects of torsional stress on loop configuration and global topology. For instance, 2471-Pos altering the position or orientation of the end constraints provides insight Evidence for Conformational Capture Mechanism for Damage Recogni- into mechanisms that proteins may use to control DNA folding. Alternatively, tion by DNA Repairprotein Rad4 changing the chain length of the constrained DNA molecule alters the total Sagnik Chakraborty1, Saroj Baral1, Debamita Paul2, Peter J. Steinbach3, twist, which is reflected in the supercoiling of the molecule. Adjusting the Phoebe A. Rice4, Jung-Hyun Min2, Anjum Ansari1. intrinsic helical repeat in the optimization simulates changes in the cellular 1Department of Physics, Univ. of Illinois at Chicago, Chicago, IL, USA, environment such as found with increased salt content. Using knowledge of 2Department of Chemistry, Univ. of Illinois at Chicago, Chicago, IL, USA, the cost of deforming base-pair steps of different types we simulate the effect 3Center of Molecular Modeling, Center of Information Technology, National of nucleotide sequence on the overall structure of constrained DNA chains. Institutes of Health, Bethesda, MD, USA, 4Department of Biochemistry & 2474-Pos Molecular Biology, Univ. of Chicago, Chicago, IL, USA. Binding of CTAB to Single Stranded DNA XPC protein recognizes diverse DNA lesions including ultraviolet- Pamela St. John, Tetsuya Kawakita. photolesions and carcinogen-DNA adducts, initiating nucleotide excision Chemistry, State Univ New York New Paltz, New Paltz, NY, USA. repair. Structural studies showed that Rad4 (yeast ortholog) bound specifically Oligonucleotides will aggregate in the presence of a cationic surfactant in a to DNA flips out damaged nucleotides away from the protein, indicating that it similar way to DNA condensation, which occurs during the transportation of relies on indirect readout for damage recognition. However, characterizing nucleic acids through the cell membrane. Aggregates formed from oligonucle- intrinsic DNA deformability has been a significant challenge. Using fluores- otides of 20 to 100 bases in length in the presence of the surfactant, CTAB (ce- cence lifetime measurements on DNA containing model lesions sandwiched tyl trimethylammonium bromide) have been studied using UV spectroscopy by tCo and tC -cytosine-analog FRET pair exquisitely sensitive to local dis- nitro and fluorescence polarization by titrating oligonucleotide solutions with tortions, we unveiled the conformational heterogeneities of DNA with varying CTAB. Results indicate that the onset of a signal change (optical density or po- Rad4-binding specificities and revealed a direct connection between intrinsic larization) occurs when the ratio of CTAB to DNA nucleotides reaches approx- DNA distortions/deformability and Rad4 recognition: high-specificity CCC/ imately 0.7. This ratio is consistent with previous literature studies and has been CCC mismatch sampled conformations that deviated significantly from attributed to the formation of aggregates triggered by the presence of a surfac- B-DNA-like, even in the absence of Rad4; nonspecific TAT/TAT mismatch tant such as CTAB. Measurements from UV spectroscopy showed a second was largely homogeneous and B-DNA-like. We employed laser temperature- onset occurring at a ratio of about 2 that may be attributed to aggregate precip- jump perturbation to measure the rates of these distortional dynamics and found itation. The saturation binding curves obtained using both techniques were that these rates in CCC/CCC-containing DNA remained essentially unchanged sigmoidal in shape implying that the process is cooperative. A variety of with and without Rad4, pointing to a conformational capture mechanism for models were explored to glean additional information on the binding including Rad4. the association constant between CTAB and a DNA nucleotide, the Hill coef- These initial studies were done with short, linear DNA oligomers, while DNA ficient, and the number of CTAB molecules bound per nucleotide. in our cells is typically bent and supercoiled, which is expected to have a pro- found impact on DNA damage recognition. We examined the effect of DNA 2475-Pos bending strain on the intrinsic deformability at the CCC/CCC site by extending Label-Free Chromatin-DNA Imaging by Circular Polarized Light Scat- the fluorescence lifetime studies in the context of 126-bp DNA minicircles. tering Scanning Microscopy These minicircles amplified DNA distortions and exhibited >100-fold binding Aymeric Le Gratiet1, Riccardo Marongiu1,2, Luca Pesce1,2, Michele Oneto1, affinity for Rad4 compared with linear DNA. Taken together, these results Paolo Bianchini1, Giulia Zanini1,2, Alberto Diaspro1. show that lesion-containing DNA has the propensity to undergo spontaneous 1Nanoscopy and NIC@IIT, Istituto Italiano di Tecnologia, Genova, Italy, unwinding fluctuations to adopt pre-distorted conformations that Rad4 recog- 2Fisica, Universita` degli Studi di Genova, Genova, Italy. nizes, and bending deformations amplify these effects. Circular Dichroism (CD) resolved scanning microscopy has proven to be an interesting label free technique to study chiral biological systems. This tech- 2472-Pos nique is based on the measurement of the difference of intensity between cir- Kinetics of DNA Strand Displacement cular left and right polarization states after interaction with the sample. It Alexander W. Cook, Bo Broadwater, Harold Kim. was demonstrated in early works that far from the absorption bands, the CD Physics, Georgia Institute of Technology, Atlanta, GA, USA. signal is attributed only to scattering effects and is thus referred to as Circular DNA strand displacement is crucial to many fundamental biological processes Intensity Differential Scattering (CIDS), which is sensitive to the higher-order and is a vital tool in DNA nanotechnology. This process begins as a single nu- chiral conformation of biopolymers.We have developed a scanning microscope cleic acid strand locally invades a partial duplex and is thought to proceed based on the measurements of the CIDS signal, using a 50 kHz Photoelastic through branch migration. However, little is known about its kinetics and Modulator (PEM) and two channels detection via a lock-in amplifier. In this mechanism because for most studies performed in bulk, the reaction is domi- way, a differential image extracted from the Mueller Matrix formalism gives nated by the association kinetics between the invader and the partial duplex. access to information at the single molecular level using a high-numerical aper- In this study, we measured the distribution of the actual displacement times for ture objective (Nikon 100X oil objective) in a time compatible with the scan the first time. The highlights of this study are: time (a few seconds for a complete 512x512 image). As a proof of principle It takes 37 milliseconds to displace a 14-nucleotide strand, which corre- of the technique, the CIDS configuration has been coupled with a modified sponds to 350 microseconds per step. confocal scanning microscope allowing a multimodal acquisition of the The displacement rate is very sensitive to DNA sequence and the side of CIDS and the fluorescence contrasts simultaneously. We have studied the invasion. chromatin-DNA organization of fixed isolated Hek-cell nuclei, which has The side dependence is reversed for RNA invasion. shown our capability to distinguish the different order of compaction in The mean displacement time shows a stronger correlation with the thermody- DNA, i.e. the more compacted heterochromatin areas from the euchromatin namic stability of distal base pairs than proximal ones. areas. The salt and temperature dependence of strand displacement are also explored. 2476-Pos To Kink or Not to Kink: Sequence-Dependent DNA Flexibility Unveiled in 2473-Pos Complex with DNA-Bending Protein IHF Investigating Structure and Topology of Protein-Mediated DNA Loops via Mitchell Connolly1, Aline Arra1, Viktoriya Zvoda1, Peter J. Steinbach2, Computational Modeling of Elastic Energy Phoebe Rice3, Anjum Ansari1. Pamela J. Perez, Wilma K. Olson. 1Department of Physics, University of Illinois at Chicago, Chicago, IL, USA, Wright-Rieman Labs, Rutgers Univ, Piscataway, NJ, USA. 2Center for Molecular Modeling, Center for Information Technology, Many biological processes involve deformations of DNA 3D structure. DNA National Institutes of Health, Bethesda, MD, USA, 3Department of looping generated by a protein bound to two distant sites on the double helix Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, is a familiar mechanism employed in regulation of cellular processes. We USA. employ a computational method to obtain minimum energy structures of Site-specific recognition by DNA-binding proteins often involves severe DNA DNA loops mediated by the Lac repressor protein, whereby the potential en- deformations including sharp kinks. Yet, it has been unclear how rigid or flex- ergy of elastic deformation is optimized. By evaluating the energy required ible these protein-induced kinks are. Here we investigated the dynamic nature to deform DNA, we can predict the likely spatial pathway of the DNA strand. of DNA in complex with integration host factor (IHF), a nucleoid-associated

BPJ 9459_9462 500a Wednesday, March 6, 2019 architectural protein known to bend one of its cognate sites (35 base pair H’) previously proposed that DNA is not a passive substrate of bacteriophage pack- into a U-turn by sharply kinking DNA at two sites by 90. We utilized fluo- aging motors but is, instead, an active component of the machinery. Computa- rescence lifetime-based FRET spectroscopy and revealed a surprisingly dy- tional studies on dsDNA in the channel of viral portal proteins reported here namic IHF-H’ specific complex: while 78% of the IHF-H’ population reveal DNA conformational changes consistent with that hypothesis. dsDNA exhibited FRET efficiency consistent with the crystal structure, 22% exhibited becomes longer ("stretched") in regions of high negative electrostatic potential, FRET efficiency indicative of partially bent DNA, with one side or the other and shorter ("scrunched") in regions of high positive potential. These results unkinked. This conformational flexibility was modulated by sequence varia- suggest a mechanism that couples the energy released by ATP hydrolysis to tions in the cognate site. Notably, IHF bound to another cognate site, H1, DNA translocation: The chemical cycle of ATP binding, hydrolysis and prod- that differs from H’ primarily by the lack of an A-tract on one side, exhibited uct release drives a cycle of protein conformational changes. This produces a significantly higher population (68%) in the partially bent conformations. changes in the electrostatic potential in the channel through the portal, and These differences in their dynamics reflect the different roles of these com- these drive cyclic changes in the length of dsDNA. The DNA motions are plexes in their natural context, in the phage lambda ‘‘intasome’’. The popula- captured by a coordinated protein-DNA grip-and-release cycle to produce tion distribution between bent/unbent conformations could be further tuned DNA translocation. In short, the ATPase, portal and dsDNA work synergisti- by introducing mismatches in the DNA at one of the kink sites, to enhance cally to promote genome packaging. DNA ‘‘kinkability’’ at that site. We propose to take advantage of this FRET assay to further investigate sequence-dependent flexibility at the kink sites 2479-Pos by performing SELEX studies for tight-binders to IHF, selected from a pool Measurement of the Length Dependence of DNA Cyclization using Next of semi-randomized sequences, with segments that encompass the kink sites Generation Sequencing chosen to be randomized for selection of the most ‘‘kinkable’’ (by IHF) Jason D. Kahn, Jason M. Hustedt. DNA sequences. These SELEX studies are expected to go beyond similar Dept Chem/Biochem, Univ Maryland College Park, College Park, MD, USA. studies on nucleosomes and reveal sequence patterns that significantly enhance DNA flexibility is important both for fundamental biophysics and also because DNA flexibility. DNA flexibility affects DNA packaging and the regulation of gene expression through DNA looping. Historically DNA flexibility has been studied with ex- 2477-Pos periments ranging from biochemical ring closure or DNA looping experiments Molecular Transport through Self-Assembled DNA Nanofluidic Channels to AFM, crystallography, and tethered particle microscopy. Even so, the flex- in vitro in vivo Yi Li1, Rebecca Schulman2. ibility of DNA and remains controversial. We have constructed 1Chemical and Biomolecular Engineering, Johns Hopkins University, a library of DNA molecules ranging from 125 to 225 base pairs, via ligation of Baltimore, MD, USA, 2Dept Chem/Mol Eng, Johns Hopkins Univ, pools of synthetic DNA of different lengths and PCR, yielding 1023 distinct Baltimore, MD, USA. sequences. The design incorporated barcoding for redundant identification of Nanoscale channels are a fundamental mechanism for directed transport each molecule, allowing for a ligation reaction to be performed on the entire within living systems. Confinement of transport to one dimension makes library in the same reaction mixture. Two different DNA concentrations transport rapid, and gating at channel entrances can make transport selective. were used to promote either unimolecular cyclization or bimolecular ligation However, little is known about the mechanisms and rates of transport of small and thereby explore a wide range of cyclization efficiencies (J factors). A molecules within nanoscale channels, particularly those that extend beyond portion of each reaction mixture was treated with BAL-31 to destroy non- the dimensions of lipid membranes and across hundreds of nanometers or cyclized molecules. All products were linearized by restriction digestion and microns. Illumina indices were added to the four mixtures of cyclized and non- We have used techniques from structural DNA nanotechnology, which makes cyclized products. The initial library and all of the reaction mixtures were it possible to precisely control the geometry and chemical functionality of sequenced in a single Illumina MiSeq 300 base pair paired end run. From the self-assembled structures, to construct synthetic nanoscale channels for mo- roughly 30 million sequence reads obtained, we expect to extract J factors lecular transport. The channel we have designed and constructed has an inter- for each of the 1023 molecules in the library, giving an overlapping set of J fac- nal diameter of 4-6 nm and a length that can extend for multiple microns. It is tors as a function of length. The analysis of the length dependence of ring composed of a DNA origami pore that penetrates and spans a lipid membrane closure will be presented. We believe this data set will be valuable for and a DNA nanotube self-assembled from DAE-E double crossover tiles, con- improving course grain modeling of DNA. The same methodology should be sisting of five DNA oligomers that grows from the DNA origami pore. Mem- applicable to DNA loops anchored by proteins. brane incorporation is facilitated by hydrophobic functionalization of DNA 2480-Pos origami pore via 12 cholesterol moieties. These self-assembled channels Dynamics of Supercoiled Knotted DNA: Large Scale Rearrangements and can self-repair and can be grown such that their endpoints specifically attach Persistent Multi Strand Interlocking at molecular landmarks, making them a promising biomolecular device for Lucia Coronel1, Antonio Suma2, Cristian Micheletti1. single-molecule biosensing, for studying intercellular signaling, and for 1Dept Physics, SISSA, Trieste, Italy, 2Temple University, Philadelphia, PA, drug delivery. USA. We characterized molecular transport through these DNA channels by confocal In bacterial plasmids, catalytic processes, involving DNA strand passages, microscopy and a dye influx assay, in which synthesized DNA channels spon- introduce knots and supercoiling. The effect of the latter has been separately taneously inserted onto giant unilamellar vesicles. and extensively studied, however much less is known about their concurrent The rates of fluorescent dye transport through the channels were quantified by action. Thus, to study the kinetic and metric changes introduced by complex time-lapse observation of intensities inside many vesicles through a confocal knots and supercoiling in 2kbp long DNA rings, we use molecular dynamics microscope and comparing to a diffusion-dominated molecular transport simulation and oxDNA, a mesoscopic DNA model, finding several unex- model. Our results indicate that transport of small molecules through these pected results. First, two distinct states dominate the conformational channels is diffusive and that molecule size determines the degree to which ensemble, they differ in branchedness and knot size; secondly, fluctuations be- transport may occur. tween these states are as fast as the metric relaxation of unknotted rings. Nevertheless, certain boundaries of knotted and plectonemically wound re- 2478-Pos gions can persist over much longer timescales. These regions involve multiple DNA Conformational Changes Play a Force-Generating Role during strands that are interlocked by the cooperative action of topological and super- Bacteriophage Genome Packaging coiling constraints. Their long liver character may be relevant for the simpli- 1 2 3 1 Kim A. Sharp , Xiang-Jun Lu , Gino Cingolani , Stephen C. Harvey . fying action of topoisomerases. 1Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA, 2Department of Biological Sciences, Columbia 2481-Pos University, New York, NY, USA, 3Department of Biochemistry and Long-Range Slippery Hairpin Reconfiguration and its Mechanism in Molecular Biology, Thomas Jefferson University, Philadelphia, PA, USA. Trinucleotide Repeats Revealed by Single-Molecule Spectroscopy Motors that move DNA, or that move along DNA, play essential roles in DNA I-Ren Lee, Cheng-Wei Ni, Yu-Jie Wei, Yang-I Shen, Chien Chen. replication, transcription, recombination, and chromosome segregation. The Dept Chemistry, Natl Normal Univ, Taipei, Taiwan. mechanisms by which these DNA translocases operate remain largely un- Stem-loop hairpins, a common DNA and RNA motif that are involved in many known. Some double-stranded DNA (dsDNA) viruses use an ATP-dependent biological functions, are usually thermodynamically stable. Meanwhile, these motor to drive DNA into preformed capsids. These include several human path- hairpins can also be very dynamic microscopically with rapid conformational ogens, as well as dsDNA bacteriophages – viruses that infect bacteria. We changes, which connect to the genomic instabilities such as trinucleotide

BPJ 9459_9462 Wednesday, March 6, 2019 501a repeat expansions that responsible for several neurodegenerative diseases. 2484-Pos Here, we studied the structural dynamics of tandem dCTG repeats at the CpG Methylation of the C9orf72 Nucleotide Repeat Expansion Alters single-molecule level. We found that the odd-numbered d(CTG)n (n denotes G-Quadruplex Topological Distribution the repeat number, n = 13-37) hairpins undergo stochastic conformation inter- Kadir Ozcan, Aaron Haeusler. conversions between blunt-end and overhang configurations. Throughout the Neuroscience, Thomas Jefferson University, Philadelphia, PA, USA. repeat-number-dependent kinetic analysis, we proposed a loop-initiated bulge A C9orf72 nucleotide repeat expansion (NRE) mutation has been implicated as diffusion model, where a bulge is initially prepared at the loop followed by the most prevalent genetic cause of amyotrophic lateral sclerosis and fronto- the diffusion through the stem, and ultimately complete the conformation temporal dementia. The NRE mutation is a hexanucleotide sequence, change. This model was further supported by our Monte Carlo simulations. (GGGGCC)n, that has the ability to adopt parallel and antiparallel G-quadru- With this model, we can reasonably extrapolate the dynamic slippery hairpin plex structures. It has been shown that the NRE is CpG methylated, and this reconfiguration, which is believed to be crucial in repeat expansion, to a longer methylation may correlate with disease onset and/or progression. The structural and pathological-relevant hairpins. consequences of NRE CpG methylation are unknown. Here we used molecular dynamic simulations and spectroscopic analyses to characterize the structures 2482-Pos formed by the sequence d(GGGGCC)3GGGG with and without 5-methylcyto- Enhancement of Dissociation Kinetics of Biomolecules through the Use of sine (5mCpG). In silico, CpG methylated anti-parallel G-quadruplex is destabi- Alternating Electric Fields lized by CpG methylation. Whereas, we show that the CpG methylated parallel Sebastian Sensale1, Zhangli Peng1, H.C. Chang2. G-quadruplex remained stable throughout 1 ms of simulation. Spectroscopic 1 Dept Aerospace/Mechanical Eng, Univ Notre Dame, Notre Dame, IN, USA, analyses of d(GGGGCC)3GGGG support that CpG methylation differentially 2Dept Chemical/Biomolecular Engineering, Univ Notre Dame, Notre Dame, alters the stability of specific G-quadruplex topologies. In summary, our results IN, USA. suggest that the predominant G-quadruplex species formed by the C9orf72 In this work we verify both theoretically and by simulation that an AC electric NRE DNA is dependent on methylation status, which has important implica- field, with a frequency much higher than the dissociation rate, can signifi- tions in strategies targeting G-quadruplexes as a therapeutic intervention for cantly accelerate the dissociation of biological molecules, such as DNA, under a spectrum of neurodegenerative disorders. isothermal conditions. The cumulative effect of the AC field is shown to break a key bottleneck by reducing the entropy (and increasing the free energy of Posters: Protein-Nucleic Acid Interactions II the local minimum) via alignment of the molecular dipole with the field. For DNA molecules, this dissociation mechanism is shown to follow a univer- 2485-Pos sal pathway of successive molecular alignment and base-pair breakage over Cellular Distribution and Diffusivity of Hfq with Interacting RNAs each cycle. It is an activated process with a single barrier, which corresponds Seongjin Park1, Karine Prevost2, Matt Reyer3, Emily Heideman1, Wei Liu1, to the breaking of the first couple of base pairs at one end, and the cumulative Eric Masse2, Jingyi Fei1. work done by the AC field in repeated alignments of the DNA dipole is to 1Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, reduce this barrier. We were able to derive frequency and amplitude depen- USA, 2Biochemistry, University of Sherbrooke, Sherbrooke, QC, Canada, dence of the enhanced dissociation that is consistent with All-Atomic simula- 3The Institute for Biophysical Dynamics, University of Chicago, Chicago, IL, tions, to demonstrate the existence of an optimum frequency with the highest USA. dissociation rate (roughly corresponding to the inverse dipole decay time) and In bacteria, Hfq plays key roles in small RNA (sRNA) mediated translational to show that the dissociation rate at high enhancement is a universal one with repression by either stabilizing the sRNA or mediating mRNA to sRNA inter- a weak logarithm dependence on the DNA length and sequence, in concor- action, leading translational repression and/or mRNA degradation by RNAse E dance with experiments. This suggests that the high electric field at gap junc- (RNE) as one of the most general mechanisms. However, the cellular localiza- tions and ion channels may have a universal transient time to facilitate their tion or distribution of Hfq remain controversial in the literature, and how such synchronization. distribution is related to its interactions with mRNA or sRNA remain largely unexplored. To address these questions, we developed a rigorous immunofluo- 2483-Pos rescence (IF) combined with fluorescence in situ hybridization (FISH), over- An Algorithm for Reconstructing the Dynamics of Supercoiled DNA coming fixation or permeabilization artifacts. Using super-resolved IF and 1 2 Todd D. Lillian , Saeed Babamohammadi . FISH images, we find RNE resides mostly on the membrane but Hfq and 1Department of Mechanical Engineering, University of South Alabama, 2 RNAs sRNA stays in the cytoplasm in the diffused manner. Then to further Mobile, AL, USA, Islamic Republic of Iran. study the cellular dynamics of Hfq, we conducted single particle tracking pho- The level and distribution of DNA supercoiling changes continuously in toactivated localization microscopy (sptPALM) on Hfq. By introducing point response to thermal fluctuations and important cellular processes (e.g., tran- mutations to Hfq at different RNA binding faces, We discovered that Hfq diffu- scription). Interestingly, such processes are sensitive to the level of super- sivity is dependent upon its interacting RNAs. coiling such that topoisomerases are required to regulate the level of supercoiling. Clearly, we must characterize and understand supercoil dy- 2486-Pos namics to better understand cellular processes. Unfortunately, previous Structural Characterization of a Peptide Derived from a Lab-Evolved experimental and computational techniques offer only limited access to these Protein that Targets HIV-1 TAR RNA dynamics. We believe the marriage of next generation imaging systems with Sai Shashank Chavali, Ivan Belashov, Jermaine Jenkins, Joseph Wedekind. novel computational algorithms will alleviate these limitations. Therefore, Biophysics, Univ Rochester, Rochester, NY, USA. our long term research objective is to develop computational algorithms to Innovative approaches are needed to create new therapeutics that target HIV, facilitate this marriage. We present an algorithm intended to be paired especially those that act on novel facets of the viral life cycle. The HIV-1 with experiments in which several identical fluorescent labels are distributed TAR (trans-activation response) element RNA is a validated drug target that around supercoiled circular DNA molecules. The 3-dimensional trajectory resists mutations to maintain interactions with the viral protein Tat and the through time of each fluorophore could be obtained by a next generation host pTEFb complex, giving rise to a protein-RNA complex that is essential super-resolution fluorescent imaging system. Given these trajectories, our al- for efficient proviral transcription. So far, TAR has evaded discovery of com- gorithm reconstructs the arrangement of fluorophores around circular DNA pounds with sufficient affinity and selectivity to warrant pharmaceutical devel- much like a game of connect the dots. However, this isn’t an easy game opment. To meet this challenge, we pursued a ‘semi-design and protein because the ‘dots’ aren’t numbered (the fluorophores are indistinguishable), evolution’ approach that yielded many high-affinity TAR Binding Proteins the ‘dots’ are spaced several persistence lengths apart along the DNA, and (TBPs) derived from RRM1 of the U1A spliceosomal protein. Here we describe supercoiling implies the connections cross in 3-dimensions. Our approach the lab-evolution results and present the structural analysis of TBP6.7 in com- maps reconstruction into the well-known traveling salesman problem for plex with TAR at 1.8A˚ . TBP6.7 recognizes conserved guanines in the TAR which there are efficient solvers. To demonstrate the utility of our algorithm, major groove using a constellation of arginines within the beta2-beta3 loop. we simulate fluorophore trajectories with our discrete wormlike chain model We demonstrate the feasibility of reducing TBPs to short a cyclic peptide (pep- for DNA. We show that (i) our algorithm converges to the correct arrange- tide 1) that retains TAR binding and inhibit Tat-mediated transcription in HeLa ment as observation time increases, (ii) increasing the number of fluoro- cell nuclear lysate. In addition, TBP6.7(Q48T) shows that a mutation in the phores on a supercoiled DNA reduces the observation time required for beta2-beta3 loop yields additional interactions between K20, K22 and N15 correct reconstruction, and (iii) the required observation time increases of the b1-a1 loop and the TAR bulge. Concurrently, we also present a crystal- with the level of supercoiling. lization module validated by isothermal titration calorimetry (ITC), which will

BPJ 9459_9462 502a Wednesday, March 6, 2019 be used for co-crystallizing TAR RNA with peptides and small molecules. silencing and chromatin compaction in both plants and animals. However, Future efforts will involve optimization of peptide 1, biophysical characteriza- the functional mechanism by which MORCs act is poorly understood. Here tion of optimized peptides and co-crystallization with TAR RNA. our single-molecule experiments show that C. elegans MORC-1 protein binds to and can robustly compact naked DNA in a mildly ATP-dependent manner. 2487-Pos After initial binding to DNA, MORC-1 forms multimeric assemblies that grow Nucleotide-Dependent Stability of Nucleosome-Chd1 Complexes as DNA compaction proceeds, which is consistent with in vivo observations 1 2 1 Samaneh Ghassabi Kondalaji , Ren Ren , Ilana M. Nodelman , that MORC-1 forms discrete nuclear puncta adjacent to heterochromatin. 1 Gregory D. Bowman . Tracking multiple positions of site-specifically labeled DNA indicates that 1TC Jenkins Dept of Biophysics, Johns Hopkins University, Baltimore, MD, 2 this DNA condensation activity appears to act via a loop trapping mechanism. USA, Department of Molecular & Cellular Oncology, The University of Furthermore, the multimeric assemblies do not leave the flow-stretched DNA Texas MD Anderson Cancer Center, Houston, TX, USA. upon high salt wash when the free end of the DNA is tagged with a bulky quan- Chromodomain helicase DNA-binding protein 1 (Chd1) contains an ATP hy- tum dot, which suggests that they topologically entrap the DNA. These results drolyzing motor domain that repositions nucleosomes across the genome. Pre- highlight several aspects of MORC-1 complex assembly on DNA that are rele- vious biochemical and cryoEM work has shown that the ATPase domain makes vant to understanding the fundamental mechanism of MORC action in a variety site-specific interactions on each side of the nucleosome, forming both 1:1 and of organisms. 2:1 Chd1-nucleosome assemblies. During its ATP hydrolysis cycle, Chd1 must remain in stable contact with nucleosomal DNA to stimulate translocation, yet 2490-Pos also easily release and reset its interactions with DNA. Here, we describe the An Effective Scoring Function with Atomic and Coarse-Grained Hybrid nucleotide-dependence of Chd1-nucleosome complex stability. We measured Representation for Protein-RNA Interactions rates of dissociation for different nucleotide-bound states using a 100-fold Jiahua He, Shengyou Huang. excess of competitor nucleosomes to remove rapidly dissociating Chd1. We Sch Physics, Huazhong Univ Sci & Tech, Wuhan, China. find that Chd1-nucleosome complexes are relatively unstable in the absence RNA-protein complexes (RNPs) play essential roles in many biological pro- of nucleotide or with ADP, with no complexes detected after a 10 second cesses such as protein synthesis and regulation of co-transcriptional and incubation with competitor. In contrast, in presence of AMP-PNP, Chd1-nucle- post-transcriptional gene expression. Three dimensional structures of RNP osome complexes are much more stable, possessing a mean lifetime of complexes can provide insights into the biological mechanism of protein- approximately 8 minutes. Remarkably, the transition state analogs ADP- RNA interactions at the atomic level. However, experimental determination Be3 , ADP-AlFx and ADP-MgFx produce strikingly stable Chd1-nucleosome of RNP structures is a costly and technically difficult process. One of the al- complexes that resisted dissociation even after a 24-hour incubation period ternatives is computational prediction of the binding complexes from individ- with a 100-fold molar excess of competitor nucleosomes. We will present a ual protein and RNA structures, in which a reliable scoring function is one of model for how these different nucleotide-dependent stabilities help drive the decisive factors. In this study, based on our double iterative method, we conformational changes in the nucleosome in coordination with the ATP hydro- have developed a knowledge-based scoring function with atomic and lysis cycle. coarse-grained hybrid representation for protein-RNA interactions from a 2488-Pos non-redundant diverse training set of 144 RNP complexes. For validation, A High-Throughput Platform for Probing Mechanisms of Transcription we have compared our scoring function with three other scoring functions Factor-DNA Binding including QUASI-RNP, DARS-RNP and dRNA, on a diverse test set of Arjun Aditham, Polly M. Fordyce. 126 non-redundant RNP complexes. According to the CAPRI criterion, for Stanford University, Stanford, CA, USA. bound docking, our scoring function shows a good performance in binding Transcription factors (TFs) bind regulatory DNA sequences to control gene mode prediction and yielded a success rate of 77.0% if the top prediction expression. An enhanced understanding of the relationship between TF was considered, compared to 50.0% for QUASI-RNP, 53.2% for DARS- sequence and DNA specificity would allow for rational TF design for synthetic RNP and 66.7% for dRNA. Our scoring function also demonstrated its signif- biology and facilitate identification of benign and deleterious TF mutations in icant advantage in unbound docking and achieved a success rate of 27.7% if precision medicine. To date, however, we have a very limited understanding of the top prediction was considered, compared to 20.6% for QUASI-RNP, how TF amino acid sequence dictates specificity. This lack of understanding 21.4% for DARS-RNP and 22.2% for dRNA. The results showed the effec- stems largely from a paucity of experimental data: although many technologies tiveness of our double iterative method and hybrid structural representation map DNA specificities for a single known TF, current techniques cannot simul- in deriving a robust scoring function. taneously mutate TF and DNA sequences in tandem and quantitatively assess how these mutations affect affinity. 2491-Pos To address this, we have developed a microfluidic platform that automates the Investigating the DNA Binding Activity of the Polybromo-1 Bromodo- expression, purification, and measurement of affinities and binding kinetics for mains 1 2 3 hundreds of TF variants interacting with tens of distinct DNA sequences in a Saumya M. De Silva , Yangtian Shangguan , Tyler M. Weaver , Brianna E. Lupo3, Catherine A. Musselman3. single experiment. Using this platform, we have mapped how amino acids 1 2 S. cerevisiae Department of Chemistry, University of Iowa, Iowa City, IA, USA, West within Pho4, a model basic helix-loop-helix TF from , dictate spec- 3 ificity for its cognate E-box response element 5’-CACGTG-3’. As expected, High School, Iowa City, IA, USA, Department of Biochemistry, University mutating amino acids in contact with the DNA have strong effects on affinity. of Iowa, Iowa City, IA, USA. In addition, we have found that residues outside of the DNA-binding domain DNA in eukaryotic cells is packaged into chromatin, which is made up of re- (and not present in the crystal structure) can affect binding affinity as much peats of the basic subunit, the nucleosome. Each nucleosome is composed of as mutations within the DNA-binding domain. 147 base pairs of DNA wrapped around an octamer of histone proteins that To understand the mechanisms by which individual amino acids exert their contains two copies of each H2A, H2B, H3, and H4. Modulation of chromatin effects, we are now systematically manipulating the Debye length of charge structure is critical for all DNA templated processes, and occurs through screening, the spine of hydration, nucleotides within the E-box, and nucleotides several mechanisms including post-translational modifications of flanking the E-box while measuring affinities and dissociation rates. Together, and remodeling of the nucleosome core. The ATP-dependent remodeling of nu- these experiments bridge a critical experimental gap surrounding TF protein cleosomes is mediated by several families of remodeling complexes. These sequence and function and will broadly impact several fields—from bioengi- complexes must navigate a complex chromatin landscape such that the proper neers rationally designing TFs to biophysicists building computational models chromatin structure is formed both spatially and temporally. of protein function. One such remodeling complex is PBAF (Polybromo-associated BRG1 or hBRM-associated factors), a member of the SWI/SNF family of remodeling 2489-Pos complexes. This complex is distinguished by the presence of Polybromo-1 Caenorhabditis elegans MORC-1 Topologically Traps and Compacts DNA (PBRM1). PBRM1 contains six tandem bromodomains (BDs) and is thought HyeongJun Kim1, Linda Yen2, Somsakul Wongpalee2, Joseph Loparo3, to be important in nucleosome recognition by the PBAF complex. Bromodo- Steve Jacobsen2. mains are a highly conserved domain of 110 amino acids, composed of a 1Physics and Astronomy, The University of Texas Rio Grande Valley, bundle of four alpha-helices linked by two loops. Canonical BDs associate Edinburg, TX, USA, 2The University of California at Los Angeles, Los with acetylated lysines on histone tails and BD1 through BD5 of the PBRM1 Angeles, CA, USA, 3Harvard Medical School, Boston, MA, USA. have been shown to associate with histone H3 peptides containing single acet- Microrchidia (MORC) ATPases are a highly conserved family of GHKL ylated sites. Recently, our laboratory demonstrated that certain BDs can also (Gyrase, HSP90, Histidine Kinase, MutL) ATPases that are critical for gene associate with DNA, and have predicted that some of the PBRM1 BDs harbor

BPJ 9459_9462 Wednesday, March 6, 2019 503a

DNA binding activity. Here, we investigate the interaction of the PBRM1 BDs existing structural data that can infer structural effects on specificity from with DNA using gel shift assays and NMR spectroscopy. sequence alone could therefore provide novel insights into the effects of un- characterized mutations and de novo TF binding specificity at low cost and 2492-Pos high throughput. To generalizably model TF binding specificity from protein Determination of the Mechanism of RNA Regulation by CPSF30 Utilizing sequence, we are applying state-of-the-art deep learning models to integrate Both Biophysical and Structural Approaches these structure and specificity data. As a proof of concept, we constructed a Jordan D. Pritts, Abdulafeez A. Oluyadi, Daniel Deredge, model to predict binding specificities of 168 mouse homeodomain TFs as Patrick L. Wintrode, Sarah L.J. Michel. measured by protein binding microarrays (PBMs) from TF sequence alone. Pharmaceutical Sciences, University of Maryland Baltimore, Baltimore, MD, We find that this model performs exceptionally well, often approximating the USA. reproducibility of the experiment itself, even on unseen test-replicate pairs. Zinc finger proteins are metal regulated transcription factors that utilize zinc to These results are competitive with best-in-class regression-based models while fold and function. ZFs provide high specificity DNA, RNA, and protein binding eliminating the need for non-linear normalization across experiments, indi- resulting in gene regulation at both the transcriptional and translational levels. cating that this strategy may generalize to a variety of TF binding modalities. ZF proteins utilize 4 cysteine/histidine ligands to bind Zn ions and can consist To extend this work, we have trained models to predict intra- and intermolec- of 1 or more domains. These proteins are commonly classed based on the ratios ular chemical contacts, allowing for integration of structural information into of cysteine to histidine ligands as well as their spacing in the amino acid the binding affinity modeling task. Here, we present our progress toward an in- sequence. We are investigating Cleavage and Polyadenylation Specificity Fac- tegrated model for the multitask prediction of TF-DNA complex formation and tor 30 (CPSF30). Mammalian CPSF30 contains 5 CCCH type ZF domains and the effects on DNA sequence binding specificity. In future work, we plan to 1 ‘zinc knuckle’ domain and is vital to proper polyadenylation. We have experimentally test predictions from these models via microfluidic affinity recently shown that CPSF30 is an RNA binding protein involved in recognition measurements. of the AAUAAA polyadenylation signal (PAS) found in the majority of eukary- otic pre-mRNAs. Interestingly, we found CPSF30 to harbor a 2Fe-2S cluster as 2495-Pos well as traditional zinc binding sites. This is one of a few emerging examples of RRM2 of CELF1 Protein from Plasmodium falciparum Preferentially improper ZF annotations following the whole genome sequencing efforts at the Binds to UG Repeats RNA turn of the century. Current work aims to characterize the RNA binding recog- Garima Verma, Neel Sarovar Bhavesh. nition mechanism of CPSF30 as well as the role and redox properties of the Transcriptional Regulation Group, International Centre for Genetic incorporated 2Fe-2S cluster. Fluorescence anisotropy is being utilized to Engineering and Biotechnology, New Delhi, India. look at RNA mutational effects on binding and recognition while hydrogen/ Malaria causes high mortality (445,000 casualties) despite continued efforts deuterium exchange coupled mass spectrometry is being employed to deter- for its eradication. Plasmodium falciparum is the prime cause of lethal malaria mine which ZF domains of CPSF30 are involved in binding. Additionally, in human. In spite of having a small genome, it exemplifies a vast proteome, these studies are tied together with ongoing crystallography efforts for the which leads to its widespread resistance against various anti-malarial drugs. interaction. The process of alternative splicing is mostly responsible for this proteomic 2493-Pos diversity. RNA G-Quadruplex is Resolved by Repetitive and ATP Dependent Mech- CELF (CUGBP Elav like family member) proteins play an active role in pre- anism of DHX36 mRNA alternative splicing, translational regulation, de-adenylation, mRNA Ramreddy Tippana1, Michael C. Chen2, Natalia A. Demeshkina3, stability and RNA editing among various eukaryotes. PfCELF (P. falciparum Adrian R. Ferre-D’Amare3, SuA Myong1. CELF) has three RNA recognition motifs (RRMs), of which second and third 1Biophysics, Johns Hopkins University, Baltimore, MD, USA, 2Chemistry, are separated by a WW domain. To understand the molecular basis of RNA University of Cambridge, Cambridge, United Kingdom, 3Biochemistry and recognition by PfCELF we have undertaken extensive structural and biochem- Biophysics Center, National Heart, Lung and Blood Institute, Bethesda, MD, ical studies. We have completed the sequence-specific NMR resonance assign- USA. ment of RRM2 of PfCELF and the initial model generated using torsion angles b a b b a b Guanine-rich sequences in DNA or RNA have a potential to fold into stable G- derived from chemical shift shows it of having a characteristic - - - - - quadruplex (G4) structures. Strongly folded G4s can pose challenges to vital fold. Our calorimetric studies and NMR titration show that PfCELF-RRM2 cellular functions like transcription, translation, and replication. DHX36, an has a strong affinity towards UG repeats RNA that possibly interact in a canon- RNA helicase is a part of DEAH family and binds to both RNA and DNA ical manner. We have also prepared selenomethionine-enriched crystals of this protein and collected X-ray diffraction data at 2.2A˚ resolution. G4s with an extremely high affinity with Kd in a picomolar range. We reported previously that DHX36 induces partial unfolding and refolding of G4-DNA in ATP independent manner. Recent co-crystal structure of DHX36 bound G4- 2496-Pos DNA revealed an intimate contact between the two RecA domains and single Single-Stranded DNA Binding and Crosslinking Activities of the Viral Re- strand DNA tail, raising a question about the role of ATP hydrolysis in G4 striction Factor Apobec3G Characterized by Force Spectroscopy 1 2 3 4 resolving activity. Here, we demonstrate that unlike on G4-DNA, DHX36 Ioulia F. Rouzina , Michael Morse , Nabuan Naufer , Yuqing Feng , Linda Chelico4, Mark C. Williams5. uses ATP hydrolysis to repetitively refold G4-RNA and subsequently dislodges 1 2 from the RNA. The ATP independent unfolding of G4-RNA followed by ATP Ohio State Univ, Columbus, OH, USA, Department of Physics, Northeastern, Boston, MA, USA, 3Northeastern, Boston, MA, USA, dependent refolding generates highly asymmetric pattern of activity whose rate 4 5 depends on ATP concentration. Interestingly, DHX36 refolds G4-RNA in University of Saskatchewan, Saskatoon, SK, Canada, Northeastern several steps, reflecting the discrete steps in refolding the G4 structure. The University, Boston, MA, USA. ATP dependent activity of DHX36 arises from the RNA tail rather than the APOBEC3G (A3G) is a human deaminase able to inhibit human immunodefi- G4 as the same activity is recapitulated on G4-DNA with RNA tail, but not ciency virus type 1 (HIV-1). A3G reduces HIV-1 infectivity via two indepen- on G4-RNA with DNA tail. Mutations which perturb G4 contact results in a dent mechanisms, including enzymatic hyper-mutation of the viral genome, quick dissociation of the protein from RNA upon ATP hydrolysis while the mu- and non-enzymatic stalling of the reverse transcription. However, the physical tations which interfere with binding RNA tail induces dysregulated activity. mechanism of these A3G activities remain unclear. Here we use force spectros- We propose that such activity of DHX36 may be useful for dynamically copy to show that the ssDNA-bound A3G exists in the rapid (1-10 s) conforma- resolving various G4-RNA structures in cells. tional equilibrium between its ‘‘globular’’ state with its NTD and CTD bound to each other, with free energy 2.4 kBT, and its extended ‘‘dumbbell’’ state with 2494-Pos its domains unbound, but connected by 0.6 nm flexible 7 aa linker. Low free Deep Learning Models Explore the Structural Effects of Transcription energy and fast kinetics of this conformational transition in ssDNA-bound A3G Factor-DNA Complexes on Binding Specificity suggest that both of these states are functionally important. We also measure Tyler C. Shimko, Polly M. Fordyce. much slower rates of monomeric A3G binding to and unbinding from ssDNA, Genetics, Stanford University, Stanford, CA, USA. which are of 107 M 1.s 1 and 0.014 s 1, respectively. However, while all of Transcription factors (TFs) are primary regulators of gene expression, modu- dimerization deficient FW A3G mutant dissociates with that rate, the a majority lating activity through sequence-specific DNA binding. TF binding interactions of the WT A3G oligomerizes upon binding within 10 s, and does not dissociate can be modified through oligomerization of TFs or mutational/chemical modi- for over 10 min. We show that the A3G monomers can crosslink two DNA fication of either binding partner. Since the effects of many modifications man- strands, leading to major construct shortening. For the WT A3G oligomerized ifest as structural changes to the TF-DNA complex, the ability to predict on ssDNA the strand crosslinking process takes 300 s, while it only takes structural effects on binding specificity is of great value. A model trained on 10 s for the FW A3G mutant. In both cases crosslinking is rate-limited by

BPJ 9459_9462 504a Wednesday, March 6, 2019 one of the A3G domain unbinding one ssDNA strand and re-binding another PU.1 with corresponding sections from Ets-1, a series of chimeric constructs strand. Taken together with other published data, our results suggest that the were designed. These constructs display significantly reduced osmotic sensi- ssDNA-crosslinking A3G monomers are calalyticalycatalytically active, while tivity in a manner correlated with proximity/contact to DNA. With the the oligomerized A3Gs are catalytically inactive, and present a reverse tran- decrease in osmotic sensitivity, chimeric constructs retain wildtype PU.1- scription road block. like binding within 5-fold at normo-osmotic pressure, excluding the a-helix 3 (H3) construct which suffered a 50-fold decrease. Closer examination of 2497-Pos the recognition helix H3 indicates N236 is crucial for wildtype PU.1/HA bind- NMR Studies of Conformational Selection of hnRNP H on RNA Recogni- ing. Mutation of N236 to its counterpart in Ets-1 reproduced the affinities tion and its Interaction with the HIV Exonic Splicing Silencer ESS2P RNA observed with chimera H3. Molecular dynamics simulations indicated that 1 1 1 2 Liang-Yuan Chiu , Srinivas Penumutchu , Niyati Jain , Andrew Sugarman , replacement of N236Y results in a tilt of PU.1 on DNA by 40. Constructs Blanton S. Tolbert1. 1 2 bound to low-affinity (LA) DNA exhibited increased binding affinities, with Dept Chemistry, Case Western Reserve Univ, Cleveland, OH, USA, Dept S3 displaying a reduced difference in affinity between high and low-affinity Chemistry, Oberlin College, Cleveland, OH, USA. DNA. The separation of high-affinity binding from osmotic sensitivity and Alternative splicing in human immunodeficiency virus type 1 (HIV-1) is the decrease in differences of high and low-affinity binding suggests the hy- tightly regulated by the equilibrium between the spliced and unspliced pri- dration network found in PU.1 serves as a mechanism to aid in sequence mary transcripts. This equilibrium is maintained by the presence of inefficient selectivity. splice sites that are further regulated by hnRNP and SR proteins. The A3 3’- splice site of HIV-1 is required for Tat mRNA production. The inefficient uti- 2500-Pos lization of this splice site has been attributed to the presence of a second Cryo-EM Structure of the P-Element Transposase Strand-Transfer exonic splicing silencer (ESS2p), which acts to repress splice site A3. HnRNP Complex H binds to ESS2P element to inhibit the splicing process; however, there is no Elizabeth H. Kellogg1, George Ghanim2, Eva Nogales3, Donald C. Rio2. structural and mechanistic information about how hnRNP H recognizes 1MBG, Cornell University, Ithaca, NY, USA, 2MCB, Univ CA Berkeley, ESS2P to inhibit the splicing. In our studies, we aim to characterize the Berkeley, CA, USA, 3Univ CA Berkeley, Berkeley, CA, USA. three-dimensional structure of hnRNP H and ESS2P. The domain organization The P element transposase (TNP) is a unique class II DNA transposase. of hnRNP H/F proteins is modular consisting of N-terminal tandem quasi- Originally identified as an invasive mobile element in Drosophila,TNP RNA Recognition Motifs (HqRRM1,2) and a third C-terminal qRRM3 has historically been leveraged as a genetic tool to modify the Drosophila embedded within glycine-rich repeats. The tandem qRRMs are connected genome. Interestingly, the P element encodes a truncated repressor protein through a 10-residue linker with most of the amino acids strictly conserved via alternative splicing. With the piRNA pathway, this results in complete between hnRNP H and F. We probed the structural dynamics of its repression of P element mobility in wild Drosophila populations, ensuring HqRRM1,2 domain with x-ray crystallography, NMR spectroscopy, and propagation without compromising host genomic integrity [1,2]. The full- Small Angle X-ray Scattering (SAXS). We observed that HqRRM12 contains length, 87kDa, TNP is known to have a number of unique biochemical multiple structures in solution by SAXS. These exchangeable conformations and mechanistic features that distinguish it from other transposase superfam- are located on the linker region and RNA recognition sites. Moreover, the ilies. TNP generates unusually long 17-nucleotide, 3’-extensions upon cleav- three-dimensional structure of ESS2P has been determined using NMR spec- age of P element donor DNA, which is required for integration into target troscopy and SAXS. Collectively, this work provides structural insight how DNA [3]. Interestingly, TNP uses GTP binding, but not hydrolysis, as a hnRNP H recognizes the HIV ESS2P element. cofactor for synaptic complex formation and transposition [4]. Though 2498-Pos TNP has similarities to other class II transposases, it has very limited homol- Using Single Molecule Methods to Study Mechanisms of Site Specific DNA ogy to other class II transposases which obfuscates clear identification of catalytic residues, correspondingly assignment of functional domains re- Cleavage ˚ Allen C. Price1, Stephen D. Parziale2, Karissa Mehrtens1, Anna D. Ware3, mains unconfirmed. We have recently obtained a high-resolution (4.5 A) Emily K. Matozel3, Nathaniel Dale3. cryo-EM reconstruction of the P element TNP synaptic complex, sufficient 1Dept Chem/Phys, Emmanuel College, Boston, MA, USA, 2Dept to identify protein folds and nucleic acid structure. This dimeric complex Mathematics, Emmanuel College, Boston, MA, USA, 3Dept Biology, contains characteristic features of transposases, such as highly distorted Emmanuel College, Boston, MA, USA. target DNA. In addition, both monomers make crucial cis- and trans- con- We study the target search and cleavage mechanisms of type II restriction en- tacts with the P-element ends (i.e. self and swapped), immediately suggest- donucleases. In our single molecule technique, micron sized beads are teth- ing a mechanism for ensuring fidelity of synaptic complex formation. ered with single DNAs in a microfluidic flow cell and imaged using video Through homology modeling we have identified the location of the RNAse microscopy. We have characterized the salt dependence of DNA cleavage H domain and are in the process of interpreting the density in more detail. by both NdeI and EcoRI and found that both enzymes exhibit a peak in cleav- References 1. Castro JP. et al. Genetica 2004, 121(2):107-18. 2. Kelleher age rates near 80 mM under diffusion limited conditions, an observation ES. Genetics 2016 203(4):1513-31. 3. Kaufman et al. Cell 1992, 69(1):27- consistent with the theory of facilitated diffusion. Data on cleavage rates at 39. 4. Majumdar et al. Micro. Spectrum. 2014, 3(2):MDNA3 high protein concentration suggest there is a rate limiting step which occurs after target search. In one hypothesis, a conformational change after target 2501-Pos binding occurs before cleavage can proceed. In another, it is the divalent Functional Implications of the RecQ Helicase - Topoisomerase III - SSB cation dependent hydrolysis which is rate limiting. We are currently under Complex: Insights from Single Molecule Measurements way testing these competing hypotheses. In another project, we are devel- K. Maria Mills, Yeonee Seol, Keir C. Neuman. oping dCas9 as a programmable roadblock to interfere with DNA target Lab Molec Biophys, NHLBI NIH, Bethesda, MD, USA. search. We plan on using this technique to test models of target search in RecQ helicases are a family of DNA helicases that are critical for maintaining E. coli our single molecule assay. genome stability in organisms from bacteria to humans. In , RecQ is known to functionally interact with topoisomerase III to resolve DNA struc- 2499-Pos tures that arise at late stages of recombination. Similar interactions have been The Role of Interfacial Hydration in the Transcription Factor PU.1/DNA demonstrated in homologous proteins in other organisms, including the hu- Complex man BLM helicase and topoisomerase IIIa, and the yeast SgsI helicase and Amanda V. Albrecht, Hye Mi Kim, Gregory M.K. Poon. Topo III. There is also evidence that single-stranded DNA binding protein Chemistry, Georgia State University, Atlanta, GA, USA. (SSB) interacts with RecQ and is a necessary component of this complex. Interfacial hydration is a mechanism by which proteins may recognize specific Whereas the genetic and functional interactions among RecQ, Topo III, and DNA sequences. The ETS family of transcription factors presents a useful SSB have been established, the degree, and functional consequences, of phys- model to explore the role of hydration in sequence specificity due to its struc- ical interactions among these proteins has not been established. To identify turally conserved, sequence divergent DNA binding domain. ETS family physical and functional interactions among RecQ, Topo III and SSB, we members PU.1 and Ets-1 bind high-affinity (HA) DNA with opposing used a single-molecule magnetic tweezers assay to measure DNA hairpin un- behavior of interfacial hydration- increased osmotic pressure destabilizes winding and refolding in the presence of different combinations of individual the PU.1/HA DNA complex, a phenomenon not observed in the less hydrated proteins, pairs of proteins, and all three proteins. Our results suggest that the Ets-1. The osmotic sensitivity of PU.1 in conjunction with its hydrated inter- three proteins directly interact with each other during DNA-processing and face with DNA suggests interfacial hydration may play a role unrelated to af- provide a framework for understanding the mechanism of the resolvase activ- finity. By substituting individual structural elements that contact DNA from ity of the RecQ-Topo III- SSB complex.

BPJ 9459_9462 Wednesday, March 6, 2019 505a

2502-Pos Arginine-glycine-glycine (RGG) box containing RNA binding proteins Establishing a Single-Molecule FRET System for Studying DNA-Protein have been shown to interact with G-quadruplexes. One specific RGG Interactions box containing protein, fragile-x mental retardation protein (FMRP), has Dacheng Zhao, Ishita Mukerji, Candice Etson. also been shown to colocalize with C9orf72 repeat RNA foci in the nu- Wesleyan University, Middleotown, CT, USA. cleus and cytoplasm, making this protein a possible player in the develop- Integration host factor (IHF) is an E.Coli architectural protein that exists ment of ALS and FTD. In this study we used computational chemistry to as a heterodimer, composed of an a and b subunit. Originally identified in examine the interactions of FMRP with the (G4C2)n repeat expansion the integration of bacterial phage DNA, IHF has been implicated in regu- RNA G-quadruplexes and hairpin structures using molecular docking. lating replication, transcription, recombination and nucleoid compaction. FMRP was docked to (G4C2 )4 and (G4C2)8 quadruplex and hairpin IHF binds to consensus sequences in the genome with high specificity models using ZDOCK with Amber03 forcefield parameters incorporated and induces a bend of 160 in the DNA upon binding. This process into the scoring function. Docking results were further refined using mo- helps package the genome and facilitate DNA protein interactions. Previ- lecular dynamics to equilibrate the structures and find a lower energy ous studies performed in our laboratory have shown that IHF binds to conformation of the protein:RNA complex. Solvated and ionized RNA DNA four-way Holliday Junction with tight affinity and induces the junc- and protein systems underwent 100 ns of simulation time. Predicted tion to adopt the open conformation. In this study, we are interested in hydrogen bonds of the complexes, as well as the RMSD of the change exploring the population distribution of different conformations of the in structure over the simulation was determined using VMD. Preliminary DNA-junction complex using single-molecule Fo¨rster resonance energy results suggest that sequences with a larger number of repeats have transfer (FRET)system methodologies. To refine our single molecule distinct binding sites and interactions. methodologies for our system, our initial experiments are performed with 34 bp duplex DNA containing the IHF consensus sequence. Consis- 2505-Pos tent with earlier studies, we find that IHF binds to consensus DNA with Single-Molecule Study of TRF2 Mediated DNA Compaction using Physio- high affinity (3-10nM) and introduces a bend in the duplex DNA struc- logically Relevant Long Telomeric DNA ture, as measured by steady state FRET. Preliminary data also show Parminder Kaur1, Ryan Barnes2, Hai Pan1, Patricia Opresko2, that we can observe IHF induced DNA bending event under the total in- Robert Riehn1, Hong Wang1. ternal reflection fluorescence (TIRF) spectroscopy. Our future work will 1Dept Physics, North Carolina State Univ, Raleigh, NC, USA, 2Department focus on optimizing our conditions to perform similar measurements of Environmental and Occupational Health, University of Pittsburgh, with the IHF-junction complex. Pittsburgh, PA, USA. Telomeres are nucleoprotein structures that prevent the degradation or fusion 2503-Pos of the ends of linear chromosomes by shielding them from activating DNA Cohesin SA2 and EWSR1 in R-Loop Regulation damage response (DDR) and DNA double-strand break (DSB) repair. Hu- Hong Wang1, Ashwin Ghadiyaram1, Hai Pan1, Yanlin Fan2, man telomeres contain 2to20kbofTTAGGGrepeatsandaG-rich3’ Parminder Kaur1, Aparna Gorthi3, Robert Riehn1, Alexander J.R. Bishop3, overhang. A specialized six-protein shelterin complex, including TRF1, Yizhi Jane Tao4. TRF2, RAP1, TIN2, TPP1 and POT1, binds and prevents telomeres from be- 1Dept Physics, North Carolina State Univ, Raleigh, NC, USA, 2Dept Physics, ing falsely recognized as double strand breaks, and regulates telomerase and Rice University, Houston, TX, USA, 3Department of Cell Systems and DNA repair protein access. Extensive telomere shortening or dramatic telo- Anatomy, University of Texas Health at San Antonio, San Antonio, TX, mere loss due to DNA damage causes chromosome ends to be recognized as USA, 4Department of BioSciences, Rice University, Houston, TX, USA. DNA breaks, which triggers cell senescence and aging-related pathologies. R-loops are three-stranded nucleic acid structures consisting of an RNA- Human telomeric DNA is arranged into T-loops, in which the 3’ single- DNA hybrid and a displaced single-stranded DNA (ssDNA) loop. R-loops stranded overhang invades the upstream double-stranded region. A recent collectively occupy up to 5% of the mammalian genome, which occur at study suggests that T-loops at telomeres function as conformational switches conserved hotspot including promoter and terminator regions of poly(A) that regulate ATM activation [1]. Further, it was also shown that TRF2 medi- dependent genes. R-loops are proposed to be the ‘‘double-edged sword’’ ated DNA compaction drives T-loop formation [2]. While it has been shown that functions as powerful regulators of gene expression and induces that TRF2 protein is required for the T-loop formation, essential information genome instability if it is un-regulated. Despite the importance of R-loops regarding the dynamics of its formation still remains unknown. Moreover, in a wide range of biological pathways, proteins that mediate R-loop most of the previous studies were done using short telomeric DNA se- dependent cellular function are not fully understood. Recently The Bishop quences 20 kb. To elucidate the dynamics of TRF2 mediated DNA group showed that Ewing sarcoma cells displayed a higher level of R-loops compaction and T-loop formation, we used single molecule methods which compared to control cell lines. EWSR1 depletion induces R-loop accumu- frequently require long DNA substrates. We established a new method for lation. In addition, the second most common mutation in Ewing sarcoma extracting and purifying long telomeric DNA from mouse liver cells using cells was found in cohesin SA2. These observations raise the important Region specific Extraction (RSE) [3]. question of whether EWSR1 as well as cohesin SA1 and SA2 subunits are sensors of R-loops that regulate R-loop processing. Using atomic force 2506-Pos microscopy (AFM) and bulk fluorescence anisotropy, we tested the hypoth- Structural Rearrangement of DNA for CRISPR-Cas9 Nuclease Specificity esis that cohesin SA1, SA2, and EWSR1 directly bind to R-loops. We Regulated by the REC2 Domain observed that cohesin SA1/SA2, and EWSR1 specifically recognize Keewon Sung1, Jinho Park1, Younggyu Kim2, Nam Ki Lee1, R-loops. These observations suggest direct roles of cohesin SA1/SA2 and Seong Keun Kim1. EWSR1 in R-loop regulation and how the loss of SA2 in Ewing sarcoma 1Department of Chemistry, Seoul National University, Seoul, Republic of may provide some benefit to these cancers. Korea, 2Department of Research and Development, LumiMac, Inc., Seoul, Republic of Korea. 2504-Pos The CRISPR-Cas9 nuclease system has been widely applied to genome editing, Characterization of the Interactions of Fragile-X Mental Retardation for which understanding the underlying principles for its target-specific Protein with C9orf72 Repeat Expanded RNA nuclease activity is a prerequisite to overcome off-target cleavage. The Kendy A. Pellegrene1, Mihaela Rita Mihailescu2, Jeffrey D. Evanseck1. RNA-guided CRISPR-associated nuclease Cas9 recognizes target DNA using 1Center for Computational Sciences, Duquesne Univ, Pittsburgh, PA, USA, base-pairing between the RNA and DNA sequences, but the detailed regulatory 2Chemistry and Biochemistry, Duquesne Univ, Pittsburgh, PA, USA. mechanism of its target-specific cleavage remains still unclear. In this work, we Since the discovery of the C9orf72 (G4C2) repeat expansion, accounting show that a non-catalytic domain of the Cas9 nuclease called REC2 plays a for the largest number of familial amyotrophic lateral sclerosis (ALS) crucial role in enhanced off-target discrimination. Using single-molecule fluo- and frontotemporal dementia (FTD) cases to date, research has been rescence resonance energy transfer (smFRET) assays, we investigate real-time focused on elucidating the mechanisms by which the expansion leads to conformational dynamics of the non-target strand (NTS) of DNA along the re- disease. One such mechanism involves RNA gain of function through action pathway toward DNA cleavage upon Cas9 binding. Together with muta- the formation of the non-canonical secondary structure, the G-quadruplex. tional analysis of the REC2 domain, we identify that REC2 regulates NTS This secondary structure provides unwanted targets for the binding of spe- rearrangement for the cleavage reaction with the help of positively-charged cific RNA binding proteins, potentially disrupting their cellular function. surface residues, by driving a non-cleavable intermediate conformation

BPJ 9459_9462 506a Wednesday, March 6, 2019 particularly for off-target DNAs. This result unravels dynamical and structural various concentrations of Doxorubicin. Preliminary results suggest that aspects of the NTS regulation for the target-specific cleavage and thereby pro- DNA melting facilitates the intercalation process in the nanomolar range, vides molecular insights into rational design of engineered Cas9 with improved implying a higher binding strength than the previously reported micromolar specificity. range. 2507-Pos Alpha-Synuclein Binds to DNA and Modulates its Physical Properties Posters: Membrane Physical Chemistry II Kai Jiang1, Sandra Rocha1, Alvina Westling1, K.K. Sriram1, Kevin D. Dorfman2, Pernilla Wittung-Stafshede1, Fredrik Westerlund1. 2510-Pos 1Biology and Biological Engineering, Chalmers University of Technology, Intrinsic Curvatures from Global X-Ray Scattering Data Analysis of In- 2 verted Hexagonal Phases Gothenburg, Sweden, Dept Chem Eng/Mat Sci, Univ Minnesota, 1 1 2,3 1 Minneapolis, MN, USA. Moritz P.K. Frewein , Johannes Kremser , Primoz Ziherl , Georg Pabst . 1Institute of Molecular Biosciences, Biophysics Division, University of Graz, Parkinson’s disease (PD) is a neurodegenerative disease that is estimated to 2 a Graz, Austria, Faculty of Mathematics and Physics, University of Ljubljana, affect 2 % of the population older than 60 years. -synuclein (aS) is a 140-res- 3 idue protein, and its assembly process into amyloid fibers is directly related to Ljubljana, Slovenia, Department of Theoretical Physics, Jozef Stefan Institute, Ljubljana, Slovenia. PD. Research on Parkinson’s disease (PD) mostly focuses on the ability of aS to C form oligomers and amyloids, and how such species promote brain cell death. The intrinsic lipid curvature 0 is an important parameter for the stored elastic However, there are indications that aS also plays a gene-regulatory role in the energy strain in membranes and plays a substantial role in lipid/protein inter- actions. We explored a model-based approach for C0 determination applying a . q In this study, we investigate the interaction between monomeric aS and DNA in full -range small-angle X-ray scattering data analysis of inverted hexagonal vitro with single molecule techniques. Using a nanofluidic channel system, we phases. The technique was tested on different phosphatidylethanolamines and discovered that aS binds to DNA, and by studying the DNA-protein complexes mixtures of dioleoyl phosphatidylethanolamine with diverse lamellar phase in nano-channels of different size, we found that the binding of aS increased the forming lipids, including phosphatdiylcholines of various hydrocarbon chain stiffness of DNA, where the persistence length is increased by 30% at high composition, as well as sphingomyelin. For these mixtures, we included a coverage. In addition, using atomic force microscopy, we observed that the non-linearity term for lipid mixtures based on the effective lipid headgroup aS binding occurs as small protein clusters scattered along the DNA at low size. We discuss advantages and challenges of our approach and present re- protein-to-DNA ratio, and the DNA is fully covered by aS at high protein-to- sults for intrinsic curvatures of lipids previously not accessible from DNA ratio. experiment. Recent studies have shown that DNA interactions may be a common property 2511-Pos of proteins associated with neurodegenerative disorders such that, in addition Statistical Analysis of Acyl Chain Confinement in Lipid Membranes toxic amyloid formation, these proteins may also alter expression profiles of Abhinav Ramkumar, Xiaoling Leng, Horia I. Petrache. disease-modifying genes. Single DNA molecule techniques, such as the Department of Physics, IUPUI, Indianapolis, IN, USA. approach in our study here, may be useful to characterize putative DNA binding One measure of acyl chain confinement in lipid membranes is provided by or- of many other proteins involved in neurodegeneration. der parameters that are obtained from 2H NMR spectroscopy. A statistical 2508-Pos model that uses a mean-filed approach can be used to relate the measured pa- Simulation of H2A.B Containing Histone Variant Nucleosome rameters to a torque field experienced by the various carbon segments as a Havva Kohestani1, Jeffery M. Wereszczynski2. function of position along the membrane normal [1]. We use Molecular Dy- 1Physics, Illinois Institute of Technology, Chicago, IL, USA, 2Dept Physics, namics (MD) simulations to determine to what extent acyl chain conforma- Illinois Inst Tech, Chicago, IL, USA. tions can be captured by mean-field approximations especially for carbon The H2A.B histone is a highly evolving vertebrate specific variant of the H2A segments closer to the bilayer center where chain disorder is high. We histone family. It has been implicated in increased gene expression, and ex- show that the mean-field approximation holds surprisingly well and it can periments have shown that incorporation of H2A.B into nucleosomes results therefore be used to characterize the confinement (stress) field across the in more extended structures with fewer wrapped DNA basepairs. To study entire bilayer. [1] Petrache et al., Biophys. J. 2000. the molecular mechanisms of H2A.B, we have performed a series of conven- 2512-Pos tional and enhanced sampling molecular dynamics simulation of H2A.B and Parameterization of the Charmm Force Field for Ether Lipids and Model canonical H2A containing nucleosomes. Results of adaptively biased molec- Linear Ethers ular simulations show that, substitution of canonical H2A with H2A.B results Alison Leonard1, Richard W. Pastor2, Jeffery B. Klauda3. in geometrical changes such as unwrapping of 10 to 15 basepairs of DNA on 1Univ Maryland, College Park, MD, USA, 2Lab Compu Biol, NHLBI NIH, each side of the nucleosome and an increase in the diameter and radius of Bethesda, MD, USA, 3Dept Chem/Biomolec Eng, Univ Maryland, College gyration, which is in agreement with previous AFM, FRET and SAXS exper- Park, MD, USA. iments. In addition, ensemble fitting to experimental SAXS data, demonstrates Linear ethers such as polyethylene glycol (PEG) have extensive industrial that the histone octamer is largely intact for both H2A.B and canonical con- and medical applications. Additionally, phospholipids containing an ether taining nucleosomes. Finally, the H3 and H2B histone tails are specifically linkage between the glycerol backbone and hydrophobic tails are prevalent important in robustness of the nucleosome core structure and maintaining in human red blood cells and nerve tissue. This study uses ab initio results its connection to DNA. to revise the CHARMM additive (C36) partial-charge and dihedral parame- ters for linear ethers and develop parameters for the ether-linked phospho- 2509-Pos lipid 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine (DHPC). The new Quantifying Anticancer Drug Doxorubicin Binding to DNA using Optical force field, called C36e, more accurately represents the dihedral potential en- Tweezers ergy landscape and improves the densities and free energies of hydration of Zachary Ells, Brian Dolle, Thayaparan Paramanathan. linear ethers. C36e allows more water to penetrate into a DHPC bilayer, Bridgewater State University, Bridgewater, MA, USA. increasing the surface area per lipid compared to simulations carried out Doxorubicin is a successful anticancer drug approved for use in the 1970s with the original C36 parameters, and improving agreement with X-ray and is considered to be one of the most effective cancer treatment methods and neutron scattering data. Preliminary results for the densities and free en- today. Although Doxorubicin has positive survival statistics it has very nega- ergies of hydration of linear ethers in the polarizable Drude FF are presented. tive side effects in many cases. Bleeding from the soles of the palms and New Drude linear ether models are developed based on C36e and compared feet, along with excruciating pain is often exhibited through the administra- with the current Drude models. tion of this drug. Based on the preliminary findings utilizing optical tweezers we anticipate that this study will provide critical information about the drug 2513-Pos binding mechanism. Single molecule biophysics techniques have provided Protonation States and Conformations of Inositol and Phosphoinositol useful insight into the DNA-binding mechanisms of small molecules. For Phosphates from Molecular Simulations this reason, in this study we quantify the binding kinetics and affinity of Brian Radak. Doxorubicin to DNA using optical tweezers. The optical tweezers allow Beckman Institute for Advanced Science & Technology, University of us to trap a single DNA molecule and manipulate it in the presence of Illinois at Urbana-Champaign, Urbana, IL, USA.

BPJ 9459_9462 Wednesday, March 6, 2019 507a

Phosphoinositide phosphates (PIPs) are ubiquitous components in numerous 2516-Pos cell signaling pathways. However, there is currently a considerable lack of Lipid Charge Increases the Bending Rigidity of Bilayer Membranes detailed atomistic models for how PIPs interact with their environment, Hammad Ali Faizi1, Jan Steinkuhler€ 2, Shelli L. Frey3, Rumiana Dimova2, especially with kinase and phosphatase proteins. While it is well- Petia M. Vlahovska4. documented that molecular recognition of PIPs depends on the specific num- 1Department of Mechanical Engineering, Northwestern University, Evanston, ber of phosphorylated sites (one to three), the protonation state of each phos- IL, USA, 2Department of Theory and Biosystems, Max Planck Institute of phate is also a critical component. Indeed, this clearly has an effect on the Colloids and Interfaces, Potsdam, Germany, 3Dept Chemistry, Gettysburg specific protein-lipid binding pose. Nonetheless, there have been very few Coll, Gettysburg, PA, USA, 4Department of Engineering Sciences and studies of how these protonation states change between bound/unbound Applied Mathematics, Northwestern University, Evanston, IL, USA. states or even when a PIP is in a membrane versus free in solution. Here Understanding the electrostatic contribution to membrane bending rigidity is we present constant-pH molecular dynamics simulations of various PIP- a key step in probing the membrane functionality and structure in physiolog- related compounds in order to analyze correlations between their pKa ical conditions. We report a systematic experimental study on the effect of values, structure, and environment. electric charge on the bending rigidity of bilayer membranes by considering membranes made of a mixture of POPC and POPG with variable composi- 2514-Pos tions 0-100% of charged lipids. We analyze the shape fluctuations of giant Lipid Nanomaterials for Delivery in Cancer Therapeutics: unilamellar vesicles with two independent approaches, equilibrium shape Effect of Pegylation and Charge on the Morphology and Efficacy fluctuations and time correlations, to extract the bending rigidity of charged Victoria Steffes1, Zhening Zhang2, John Crowe1, Scott MacDonald1, membranes. Both approaches show good agreement for the same vesicle Kai K. Ewert1, Bridget Carragher2, Clinton S. Potter2, Cyrus R. Safinya1. 1 2 analyzed. Time correlations functions decay as a single exponential demon- University of California Santa Barbara, Santa Barbara, CA, USA, The strating no additional non-equilibrium behavior unlike active membranes or National Resource for Automated Molecular Microscopy, Simons Electron neutral lipid membranes in electric field. Our results show that membrane Microscopy Center, New York Structural Biology Center, New York, NY, charge increases the bending rigidity by as a much as 80 % relative to the USA. charge-free membrane. The surface charge effect is diminished when salt Liposomes have advantages as drug delivery nanomaterials including low is added to the suspending solutions. High salt concentration of 5 mM toxicity, compatibility with a variety of therapeutics, ease of modular pro- NaCl screens out the membrane surface charge and membrane bending rigid- cessing, and a diverse set of lipid building blocks to tune their physical ity of the charged membranes approaches the value for the charge-free mem- and chemical properties. Polyethylene glycol (PEG) is a polymer widely brane. The experimental study is in good agreement with the Winterhalter used to create ‘‘stealth’’ nanoparticles (NPs). This steric coating increases and Helfrich theory. The trend shown by the membrane bending rigidity in vivo the circulation time of NPs , delaying immune clearance; additionally, matches well with the zeta potential measurements confirming the screening short peptide sequences and other moieties can be installed on the distal end of the charges in high salt concentration buffer solutions. These experi- of PEG to target specific tissues. When PEG-lipids are inserted into charge- mental results have profound implications for natural membranes where > neutral vesicles at very high PEG-lipid mol fraction ( 0.25), they are most of them are negatively charged. known to induce a structural transition to micelles. Here we examine shape changes induced by the incorporation of PEG-lipid, from low to high mol 2517-Pos fraction, to a series of liposomes ranging from neutral to high cationic Charge Melting of Liposome Colloidal Crystals charge. Significantly, we report on how these morphological changes corre- Joel Cohen1, Andrew Ford2. late to drug delivery efficacy of paclitaxel (PTXL)—a hydrophobic cancer 1Dept Physics, Univ Massachusetts, Amherst, MA, USA, 2Univ drug that acts as a mitotic inhibitor, inducing apoptosis and halting the pro- Pennsylvania, Philadelphia, PA, USA. liferation of tumor cells. Due to its hydrophobic nature, PTXL must be well Recently we measured the charge dependence of electrostatic interparticle solubilized for administration. In lipid bilayers and micelles, PTXL loads repulsions in liposome colloidal crystals (Cohen, Wei & Ou-Yang, ms. in into the hydrophobic core (Steffes et al. Biomaterials 2017, 145, 242 preparation). The liposomes were made from binary mixtures of charged https://doi.org/10.1016/j.biomaterials.2017.08.026). Here we show how lipo- and neutral phospholipids at various ratios. The shear modulus (rigidity) some composition correlates to shape changes and to interactions of the par- of each liposome suspension in deionized water was measured by laser- ticles with cells, ultimately dictating PTXL delivery. Next-generation tweezer methods. As a function of increasing liposome charge, suspension nanomaterial delivery systems will build upon the ‘‘stealth’’ properties of rigidity first increased, peaked at 20% charged-lipid content, then PEGylated NPs and incorporate components such as receptor-specific pep- decreased, becoming watery near 100% charged lipids. By analogy to tide sequences for tumor targeting and penetration. colloidal-crystal ‘‘shear melting,’’ we call this phenomenon ‘‘charge melting.’’ The effect was calculated quantitatively by use of the Wigner- 2515-Pos Seitz (WS) model, which requires solving the electrostatics problem of a Modeling Pseudomonas aeruginosa Inner Plasma Membrane in Planktonic radially symmetric charged sphere and its counterions in an electroneutral and Biofilm Modes spherical WS shell whose radius R is determined by the liposome concentra- Yalun Yu1, Jeffery B. Klauda2. tion. Electroneutrality means the entire Hþ screening cloud surrounding each 1Biophysics Program, University of Maryland, College Park, MD, USA, 2 liposome is compressed into its WS cell. The compression creates an os- Dept Chem/Biomolecular Eng, Univ Maryland, College Park, MD, USA. motic pressure related to the electrostatic potential c(R) at the WS-cell sur- Pseudomonas aeruginosa changes its life modes under different conditions. face. The osmotic pressure is a measure of the interparticle interaction The biofilm mode is more resistant to environmental stress and antibiotics strength, which is what stiffens the sample. ‘‘Melting’’ as a function of par- compared to the planktonic mode. Experiments have seen noticeable differ- ticle charge Z arises because electroneutrality requires (a) as Z increases, so ence between the two modes in terms of the lipid composition of its inner does the number of counterions, which increases osmotic pressure and c(R). plasma membrane. In our study, molecular dynamics (MD) simulation is This effect is linear in Z. (b) More counterions also cause more screening of applied to study the membrane. The lipid compositions are decided based Z, which decreases c(R) and osmotic pressure. This effect is exponential in on the experimental data. Membrane properties studied are surface area Z, so it ultimately dominates. We solve the WS electrostatics problem per lipid, area compressibility modulus, deuterium order parameters, elec- analytically in the Debye-Huckel€ approximation and numerically with the tron density profile, bilayer thicknesses, two-dimensional radial distribution full Poisson-Boltzmann equation. The problem must be solved self- functions, hydrogen bonding, lipid clustering, and correlation time of lipid consistently for a closed system with no reservoir, a counterion-only electro- wobble. The CHARMM36 lipid force field is used and tested against exper- lyte, surface Hþ binding with charge regulation, and zero electric fields at imental data. Based on our results, the fraction of lipids containing cyclopro- the WS-cell surface and liposome center. pane may play an important role in influencing some of the membrane properties. Compared to previous model membranes built for Escherichia 2518-Pos coli, the inner membrane of P. aeruginosa has a longer averaged lipid tail Measurements of Lipid Vesicle Charge in Solutions of Zwitterions length and a higher percentage of PG lipids, which might be responsible Azam Shafieenezhad, Rania Ousman, Ryan Z. Lybarger, Bruce D. Ray, for the changes in membrane properties like membrane thickness and stiff- Horia I. Petrache. ness. Finally, the comparisons to experimental data show good agreements Department of Physics, IUPUI, Indianapolis, IN, USA. and encourage the model’s use to study the proteins and other molecules Binding of water soluble molecules to biological membranes is an important associated with the membrane. step in cellular signaling. We investigate the physical nature of such

BPJ 9459_9462 508a Wednesday, March 6, 2019 interactions using model lipid membranes. In particular, neutral lipid vesicles tion and formation of domains (rafts) in bilayers composed of different can acquire electrostatic charge by binding free ions from solution. Interest- lipids. Fluorescence depolarization experiments can report on the local ingly, they can also acquire charge from zwitterionic molecules that have average reorientation mobility and restriction of the probe group. The dipole character. Examples are amino acids, pH buffers such as MOPS and probe molecules mutually interact by electronic energy transport and the HEPES, as well as (ATP) and its hydrolysis products. observed fluorescence depends on their spatial distribution. The pairwise en- By using x-ray scattering we have determined that the presence of zwitterions ergy transfer/migration of probes within and across bilayers is detected by affects the lattice spacing of multilamellar phosphatidylcholine vesicles in a the fluorescence depolarization experiments and the fittings of data to manner that is consistent with the presence of electrostatic repulsion between Baumann-Fayer (B-F) model [The Journal of Chemical Physics, 1986. neighboring membranes. The remaining question is how to quantify the surface 85(7): p. 4087-4107]. The results reveal values of reduced surface concentra- electric charge and, equally interesting, how to determine the sign of the charge tion. Additionally, molecular dynamic simulation(s) aims at creating accept- conferred by zwitterions on membranes. Here we report measurements by dy- able synthetic data following the same theoretical prerequisites of the B-F namic light scattering (DLS) and by diffusion of phosphatidylcholine lipid ves- model. icles in constant electric fields for a class of zwitterionic solutes. We find that the measured electrostatic charging can qualitatively account for the swelling 2521-Pos of multilamellar structures and is consistent with NMR measurements of mo- Mitochondrial Membrane Organization under Oxidative Stress: Insight lecular binding. However, the results also show that more theoretical work is by Solid-State NMR and Neutron Reflectometry needed for quantitative analysis of electrostatic and van der Waals forces in Artur P. Dingeldein1, Tobias Sparrman1,Jo¨rgen A˚ den1, Hanna P. Wacklin2, these systems and for explanation of zwitterion affinities to biological Luke A. Clifton3, Gerhard Grobner1. membranes. 1Dept Chemistry, Umea Univ, Umea, Sweden, 2ESS, Lund, Sweden, 3ISIS Spallation Neutron Source, STFC, Didcot, United Kingdom. 2519-Pos Membrane Deformation Under Isotropic External Stress Virtually every biological process involves membranes. Especially, the mitochondrial outer membrane (MOM) plays a key role in programmed K.J. Mallikarjunaiah1,2, Jacob J. Kinnun3, Horia I. Petrache3, cell death. Opposing members of the Bcl-2 protein family meet at this mito- Michael F. Brown1,4. 1Department of Chemistry and Biochemistry, University of Arizona, Tucson, chondrial membrane system and decide about the fate of the cell. High intra- AZ, USA, 2Department of Physics, Indian Institute of Science, Bengaluru, cellular oxidative stress levels trigger this cell death process and initiate per- 3 4 meabilization of the MOM and the release of cytochrome c. In recent years India, Department of Physics, IUPUI, Indianapolis, IN, USA, Department of Physics, University of Arizona, Tucson, AZ, USA. we have developed a realistic, MOM-like membrane system based on PC/ Studies of lipid membranes have revealed that lipid bilayers deform aniso- PE/CL lipid mixed, an ideal system for elucidating the role of oxidized lipids tropically under osmotic stress. Here we show that solid-state 2H NMR spec- on the regulation of mitochondria-mediated apoptosis. By using solid state troscopy is a powerful method in addressing such questions, because it NMR approaches in combination with neutron reflectometry approaches provides information over a range of length scales [1]. Despite that they we could generate a comprehensive model of the dynamic and structural or- ganization of these membranes under oxidative stress conditions. In partic- are volumetrically incompressible, the membrane material undergoes a sig- ular we provided novel insight into the overall architecture behind the nificant reduction of area per lipid (up to 20%) when exposed to osmotic membrane’s basic function, but also about its dynamics across the entire pressures in the range of 0-200 atm [2]. Such a reduction is compensated 13 by an opposite increase in volumetric thickness. The pronounced anisotropic membrane using C MAS NMR and dynamic filters such as CP and IN- deformation is described by the Young’ modulus and Poisson ratio (e.g., the EPT.We observed that the MOM environment is severely altered structurally thickness can increase by the projected length of 3-4 methylene segments) and dynamically in the presence of oxidized lipids (OxPls). To obtain a more [3]. Yet how membranes respond under isotropic external stress is less un- comprehensive view about the overall organization across these membranes and changes due to oxidative stress and the presence of apoptosis regulating derstood. Are these hydrostatically induced membrane deformations related to those under osmotic stress? Experimentally these questions can be ad- proteins of the Bcl-2 family we also employ neutron reflectometry (SURF at dressed by measurements of bilayer structural properties as a function of hy- ISIS, UK). By this research we provide a basic understanding of the molec- drostatic pressure. By analyzing 2H NMR data from both osmotic and ular mechanism by which OxPls are involved in regulation of apoptosis at hydrostatic conditions, we show that bilayer deformations are anisotropic the mitochondrial outer membrane and how apoptotic proteins such as even under isotropic external stress. By their nature, lipid interactions are Bax or its counterplayer Bcl-2 interact with them. anisotropic because of the chemical composition and geometry. This leads to differences in compressibility along the bilayer normal and in the mem- 2522-Pos brane plane as described by the Poisson ratio. Material anisotropy explains The Role of Hydrodynamic Forces in Nuclear Pores Assembly why the area per lipid is expected to change not only under osmotic pressure Vasily V. Kuvichkin. but also under isotropic hydrostatic pressure. This viewpoint is informative, Mechanisms of Reception, Institute of Cell Biophysics, Pushchino IBC RAS, not only in theoretical studies of lipid-protein interactions, but also in setting Russian Federation. up and testing atomistic molecular-dynamics simulations. [1] H.I. Petrache The nucleoporins are believed basic constructional element in modern et al. (2001) J. Am. Chem. Soc. 123, 12611. [3] J. J. Kinnun et al. (2015) models of nuclear pores (NP). The creation of pores between spaced lipid Biochim. Biophys. Acta 1848, 246. [2] K. J. Mallikarjunaiah et al. (2011) bilayers is the main obstacle of such models, but this problem easy over- Biophys. J. 100,98. comes in our nuclear pore model. In current models of NP, the participation of DNA and lipids is not assumed. The task of this study was to select 2520-Pos ternary complexes (TCs): (zwitterionicþanionic) liposomes, DNA and Fluorescence Studies of Lipid Distribution in Bilayers under Oxidative Mg2þ to create holes in a double lipid bilayer similar to NP but without pro- Stress teins. The nucleoporins are believed basic constructional element in modern Md Khorshed Alam1, Ivo Vinkla´rek2, Gerhard Gro¨bner1, models of NP. The creation of pores between spaced lipid bilayers is the Lennart B-A˚ Johansson1, Radek Sachl2. main obstacle of such models but this problem easy overcomes in our nu- 1Chemistry, Umea˚ University, Umea˚, Sweden, 2Biophysical Chemistry, J. clear pore model. In the system: Large Unilamellar Vesicles (LUVs)þ Heyrovsky´ Institute of Physical Chemistry, Prague, Czech Republic. DNAþMg2þ inside Giant Unilamellar Vesicles (GUV). The one case, The present work aims at demonstrating a biophysical method to detect the when TCs located inside GUV and LUV2 is outside GUV, but near of the heterogeneity or homogeneity in membrane organization under oxidative TCs. By microscopy, we observed the formation of short-term channels be- stress by combining fluorescence spectroscopy with molecular simulations. tween the TCs and the liposome outside of the GUV but close of TCs. In Since the presence of oxidized lipids in phospholipid bilayers can alter mem- addition, we observed emissions of particles containing DNA from GUV, re- brane’s organization as well as its working function, we have tested the sulting in the movement of the GUV on the distance at a multiple of their effect of different oxidized lipids on DOPC (1,2-dioleoyl-sn-glycero-3- size. This indicates that a high energy of fusion between TCs and the lipid phosphocholine) and POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho- bilayer unleash in this process. Nuclear pores serve as sites of initiation of line) bilayers. It is assumed the unilamellar vesicles composed of DOPC transcriptions in a cell, because ssDNA is the best site of transcription initi- or POPC lipids can work as a simple system mimicking basic features of ation. Not all TCs reach nuclear membrane and transform into pores; many the organization of mitochondrial membranes prior and upon oxidative of them located far from the nuclear envelope also enhance transcription. stress. We use various fluorescent probes to investigate the spatial distribu- The TCs can form aggregates that are the basis ‘‘transcription factories’’.

BPJ 9459_9462 Wednesday, March 6, 2019 509a

2523-Pos are vividly amphipathic molecules. The questions of whether these substances Microscopic Insights into Biological Relevance of Membrane Channels in prefer a membrane-bound state, and whether diffusion across the membrane Gas Transport across Lipid Membranes can be the natural mechanism of release have not been addressed. Here we Paween Mahinthichaichan, Emad Tajkhorshid. study the partitioning of CAI-1, its two nitrogen-containing C9 precursors Biochemistry, NIH Center for Macromolecular Modeling and and two shorter-chain analogs (C7-CAI-1 and C5-CAI-1), into lipid mono- Bioinformatics, Beckman Institute, University of Illinois at Urbana- layers and into the inner bacterial membrane using the mechanosensitive chan- Champaign, Urbana, IL, USA. nel MscS as a sensor of lateral pressure asymmetry. CAI-1 and its analogs all Membrane transport of small, non-polar gas molecules, such as O2 and CO2, show pressure-dependent partitioning into monolayers, with C9 compounds is among fundamental processes of living cells. Because of their size and residing in the monolayer at the monolayer-bilayer equivalence pressure (35- chemical properties, these molecules may unimpededly transit through the 40 mN/m), whereas the shorter-chain analogs largely partition out of the mono- lipid phase of the membrane. However, varying degrees of gas permeability layer. The slope of repartitioning on pressure gave an estimate of the in-plane in membranes with different lipid compositions have been reported experi- area of these molecules in the monolayer as 22-35 A2, corroborating with MD mentally, suggesting involvement of protein channels, particularly aquapor- simulation data. The CAI-1 lipid-water partitioning coefficient estimated from ins (AQPs), in gas transport across membranes. To account for membrane p-A isotherms is 2106. Patch-clamp experiments demonstrated large shifts heterogeneity and to probe potential roles of AQPs in gas transport, we per- of MscS tension activation curves confirming unilateral partitioning for all C9 formed extensive MD simulations of gas diffusion through lipid membranes. compounds, however with a diminished magnitude of shift for CAI-1. The data The systems simulated included POPC bilayers embedded with an AQP suggests that CAI-1 does not hold a strongly asymmetric profile and quickly tetramer (i.e., AQP1, AQP5 or AQP7), and protein-free lipid bilayer mix- partitions to the opposite leaflet, signifying permeation. MD simulations tures of POPC, cholesterol (CHL) and sphingomyelin (SM) or DPPC. confirm stable residence in one monolayer for both C9 amino precursors, but Each of them contained an excess amount (125 molecules) of a gas species fast permeation across the bilayer for CAI-1. We conclude that the precursors and was simulated for several hundred nanoseconds. Since the gas mole- are less permeable across the membrane, whereas the final conversion makes cules were allowed to diffuse freely, their permeability coefficients were CAI-1 more permeable, which supports the direct diffusion mechanism of its directly estimated by measuring their flow through the lipid and protein release. phases. The results showed that gas molecules diffused through the mem- brane via monomeric water and hydrophobic central pores of AQPs, and 2526-Pos via the lipid phase. For fluid-like membranes (e.g., 100% POPC) which Binding of Human Beta Defensin Type 3 with Negatively Charged Lipid exhibited high gas permeability, AQPs would not facilitate the permeation. Membranes Highly-ordered or gel-like membranes (e.g., 100% SM and 50:50 SM:CHL Liqun Zhang. or DPPC:CHL), on the other hand, exhibited reduced gas permeability as Physiology and Biophysics, Tennessee Technological Univ, Cookeville, TN, low as or even lower than through AQPs, suggesting that in such conditions, USA. AQPs would become imminent in facilitating gas transport. Consistently Defensins are cationic cysteine-rich small molecules with molecular masses b with experimentally observed low gas permeability in high SM-CHL con- ranging from 3 to 5 kDa. Human defensin (hBD) type 3 (hBD-3) is mainly taining membranes, such as those of erythrocytes and ocular lens, our study secreted from epithelial cells, and it is important to the human innate immune suggests biological significance of protein-facilitated gas permeation across system. It has broad-spectrum of antimicrobial activities on virus, fungi, and membranes. both Gram-positive and Gram-negative bacteria. hBD-3 has a charge density of þ11. It can selectively interact with negatively charged bacteria lipid mem- 2524-Pos brane without disrupting the mammalian cell membrane at the same concentra- Understanding How Alpha-Synuclein Modifies Steric Interactions of Silica tion attacking bacterial lipid membrane. Supported Lipid Bilayers in Crowded Environments In this project, the interaction and binding of hBD-3 with different kinds of Hyeondo (Luke) Hwang, Peter J. Chung, Alessandra Leong, Ka Yee C. Lee. negatively charged lipid bilayers was investigated using molecular dynamics Chemistry, Univ Chicago, Chicago, IL, USA. simulations. In total, three kinds of lipid bilayers: POPS, POPG, POPCþ10% It has been observed that the first 100 residues of the intrinsically disordered PIP2 were set up. In order to consider the hBD-3 structure effect on the binding, alpha-synuclein adopt an ordered helical conformation upon membrane besides hBD-3 wildtype, hBD-3 analog which has three disulfide bonds binding, but the remaining C-terminal 40 residues retain their structural dis- removed, was also studied. Since hBD-3 can form a dimer, the binding of order, prompting questions on the role of this partial order in its physiolog- hBD-3 in both monomer and dimer forms with lipid membranes were investi- ical function. Characterizing the disordered C-terminal domain that projects gated as well. The structure of hBD-3 on lipid membranes and the key residues from the membrane surface is important for elucidating how alpha-synuclein on the binding interface between hBD-3 and lipid membranes were explored affects steric interactions between membranes— and consequential in the and compared. hBD-3 analog shows more interaction with the lipid membrane biological context of synaptic vesicle regulation, as it is thought to affect than the wildtype, and it has a more compacted structure around lipid mem- SNARE complex-mediated synaptic vesicle fusion to the neuronal mem- branes than in solvent. Moreover, in different lipid bilayers, the dynamics brane. In this study, we characterize the colloidal states of alpha-synuclein and the complex structures of hBD-3 and lipid membranes are different. in complex with silica nanoparticle-supported lipid bilayers under various Thus, it is expected that lipid head groups can influence the antibacterial func- macromolecular crowding conditions produced by polymer-induced deple- tion of hBD-3. tion forces. SAXS measurements of alpha-synuclein bound membranes as a function of protein-lipid ratio and depletant concentration show that 2527-Pos alpha-synuclein definitively disrupts attractive colloidal interaction between Amino Acids Bind to and Influence the Structure of Fatty Acid Vesicles nanoparticle-supported lipid bilayers, and this attractive interaction is Zachary R. Cohen, Andrew Ramsay, Caitlin E. Cornell, Roy A. Black, restored only under highly crowded conditions. We have also mapped out Sarah L. Keller. a phase diagram of colloidal gel, fluid, and non-interacting ‘‘gas’’ phases Department of Chemistry, University of Washington, Seattle, WA, USA. of supported lipid bilayers, shedding light on the multiple states created The first protocells on the early Earth were likely encapsulated by a self- by the presence of protein and depletant. Our results suggest that alpha- assembled fatty acid membrane. Our group explores physical interactions synuclein at certain surface densities gives rise to repulsive membrane between membranes of decanoic acid (a prebiotic fatty acid with a chain interactions. Current work examines alpha-synuclein variant with truncated of 10 carbons) and the building blocks of RNA and proteins (namely, nucle- C-terminal domain to gain a more refined understanding of how the disor- obases, sugars, and amino acids). Our goal is to understand how RNA, pro- dered domain affects membrane-membrane interactions. teins, and membranes came to be co-localized within protocells. We have already established that nucleobases and ribose spontaneously bind to self- 2525-Pos assembled fatty acids and stabilize fatty acid membranes [Proc. Natl. Partitioning of Vibrio Cholerae Autoinducer Cai1 and its Precursors into Acad. Sci. USA 110:13272 (2013)]. Here, we demonstrate that amino acids Lipid Membranes Suggests the Direct Release Mechanism also bind and stabilize fatty acid membranes. To demonstrate binding, we Hannah Cetuk. use a filtration assay to show that amino acids are retained with decanoic Biology, University of Maryland College Park, College Park, MD, USA. acid vesicles, and we use diffusion NMR to show that a second, slow diffu- CAI-1, an autoinducer which regulates virulence and biofilm formation in sion coefficient arises when amino acids are in the presence of vesicles. To Vibrio cholerae, is synthesized in the form of amino EA-CAI-1 or Am- demonstrate stabilization, we use light scattering and cryo electron micro- CAI-1 precursors. Conversion to CAI-1 leaves a moderately polar methyl- scopy to show that serine increases the number of lamellae in decanoic hydroxyl-carbonyl group on a C9 aliphatic tail. Both CAI-1 and its precursors acid vesicles. Furthermore, amino acids prevent the disruption of fatty

BPJ 9459_9462 510a Wednesday, March 6, 2019 acid vesicles by divalent magnesium cations. This feature is critical to brane repair and trafficking as well as endocytosis. Proteins such as dynamin, membrane-centric theories of the origins of life because high concentrations coat proteins, and ESCRTs are usually involved in the fission of cellular mem- of magnesium are required for RNA polymerization. Overall, our results branes. Here, using coarse-grained molecular dynamics simulations, we imply that amino acids spontaneously bind to and influence the structure observe spontaneous budding and fission of a 30-nm vesicle in a solution of of fatty acid membranes. These results contribute to an explanation for the small solute molecules, which are completely miscible with water and adsorb prebiotic selection, concentration and co-localization of the essential compo- onto the membranes. We start from a spherical vesicle that is exposed to a nents of cellular life. very low solute concentration in the exterior solution. Even for these low con- centrations, we observe the formation of a metastable membrane neck connect- 2528-Pos ing the membrane bud to the mother membrane when we decrease the vesicle Dopamine Interaction with Membrane Surfaces volume. The membrane neck repeatedly opens and closes along the simulation Adhitya Ramkumar, Samuel Canner, Bruce D. Ray, Horia I. Petrache. trajectory, reminiscent of ‘kiss-and-run’ events as observed for synaptic vesi- Department of Physics, IUPUI, Indianapolis, IN, USA. cles, until it is eventually cleaved by membrane scission. This fission process Dopamine is a neurotransmitter that plays a central role in the brain’s reward is driven by the concerted action of spontaneous curvature and the preferential system. It is transported by and released by membrane vesicles in neuronal adsorption of solutes around the neck which then act as crosslinkers between cells. In this process, dopamine interacts with lipid molecules that are the the two opposing membrane segments adjacent to this neck. This solute- scaffold of cellular membranes. Current knowledge on the direct interaction mediated membrane-membrane adhesion leads to a significant contact area be- of dopamine with lipids and membrane surfaces as a whole is limited due tween the two membrane segments prior to the fission event. For solutes that to lack of appropriate experimental methods. Relatively recently, a biophysi- adhere less strongly to the membrane, membrane budding and neck formation cal method using Nuclear Magnetic Resonance (NMR) spectroscopy has been occur only at high particle concentration and do not lead to fission. Our results developed and applied to measurements of dopamine interaction with neutral reveal a generic fission process induced by the adsorption of small solute mol- and charged lipid vesicles [1, 2]. Although NMR measurements provide infor- ecules from the aqueous solution, irrespective of the detailed molecular struc- mation on dopamine affinity to various membrane surfaces, computer simula- ture of these solutes. tions are needed in order to fully interpret experimental data. In this project, we use Molecular Dynamics (MD) simulations to determine the interaction 2531-Pos and dynamic of dopamine molecules in the vicinity of lipid bilayers. This Interfacial and Hydrophobicity Scales for Small Drug-Like Molecules approach shows with atomic resolution how dopamine is oriented in the vicin- from Atomistic Free Energy Calculations ity of lipid headgroups and allows a calculation of dopamine residence time W.F. Drew Bennett, Stewart He, Helgi Ingolfsson. within membranes. This study can help develop new experimental methods BioScis and BioTech, Lawrence Livermore Natl. Lab, Livermore, CA, USA. for neurotransmitter studies and can possible guide studies at cellular level. A key parameter for drug development is small molecule lipid membrane par- [1] Y. Matam, B. R. Ray, and H. I. Petrache, Direct affinity of dopamine to titioning. Drugs must be able to cross the membrane to reach their intracel- lipid membranes investigated by Nuclear Magnetic Resonance, Neuroscience lular targets and hydrophobicity is an important driving force for drug- Letters 618, 104-109, 2016. [2] M. A. Johnson, B. D. Ray, S. R. Wassall, H. I. protein binding. We calculated water-cyclohexane transfer free energies for Petrache, Equivalent isopropanol concentrations of aromatic amino acids in- 30,000 drug-like small molecules, including world-approved drugs and teractions with lipid vesicles, Journal of Membrane Biology 248, 695-703, drugs currently in clinical trials. By having such a large data set we can char- 2015. acterize trends in molecular structure that result in particular transfer free en- 2529-Pos ergies. Atomistic level details, including the number of hydrogen bonds, Binding and Competition of Be2D and Ca2D with Model Phospholipid dipole, and molecular size are calculated in each chemical environment. A Membranes 3D convolutional neural network is trained to predict atomistic free energies Sergei Sukharev1, Curtis W. Meuse2. of transfer. These results show that high-throughput molecular dynamics sim- 1Dept Biol & IPST, Univ Maryland, College Park, MD, USA, 2NIST and the ulations for machine learning molecular thermodynamics is achievable and University of Maryland, Institute for Bioscience and Biotechnology provides numerous avenues for future work. This work performed under the Research, Rockville, MD, USA. auspices of the U.S. Department of Energy by Lawrence Livermore National Membrane surfaces, carrying multiple electronegative groups, attract metal Laboratory under Contract DE-AC52-07NA27344 and was supported by the ions and provide a rich environment for multiple ion-mediated signaling LLNL-LDRD Program under Project No. 18-ER-035. Release number: events. Be2þ, a small divalent ion known for its toxicity and ability to activate LLNL-ABS- 758788. abnormal immune responses is recognized as a strong ligand for anionic lipids. Be2þ’s tight binding to the surface of phosphatidylserine-containing 2532-Pos (PS) membranes was recently shown to diminish the recognition of phospha- NMR Study of Partition and Permeation Properties of Ga(III) Chelates 1 2 2 2 tidylserine by macrophages. The major hypothesis for the mechanism of this Maria Rangel , Silvia Vinhas , Galya Ivanova , Silvia Lopes , 2 process is that Be2þ is able to outcompete and partially displace Ca2þ from its Sofia Ferreira . 1REQUIMTE, Instituto de Cieˆncias Biomedicas Abel Salazar, Porto, binding sites. This displacement may be due to a very different 4-fold coor- 2 dination of Be2þ by oxygens, and the inability of small Be2þ to substitute Portugal, REQUIMTE, Faculdade de Cieˆncias, Porto, Portugal. þ tris for six-coordinated Ca2 in physiological reactions. FTIR is a highly informa- We found that (3-hydroxy-4-pyridinonate) Fe(III) complex [Fe(mpp)3] tive technique in studies of ligand binding and ion coordination at membrane appears to be a strong candidate for iron fertilizer [1]. Further studies in interfaces. Despite ITC studies showing that Be2þ has the capacity to compete which three Fe-chelates of the same family of ligands were compared þ tris with Ca2 on the surface of PS liposomes, there have been no spectroscopic (3-hydroxy-4-pyridinonate) Fe(III) complexes [Fe(mpp)3, Fe(dmpp)3 þ studies of Be2 interactions with phospholipids. In this work, we apply the and Fe(etpp)3], were indicative that the first chelate presents a superior abil- ATR-FTIR technique and present the spectral ‘signature’ of Be2þ binding ity to be translocated from the roots to the shoots. The three complexes to PS manifested as a dominant 1095 cm-1 line apparently arising from ion exhibit variable lipophilicity and for that reason will interact differently binding to phosphate oxygens. Be2þ produces no visible alteration of with membranes. To get insight on the permeation properties of Fe(III)- carbonyl bands and appears to partially suppress carboxyl vibrational bands. chelates, we performed NMR studies with parent diamagnetic/isostructural Dialysis experiments show that FTIR can be used to watch the ionic equilibria Ga(III)-chelates. The affinity of the complexes for the phospholipid bilayer and determine binding constants for specific atomic groups in the phospho- of POPC/POPE liposomes and permeation properties of the Ga-chelates lipids. We show that Be2þ tightly binds not only to anionic PS, but also to were estimated on the basis of the induced alterations of a number of zwitterionic PC. We also observe Be2þ readily displacing more abundant NMR parameters, such as chemical shifts, line shape, spin-lattice relaxation Ca2þ from phosphate head groups. time (T1), and translational diffusion coefficient of the lipids and the lipo- somes. Acknowledgements: This work received financial support from 2530-Pos FCT/MEC through national funds and co-financed by FEDER, under the Budding and Fission of Vesicles Induced by Small Solute Molecules Partnership Agreement PT2020, through the projects UID/QUI/50006/ Rikhia Ghosh, Andrea Grafmueller, Reinhard Lipowsky. 2013-POCI/01/0145/FEDER/007265 (LAQV/REQUIMTE) and project Theory and Biosystems, Max Planck Institute of Colloids and Interfaces, PTDC/AGRPRO/3515/2014-POCI-01-0145-FEDER-016599. [1] Santos, Potsdam, Germany. C.S., Carvalho, S.M.P., Leite, A., Moniz, T., Roriz, M., Rangel, A.O.S.S., Membrane budding and fission, which produce new membrane compartments, Rangel, M., Vasconcelos, M.W., Plant Physiology and Biochemistry, are essential cellular processes during organelle division, cytokinesis, mem- 2016, 106, 91-100.

BPJ 9459_9462 Wednesday, March 6, 2019 511a

2533-Pos to prey cells, including E. coli, as part of its overall lifecycle to hunt and Membrane Lipids Alter Uncoupling Effect of 2,4 Dinitrophenol consume prey. In this study, atomic force microscopy (AFM) with force mea- Olga Jovanovic1, Lars Gille2, Mario Vazdar3, Elena E. Pohl1. surement modes is the technique utilized to quantitatively measure the force 1Institute of Physiology, Pathophysiology and Biophysics, University of and timescales of adhesion between Bdellovibrio and E. coli as well as a series Veterinary Medicine Vienna, Wien, Austria, 2Institute of Pharmacology and of chemically distinct surfaces. Our study echoes previously published work Toxicology, University of Veterinary Medicine Vienna, Wien, Austria, [Xu, H. et al. Langmuir, 2013, 29 (9), pp 3000–3011], where AFM force mea- 3Division of Organic Chemistry and Biochemistry, Rudjer Boskovic Institute, surement data are collected for live bacterial cells interacting with specifically Zagreb, Croatia. characterized chemical surfaces under buffer solution. Interpretation of these 2,4 dinitrophenol (DNP) is an artificial uncoupler of oxidative phosphorylation force data will be presented, leading to a better understanding of the prey in mitochondria and was used in therapy against obesity in the mid-1930s. Due sensing mechanisms of this enigmatic bacterial predator. to severe side effects and even death cases DNP was prohibited for the thera- peutic applications. A renewed interest for DNP originates from the intent to Posters: Membrane Active Peptides and Toxins II re-use it in small doses for the treatment of obesity, diabetes, hepatic steatosis and neuronal dysfunction. However, several aspects of its action mechanism in 2536-Pos mitochondria are not understood. Considering an important role of membrane 1 Membranes Matter: Predicting Drug Toxicity lipid composition for the mitochondrial uncoupling , we compared the uncou- R Lea Sanford1, Jeanne Chiaravalli-Giganti2, Wesley Chao1, Anotonio Luz2, pling effect of DNP in bilayer lipid membranes composed of (i) 1,2-dioleoyl- J. Fraser Glickman2, Olaf S. Andersen1. sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoe- 1Dept Physiology, Biophysics, and Systems Biology, Weill Cornell thanolamine (DOPE) and cardiolipin (CL), which mimic inner mitochondrial Medicine, New York, NY, USA, 2High Throughput and Spectroscopy þ membrane and (ii) DOPE-free membranes (DOPC CL). Resource Center, The Rockefeller University, New York, NY, USA. Measurements of total membrane conductance, G, and membrane order param- It remains a challenge to predict whether a new drug candidate will have undesir- eter, S, revealed that DNP decreases G in concentration-dependent manner in able side effects. Here we explore a general mechanism that may cause side effects, DOPE-containing membranes. In contrast, S was more affected by DNP in namely that many biologically active molecules, including drugs and drug-leads, DOPC-membranes. MD simulations revealed that (i) DNP-anions are localized are amphiphiles that partition into lipid bilayers. This may alter bilayer physical in lipid headgroup region whereas protonated DNP were found to be shifted to 2 properties, which may cause undesirable changes in cell function that, if large the membrane centre as shown previously for fatty acids , (ii) maxima of num- enough, may cause toxicity. Thus, it may be possible to predict whether a com- ber density profiles for DNP-anion and DOPE overlap and (iii) the average dis- pound will have important off-target effects based on the compound’s bilayer- tance between DNP-anion and DOPE headgroup corresponds to those of modifying potential. Using a gramicidin-based fluorescence assay (GBFA), which hydrogen bond. The molecular mechanism is discussed. reports how a compound alters the gramicidin monomer <-> dimer equilibrium, REFERENCES we have shown that many drugs and drug-leads alter lipid bilayer properties at the et al. [1] Jovanovic, O. The molecular mechanism behind reactive aldehyde action concentrations where these compounds become indiscriminate modifiers of mem- Free Radic Biol on transmembrane translocations of proton and potassium ions. brane protein function and are likely to have off target effects. We pursued this Med 89, 1067-1076, https://doi.org/10.1016/j.freeradbiomed.2015.10.422 (2015). question in a blinded study on a library of 488 compounds (289 non-toxic, 199 [2] Pashkovskaya, A. A.; Vazdar, M.; Zimmermann, L.; Jovanovic, O.; Pohl, toxic) that had been tested for cytotoxicity with ‘‘high-content’’ screening. We P.; Pohl, E.E. Mechanism of long-chain free fatty acid protonation at the found that the GBFA can be used to predict cellular toxicity, with a false positive Biophysical Journal membrane-water interface. 114 (2018). rate of 5%. We are currently investigating why these false positives arise. We hy- pothesize that their toxicity is due to direct protein interactions and are examining 2534-Pos the impact of albuminon their bilayerperturbing effects. We also explore a compu- Computational Mechanical Studies of E. coli Type-1 Pili Adhesion with tational approach to gain insight into which physicochemical parameters drive a Homogeneous Surfaces compound’s bilayer-perturbing propensity. We can predict which compounds Jeremy M.G. Leung, Eileen M. Spain. will produce bilayer-modifying effects at either extreme (i.e. little membrane ef- Deptartment of Chemistry, Occidental College, Los Angeles, CA, USA. fect or significant membrane perturbation) with 90% accuracy. Our results support Prior research [Xu, H. et al. Langmuir, 2013, 29 (9), pp 3000-3011] showed that a mechanism by which amphiphiles exert their toxicity, namely by altering lipid E. coli adhered to distinct, chemically well-characterized homogeneous sur- bilayer physical properties and that this in vitro measurement could be used as a faces at various strengths using atomic force microscopy. In this work, the in- warning sign for off-target biological effects in drug discovery efforts. teractions between the E. coli FimH pili protein and various surfaces are modeled and simulated to understand the chemical mechanisms of the adhe- 2537-Pos sion. Scripps Research Institute’s AutoDock Vina software was used to screen Biophysical Approaches Toward Understanding the Molecular Mecha- for the optimal binding sites and that data are subsequently used in steered mo- nism of Action of the Mitochondrial Therapeutic SS-31 (Elamipretide) lecular dynamics to replicate Xu et al.’s experiment in silico. From the docking Nathan N. Alder1, Wayne Mitchell1, Emily Ng1, Kevin Boyd1, data, we observed major bindings sites for fluorosilane, polyethylene glycol, Jeffrey Tamucci1, Eric May1, Nicholas Eddy2, Hazel Szeto3. aminosilane, and methyl a-D-mannopyranoside (MADM), as well as specu- 1Dept Molec/Cell Biol, Univ Connecticut, Storrs, CT, USA, 2Inst Mat Sci, lated on the mechanism of which MADM inhibits fluorosilane adhesion. Univ Connecticut, Storrs, CT, USA, 3Burke Institute, White Plains, NY, USA. With those binding conformation data in hand, CHARMM General Force Field Mitochondria orchestrate energy metabolism within eukaryotic cells. Mito- data was modified to parameterize physical interactions within the surface li- chondrial disorders can arise from genetic defects, and are also associated gands. Using a constant force pull in Nanoscale Molecular Dynamics with aging-related declines in cellular bioenergetic capacity and with complex (NAMD) software to simulate a fast-paced version of what happens when diseases including cardiomyopathy, neurodegeneration, diabetes, and cancer. E. coli detaches from the surface, mechanisms are observed such that single To date, there are no cures for mitochondrial diseases. Szeto-Schiller (SS) pep- or multiple strands of the surface detach from the protein. Data generated by tides are among the most promising mitochondrial therapeutics currently under steered molecular dynamics experiments are reminiscent of measurements ob- investigation. They comprise a novel class of tetrapeptides characterized by tained by Xu et al., particularly the pull-off features. These findings indicate aromatic-cationic motifs that selectively bind to mitochondria and display that there is accuracy and precision in replication, allowing further step-by- broad intrinsic therapeutic potential. Among SS peptides, the lead compound step analysis of these mechanics and processes. SS-31, under the proprietary name Elamipretide, is currently in advanced clin- ical trials. However, the molecular mechanism of action (MOA) of SS peptides 2535-Pos remains virtually unknown. We are conducting a highly interdisciplinary study Mechanisms of Adhesion of a Bacterial Predator to Surface with Quanti- to elucidate how SS peptides interact with the cardiolipin-rich mitochondrial tative Force Measurements inner membrane and the effects that they have on membrane physical proper- Yuyu Chen1, Eileen M. Spain2. ties and structural assembly of oxidative phosphorylation (OXPHOS) com- 1Occidental College, Los Angeles, CA, USA, 2Dept Chemistry, Occidental plexes. With model membrane systems, we have used a host of approaches Coll, Los Angeles, CA, USA. including fluorescence spectroscopy, calorimetry, solid state NMR, and small The Gram-negative bacterial predator Bdellovibrio bacteriovorus has the capa- angle x-ray scattering to understand: (i) the driving forces, affinities, and ther- bility to consume other Gram-negative prey bacteria including pathogens in modynamics of the peptide-bilayer interaction, and (ii) the effects that SS pep- biofilms. As a result, utilizing Bdellovibrio bacteriovorus as an approach for tides have on lipid packing and order parameters. We have complemented these drug-free biofilm control is a robust area of current research. Our research is measurements with molecular dynamics approaches to analyze the conforma- aimed at elucidating the physiochemical mechanisms of Bdellovibrio adhesion tions and lipid interactions of SS peptides in the bilayer-docked state. Finally,

BPJ 9459_9462 512a Wednesday, March 6, 2019 using disease models, we show that SS-31 restores the structural assembly of key Gramicidin A (formyl-L-Val-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-L- OXPHOS complexes. With our unprecedented insights into the MOA of these Trp9-D-Leu-L-Trp11-D-Leu-L-Trp13-D-Leu-L-Trp15-ethanolamine) functions compounds, we present models for how they may restore cellular bioenergetics as a membrane-spanning channel for monovalent cations. Each channel is by affecting membrane properties. These insights will be used to inform the composed of two subunits joined by six hydrogen bonds. The four Trp residues development of libraries of related compounds with increased efficacy. anchor each subunit to the aqueous interface and prevent subunits from crossing a lipid-bilayer membrane. Nevertheless, it has been observed that re- 2538-Pos placing Trp residues 13 and 15 with Phe results in subunits (designated Kink in Helical Peptides Affects Membrane Pore Formation [Phe13,15]gA) that are capable of crossing the membrane, presumably in a Alzbeta Turkova1, Ivo Kabelka1, Tereza Kralova1, Lukas Sukenik1, double-stranded conformation, to engage in channel formation. Our goal is Sarka Pokorna2, Martin Hof3, Robert Vacha1. to modify [Phe13,15]gA to increase the channel lifetime in biological mem- 1CEITEC MU, Masaryk University, Brno, Czech Republic, 2J. Herovsky branes, while retaining its ability to cross membranes. To this end, the length Institue of Physical Chemistry AVCR, Prague, Czech Republic, 3Biophysical of [Phe13,15]gA was increased to allow for better matching in thicker biological Chemistry, J.H. Inst. Phys. Chem., Prague, Czech Republic. membranes, and Ala was introduced near the N-terminus to increase channel Antimicrobial peptides (AMPs) can kill pathogenic cells via the formation of stability. Comparative biophysical experiments with three novel peptides, membrane pores. However, the connection between peptide properties and their endo-Gly0B-Gly0A-[Phe13,15]gA, endo-Gly0B-D-Ala0A-[Phe13,15]gA and endo- effect on pore formation remains elusive. In particular, the role of proline/ D-Ala0A-[Phe13,15]gA, were performed in DMPC and DOPC bilayers. Deuter- glycine kink in helical AMPs has been reported with contradictory effects on ated alanines were included to facilitate analysis by solid-state deuterium NMR antimicrobial activity. Using computer simulations and fluorescence leakage ex- spectroscopy. The 2H NMR spectra in mechanically aligned DMPC bilayers periments we show the relationship between alpha-helical kink and the structure suggest a single major membrane orientation for each peptide, with slightly of the formed pore. Reconciling previous data, the presence of kinks was found to different backbone torsion angles for Ala3 and Ala5. By contrast, the spectra have both stabilizing or destabilizing effect, depending on the pore structure. in DOPC are poorly resolved. Circular dichroism spectra in DMPC and Moreover, the position of proline/glycine kink in AMP sequence controls the DOPC vesicles suggest mixtures of single- and double-stranded conformations. peptide arrangements in the stabilized pores. The provided knowledge can be uti- The Trp fluorescence emission l-maxima for endo-Gly0B-D-Ala0A-[Phe13,15] lized to rationally design peptides with different ability to form membrane pores gA (335 nm) and endo-D-Ala0A-[Phe13,15]gA (336 nm) in DOPC vesicles are useful for the development of new antibacterial agents. shifted to higher wavelengths compared to [Phe13,15]gA (330 nm). Experiments to assess channel lifetimes and membrane-crossing ability of each peptide are 2539-Pos underway. Sphingomyelin Plays a Critical Role in Membrane-Related Effects Induced by the Steroid Saponin Ginsenoside Rh2 2541-Pos Sandrine L. Verstraeten1,2, Maria Janikowska-Sagan3, 4,5 4 4 Conformational Sampling of the pH Low Insertion Peptide is Tuned by pH Emily J.S. Claereboudt , Laurence Lins , Magali Deleu , Nicolas C. Frazee, Blake Mertz. Donatienne Tyteca2, Marie-Paule Mingeot-Leclercq1. 1 Chemistry, West Virginia University, Morgantown, WV, USA. Louvain Drug Research Institute, Universite Catholique de Louvain, The pH Low Insertion Peptide (pHLIP) is a 36 residue, pH-sensitive mem- Brussels, Belgium, 2de Duve Institute, Universite Catholique de Louvain, 3 brane-active peptide that is coiled both in solution and when bound to a mem- Brussels, Belgium, Faculty of Physics, Astronomy and Applied Computer a 4 brane at physiological pH. Upon acidification, pHLIP folds into an -helix and Science, Jagiellonian University, Krakow, Poland, Gembloux-Agro-Bio inserts unidirectionally into the bilayer. Thus, pHLIP could act as a potential Tech, Universite de Lie`ge, Gembloux, Belgium, 5Research Institute for vector for targeted drug delivery to or diagnostic imaging of tissues associated Biosciences, Universite de Mons, Mons, Belgium. with acidosis, such as cancer or heart ischemia. However, without a detailed Cholesterol (Chol) and sphingomyelin (SM) are known to cluster into transient description of the biophysical interactions of pHLIP that lead to function, it nanometric domains, forming a liquid ordered (Lo) phase. These domains serve will be difficult to design variants with increased targeting abilities. A recent as a platform that contributes to cellular signaling including cell survival or pro- study utilizing pH probes on cancerous cells has determined an increased radius liferation with potential implications in cancer development. Saponins, amphi- of extracellular space is affected by acidosis [1], and therefore a peptide ap- philic compounds widely found in plants, attract more and more attention based proaching the bilayer would experience an acidic environment before interact- on their biological activities including anticancer properties. Although the ac- a ing with the membrane. With this in mind, we employed constant pH molecular tivity for most saponins (e.g. digitonin, -hederin) is usually attributed to their dynamics (MD) simulations to determine potential changes in conformation of interactions with membrane Chol, our recent publication showed that Chol, pHLIP as it transitions into the acidic extracellular space. Surprisingly, we contrary to SM, delays the cytotoxicity of the saponin ginsenoside Rh2 in hu- found that a single amino acid residue, D31, has a noticeably higher pK man cancer cell lines (A549, THP-1, and U937)[1], renewing the idea that a than what is expected from the experimentally determined pKa of aspartic saponin activity is only ascribed to an interaction with Chol. In this study, acid residues. In addition, we observe formation of a stable helical fold from we elucidated the respective importance of Chol and SM in the membrane- D14 to T18 at pH values below 4.5, suggesting pHLIP has the potential to sam- related effects of Rh2 using a large panel of biophysical approaches while care- a i.e. ple conformations (i.e., -helices) that facilitate partitioning into the membrane fully modulating their lipid compositions, the presence or absence of Chol while still in solution. Our results provide a unique and essential perspective on or SM. Using lipid monolayers, we observed that Rh2 preferentially interacted the behavior of pHLIP as it transitions into acidic microenvironments such as with eSM:ePC, then eSM:ePC:Chol followed by ePC:Chol. Using Large Uni- the extracellular region of cancerous cells. [1] Luo (2018) Anal. Chem. lamellar Vesicles (LUVs), we found that Rh2 increased the vesicle size, 90:5803-5809. decreased membrane fluidity and induced membrane permeability with the same ranking, i.e eSM:ePC>eSM:ePC:Chol>ePC:Chol. Using Giant Unila- mellar Vesicles (GUVs), Rh2 generated positive curvature in SM-containing 2542-Pos membrane and small buds followed by intra-luminal vesicles in the absence Using pH Sensitive Peptides for the Endosomal Release of Antibodies of SM. Altogether, our results indicate that SM promotes and accelerates Eric Wu1, Sarah Y. Kim2, Kalina Hristova2, William C. Wimley3. membrane-related effects induced by Rh2. To the best of our knowledge, 1Biochemistry, Tulane University School of Medicine, New Orleans, LA, this study is the first to reveal the critical role of SM in membrane-related ef- USA, 2Materials Science and Engineering, Johns Hopkins University, fects induced by a saponin reconsidering the theory that Chol is the only Baltimore, MD, USA, 3Biochemistry and Molecular Biology, Tulane responsible for the activity of saponins. [1] Toxicol Appl Pharmacol 352, 59- University School of Medicine, New Orleans, LA, USA. 67(2018). Most therapeutic antibodies target cell surface epitopes. However, there are many appealing cytosolic targets that have not been fully explored, largely 2540-Pos due to the difficulty in delivering antibodies into the cell. While endosomal Novel F13,F15 Gramicidin Subunits Predicted to Cross Bilayer Mem- uptake can be achieved, the fate of endocytosed proteins is mostly degrada- branes and form Ion Channels tion by the endosome/lysosome pathway. Development of a method that en- Matthew Brownd1,2, Matthew J. McKay2, Denise V. Greathouse2, ables antibodies to escape this hydrolytic fate and be released into the Olaf S. Andersen3, Roger E. Koeppe2. cytosol would open up an entirely new set of cancer therapeutic targets 1Dept of Chemistry & Biochemistry, John Brown University, Siloam Springs, for exploration. We are developing pH-sensitive pore-forming peptides to AR, USA, 2Dept of Chemistry & Biochemistry, University of Arkansas, enable endosomal escape of endocytosed protein cargo. The pH sensitive, Fayetteville, AR, USA, 3Dept of Physiology & Biophysics, Weill Cornell pore forming peptide pHD108 is a 26-residue peptide that is a gain-of- Medical College, New York, NY, USA. function variant of melittin, a membrane permeabilizing peptide from bee

BPJ 9459_9462 Wednesday, March 6, 2019 513a venom. Multiple acidic glutamate residues in pHD108 confer the pH- 2545-Pos triggered membrane permeabilization activity. In synthetic unilamellar ves- Peptide-Enhanced Cargo Transport across 2D and 3D Epithelial Barriers: icles, pHD108 has no poration activity at pH 7 but at pH % 5.5 it releases A Structure-Function Investigation macromolecules from lipid vesicles at very low peptide concentration. In Alexander Komin1, Max I. Bogorad1, Ran Lin2, Honggang Cui2, this work, we are i) engineering pHD108 to bind strongly to cell membranes Peter C. Searson1, Kalina Hristova1. at pH 7 to promote endosomal uptake, and ii) determining the effect of 1Department of Materials Science and Engineering, Johns Hopkins increased membrane binding on the activity profile of pHD. Specifically, University, Baltimore, MD, USA, 2Department of Chemical and we are modifying pHD108 by conjugation to fatty acids and cholesterol, Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA. and testing the effects of lipidation on small molecule and macromolecule There is a great need to enhance the delivery of therapeutics across biological bar- release from lipid vesicles. Ultimately, we will study the effect of lipidated riers, such as the blood-brain barrier. We have identified a cationic peptide, called pHD108 on cells, measure their endosomal uptake, and determine their abil- the CL peptide, which can deliver a small-molecule cargo across a 2D epithelial ity to enable release of proteins from endosomes. Success in this research barrier model at a moderately high rate. In this study, we measure cargo delivery would result in identifying a potential cancer therapeutic adjuvant as well in a novel 3D microvessel platform. Furthermore, we investigate the effect of as providing a novel means to study the blocking/neutralizing effects of an- arginine spacing, number of arginines, secondary structure, stereochemistry, tibodies against intracellular targets. and other parameters on the cargo permeability. These studies can help provide design rules for developing novel peptides for transcellular cargo delivery. 2543-Pos The pH Sensitive Atram Peptide Hitchhikes on Human Serum Albumin En 2546-Pos Route to Target Diseased Acidic Tissues Translocation of CPP-Cargo Protein Fusions into Candida albicans Cells Vanessa P. Nguyen1, Stephen J. Kennel2, Jonathan S. Wall2, and Designing for Enhanced Translocation with Simulations 1 1 2 1 Francisco N. Barrera1. Sayanee Adhikari , Mahdi Ghorbani , Katherine Dura , Jeffery B. Klauda , 1 Amy J. Karlsson3. Biochemistry & Cellular and Molecular Biology, University of Tennessee, 1 Knoxville, TN, USA, 2Medicine & Radiology, University of Tennessee, Chemical and Biomolecular Engineering, University of Maryland, College Park, MD, USA, 2Fischell Department of Bioengineering, University of Knoxville, TN, USA. 3 Our lab developed the acidity triggered rational membrane (ATRAM) peptide Maryland, College Park, MD, USA, Chemical and Biomolecular to target the acidic extracellular environment of tumors. Biophysical and Engineering; Fischell Department of Bioengineering, University of cellular experiments have shown that the membrane interaction of ATRAM Maryland, College Park, MD, USA. Candida albicans is pH-dependent. Often rapid enzymatic degradation and renal clearance of Fungal infections caused by the opportunistic pathogen are peptides in vivo restricts their successful application as a drug delivery sys- a concern in immunocompromised patients. To address limitations of current tem. However, our previous mice studies demonstrated that ATRAM has an antifungal therapies, we propose using cell-penetrating peptides (CPPs), extended circulation half-life. We hypothesized that ATRAM might be inter- which have the ability to carry biomolecules across cell membranes. The acting with a blood component that shields the peptide from degradation. CPPs used in this study include histatin 5 (Hst5), pVEC, MPG and SynB, Binding assays confirmed that ATRAM binds to human serum albumin which were fused to different biomolecular cargoes. As an example, we (HSA), a plasma protein, with higher affinity than insulin detemir, a clinically explored the intracellular delivery of purified Hst5-G4S-GFP and purified C. albicans used drug, suggesting that it might bind to this protein in the blood stream. GFP into . Various concentrations of GFP and Hst5-G4S-GFP Fluorescence studies established that ATRAM interacts reversibly with were tested, and initial results show that higher concentration of the fusion m HSA, as the peptide partitioned to lipid membranes after pre-incubation led to higher uptake. At 100 M, Hst5-G4S-GFP translocated into nearly C. albicans with HSA. This result suggests that while that peptide can use albumin as a 5% of cells, whereas GFP translocated into 1% of cells. We < carrier in the blood stream, it will still target and transfer to the cell mem- observed low levels of dead cells ( 1% via PI staining) following incubation brane. Understanding how ATRAM is able to avoid immediate degradation with Hst5-G4S-GFP, indicating the fusion protein does not significantly affect and renal clearance in vivo can broaden the design and application range of membrane integrity or viability of the cells. To improve the translocation of peptides as therapeutics. the fusions, we are designing changes to our CPP-cargo protein fusions using experiments and simulations. We are looking at molecular dynamics simula- 2544-Pos tions of these peptides using the highly mobile membrane model that will give The pH-Specific Thermodynamic Intermediates of Phlip Membrane insight about which end of the peptide to link to the cargo protein to achieve Insertion higher transport. In these simulations, peptides are placed in an aqueous phase Sarah A. Otieno1, Lukas M. Klees1, Anqi Zhang1, Heather M. Giza1, at different orientations with respect to the membrane, enabling us to explore Samuel Z. Hanz1, Bianca Chakravorty1, Lan Yao2, Ming An1, Wei Qiang1. regions of the wild-type peptide contributing most to translocation. We are 1Department of Chemistry, State University of New York, Binghamton also mutating specific residues in the CPPs, both by simulation and experi- University, Binghamton, NY, USA, 2Department of Physics, Applied Physics ments and using these mutated CPP-cargo fusions to determine experimen- and Astronomy, State University of New York, Binghamton University, tally whether these changes in the CPPs affect translocation. These experimental and simulation results will help us design better CPP-cargo con- Binghamton, NY, USA. C. albicans . The applications of the pH-Low Insertion Peptide (pHLIP) in cancer diag- structs to improve translocation into cells nosis and cross-membrane cargo delivery have drawn increasing attention 2547-Pos in the past decade. With its origin as the transmembrane (TM) helix C of Energetics and Kinetics of Membrane Protein-Detergent Interactions bacteriorhodopsin, pHLIP is also an important model for understanding Aaron Wolfe, Liviu Movileanu. a how pH can affect the folding and topogenesis of a TM -helix. Proton- Dept Physics, Syracuse Univ, Syracuse, NY, USA. ations of multiple D/E residues transform pHLIP from an unstructured Interfacial interactions of the membrane protein-detergent complex (PDC) has State II > a coil at membrane surface (known as ,atpH 7) to a TM -helix paramount importance to protein function, structure, and dynamics. Current State III < ( ,pH 5.3). While these initial and end states of pHLIP insertion methods are low throughput, are not amenable to a widespread adoption, or have been firmly established, what happen at the intervening pH values are necessitate high amounts of protein. Here, we used steady-state fluorescence less clear. Yet the intervening pH range is most relevant to pHLIP-cell in- polarization anisotropy, along with membrane protein engineering, to probe teractions in the acidic extracellular tumor environment (and in the endo- the specific kinetic reads of interfacial PDC interactions. These studies were somes within cells). Here, using advanced solid-state NMR spectroscopy, conducted at detergent concentrations below the critical micelle concentration. as well as Trp fluorescence methods, with POPC unilamellar vesicles as Under these conditions, prevailing spectroscopic and calorimetric approaches the model membrane, we systematically examined the state of pHLIP- fail to provide a satisfactory signal-to-noise ratio due to unproductive protein membrane interactions (in terms of the membrane locations of D/E residues, aggregation. This method was formulated in a high-throughput, microplate as well as lipid dynamics) at the intervening pH values of 6.4, 6.1, and 5.8, reader setting for the massive screening of membrane proteins and detergents. along with the known states at pH 7.4 and 5.3. Thermodynamic intermediate Our approach was validated using several beta-barrel membrane proteins and states distinct from the initial and end states were discovered to exist at each numerous detergents of varying physicochemical properties. We showed that of the intervening pH examined. They support a new multi-stage model of the kinetic rate constants of association and dissociation of the proteomicelles pHLIP insertion in which the D/E titrations occur in a defined sequence at depend on both the interfacial hydrophobic and electrostatic interactions. distinct intermediate pH values. This multi-stage model has important ram- Further developments of this semi-quantitative method will impact physical ifications in pHLIP applications. and chemical biology of membrane proteins.

BPJ 9459_9462 514a Wednesday, March 6, 2019

2548-Pos Listeriolysin O (LLO) falls in the sub-class of cholesterol dependent cytolysin Determining the Essential Unfolding Step in Protein Translocation using (CDC) pore-forming protein. It is secreted by Listeria monocytogenes which Anthrax Toxin causes listeriosis, a fatal disease to immune-compromised individuals as well Koyel J. Ghosal, Bryan A. Krantz. as infants. Pore formation has been studied and found to be initiated by the bind- Department of Microbial Pathogenesis, University of Maryland Baltimore, ing of LLO to cholesterol, followed by oligomerization of the monomers and Baltimore, MD, USA. insertion of transmembrane segments inside the bilayer to form a pore. Studies Protein translocation across membranes is crucial to many cellular processes, suggest that LLO transitions through an inactive intermediate state, called a pre- which includes bacterial pathogenesis. However, its exact molecular mechanism pore, in the pore formation process. Although LLO has been widely studied, is not well understood. An established model to study this process is the anthrax there is very little information in the literature that connects the manner in which toxin, a three-protein virulence factor that intoxicates cells via transmembrane membrane lipid dynamics are modulated during pore formation. To address protein translocation. Following endocytosis of the toxin, the protective antigen some these unresolved issues pertaining to LLO interaction and assembly on (PA) forms a transmembrane channel, triggered by the acidic endosomal pH, phospholipid membrane, we used fluorescence correlation spectroscopy (FCS) through which the two active enzyme components, the lethal factor (LF) and and FRET on artificial membrane systems. Giant unilamellar vesicles (GUVs) the edema factor (EF) are translocated to the cytosol of the host cell. Two key were used as model systems to study LLO interactions with membranes. LLO non-specific polypeptide binding sites – the a-clamp and F-clamp – catalyze induced dye leakage of GUVs, revealed two distinct population of vesicles: protein translocation across the PA channel. The F-clamp is a very narrow open- leaked and unleaked. Interestingly, LLO was found to preferentially bind to ing, thus requiring proteins to unfold during translocation. We hypothesize that the Ld region of GUVs on both leaked and unleaked vesicles. FRET was moni- the PA channel can be used to translocate small heterologous proteins, such as tored between Alexa488-tagged-LLO and DiI-labelled-lipid. We observed sig- Im7, and that translocation occurs by protein unfolding through a rate-limiting nificant FRET efficiency on leaked vesicles whereas it was rarely observed in transition state. We expressed and purified small proteins composed of Im7 unleaked vesicles supporting the connection between the pore states and leakage. wild type attached to the first 30 amino acids of LF (LF1-30.Im7 WT), and its Interestingly, lipid diffusivities as measured from FCS, also showed correspond- 10 single amino acid mutants. Planar lipid bilayer electrophysiology was used ing difference between leaked and unleaked vesicles. Leaked vesicles demon- to perform single channel translocation assays of the WT and its mutants. Our strated enhanced lipid diffusivity in comparison to the unleaked vesicles. results revealed that the mutants translocated faster and at lower voltages than These results are attributed to the different structural changes that happen during the wild type, indicating protein destabilization due to mutation of the core res- the pore formation. Based on our results, lipid dynamics can potentially be used idues. This destabilization facilitated the unfolding process, which reduced the as a marker to distinguish between oligomeric states. driving force necessary for their translocation. Moreover, the critical step in translocation can be determined by mapping the transition states through dena- 2551-Pos turation studies using guanidine hydrochloride. Understanding the mechanism Recombinant Expression and Refolding of a Potassium Channel- of protein unfolding can significantly contribute to the development of a new Activating Three-Finger Toxin model for targeted drug delivery. Jamye Moya1, Adel K. Hussein1,2, Sebastien F. Poget1,2. 1Department of Chemistry, College of Staten Island, CUNY, Staten Island, 2549-Pos NY, USA, 2Program in Biochemistry, Graduate Center, CUNY, New York, Determining the High-Resolution Structures of the Anthrax Toxin Protec- NY, USA. tive Antigen Pore Bound to its Lethal and Edema Factors Tx7335 is a novel three-finger peptide toxin that was originally isolated from the Nathan J. Hardenbrook1, Shiheng Liu2,3, Kang Zhou2,3, Jiansen Jang2,3, Eastern Green Mamba venom. A unique characteristic of Tx7335 is the ability to Z. Hong Zhou2,3, Bryan Krantz1. increase duration and frequency of openings in the potassium channel KcsA. Po- 1Department of Microbial Pathogenesis, University of Maryland, Baltimore, tassium channels play an important role in electrical signaling of excitable cells. Baltimore, MD, USA, 2Department of Microbiology, Immunology and Dysfunction of the channels can lead to major health problems such as certain Molecular Genetics, University of California, Los Angeles, Los Angeles, CA, heart and autoimmune diseases. Potassium channels are natural targets for USA, 3California NanoSystems Institute, University of California, Los venomous animals seeking to paralyze their prey. As a result, toxins have Angeles, Los Angeles, CA, USA. been shown to be an effective method of modulating and studying potassium Anthrax toxin, one of the virulence factors of Bacillus anthracis, consists of channels. To obtain sufficient amounts of Tx7335 for future studies into its three individual protein factors: the cell binding/pore-forming protective anti- mechanism of action, we are recombinantly expressing and purifying this toxin. gen (PA), and the enzymatic factor lethal factor (LF), and edema factor (EF). A synthetic gene encoding for Tx7335 was cloned into an expression vector ex- Due to its formation of a protein-conducting pore, PA provides an excellent pressing the toxin as a thioredoxin fusion protein with a TEV protease cleavage system to study protein translocation. To date, the only structural information site in between them. The fusion protein was overexpressed in E coli. available of the PA bound to its enzymatic substrate is of the amino terminal BL21(DE3) C43 expression strain and purified by Nickel affinity chromatog- domain of LF, LFn, bound to the pre-pore octamer. This structure has shown raphy. The toxin was released from the fusion protein by cleavage with TEV pro- that the binding of LF to the PA pre-pore serves to stabilize its unfolding before tease and refolded by addition of reduced and oxidized glutathione in 1 to 10 being translocated through the channel. Using cryo-EM, we determined high- ratio. Tx7335 was further purified through Cation exchange chromatography. resolution structures of the heptameric PA in its pore conformation bound to Refolding was assessed by mass spectrometry, and the recombinant toxin will ˚ ˚ LF at 4.6-A, and bound to EF at 3.6-A. The PA7LF3 and PA7EF3 pre-pore com- be used for future functional and structural studies of toxin-KcsA interactions. plexes were inserted into lipid nanodiscs, causing them to take random orien- tation on cryo-EM grids and allowing us to perform single-particle analysis. 2552-Pos The structures contain the pore form PA heptamer bound to a single LF or Ebola Virus Delta-Peptide Acts as an Enterotoxic Viroporin In Vivo Shantanu Guha1, Lilia Melnik2, Robert F. Garry2, William C. Wimley1. EF, respectively. The C-terminal domains of LF is disordered in the PA7 LF 1Biochemistry & Molecular Biology, Tulane University, New Orleans, LA, structure, resulting in no visible electron density. When comparing the LFn 2 domain when bound to PA in the pore state versus the pre-pore complex, there USA, Microbiology & Immunology, Tulane University, New Orleans, LA, appears to be no significant changes in structure. The first a-helix in LF its USA. Ebola virus (EBOV), part of the filovirus family, has a genome that encodes for unfolded and docked within the a-clamp site. In the structure of PA7EF, the full length of EF protein appears to be ordered, showing domain reorganization a partly conserved, 40-residue polypeptide, called the delta peptide, which is within EF upon binding to PA. In addition, the a-clamp is also populated by the produced during Ebola virus disease pathogenesis. Sequence-structure- in vitro first a-helix in EF. These structures can help in our understanding of the mech- function analysis and data suggest that the delta peptide is a viroporin, anisms involved in protein translocation. a term used to describe a diverse family of virally encoded pore-forming pep- tides and proteins involved in the replication and pathogenesis of numerous vi- 2550-Pos ruses. Full-length and conserved C-terminal EBOV delta peptide fragments Elucidating the Effect of Listeriolysin O Structural Intermediates on Lipid permeabilize synthetic lipid bilayers and multiple cell types in vitro. Activity Diffusivity requires that the two conserved cysteines are connected by a disulfide linkage. Ilanila Ilangumaran Ponmalar1, Ganapathy Ayappa2, Here, we follow up those results by testing delta peptides using an established Jaydeep Kumar Basu3. mouse model of diarrheal pathology focusing on the small intestine. We hy- 1Center for BioSystems Science and Engine, Indian Institute of Science, pothesized that the cell permeabilization observed against numerous cell types Bangalore, India, 2Department of Chemical Engineering, Indian Institute of in vitro would correspond to intestinal pathology, in vivo. We found that the Science, Bangalore, India, 3Department of Physics, Indian Institute of delta peptide exerts potent enterotoxic activity against mouse intestine upon Science, Bangalore, India. introduction to a closed intestinal ileal loop, resulting in a diarrheal syndrome

BPJ 9459_9462 Wednesday, March 6, 2019 515a in the mice. The peak activity of the peptide occurs 9-12 hours after introduc- nanopore-based endopeptidase assay for assessing the peptidase activity of tion of peptide into the intestinal loop. Histological examination of the intesti- the toxin. When the BoNT-containing sample is introduced into the assay, nal architecture showed severe damage and loss of goblet cells. Our in vivo the BoNT heavy chain (HC) binds to its target receptors before forming a trans- results demonstrate that the EBOV delta peptide is a potential novel therapeutic membrane channel that then transports the light chain (LC) across the lipid target, and neutralizing it could ameliorate some of the severe enterotoxic bilayer. Once the LC has traversed the membrane and is released from the burden characteristic of EBOV disease pathogenesis. Accordingly, our focus HC, it then cleaves and inactivates its respective target protein, i.e., its soluble is to generate a high-affinity neutralizing antibody to block the lytic activity N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) protein of the delta peptide in vitro and in vivo as a potentially efficacious therapeutic target. In the BBA, both the rate of HC insertions into the planar lipid bilayer agent against EBOV disease. and LC peptidase activity can be independently, but simultaneously, moni- tored, with the rate of SNARE protein cleavage being directly proportional 2553-Pos to the potency of the sample introduced. While the assay is being specifically Mechanism of Catechin-Mediated Inhibition of RTX Toxin Activity developed for BoNT testing, the platform will be generally applicable to a large En Hyung Chang, Angela C. Brown. number of other biomolecules with transmembrane activity for which there is Dept Chem/Biomol Eng, Lehigh Univ, Bethlehem, PA, USA. currently no standard in vitro characterization technique. The leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans,an oral pathogen, plays a key role in the ability of this organism to colonize the host. 2556-Pos This toxin specifically and effectively kills human white blood cells, thus sup- Artificial Membrane Attack Complex through DNA-Guided Self-Assem- pressing the local immune response to the bacteria and promoting infection. Typi- bly of Pore-Forming Peptides: Biological Nanopores with Programmable cally, this infection is treated with systemic antibiotics, but in recent years, Diameter resistance to the most effective antibiotics in this organism has been increasing. Aziz Fennouri1, Jonathan List1, Julie Ducrey1, Laura Pascual1, For this reason, we and others have sought alternative strategies to treat this dis- Frederick Bertani1, Sandra Rodriguez Gonzalo1, Simon F. Mayer1, ease. Our group designs targeted agents to inhibit LtxA activity as a way of Jerry Yang2, Michael Mayer1. limiting the growth of the bacteria, with the idea that impaired LtxA function 1Adolphe Merkle Institute, Fribourg, Switzerland, 2Department of Chemistry will restore clearance of the bacteria by the immune system. Several reports and Biochemistry, University of California, San Diego, San Diego, CA, USA. have demonstrated that polyphenols, particularly certain catechins, have antibac- The membrane attack complex (MAC) of the complement system is an essential terial and anti-LtxA activities, but the mechanism of this activity has not yet been part of the innate mammalian immune response. Invasion of a host by a path- uncovered. We therefore undertook this study to investigate the catechin- ogen triggers a proteolytic cascade leading to the activation of various proteins mediated changes in host membrane and LtxA properties to better understand that assemble on the pathogen cell membrane and form a transmembrane chan- how these molecules prevent cell damage by the toxin. We found that most of nel. These large MAC pores induce osmotic lysis of the pathogen cell. Cell the catechins we tested inhibited LtxA activity against white blood cells, with killing applications would benefit from such large pores than insert upon pro- the galloylated catechins exhibiting the greatest effect. Pre-incubation of the toxin grammable signals. Here, we developed DNA-templated assemblies forming with the catechins increased the inhibitory action, indicating that the catechins act large and defined pores, that insert into membranes in response to stimuli, to on the protein, rather than the cell. We demonstrated using CD spectroscopy that mimic the function of the MAC. To this end, we covalently linked a nucleic the catechins alter the secondary structure of the toxin, and as a result, prevent the acid to a pore-forming antimicrobial peptide, Ceratotoxin A (CtxA). This toxin from binding to cholesterol on the host cell, a key step in the toxic mecha- CtxA-nucleic acid conjugate hybridizes to complementary DNA template struc- nism of LtxA. Together, our results demonstrate that the anti-toxin activity of cat- tures possessing multiple hybridization sites, which define the number of pep- echins is a result of catechin-mediated structural changes in LtxA which alter the tide monomers involved in pore formation. A simple DNA strand allowed interaction of the toxin with the cell membrane lipids. templating tetra-, octa or dodecameric assemblies, while rigid DNA origami 2554-Pos structures further increased the assembly size and made it possible to template Characterization of Combined Cholesterol and Integrin Interactions for up to 40 peptide-DNA monomers. We observed direct incorporation of these RTX Toxin Activity templated assemblies into planar lipid bilayer membranes as opposed to sequen- Eric Krueger, Angela C. Brown. tial peptide association and dissociation typically seen with native CtxA. Such Department of Chemical & Biomolecular Engineering, Lehigh University, pores with tunable diameters are highly beneficial for resistive pulse sensing ap- Bethlehem, PA, USA. plications. The modularity of this design allowed us to functionalize the The leukotoxin (LtxA), secreted by the bacterium Aggregatibacter actinomyce- construct with hydrophobic or receptor-binding moieties. This straightforward temcomitans, is a member of the repeats-in-toxin (RTX) family of toxins and modification increased the affinity of the construct for the membrane, without targets leukocytes through an affinity for cholesterol and a specificity for the modifying the peptide’s amino acid sequence. Folic acid-modified constructs, lymphocyte function-associated antigen 1 (LFA-1) integrin. The goal of this for instance, exhibited a 10-fold greater cytotoxicity than non-specific peptide project is to investigate these individual interactions and study how their com- assemblies. Ultimately, employing DNA-based recognition mechanisms or bio- bined effects facilitate the cytotoxic activity of LtxA. Specifically, we have molecular targets such as cancer-derived microRNAs may trigger templated developed a series of Fo¨rster resonance energy transfer (FRET) and fluores- pore formation for targeted killing of pathogenic cells. cence colocalization assays to investigate the roles of these interactions in LtxA activity in cells and model membranes. We have demonstrated that 2557-Pos LFA-1 is not required for LtxA-cholesterol binding and further investigations Distinct Roles of Snare-Mimicking Lipopeptides during Initial Steps of revealed that complexes of toxin bound with its targeted LFA-1 domains retains Membrane Fusion its propensity for cholesterol, indicating that the these two interactions are in- Alexander Kros. dependent. We are currently applying these biophysical methods to investigate Leiden Inst Chem, Leiden Univ, Leiden, Netherlands. how the organization of cholesterol and LFA-1 in the cell membrane contrib- A model system for membrane fusion, inspired by SNARE proteins and based utes to LtxA activity. By characterizing the role of these critical individual on two complementary lipopeptides CPnE4 and CPnK4, has been recently and combined elements, we will better understand the mechanism of its cyto- developed. It consists of cholesterol (C), a poly(ethylene glycol) linker (Pn) toxicity, enabling the development of unique anti-virulence strategies against and either a cationic peptide K4 (KIAALKE)4 or an anionic peptide E4 (EIAA- A. actinomycetemcomitans infections. LEK)4. In this paper, fluorescence spectroscopy is used to decipher distinct but complementary roles of these lipopeptides during early stages of membrane 2555-Pos fusion. Molecular evidence is provided that different distances of E4 in A Bilayer-Based In Vitro Assay for Botulinum Holotoxin Potency Assess- CPnE4 and K4 in CPnK4 from the bilayer represent an important mechanism, ments which enables fusion. Whereas E4 is exposed to the bulk and solely promotes Runzhi Lai, Eric N. Ervin. membrane binding of CPnK4,K4 loops back to the lipid-water interface where it Electronic BioSciences, Salt Lake City, UT, USA. fulfills two distinct roles: it initiates bilayer contact by binding to CPnE4 con- The standard procedure for botulinum toxin potency assessments is the mouse taining bilayers; and it initiates fusion by modulating the bilayer properties. lethality assay by intraperitoneal injection. However, the mouse assay is labo- The interaction between CPnE4 and CPnK4 is severely down-regulated by bind- rious, expensive and ethically controversial. We have developed and are ing of K4 to the bilayer and possible only if the lipopeptides approach each refining an in vitro, synthetic bilayer-based assay (BBA) for characterizing other as constituents of different bilayers. When the complementary lipopepti- the potency of botulinum neurotoxin (BoNT) containing therapeutics, as a des are localized in the same bilayer, hetero-coiling is disabled. These data pro- replacement for the mouse lethality assay. The BBA employs interfacial planar vide crucial insights as to how fusion is initiated and highlight the importance lipid bilayer, preconfigured to mimic a neuronal cell membrane, along with a of both peptides in this process.

BPJ 9459_9462 516a Wednesday, March 6, 2019

Posters: Protein-Lipid Interactions: Structures our strictly outlined peptide-lipid system, we substitute a glutamic acid residue in the L14 position of the helical model peptide GWALP23 (acetyl-GGALWLA- 14 2558-Pos LALALAL ALALWLAGA-amide). The substitutions of glutamine and as- Live Action of Escrt III Machineries in Membrane Remodelling: Mem- partic acid will serve as controls. The GWALP23 derivatives are placed in brane Deformation & Membrane Scission different lipid environments containing negatively or positively charged lipids Sourav Maity1, Christophe Caillat2, Nicola De Franceschi3, among the primary zwitterionic lipids of the membrane. The purpose is to create Maryam Alqabandi3, Nolwenn Miguet2, Patricia M. Bassereau3, an environment similar to a cell membrane that can also accommodate the gluta- Winfried Weissenhorn2, Wouter H. Roos1. mic acid side chain. Core alanine resides are labeled with deuterium to determine 1Molecular Biophysics, Zernike Institute, University of Groningen, helix characteristics by solid-state NMR. The peptide-lipid samples will include 2 DMPC doped with 10% charged lipid, as lipid bilayer formation has been Groningen, Netherlands, Institut de Biologie Structurale (IBS), Univ. 31 Grenoble Alpes, CEA, CNRS, Grenoble, France, 3Institut Curie, Paris, France. confirmed for these lipid mixtures by solid state P NMR. The charged lipids Endosomal sorting complexes required for transport (ESCRT) are involved in will consist of the negatively charged lipid, DMPG, and the positively charged many cellular membrane remodelling processes including membrane deforma- lipid DMTAP. Samples with a 20% charged lipid mixture do not form adequately tion and fission. Examples of such processes are the biogenesis of endosomal oriented bilayers with the E14 peptide present. Initially, these experiments will vesicles, virus budding, cytokinesis and nuclear envelope closure. These ma- monitor changes in the quadrupolar splittings of the neighboring deuterated chineries are also vulnerable to cancerous growth and neurodegenerative dis- alanine residues at positions 15 and 13 as the sample pH is varied. Samples con- taining E14 and 10% cationic lipid buffered between a pH range of 5.5-8.1 form eases. In membrane fission, first a budded neck is formed assisted by ESCRT 31 2 III filaments that stabilize the highly curved membrane neck. In the final stage, isotropic peaks in both Pand H-NMR observables. Additional experiments are these filaments accumulate to set the stage for membrane fission, in cooperation underway in different lipid mixtures to monitor changes in helix behavior. with vacuolar protein sorting-associated protein 4 (VPS4). The small size and 2561-Pos the dynamic nature of these machineries present challenges for scientists to Position Dependent Orientation Difference of Transmembrane Peptides gain a comprehensive understanding of these processes. Therefor, we sought Flanked by Single or Multiple Histidine Residues to understand the role of such ESCRT III machineries at the single-molecule Fahmida Afrose, Denise V. Greathouse, Roger E. Koeppe. level. Specifically, we scrutinize the first and the last steps of membrane budding Department of Chemistry & Biochemistry, University of Arkansas, and fission using High Speed –Atomic Force Microscope (HS-AFM). The Fayetteville, AR, USA. unique ability of HS-AFM to study bio-molecules in near-to physiological con- GWALP23, a constructive low-dynamic model peptide framework, widens the ditions and with high spatio-temporal resolution makes it a successful tool to range of protein-lipid interactions that can be investigated when potentially study the dynamics of single molecules at millisecond resolution. Our results charged residues are present. In this context, we have examined the effects reveal, for the first time at nm resolution, the dynamics of CHMP2A/CHMP3 of single or pairs of histidine residues. To this end, we substituted residues constriction by Vps4, as a stepping stone to membrane scission. Furthermore, H8 and H16 into the parent sequence (acetyl-GGALW5LAL8ALALALA- we scrutinize the role of CHMP2B and CHMP4B in membrane remodelling L16ALW19LAGA-amide) of GWALP23. The 2H-NMR spectra of 2H-labeled and discuss possible pathways for the initiation of vesicle budding. core alanine residues show, interestingly, no titration dependency from pH 2-8, yet a difference in bilayer thickness-dependent orientation. In DLPC lipid bila- 2559-Pos C yers, a quadrupolar wave analysis indicates a tilted transmembrane helix Driving Forces Stabilizing Cellular Prion Protein (Prp ) Monomers and similar to that of the parent GWALP23 peptide. With respect to DOPC mem- Dimers on the Cell Surface branes, nevertheless, the peptide Ala residues display a wide range of 2H quad- Frances Tiffany Morden1, India Claflin1, Patricia Soto2. 1 2 rupolar splittings (about 1.5 30 kHz). The helix adopts a surface bound Biology, Creighton University, Omaha, NE, USA, Physics, Creighton orientation on DOPC membranes, however alanine A7 does not fit within the University, Omaha, NE, USA. C Sc core helix. Additional single histidine peptides, with H2 or H22 substituted The conversion of the prion protein PrP to its infectious form PrP via auto- into the parent sequence, display tilted transmembrane orientations in bilayers catalytic misfolding is critical to the development of a group of neurodegener- of DLPC or DOPC, with little or no pH dependence of the orientations from pH ative diseases known as transmissible spongiform encephalopathies (TSEs). To 2 to pH 8. Results from H2,22WALP23 will be compared with those observed gain insight on the factors that influence the PrPC to PrPSc conversion, it is C C when only H2 or H22 is present. The more central histidines 8 and 16 can lead pertinent to identify how PrP interacts with its environment. PrP is attached to a helix transition from membrane-spanning in DLPC (and most likely also in to the extracellular side of the cell membrane via a glycosylphosphatidylinosi- DMPC) to a surface orientation when the lipid bilayer is the thicker DOPC. tol (GPI) anchor and can be found in monomer or homodimer form. To inves- tigate the driving forces that stabilize PrPC monomers and dimers on the cell 2562-Pos membrane surface, we used molecular modeling techniques. From our analysis, Helix Fraying and Orientation of a Transmembrane Peptide having a we identified stable dimer conformations and characterized the dimer interface Long Hydrophobic Core and Anchored by Interfacial Arginine Residues using residue interaction network analysis and residue contact maps. We then Sara J. Sustich, Fahmida Afrose, Denise V. Greathouse, Roger E. Koeppe. implemented a series of molecular dynamics simulations to mimic the effects Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. of changing membrane lipid composition on the protein-lipid interface. Our re- Transmembrane peptides are often flanked by interfacial aromatic residues that sults indicate that PrPC dimers are stabilized by hydrophobic interactions potentially play a role in anchoring the peptide to assist in the stabilization of a along the b-sheets and that dimer stability is affected by the orientation of tilted transmembrane orientation. The peptide GWALP23 (acetyl-GG2-AW(- C 22 PrP a-helices on the cell membrane surface. We will propose a mechanism LA)6WLAG A-amide) containing two interfacial Trp(W) residues has a stable by which PrPC dimers with hydrophobic interfaces inhibit PrPSc propagation transmembrane orientation in several lipid bilayers. A related peptide 2 22 due to the mobility constraints that GPI anchors place onto the dimer structures RWALP23 (acetyl-GR -AW(LA)6WLAR A-amide) has been employed to that do not allow the presentation of a PrPSc binding domain. investigate the interplay between interfacial arginines and tryptophans. Here This work was made possible partly by grants from the National Institute for we replace the tryptophans of RWALP23 with A5 and A19, to investigate argi- General Medical Science (NIGMS) (5P20GM103427), a component of the Na- nines alone with respect to helix fraying and orientation in varying lipid bila- tional Institutes of Health (NIH), and its contents are the sole responsibility of the yers. Deuterated alanines incorporated into the core sequence of the peptide authors and do not necessarily represent the official views of NIGMS or NIH. allow the orientation and stability of the core helix to be assessed by means of solid -state 2H NMR in lipid bilayers DOPC, DMPC and DLPC. The alanines 2560-Pos located outside of the core region of the peptide are sensitive to helical integrity. Influence of Charged Lipids on Glutamic Acid Containing Transmem- The new alanines, A5 and A19, will provide new information about the length brane Helices of the core helix and the possible onset of unraveling of the terminals. In DLPC Brooke E. Nunn, Matthew J. McKay, Denise V. Greathouse, bilayers, 2H resonances from A3 and A7 reveal lower quadrupolar splitting Roger E. Koeppe. values than those for the R2,22W5,19 helix, suggesting that the R2,22A5,19 helix Chemistry & Biochemistry, University of Arkansas, Fayetteville, AR, USA. might adopt a different orientation. The 2H quadrupolar splittings for A3 and Among the crucial components of the cell are transmembrane proteins. These pro- A7 also differ for the two helices in DMPC and DOPC bilayers. Future exper- teins play major roles in many biological activities and are essential for proper cell iments will be directed toward full orientation calculations based on GALA and functioning. An important aspect of these proteins is their respective ionization Gaussian dynamic methods using all core alanine data points along with known properties. For glutamic acid, an important consideration is its interaction with A5 and A19. Further comparisons of quadrupolar splittings of A3, A5, A19 and lipids in the cell membrane, as little is known about the properties of residues A21 wave plots for the core helix may give insights into the onset of helix that can titrate from neutral to negative within the hydrophobic environment. In fraying for both N and C terminals of the RWALP23 transmembrane peptide.

BPJ 9463_9465 Wednesday, March 6, 2019 517a

2563-Pos at the surface of DOPC bilayers, with residue Ala-9 displaying multiple 2H quad- Investigating the Conformational Dynamics of the Membrane Enzyme rupolar splittings. When fitted to an alpha-helix, the quadrupolar splittings of Ala LspA residues before and after residue 9 of R12GW4,20ALP23 do not fit to the same Tracy A. Caldwell1, Linda Columbus2. quadrupolar wave plots. The N-terminal sequence up to residue 9 can be reason- 1 Molecular Physiology and Biological Physics, University of Virginia, ably fitted to a 310-helix and predicts the same surface bound orientation as the C- Charlottesville, VA, USA, 2Chemistry, University of Virginia, terminal alpha-helical residues 9-19 with only a slight variation ("kink’’) in the Charlottesville, VA, USA. average azimuthal rotation. The F4,5 helix remains transmembrane and exhibits Lipoprotein signal peptidase (LspA) is a key enzyme in the lipoprotein process- multi-state properties at residue A17 in bilayers of DOPC, although not of DMPC ing pathway and is responsible for cleaving the signal sequence of prolipopro- or DLPC. Further peptide backbone NMR experiments are underway to more teins. LspA is a membrane embedded enzyme that is hypothesized to undergo fully characterize the helix transitions and lipid interactions. conformational changes in order for the transmembrane alpha-helical substrate to enter the active site. As both the enzyme and substrate reside in the bacterial 2566-Pos inner membrane, enzyme-lipid interactions likely play a role in modulating Lipid Optimization to Improve the Solid-State NMR Spectra from these conformational changes. LspA conformation changes upon substrate Membrane-Spanning Helices with Glutamic Acid binding and in different membrane mimics are investigated using electron para- Kelsey A. Marr, Matthew J. McKay, Denise V. Greathouse, Roger E. Koeppe. magnetic resonance (EPR). Key residues involved in protein - protein interac- Chemistry & Biochemistry, University of Arkansas, Fayetteville, AR, USA. tions and protein - lipid interactions identified using evolutionary couplings are Transmembrane proteins constitute about 30% of the proteins in the cell and are explored using mutagenesis and differing lipid composition. As LspA has been involved in major biological processes. The dynamic properties of such proteins identified as a good candidate for an antibacterial drug target, these studies will and the ionization states of particular chemical components are important for bio- also aid in future structure-guided drug design. logical function. These vital properties of membrane proteins can be difficult to decipher, particularly for glutamic acid in the lipid environment of cell mem- 2564-Pos branes (see BBA Biomembranes 1859, 484-492). To study the ionization of glu- Characterizing the Structure of Styrene-Maleic Acid Copolymer-Lipid tamic acid in transmembrane peptides, guest glutamic acid residues were Nanoparticles (SMALPS) using Raft Polymerization for Membrane Pro- substituted into the well-defined helical model peptide GWALP23 (acetyl- tein Spectroscopic Studies GGALWLALALALALAL16ALWLAGA-amide). These guest residues were Benjamin D. Harding1, Gunjan Dixit1, Kevin M. Burridge1, Indra D. Sahu1, placed at position L16, with substitutions of glutamine or aspartic acid residues Carole Dabney-Smith1, Richard Edelmann2, Dominik Konkolewicz1, serving as controls. These chemically synthesized GWALP23 peptide deriva- Gary A. Lorigan1. tives were placed in lipid membrane environments with zwitterionic and nega- 1Department of Chemistry and Biochemistry, Miami University, Oxford, OH, tively or positively charged lipids. By varying the lipid head-groups, we hope USA, 2Department of Biology, Miami University, Oxford, OH, USA. to provide a favorable lipid environment for the glutamic acid-containing peptide Membrane proteins are necessary for the function of a variety of biological sys- helices, which more closely mimics the cell membrane. Specific 2H-labeled tems. They assist in transporting ions across membranes, acting as receptors alanine residues incorporated during peptide synthesis enable the detection of he- affecting down-stream signaling pathways and providing structure and assembly lix properties by means of solid-state NMR. To date, lipid bilayer formation has to cells. The function of a majority of proteins are known, but little is known been confirmed through solid-state 31P NMR of peptide in DMPC doped with 0% about their structure. Successful analysis of membrane proteins using biophysi- to 20% of a negatively or positively charged lipid (DMPG or DMTAP, respec- cal techniques is difficult to accomplish due to protein denaturization. Membrane tively). Lipid bilayer formation was less consistent in the presence of 20% proteins must be in a native environment once extracted, which poses a challenge DMPG or DMTAP. In the various lipid systems, 2H NMR spectra for labeled al- to extract the protein to analyze using spectroscopic techniques. Membrane anines in experimental GWALP23E16 and control GWALP23 Q16 are being mimetic systems such as detergent micelles, bicelles, nanodiscs, and lipodisqs investigated. Improvements are sought for the resolution of the broad 2H NMR have been shown to be successful for studying membrane proteins, but each sys- spectra which typically are observed with Glu-containing membrane peptides. tem has its limitations. Polymers have recently become a promising mechanism for stabilizing membrane proteins and form a homogeneous membrane mimetic 2567-Pos system. Recent studies have shown that a stabilizing polymer, styrene-maleic Lipid Membrane Influences Interaction between the C1 Domain of acid (StMA), can be synthesized using reversible-addition fragmentation chain Munc13-1 and the Activator 1 2 2 1 transfer polymerization (RAFT) styrene-maleic acid copolymer-lipid nanopar- Youngki You , Binhan Yu , Tatyana Igumenova , Joydip Das . 1Pharmacological and Pharmaceutic Science, University of Houston, ticles (SMALPs). This homogeneous SMALP system was characterized using 2 dynamic light scattering (DLS), transmission electron microscopy (TEM) and Houston, TX, USA, Biochemistry and Biophysics, Texas A&M University, Electron Paramagnetic Resonance (EPR) spectroscopy. Three different lipid en- College Station, TX, USA. vironments were titrated with styrene-maleic acid at a ratio of 3:1 (St:MA). This Munc13-1 is an essential presynaptic active zone protein involved in synaptic promising membrane mimetic system forms homogeneous SMALPs at a weight vesicle priming in neurons. It is activated by secondary messengers, such as diacyl- ratio of 1:1.5 (lipid:StMA) with nanoparticles 10 nm. V156C, a residue that lies glycerols (DAG). Munc13-1 contains a C1 domain where DAG/phorbol ester binds. in the hydrophobic interior of KCNQ1-Voltage Sensor Domain (Q1-VSD), was The C1 domain has a crucial role for Munc13-1 translocation to the plasma mem- successfully incorporated into SMALPs. Continuous Wave- EPR line shape brane. In this study, we aim to understand how the C1 domain of Munc13-1 could analysis shows two components of the spin label; one shows line shape broad- bind to ligands in the plasma membrane using HSQC NMR and Molecular Dy- ening, indicating a lower rigid motional component and the second indicates a namics (MD) studies. NMR studies reveal that the C1 domain partitions into the faster component. This study will open paths for researchers studying membrane DPC/DPS micelles and residues at the two loops are attenuated upon binding to proteins in more native membrane mimetic using biophysical techniques. the DOG in the presence of the micelles. The binding of phorbol ester to the C1 domain was in slow exchange regime. In the presence of ligand and the micelles, 2565-Pos Trp-588 side-chain interacts with both ligand and micelle. MD studies show that Characterization of Alpha-Helix Distortions at a Membrane Surface and a only a ligand-bound C1 domain could embed into the membrane. The complex of Partial 3(10)-Helix by Solid-State NMR the C1 with a phorbol ester maintained its position in the membrane by interacting Matthew J. McKay, Denise V. Greathouse, Roger E. Koeppe. with it during 100 ns. The C1 domain slightly changed its orientation, but the ligand Chemistry & Biochemistry, University of Arkansas, Fayetteville, AR, USA. was more dynamic than the C1. At 0 ns, Trp-588 side-chain forms a hydrogen bond This work aims to examine changes in protein-lipid interactions caused by simple with C9 hydroxy group of phorbol 13-acetate and forms another hydrogen bond with sequence mutations within a highly dynamic transmembrane peptide. We have C20 hydroxyl group via a water molecule. However at 100 ns, these two hydrogen 4,20 4 20 modified the GW ALP23 framework (acetyl-GGAW (AL)7AW AGA- bonds are lost. Trp-588 side-chain interacts with Pro-577 at both 0 and 100 ns. This [ethanol]amide) to incorporate either an Arg (R) residue at the central position study is significant for designing Munc13-1 inhibitors for CNS disorders. 12 or two Phe (F) residues at interfacial positions 4 and 5. Solid-state deuterium NMR spectroscopy was used to monitor specifically labeled Ala (A) residues 2568-Pos which serve as probes for the peptide dynamics and orientation in lipid bilayer Protein-Lipid Interactions Regulate Atg3 Activity in Autophagy 1 1 2 2 membranes. The GW4,20ALP23 helix adopts a tilted orientation within lipid bi- Erin R. Tyndall , Yansheng Ye , Zhenyuan Tang , Hong-Gang Wang , Fang Tian1. layers and exhibits moderate/high dynamic averaging of NMR observables 1 2 15 1 Biochemistry and Molecular Biology, Penn State University, Hershey, PA, such as the H quadrupolar splittings or N- H dipolar couplings (Biophys J. 2 114, 2617), attributed to competition between the interfacial Trp (W) residues USA, Pediatrics, Penn State University, Hershey, PA, USA. on opposing helical faces. The 2H NMR spectra indicate multi-state behavior Autophagy is a well-conserved cellular recycling process in eukaryotic cells for each of the new peptide variants. The R12 peptide helix adopts an orientation and is a critical pathway for mitigating cellular stresses. During autophagy,

BPJ 9463_9465 518a Wednesday, March 6, 2019 cargo is trafficked to a specially constructed organelle, the autophagosome, for anism may hold clues for understanding the regulation of efficiency in degradation. The formation of the autophagosome is dependent on a ubiquitin- exocytosis signaling. Slp-4 binds to vesicular Rab effector proteins through like conjugation system, where the final step is the conjugation of LC3 directly its N-terminal Slp homology domain, to SNARE complexes via its central to the amino group of the phosphoethanolamine (PE) lipids by the E2-like linker region, and contains C-terminal tandem C2A and C2B domains that enzyme Autophagy3 (Atg3). It has recently been shown that this lipidation re- interact with plasma membrane lipids. We have previously shown that the action requires the N-terminal of Atg3, and that the N-terminal demonstrates Slp-4 C2A domain binds with high affinity to liposomes approximating the sensitivity to membrane composition and curvature. Moreover, we have found lipid composition of the plasma membrane interior leaflet. The C2A domain that the N-terminal amphipathic helix (NAH) of Atg3 plays an essential role in has a lysine cluster that binds to phosphatidylinositol-(4, 5)-bisphosphate regulating its enzymatic activity. Biochemical assays of a series of NAH mu- (PIP2) and soluble inositol phosphates with micromolar affinity, but much tants demonstrate that membrane binding alone is insufficient for lipidation more strongly to membranes containing both PIP2 and anionic background of LC3, and that small changes in the NAH can impact the rate of catalysis lipids such as phosphatidylserine (PS). We are combining computational for Atg3 as a whole. NMR studies of the interactions between the NAH and and experimental approaches to reveal which residues of the Slp-4 C2A the membrane indicate that the NAH/membrane interaction induces a confor- domain participate in binding PS and PIP2. Using mutational analysis and mation rearrangement around its catalytic center. Together these experiments protein binding assays, here we show that mutation of specific individual demonstrate NAH is key for the regulation and activity of Atg3. amino acid residues tends to have modest effects on the domain’s membrane affinity; however, combined mutations at multiple predicted binding sites 2569-Pos have greater effects. This finding indicates that the C2A domain interacts Sticholysins, Sphingomyelin and Cholesterol: A Closer Look into a Tripar- with plasma membrane lipids through multiple binding sites that act in a tite Interaction concerted manner to produce its high membrane affinity. Juan Palacios Ortega1,2, Sara Garcı´a-Linares2, Esperanza Rivera-de-Torre2, Jose G. Gavilanes2,A´ lvaro Martı´nez-del-Pozo2, J. Peter Slotte1. 1 2 2572-Pos Dept Biochemistry, Abo Akademi Univ, Turku, Finland, Dept Lipid-Lipid and Lipid-Protein Interactions of the Matrix Domain of HIV- Biochemistry, Univ Complutense, Madrid, Spain. Gag at the Viral Assembly Site Actinoporins are a widely studied group of toxins produced by sea anemones. Viviana Monje-Galvan, Gregory A. Voth. Their mechanism of action consists of binding to sphingomyelin (SM)-contain- Dept of Chemistry, University of Chicago, Chicago, IL, USA. ing membranes. Once attached to the bilayer, they oligomerize and form Molecular dynamics (MD) studies can be critical to study specific protein-lipid cation-selective pores. Although SM is usually sufficient to recruit these toxins, interactions in a given system. The matrix (MA) domain of HIV-1 Gag poly- the presence of cholesterol in these membranes is also known to greatly facil- protein is the membrane targeting domain that mediates Gag binding to the itate the process. Despite years of research, there are still many unknown details plasma membrane (PM) of infected cells prior to viral assembly. Aggregation about the actinoporin-membrane interaction. What is the most favorable phase of MA at the membrane interface and insertion of its lipidated N-terminus, a state of the membrane for their action? What happens to SM distribution in the myristate (Myr) tail, impact membrane dynamics and lipid sorting in the bind- membrane after the toxins bind to the bilayer? What is the exact role of choles- ing leaflet. The specific role of MA and Myr in the viral assembly process re- terol and how do these proteins interact with it? mains unclear. We report microsecond-long all-atom MD simulations on the To shed some light on these questions we have tested the effect of sticholy- Anton2 machine to predict the mechanism of Myr insertion into lipid mem- sins, the actinoporins produced by the Caribbean sea anemone Stichodactyla branes and its impact in membrane ordering and structure. In this work, we helianthus, on bilayers of varied compositions. To study the behavior of SMs compare our observations from a single MA unit on the membrane surface in the events subsequent to toxin binding, we took advantage of the properties vs. a cluster of 3 MA proteins to determine protein-protein interactions that of fluorescent SM analogs labelled with pyrene. By measuring the extent of may be relevant for Myr exposure and insertion. Additionally, we explore the excimer to monomer ratio from the emission of pyrene we were able to the role of lipid co-localization to the MA binding site on Myr insertion and assess the degree of SM-SM interaction before and after pore formation. Simi- protein aggregation as well as the effect this co-localization has on the binding larly, we used a fluorescent analog of cholesterol, cholestatrienol, to gain and the opposite leaflet. Using Transitioned-Tempered Metadynamics (TTMe- further knowledge on the interaction between sticholysins and sterols. To taD), we compare the binding free energy landscapes across membrane models do this, we used a RET approach in which the tryptophan residues of sticho- to predict the most favorable environment for MA binding and aggregation. lysins acted as donors. 2570-Pos 2573-Pos A Cartographic View of Membrane Targeting and Association of the C2 A2A Adenosine Receptor Activation Studied by All-Atom Simulation Long Chen1, Edward R. Lyman2. Domain from Protein Kinase C 1,2 2,3 1University of Delaware, Newark, DE, USA, 2Dept Phys/Astron/Chem/Bioc, Muyun Lihan , Emad Tajkhorshid . 1Center for Biophysics and Quantitative Biology, University of Illinois at Univ Delaware, Newark, DE, USA. 2 G protein-coupled receptors (GPCRs) are the largest class of membrane pro- Urbana-Champaign, Urbana, IL, USA, NIH Center for Macromolecular teins and are involved in a wide variety of physiological processes, making Modeling and Bioinformatics, Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA, 3Department of Biochemistry, Center them targets of around 30-50% of medicinal drugs. The A2A adenosine receptor is activated by extracellular adenosine, and activates the stimulatory G-protein for Biophysics and Quantitative Biology, University of Illinois at Urbana- Champaign, Urbana, IL, USA. Gs. To better understand the activation cycle, 5 microsecond simulations were performed for the A adenosine receptor bound to NECA in two states: with a Protein kinase C (PKC) represents an essential family of cell signaling en- 2A zymes. The activation of PKC requires its binding to metal ions such as ‘‘mini G protein’’ bound, and without G protein. Analysis of helix RMSD dis- 2þ tributions suggests a special role for helix 3 in the return of the protein to the Ca , as well as its association with regions of cellular membranes that are inactive state. Analysis of contacts formed and broken during the simulation rich in signaling lipids including diacylglycerol, phosphatidylserine (PS) between the mini G protein and the receptor, and comparison to the crystal and phosphatidylinositol 4, 5-bisphosphate (PIP2). Here, we have performed structure identifies several new contacts formed between the third intracellular multiple replicas of sub-microsecond molecular dynamics simulations utiliz- loop and the mini G protein. Pathways of coupled side chain conformations be- ing a highly mobile membrane-mimetic (HMMM) model to investigate the a tween the mini G protein and the ligand binding site were identified by a mutual membrane association of PKC membrane-binding C2 domain. From our information analysis. novel analysis based on molecular cartography, distinct C2 domain membrane binding modes were revealed. We show that these modes can be determined þ 2571-Pos by Ca2 binding stoichiometry as well as binding of toxic metal ions such as þ þ Membrane Binding by Synaptotagmin-Like Protein 4: Site Directed Muta- Cd2 and Pb2 . Specific PS/PIP2 lipid interactions with the C2 domain were genesis of the Lipid Interaction Surface also identified as crucial factors for its membrane binding stability in our Aml A.A Alnaas, Julianna Oviedo, Abena Siriboe, Sherleen Tran, HMMM simulations. Our study provides fresh insights into the effects of Mikias Negussie, Hai Lin, Jefferson Knight. different metal ion binding, PS/PIP2 lipid recognition, and an interplay be- Chemistry and Biochemistry, Univ Colorado Denver, Denver, CO, USA. tween the two on the membrane binding process of PKCa C2 domain and Membrane-binding proteins play crucial roles in many biological process such on the structure and dynamics of its membrane-bound state. The cartographic as vesicle trafficking and exocytosis. Synaptotagmin-like protein 4 (Slp-4), method presented in our work has not only proven an effective tool for also known as granuphilin, is a Rab effector protein whose overexpression visualizing lipid-protein interactions in general but also for analyzing mem- is known to suppress insulin secretion despite increasing the number of secre- brane targeting and association process of peripheral membrane proteins in tory vesicles docked to the plasma membrane. Therefore, its structural mech- particular.

BPJ 9463_9465 Wednesday, March 6, 2019 519a

2574-Pos Cambridge, MA, USA, 3Center for Neutron Research, National Institute of Annexin V is a Sensor of Negative Plasma Membrane Curvature Standards and Technology, Gaithersburg, MD, USA. Christoffer Dam Florentsen1, Guillermo S. Moreno Pescador1, Cytochrome c (Cytc) mediates the electron transfer between the complexes Alexander K. Sonne1, Jesper Nylandsted2, Poul Martin Bendix1. involved in mitochondrial respiration, which is coupled with the ATP synthesis. 1Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark, A decline in this biological process leads to many bioenergetic diseases, such as 2Danish Cancer Society Research Center, Copenhagen, Denmark. heart failure, aging, and Barth syndrome. Cytc is anchored to the membrane by Motivated by important cellular repair mechanisms in mammalian cells, where cardiolipin (CL), a unique lipid in the inner mitochondrial membrane. The the protein family of annexins play a pivotal role, we employ optical manipu- interaction between CL and Cytc regulates the binding states of Cytc to the lation of plasma membrane (PM) vesicles to study dynamics of annexins within membrane and, therefore, regulates the electron transport function of Cytc. highly curved nanotubes.1 Membrane curvature induced recruitment of annex- This work aims to resolve the structures of CL-Cytc interactions and corre- ins and lateral interactions between proteins surrounding the site of rupture may sponding electron transport function of Cytc. X-ray and neutron reflectivity play an important role in molecular membrane repair processes.2 data show that pathological conditions involved CL’s exceeded concentration The PM comprises a mixture containing a plethora of different lipids and pro- and remodeled CL acyl chains facilitate a hydrophobic interaction between teins. It is desirable to maintain this complexity in a model system. A biomi- Cytc and CL. On the other hand, electrostatic interaction is maintained in metic model system allows quantitative investigation of protein-protein and healthy conditions where CL’s concentration is within a physiological range. protein-membrane interactions whereas reconstitution and encapsulation of Finally, capacitance-voltage profiling data prove that the hydrophobic interac- membrane proteins in synthetic vesicles is more problematic. We have devel- tion between Cytc and CL inhibits the electron transfer activity of Cytc, shed- oped an assay which allows optical manipulation of isolated PM vesicles con- ding light on a failure pathway of mitochondrial ATP synthesis. taining cell expressed fluorescent proteins. PM vesicles are formed using blebbing agents3 and isolated from cells. The cytosolic content from the mother 2577-Pos cell leaks into the vesicle and allows investigation of internal membrane- PS Membrane Asymmetry Influences the Folding and Insertion of a Trans- protein interactions. We have verified that both cytosolic and transmembrane membrane Helix 1 2 3 proteins are correctly located and oriented in the PM vesicles. Hence, these ves- Haden L. Scott , Frederick A. Heberle , John Katsaras , 4 icles provide a realistic platform for studying membrane protein dynamics. Francisco N. Barrera . 1The Bredesen Center for Interdisciplinary Research and Graduate Optical manipulation of these PM vesicles shows that annexins ANXA4 and 2 ANXA5 strongly sense negative membrane curvatures and become enriched Education, Univ Tennessee, Knoxville, TN, USA, Neutron Sciences, Oak Ridge National Lab, Houston, TX, USA, 3Neutron Sciences Directorate, Oak within nanoscale tubes. Intriguingly, a closely related protein ANXA2 does 4 not sense membrane curvature despite its closely related structure. The prelim- Ridge National Laboratory, Oak Ridge, TN, USA, Biochemistry & Cellular inary data also shows a remarkable difference in mobility within the protein and Molecular Biology, University of Tennessee, Knoxville, TN, USA. family. Several annexin family members have been shown to form oligomers The plasma membrane (PM) has an asymmetric distribution of lipids between on the PM, when Ca2þ is abundant and this feature may explain the observed the inner and outer leaflets of its bilayer. A lipid of special interest in eukaryotic difference in mobility and possibly also variability in curvature affinity.4 These cells is the negatively charged phosphatidylserine (PS). In healthy cells, PS is findings could help understanding how annexins synergistically work together actively sequestered to the inner leaflet of the PM but can redistribute to the outer to facilitate PM repair. leaflet when the cell is damaged or at the onset of apoptosis. However, mem- branes contain also proteins. Marginally hydrophobic membrane proteins 2575-Pos contain acidic residues in their transmembrane sequence and can experience to- Substrate Inducedconformational Changes of Liposomebound Cyto- pological transitions after membrane association. The pH low insertion peptide chrome C (pHLIP), which undergoes a topological reorientation and inserts into the mem- Raghed Kurbaj, Bridget Milorey, Reinhard Schweitzer-Stenner. brane at low pH, as its name implies, is a well-characterized model for studying Chemistry, Drexel University, Philadelphia, PA, USA. these transitions. Although it is known that the inclusion of PS in symmetric ves- The electron transfer protein cytochrome c can acquire peroxidase activity upon icles affects the membrane insertion process of pHLIP by lowering the pH its interaction with cardiolipin, an anionic lipid and a major ingredient of the in- midpoint of insertion, it is unclear how PS asymmetry affects these topological ner mitochondrial membrane. Thus far attempts to correlate peroxidase activities transitions. Here, we studied pHLIP’s topology using freely-floating asymmetric with protein conformation have not been very successful. The yet unanswered phosphatidylcholine (PC)/PS vesicles with PS enriched in the inner leaflet. We question arises whether guaiacol, a common substrate for determining peroxi- found that the protocol to create asymmetric vesicles had to be modified due to dase activity, causes structural changes of membrane bound cytochrome c.To the inclusion of PS. We used Annexin V labeled with an Alexa 568 fluorophore address this issue conformational changes of the protein cytochrome c binding as a new way of quantifying PS asymmetry. For pHLIP, membrane insertion was to liposomes of 20% 1,1’,1,2’-tetraoleyolcardiolipin (TOCL) and 80% 1,2-deo- affected by the surface charge difference between bilayer leaflets because of the leyol-sn-glycero3-phosphocholine (DOPC) in the presence of varying concentra- asymmetric distribution of charged lipids. We thus conclude that lipid asymme- tions of guaiacol were determined by visible circular dichroism and absorption try can have consequences for the behavior of membrane-associated proteins. A spectroscopy. We recently found that at slightly acidic pH (6.5) cardiolipin- corollary is that model studies using symmetric bilayers to mimic the PM may bound cytochrome c exists as an ensemble of conformations including a popula- fail to capture important details of protein-membrane interactions. tion of high spin species. These high-spin species should be more peroxidase active than the cardiolipin-bound low-spin proteins, but our results from perox- 2578-Pos idase assays suggest that the difference in peroxidase activity between the high Capturing Dynamic Transporter-Lipid Interactions and low spin cardiolipin-bound non-native proteins was indistinguishable. We Argyris Politis. measured changes of the positive Cotton band of the Soret band as a function Chemistry, King’s Coll London, London, United Kingdom. of TOCL concentration in the presence and absence of different guaiacol concen- Membrane proteins are dynamic biomolecules responsible for many critical trations at pH 6.5. We found that the addition of guaiacol changes the intensity of cellular functions. Transporters, a subclass of membrane proteins, play crucial the Cotton band significantly at intermediate cardiolipin/protein ratios (15) while roles for maintaining adequate conditions for life by moving diverse biomole- corresponding changes are small or negligible at low (5) and high cardiolipin/ cules across the biological membrane. Lipids surrounding transporters play key protein ratios (60). Based on an earlier modeling of spectroscopic response roles in ensuring correct protein function. Accumulating evidences suggest that data we interpret these findings as indicating that substrate binding to the protein transporters function as complexes with their surrounding membrane lipids. stabilizes the unfolded conformation of membrane bound proteins in a way that Despite advances on understanding membrane protein-lipid interactions, our allows for an unfolding of the protein at lower cardiolipin concentrations. Similar knowledge of the role of lipids in mediating protein dynamics remains limited. measurements at pH 7.4 and additional peroxidase activity measurements are un- Here, we showcase how membrane lipids impact on the oligomeric state and derway and will be presented at the conference. conformational dynamics of transporters. We show using native mass spec- trometry on a eukaryotic purine symporter that specific lipids binding to the 2576-Pos protein modulate the formation of its oligomeric states. Mechanistically, Membrane-Bound Structures and Associated Electron Transport Func- such lipid modulation is obtained through specific interactions of structural tions of Cytochrome C lipids with the protein, which has a stabilising effect in the protein interface. Minh D. Phan1, Keel Yong Lee2, Hanyu Wang1, James F. Browning1, We also show using the emerging and powerful method of hydrogen- Sushil K. Satija3, John F. Ankner1. deuterium exchange mass spectrometry (HDX-MS) that specific lipid-protein 1Neutron Scattering Div, Oak Ridge National Lab, Oak Ridge, TN, USA, interactions modulate the conformational dynamics of the homologous trans- 2Wyss Institute for Biologically Inspired Engineering, Harvard University, porters LacY and XylE from the major facilitator superfamily (MFS), the

BPJ 9463_9465 520a Wednesday, March 6, 2019 largest family of transporters. Specifically, we reveal that phosphatidylethanol- 2581-Pos amine (PE) lipids interfere with the formation of their conserved networks, crit- Determination of Heat Production in Human Skeletal Muscle from Mea- D ical for stabilising transporter states, thus unravelling a generic mechanism for surements of Basal Ca2 Movements the conformational transitions of MFS transporters between different states. Christopher J. Barclay, Bradley S. Launikonis. Overall, this work allows us to establish that the dynamics of transporter- Sch Biomed Sci, The University of Queensland, Brisbane, Australia. lipid assemblies can be captured in solution, enabling us to define changes in Skeletal muscle in mammals generates heat that is used to maintain body tem- the conformation of membrane proteins at high resolution. perature. Heat generation is largely attributable to the ATP splitting activity of the sarcoplasmic reticulum (SR) Ca2þ pump. The muscle spends most of 2579-Pos its time at rest, so harvesting heat from the muscle in this state provides an effec- Structural Basis for the Lipid-Mediated Interaction of Tubulin with tively constant generation of heat, likely dependent on RyR leak. Recently the VDAC Revealed by Neutron Reflectometry SR Ca2þ leak through the ryanodine receptor (RyR) was detected in resting hu- David P. Hoogerheide1, Sergei Y. Noskov2, Philip A. Gurnev3, man muscle fibres using a novel confocal imaging technique utilizing a Ca2þ- Tatiana K. Rostovtseva3, Sergey M. Bezrukov3. sensitive dye trapped in the sealed tubular (t-) system of skinned fibres (Cully 1Center for Neutron Research, National Institute of Standards and PNAS 2þ 2þ 2 et al 2018, ). Detection of RyR Ca leak via measurements of Ca up- Technology, Gaithersburg, MD, USA, Center for Molecular Simulation, take into the sealed t-system is the net outcome of a complex set of events. To Biochemistry Research Cluster, University of Calgary, Calgary, AB, Canada, 3 allow the prediction of heat generated by the muscle a model was devised that Section on Molecular Transport, NICHD NIH, Bethesda, MD, USA. divided the system into 7 discrete spaces across the t-system, SR, cytoplasm A preponderance of evidence suggests that the mitochondrial outer membrane and junctional space. Ca2þ movements between these spaces were linked by (MOM) binds dimeric tubulin, which blocks the flux of metabolites through the RyR, PMCA, NCX and SERCA, with EGTA buffering Ca2þ in the cyto- VDAC, the major MOM channel, by inserting its negatively charged, disordered plasm and junctional space; and SR Ca2þ buffered by calsequestrin. A system C-terminal tail into the VDAC pore. The discovery of this novel regulatory mech- of inter-related differential equations to describe the system were solved using anism of mitochondrial respiration has raised several fundamental questions, Maple software. Model optimization showed the factors that influence t-system including how tubulin binds to the MOM surface, whether its membrane-bound Ca2þ uptake were PMCA density, PMCA Ca2þ affinity, pH and t-system leak conformation is consistent with the accessibility of its C-termini to the membrane rate. The model determined a SR leak rate between 0.01 and 0.02 s1, consistent surface, and what role mitochondrial lipids assume in mediating the tubulin- with experimental data (Cully et al 2018). The model was then used to quantify VDAC interaction. Here we report neutron reflectivity (NR) studies on tubulin- SR Ca2þ efflux to estimate heat production associated with Ca2þ cycling be- coated and VDAC-containing biomimetic membranes. In combination with mo- tween SR and cytoplasm of 0.4 W kg1, which is about half of the likely lecular dynamics simulations, these reveal, first, that dimeric tubulin is anchored heat production of quiescent (or resting) human skeletal muscle. to the membrane surface by an amphipathic a-helix of the a-tubulin subunit in an orientation that presents the C-termini of both tubulin subunits to the membrane 2582-Pos surface. The structural model is supported by electrophysiological evidence using Enhancement of Sarcolemmal Calcium Influx in a Novel Mouse Model of recombinant tubulin constructs in which C-terminal tails bound to either subunit Malignant Hyperthermia are individually observed in a VDAC nanopore. Second, we demonstrate using Vikas Kaura1, JoseR.Lo´pez2, Marie-Anne Shaw1, Paul D. Allen1, NR that surface-immobilized VDAC can be reconstituted into a complete lipid Philip M. Hopkins1. membrane, thus creating a ‘‘protein-tethered bilayer lipid membrane’’ (ptBLM) 1Leeds Institute of Medical Research at St James’s, Leeds, United Kingdom, over a large sample area. The ptBLM architecture allows dynamic changes of 2Mount Sinai Medical Center, Miami, FL, USA. the solution environment, while NR reveals the corresponding structural response Malignant hyperthermia (MH) is a potential fatal skeletal muscle disorder that of the protein and its lipid environment. This allows us to experimentally observe occurs in genetically susceptible individuals on exposure to certain anaesthetic the effects of pH-induced changes in VDAC’s structure on the local lipid environ- agents. The type 1 ryanodine receptor (RYR1) missense variant c.7300G>A ment. Molecular dynamics simulations are in good agreement with NR experi- (p.G2434R) is the most common human MH variant. It is classed as a weak ments. We speculate that local curvature or lipid packing stress induced by variant and is commonly associated with genotype-phenotype discordance. membrane-embedded VDAC and its structural dynamics serves to localize cyto- The isogenetic p.G2435R variant in a novel knock-in mouse model has been solic regulators such as tubulin to the vicinity of the channel. shown to have features similar to the human MH response. We examined how the p.G2435R variant affects resting sarcolemma Ca2þ entry (RCaE) using Posters: Excitation-Contraction Coupling II the manganese-quench of Fura2 in wildtype (WT), heterozygous (HET) and homozygous (HOM) RYR1G2435R myotubes. We found a gene dose- dependent increase in RCaE with the RYR1G2435R variant. The non-specific 2580-Pos cation channel blocker gadolinium (Gd3þ) produced a profound block on sarco- Identification of Novel RyR1 Inhibitors by High-Throughput Screening us- 2D lemmal RCaE in all three genotypes while blockade using BTP2, which inhibits ing ER Ca Measurement both TRPC and Stim1/Orai1 entry pathways, had less of an effect. Administra- 1 1 1 Hiroyuki Matsukawa , Takashi Murayama , Takuya Kobayashi , tion of the TRPC3/6 specific blocker SAR7334 had a significant effect in only 1 2 2 Nagomi Kurebayashi , Mari Ishigami-Yuasa , Shuichi Mori , the HOM and HET myotubes. The gene dose-dependent increase in RCaE with Hiroyuki Kagechika2, Takashi Sakurai1. 1 2 the RYR1G2435R variant mirrors the gene dose-dependent increase in resting Juntendo University, Tokyo, Japan, Tokyo Medical and Dental University, myoplasmic [Ca2þ] we have previously observed with this variant. The mech- Tokyo, Japan. 2þ anisms behind this increased RCaE were explored and appear to be the result of Genetic mutations in type 1 ryanodine receptor (RyR1), a Ca -release channel multiple factors, all of which are sensitive to Gd3þ. on the sarcoplasmic reticulum of skeletal muscle, cause various muscle diseases including malignant hyperthermia (MH). Because the main underlying mecha- 2583-Pos þ nism of the pathogenesis is overactive Ca2 release by gain-of-function of the Mutation Analysis of the Calcium Binding Site of Skeletal Muscle Ryano- RyR1 channel, inhibition of RyR1 is expected to be a promising treatment for dine Receptor Calcium Release Channel these diseases. We have recently developed an efficient high-throughput Venkat R. Chirasani1,LeXu2, Jordan S. Carter3, Hannah G. Addis3, þ screening (HTS) platform for RyR1 inhibitors using Ca2 measurements in Daniel A. Pasek2, Nikolay V. Dokholyan1, Gerhard Meissner2, the endoplasmic reticulum (ER) and successfully identified three novel com- Naohiro Yamaguchi3. pounds from a library of well-characterized drugs (Murayama et al., Mol Phar- 1Penn State College of Medicine, Hershey, PA, USA, 2University of North macol, 94: 722-730, 2018). However, only oxolinic acid was found to be specific Carolina at Chapel Hill, Chapel Hill, NC, USA, 3Medical University of South to RyR1 and other two compounds inhibited both RyR1 and RyR2, a cardiac iso- Carolina, Charleston, SC, USA. form. To explore another RyR1-specific inhibitor, we performed HTS from a li- Skeletal muscle ryanodine receptor Ca2þ release channel (RyR1) plays a pivotal þ brary of larger numbers of compounds. ER Ca2 of HEK293 cells expressing role for muscle contraction by releasing Ca2þ from sarcoplasmic reticulum. RyR1 carrying an MH mutation (R2163C) was monitored with R-CEPIA1er, Channel activities of RyR1 are regulated by multiple molecules including þ a genetically encoded ER Ca2 indicator, using FlexStation3 fluorometer. Com- Ca2þ,Mg2þ, and ATP. Recent near-atomic level structure model of RyR1 iden- þ þ pounds which inhibit RyR1 would increase ER Ca2 by preventing Ca2 leak tified a binding site for Ca2þ, which is comprised of three direct binding amino via the mutant RyR1. We successfully identified several RyR1-specific com- acids (E3893, E3967, and T5001) and two indirect binding residues (H3895 and pounds that are structurally different from oxolinic acid or dantrolene, a known Q3970) [1]. To assess functional significance of these amino acids for Ca2þ- RyR1 inhibitor. We are currently investigating how these compounds inhibit the dependent activation of RyR1, we constructed mutant RyR1s, expressed them RyR1 channel. These compounds may be good candidates for treatment of in HEK293 cells, and characterized their function by single channel recordings RyR1-related diseases. and [3H]ryanodine binding assays. Peak channel activities of wild-type RyR1

BPJ 9463_9465 Wednesday, March 6, 2019 521a

þ were observed at 10-100 mMCa2 . However, mutations on E3893 and E3967 Liverpool, United Kingdom, 3Graduate School of Pharmaceutical Sciences, þ impaired Ca2 -dependent activation of RyR1. E3893Q/E3967Q double mutant Kyoto University, Kyoto, Japan. þ showed relatively high activity at submicromolar Ca2 and was inhibited by 10- Coupled gating of ryanodine receptor (RyR) channels is thought to play a role in þ 100 mMCa2 , while the mutant retained activation by ATP and . shaping the spatiotemporal characteristics of Ca2þ-release from the sarco- þ T5001A mutation decreased the affinity for the activation Ca2 . A mutation plasmic reticulum (SR) of striated muscle. However, the junctional SR mem- on Q3970 was reported to be associated with congenital skeletal myopathy, cen- branes are crowded with many diverse types of ion-channels. These include tral core disease (CCD) [2]. We found that CCD-linked Q3970K mutation atten- the two subtypes of trimeric intracellular cation channels, TRIC-A and TRIC- þ uated Ca2 activation of RyR1 as well as caffeine and ATP activation in single B, which are present in an approximate ratio of 5-10 TRIC-A: 1 TRIC-B: 1 channel recordings. An interesting finding is that Q3970K mutation did not alter RyR. We therefore examined the evidence for cooperative gating between and single channel conductance of RyR1, while previous studies showed decreased within different species of SR cation channels after incorporating SR vesicles single channel conductance of other loss-of-function CCD mutants of RyR1. from wild type (WT) and Tric-a knockout (KO) skeletal muscle into artificial Combined with in silico analysis of the mutant structure, our functional studies membranes. We found that the open probability (Po) of a single RyR1 channel þ reveal that the Ca2 binding site identified in the high resolution RyR1 structure (0.00450.002; n=9) was significantly lower than the average Po when multiple þ serves as a Ca2 regulatory domain. Supported by NIH. channels were present in the bilayer (0.06050.022; n=8; *p<0.05). We also [1] des Georges et al. (2016) Cell 167, 145; [2] Snoeck et al. (2015) Eur. J. Neu- observed that the average Po of SR Kþ-channels from Tric-a KO SR increased rol. 22, 1094. as increasing numbers of channels were incorporated into the bilayer, indicating that TRIC-B gates in a cooperative manner. This gating characteristic was not 2584-Pos observed in SR Kþ-channels derived from WT SR thus suggesting, since Molecular Dynamics Simulation of Ryanodine Receptor in the Presence 2D TRIC-A is the most abundant subtype in WT SR, that TRIC-A channels do and Absence of Ca Binding not functionally couple together. We also investigated whether the presence Han Wen, Wenjun Zheng. of actively gating RyR channels would influence the behaviour of SR Kþ-chan- Dept Physics, State Univ New York Buffalo, Buffalo, NY, USA. nels or vice versa. No evidence for cooperative interactions between RyR and Ryanodine receptors (RyRs), a group of homotetrameric intracellular calcium SR Kþ-channels were found in either WT or Tric-a KO SR. Thus, functional release channels, play a key role in muscular and neuronal activities. RyRs- coupling between SR cation channels appears to be restricted to RyR-RyR þ mediated calcium release can be activated and regulated by Ca(2 ) binding and TRIC-B-TRIC-B interactions. This will be important physiologically, in skeletal muscle (to RyR1) and cardiac muscle (to RyR2). Despite recent providing distinct amplification of specific ion fluxes during SR Ca2þ-release progress in structural studies of RyRs, a detailed understanding of the activa- and may be particularly important in tissues where there is little or no TRIC-A. tion mechanism of RyRs has been obscured by the lack of high-resolution struc- Supported by the BHF and JSPS. tural and dynamical information for RyRs in different functional states. To elucidate how Ca(2þ) binding impacts the dynamics and energetics of 2587-Pos RyR1, we performed molecular dynamics simulation of the C-terminal core Structure Development of Oxolinic Acid, a Novel Inhibitor of Type 1 Rya- D domain of an RyR1 Cryo-EM structure in the presence and absence of nodine Receptor (RyR1) Ca2 Release Channel Ca(2þ). Our simulation revealed elevated dynamics and specific interactions Yoshiaki Nishijima1, Takashi Murayama1, Shuichi Mori2, Hiroto Iinuma2, in the presence of Ca(2þ), which may prime RyR1 for activation. The struc- Noriaki Manaka2, Nagomi Kurebayashi1, Mari Ishigami-Yuasa2, tural and dynamic insights gained from this study will guide future functional Hiroyuki Kagechika2, Takashi Sakurai1. studies of the RyR1 Ca(2þ) activation. 1Juntendo Univesity, Tokyo, Japan, 2Tokyo Medical and Dental University, Tokyo, Japan. 2585-Pos Type 1 ryanodine receptor (RyR1) is a Ca2þ release channel on the sarcoplasmic Effects of Novel RyR2 Inhibitors on Diseased Hearts reticulum in the skeletal muscle. Mutations in RyR1 cause various muscle dis- Nagomi Kurebayashi1, Takashi Murayama1, Mai Tamura1, Shuichi Mori2, eases including malignant hyperthermia (MH) and central core disease (CCD). Mari Yuasa-Ishigami2, Hiroyuki Kagechika2, Junji Suzuki3, Although dantrolene is the only therapeutic drug for MH, it cannot be used for Kazunori Kanemaru4, Masamitsu Iino4, Sachio Morimoto5, Takashi Sakurai1. CCD due to its lower solubility and side effects. It is therefore urgent to discover 1 2 Dept Pharmacology, Juntendo Univ Sch Med, Tokyo, Japan, Tokyo novel RyR1 inhibitors. We have recently developed an efficient high-throughput 3 Medical and Dental University, Tokyo, Japan, UCSF, San Francisco, CA, screening platform for RyR1 inhibitors using Ca2þ measurements in the endo- 4 5 USA, Nihon University, Tokyo, Japan, International Univ Health Welfare, plasmic reticulum (ER). By screening a library of well-characterized drugs, we Fukuoka, Japan. successfully identified oxolinic acid as a novel RyR1 inhibitor (Murayama et 2þ The type 2 ryanodine receptor (RyR2) is the Ca release channel on the sarco- al., Mol Pharmacol, 94: 722-730, 2018). However, affinity of oxolinic acid was plasmic reticulum and plays a pivotal role in the excitation-contraction coupling much lower than that of dantrolene. In this study, we designed and synthesized in the heart. It is known that excessive activation of RyR2 by abnormal phos- a series of quinolone derivatives using oxolinic acid as a lead compound. Dose- 2þ phorylation or amino acid mutation increases spontaneous Ca release in car- dependent inhibitory effects were evaluated by ER Ca2þ measurement using diomyocytes and results in arrhythmia. Therefore, drugs that suppress activity of HEK293 cells expressing R-CEPIA1er, a genetically-encoded EC Ca2þ indica- RyR2 are expected to have anti-arrhythmic effects, but specific inhibitors of tor, and RyR1 carrying an MH mutation (R2163C). Compounds bearing a longer RyR2 have not been reported yet. Recently, we searched for novel RyR2 inhib- alkyl chain at the nitrogen atom of quinolone ring exhibited stronger RyR1 inhib- itors by high-throughput screening using HEK293 cells expressing RyR2 and iting activity. Modification at 6 and 7 positions of quinolone ring greatly affected 2þ ER Ca indicator R-CEPIA1er, and found four candidates as RyR2 inhibitors. the inhibitory activity. The most potent derivative so far exhibited >50 and 4-fold In this study, we assessed their effects on non-cardiac and cardiac cells. All four greater affinity than oxolinic acid and dantrolene, respectively. These compounds 2þ compounds suppressed spontaneous Ca release in HEK293 cells expressing may be good candidates for treatment of RyR1-related diseases. RyR2. These compounds more or less suppressed Ca2þ-dependent [3H]ryano- dine binding in microsomes obtained from RyR2 expressing cells. The effects 2588-Pos of these compounds on cardiac myocyte from dilated cardiomyopathy (DCM) Investigation of Mutant Ryanodine Receptor Channel Activity using Func- model mice were examined because cardiac muscles from this model mice tional Analysis and Molecular Dynamics show elevated RyR2 phosphorylation and frequent spontaneous activity after ac- Toshiko Yamazawa1, Haruo Ogawa2, Maki Yamaguchi1, tion potential (AP)-induced Ca2þ transients. One among the four compounds Takashi Murayama3, Hideto Oyamada4, Junji Suzuki5, was inapplicable to cardiomyocyte because it inhibited AP generation. The re- Nagomi Kurebayashi3, Kazunori Kanemaru6, Takashi Sakurai3, maining three compounds were able to suppress spontaneous activity after Iino Masamitsu6. 1 AP-induced Ca2þ transients. Our findings suggest that these compounds may Department of Molecular Physiology, Jikei University School of Medicine, 2 have anti-arrhythmic effects on RyR2-related arrhythmias. Tokyo, Japan, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan, 3Department of Pharmacology, Juntendo University 2586-Pos School of Medicine, Tokyo, Japan, 4Department of Pharmacology, Showa Cooperative Gating Among Ion-Channel Species in Junctional Sarco- University, Tokyo, Japan, 5Dept Physiol, Univ California San Francisco, San plasmic Reticulum Francisco, CA, USA, 6Department of Cellular and Molecular Pharmacology, Elisa Venturi1, Fiona O’Brien2, David Eberhardt1, Katja Witschas1, Nihon University School of Medicine, Tokyo, Japan. Sam El-Ajouz1, Tsunaki Iida3, Miyuki Takeshima3, Hiroshi Takeshima3, Membrane depolarization is translated into intracellular Ca2þ signals, and rya- Rebecca Sitsapesan1. nodine receptors (RyRs), located in the sarcoplasmic/endoplasmic reticulum 1Department of Pharmacology, University of Oxford, Oxford, United membrane, play a key role in intracellular Ca2þ release. There are three iso- Kingdom, 2Institute of Ageing and Chronic Disease, University of Liverpool, forms of RyRs, of which type 1 RyR (RyR1) is dominantly expressed in the

BPJ 9463_9465 522a Wednesday, March 6, 2019 skeletal muscle. Mutations in the RyR1 gene (RyR1) cause severe muscle dis- 35% and 60% for Dysf-W52R, -V67D, and -V68L, -A84V, and -DC2A, eases, such as malignant hyperthermia (MH), which is a disorder of Ca2þ- respectively. Thus, all mutants except Dysf-W52R were incorporated into t-tu- induced Ca2þ release via the RyR1 in skeletal muscle. So far, more than 300 bules either partially or as efficiently as wild type. Venus-C2A alone, expressed mutations have been reported in RyR1 of patients with MH, and most of those with or without the TM domain, did not support the recovery of the VICT after mutations have been found in three ‘‘hot spot’’ regions of RyR1. However, due OSI (both 50%). However, Venus-C2A lacking the TM domain significantly to lack of comprehensive analysis of the structure-function relationship of reduced Ca2þ waves after OSI (10% vs 83% of Venus- C2A-TM). We conclude mutant RyR1 Ca2þ release channels, the mechanism remains largely unknown. that C2A modulates coupling of excitation to Ca2þ release in murine skeletal Here, we combined functional studies and molecular dynamics (MD) simula- muscle, and that changes in coupling (e.g., with the Dysf-W52R and -V67D tion of RyR1 channels carrying disease-associated mutations at the N-terminal mutations) may be sufficient for pathogenicity. Supported by the Jain Founda- region. When expressed in HEK293 cells, those mutant RyR1 Ca2þ release tion, and NIH (RO1 AR064268). channels caused abnormalities in Ca2þ homeostasis. MD simulation of the mutant RyR1 revealed that hydrogen bonds/salt bridges between subdomains 2591-Pos strongly correlate with the channel function of RyR1. Especially, the mutations Measurements of Triadic Calcium in Differentiated Muscle Fibers using a of Arg402, which plays key role in connecting three subdomains (A, B, C) of Gcamp Probe Targeted to the Junctional Sr Membrane the N-terminal region, exhibit interesting result. Indeed, B and C subdomains Colline Sanchez, Christine Berthier, Bruno Allard, Vincent Jacquemond. together rotate clockwise with respect to A subdomain in the mutants. This Institut NeuroMyoGene - Universite Claude Bernard Lyon 1 - CNRS UMR 5310 - INSERM U1217, Lyon, France. movement may increase the open probability of the channel, and this increase 2þ may be the main cause of MH in the Arg402 mutants. With the aim of gaining further insights into sarcoplasmic reticulum (SR) Ca release function in skeletal muscle, we used a domain consisting of the 306 res- 2589-Pos idues (T306) of the N-terminal end of triadin to target the Ca2þ-sensitive probe Characterization of an Animal Model for Congenital Myopathies Linked GCaMP6f to the junctional SR membrane of muscle fibers. Muscles from to Recessive RyR1 Mutations mouse were electroporated with plasmid constructs encoding GCaMP6f or Moran Elbaz1, Alexis Ruiz1, Jan Eckhardt1, Susan Treves1,2, T306-GCaMP6f. Muscle fibers were isolated 1-2 weeks later and voltage- Francesco Zorzato1,2. activated fluorescence changes were studied with line-scan confocal micro- 1Dept. of Anesthesiology and Biomedicine, University Hospital Basel, Basel, scopy under voltage-clamp control. GCaMP6f fluorescence was homogenous Switzerland, 2Dept Life Sci & BioTech, University of Ferrara, Ferrara, Italy. throughout the transfected fibers and experienced large changes upon mem- The skeletal muscle sarcoplasmic reticulum calcium release channel (ryanodine brane depolarization in the absence of exogenous Ca2þ buffer in the intracel- receptor, RyR1) plays a fundamental role in excitation-contraction coupling. lular solution (e.g. mean relative change of 20 in response to a 0.5 s-long Dominant and recessive mutations in RYR1, the gene encoding the RyR1, are pulse from 80 to 15 mV). When compared with simultaneously measured associated with a range of neuromuscular disorders including the core myopa- rhod-2 Ca2þ transients, GCaMP6f transients showed obvious signs of kinetic thies central core disease and multi-minicore disease, congenital fiber type delays with, for instance, the time constant of decay after the end of short de- disproportion, centronuclear myopathy as well as the pharmacogenetic disorder polarizing pulses being 4-5 times that of the rhod-2 transients. In contrast, malignant hyperthermia. For many dominant mutations the functional effect has T306-GCaMP6f expression was localized to the vicinity of one or of a few been studied, however, to date the functional consequences of recessive RYR1 nuclei. Within these spots, fluorescence yielded a transverse banded pattern mutations have not been investigated in depth. Using the CRISPR/CAS9 tech- consistent with triadic localization. Upon membrane depolarization, T306- nology we created a mouse model knocked-in for mutations which were identi- GCaMP6f generated fluorescence transients of faster kinetics and much lower fied in a severely affected multiminicore patient, namely the RyR1A4329D and relative amplitude than T306-free GCaMP6f (e.g. mean relative change of 3.2 RyR1Q1970fsX16t mutations. In this study we compared the phenotype of the in response to a 0.5 s-long pulse to 15 mV), suggesting reduced affinity and compound heterozygous (RyR1A4329D/RyR1Q1970fsX16t) mice with that faster kinetics of the SR membrane-bound probe as compared to the free probe. of age-matched wild-type and single heterozygous RYR1 mutant littermates. In several fibers, voltage-induced fluorescence changes at different positions of The compound heterozygous mice are viable, but their body weight during the scanned line showed time-course discrepancies which may reflect distinct the first 20 weeks after birth is on average 20% lower than that of WT or behaviors of ryanodine receptor activation. Supported by CNRS, INSERM, RyR1A4329D and RyR1Q1970fsX16t heterozygous siblings. Spontaneous Universite Lyon 1 and AFM-Telethon MyoNeurAlp 2.3.1.3. motor activity assessed with a voluntary running wheel, revealed that RyR1A4329D/RyR1Q1970fsX16t compound heterozygous mice run signifi- 2592-Pos cantly less compared to age-matched WT. The mechanical properties of isolated Mathematical Model to Simulate Sr Calcium Release in Mammalian Skel- etal Muscle with Impaired T-Tubular Structure muscles from compound heterozygous mice are different from those of WT 1 2 1 mice. In conclusion the results of our study show that the phenotype of the Vincent Jacquemond , Peter Szentesi , Candice Kutchukian , Beatrix Dienes2, Laszlo Csernoch2. RyR1A4329D/RyR1Q1970fsX16t compound heterozygous mice largely reca- 1  RYR1 Institut NeuroMyoGe`ne, Universite Claude Bernard Lyon 1, Lyon, France, pitulate the clinical picture of MmD patients harboring recessive muta- 2 tions. These results also suggest that the RyR1A4329D/RyR1Q1970fsX16t Department of Physiology, University of Debrecen, Debrecen, Hungary. compound heterozygous mouse is an animal model which can be exploited for Calcium release from the sarcoplasmic reticulum (SR) in mammalian skeletal preclinical studies aimed to treat congenital myopathies linked to recessive muscle is under the tight control of the voltage sensor dihydropyridine receptors RYR1 mutations. (DHPR-s) residing in the adjacent transverse- (T-)tubular membrane. This firm control manifests in the lack of SR calcium release channels (ryanodine receptors; 2590-Pos RyR-s) to activate in response to elevations in cytosolic calcium concentration Role of Dysferlin’s C2A Domain in Voltage-Induced Calcium Release ([Ca2þ]). Nevertheless we have recently reported that in certain centronuclear After Osmotic Shock in Murine Skeletal Myofibers myopathies spontaneous local elevations in [Ca2þ], calcium sparks, occur in Valeriy I. Lukyanenko, Joaquin Muriel, Robert J. Bloch. resting, intact muscle fibers. In addition, mutations in the Mtm-1 gene, responsible Physiology, Univ Maryland, Baltimore, MD, USA. for myotubular centronuclear myopathy also result in suppressed and spatially Osmotic shock injury (OSI) decreases the amplitude of voltage-induced Ca2þ- heterogeneous SR calcium release. A hallmark of this disease is disruption in transients (VICTs) in dysferlin-null (A/J) but not control (A/WySnJ) myofibers the T-tubular network resulting in fiber areas with defective calcium signaling. (Kerr et al., Proc. Natl. Acad. Sci. USA, 2013). We recently showed using OSI Here we modelled the pathological condition by simulating the interaction be- that dysferlin modulates the coupling of excitation to Ca2þ release (Lukya- tween the DHPR and RyR together with the diffusion of calcium ions in the nenko et al., J. Physiol. 2017). Here we studied dysferlin-null A/J mouse myoplasmic space. Results indicate that introducing areas where the T-tubular FDB myofibers expressing mutated fluorescent dysferlin chimeras to examine defect would disable the interaction between the voltage sensor and the calcium the role of dysferlin’s C2A domain in that coupling. We studied dysferlin release channel cannot fully account for the observed heterogeneity in the cal- missing C2A as well as C2A point mutations (pathogenic: W52R, V67D; poly- cium release process. Furthermore, allowing calcium ions to activate RyR in morphisms: V68L; A84V), and Venus-C2A alone, expressed with and without these regions was still ineffective in describing all aspects of SR calcium dysferlin’s transmembrane (TM) domain. Dysferlin-DC2A, -W52R and -V67D release present in line-scan images recorded under voltage-clamp conditions reduced recovery of VICTs after OSI (to 37%, 43% and 31% of controls, using Rhod-2 and confocal imaging. The best results were obtained when areas respectively). Dysf-V68L (72%) and -A84V (72%) were statistically identical where RyR-s were not activatable at all were introduced together with spatially to WT Venus-dysferlin (69%). Dysferlin localization to t-tubules, examined segregated areas where the opening of RyR-s were either controlled by the with C-terminal pHluorin constructs by reducing extracellular pH to 6.5 voltage sensor or by calcium. These observations strongly argue in favor of showed 60% reduction of fluorescence for WT Dysf-pHluorin fluorescence the hypothesis that calcium-induced calcium release is present and decisive at pH6.5, 10-15% for the negative control (N-terminal pHluorin), and 10%, in myotubular myopathy.

BPJ 9463_9465 Wednesday, March 6, 2019 523a

Supported by: Hungarian National Research, Development, and Innovation Of- Exocytosis of catecholamine (adrenaline and noradrenaline) from adrenal chro- fice (NKFIH K-115461), AFM-Telethon. maffin cells is important for stress responses and insulin release from pancreatic beta cells is critical in regulating blood glucose levels. These exocytic pro- 2593-Pos cesses are highly regulated by a dozens of key proteins in eukaryotic cells. Post-Developmental Knockout of Orai1-Mediated Store-Operated Cal- While the origin, transport, and delivery of cargo-loaded vesicles (dense-core cium Entry Improves Muscle Pathology in a Mouse Model of Muscular vesicles) and the proteins involved is well studied, how these proteins are orga- Dystrophy nized with respect to dense-core vesicles (DCVs) at the nanoscale remains to be Maricela Garcı´a-Castan˜eda, Antonio Michelucci, Robert T. Dirksen. explored. Here, we directly image several proteins that are key regulators of the Pharmacology and Physiology, University of Rochester, Rochester, NY, USA. transport, docking, and priming of DCVs at the plasma membrane. Specifically, Duchenne muscular dystrophy (DMD), a genetic disorder caused by mutations correlative super-resolution (dSTORM) light and platinum replica transmission in the dystrophin gene, is characterized by progressive muscle degeneration/ þ þ electron microscopy (CLEM) imaging was performed on PC12 cells (a rat ad- weakness. Enhanced store-operated Ca2 entry (SOCE), a Ca2 influx mecha- 2þ renal chromaffin-derived neuroendocrine cell line) that were transfected with nism coordinated by STIM1 and Orai1, is proposed to potentiate Ca -medi- proteins of interest. We aligned super-resolution dSTORM images of Rab pro- ated damage in DMD. To determine the role of SOCE in promoting teins (Rab27a and Rab3a) and Rab effector proteins (granuphilin and Rabphi- muscular dystrophy, we crossed mdx mice with tamoxifen-inducible, muscle- mdx- lin) with corresponding TEM images of the plasma membrane and observed the specific Orai1 knockout mice ( Orai1 KO). Analysis of transcript levels re- precise correlation between proteins and vesicles at the plasma membrane. The vealed that Orai1 expression was 1.5x higher in muscle from mdx mice mdx nano-scale molecular architecture of DCVs generated from this correlative im- compared to WT mice, while nearly undetectable in muscle from -Orai1 aging will help us understand how key proteins guide the docking, priming, and KO mice. Levels of Orai1 transcript correlated with SOCE activity measured þ fusion of DCVs in endocrine cells. using Mn2 quench of fura-2 fluorescence in single flexor digitorum brevis muscle fibers. Specifically, SOCE was increased in mdx fibers and absent in fi- 2596-Pos bers from mdx-Orai1 KO mice. Moreover, compared to WT fibers, mdx fibers Relation between Release of Catecholamines and FFN511 Studied with exhibited: i) increased myoplasmic Ca2þ levels; ii) reduced Ca2þ store content; Electrochemical Detector Arrays þ and iii) a significant prolongation in the rate of electrically-evoked Ca2 tran- Shailendra Singh Rathore1, Meng Huang2, Manfred Lindau1. sient decay. All of these effects were normalized in fibers from mdx-Orai1 KO 1Applied and Engineering Physics, Cornell Univ, Ithaca, NY, USA, mice. Ex vivo contractility experiments revealed that extensor digitorum longus 2Materials Science and Engineering, Cornell Univ, Ithaca, NY, USA. muscles from mdx mice displayed a reduction in maximal tetanic specific force Exocytosis is an essential vesicle fusion process that mediates transmitter (150.353.5 mN/mm2) compared to WT muscles (199.6511.1 mN/mm2), a release. For high resolution spatiotemporal analysis of single vesicle fusion parameter ameliorated in muscles from mdx-Orai1 KO mice (188.1510.9 events, a combinatorial approach of amperometry and fluorescence imaging mN/mm2). When exposed to 10 consecutive eccentric contractions, mdx mus- was employed. Amperometric spikes from physically stimulated bovine chro- cles exhibited a higher damage-induced force reduction than WT muscles (re- maffin cells loaded with the Fluorescent False Neurotransmitter FFN511were sidual force after the 10th eccentric contraction was 35.553.8% and detected by microfabricated four-electrode electrochemical detector (ECD) ar- 61.851.9%, respectively). Interestingly, Orai1 ablation partially protected rays. To determine the location of a fusion event leading to amperometric spikes muscles from damage in mdx-Orai1 KO mice (residual force was with different quantal size in the different ECD array electrodes, random walk 46.152.6% after 10 contractions). Our results indicate that enhanced Orai1- simulations were performed using the actual ECD array geometry. The location dependent SOCE exacerbates the dystrophic phenotype and Orai1 deficiency of a measured release event was fitted to match the simulated data. To account promotes sarcolemmal integrity/stability during eccentric contraction. for drift of the ECD array, the pixel shift of each frame was fitted relative to the image that was used to generate the simulation map. The transparent glass sur- 2594-Pos face in the center of the ECDs allowed the concurrent fluorescence imaging of Multiple Sites of Interaction May Be Involved in the Regulation of CaV1.1 localized loss of FFN511from secretory vesicles in TIRF microscopy mode. by Stac3 1 2 1 The frequency of amperometric spikes was much higher than that of FFN511 Alexander Polster , Philip M. Hopkins , Kurt G. Beam . release events. Only the amperometric spikes that were correlated in time and 1Physiology & Biophysics, University of Colorado Denver, Aurora, CO, 2 location with a loss of fluorescence event were considered for analysis. The in- USA, Sch Med, Univ Leeds, Leeds, United Kingdom. tensity time course of a 3x3 pixel area (480 x 480 nm2) around the fusion site of Stac3, which is highly expressed only in skeletal muscle, can be subdivided into the fluorescence movie was determined and different fusion events were aligned an N-terminal region, a PKC C1 domain, a linker, and tandem SH3 domains. at the onset of their respective amperometric spikes. Time super-resolution anal- Stac3 (i) facilitates the trafficking of CaV1.1 to the plasma membrane, (ii) mod- 2þ ysis using the recently developed Event Correlated Microscopy (ECOM) ulates the function of CaV1.1 as a Ca channel, and (iii) is required for the 2þ method revealed that FFN511 release followed the amperometric spike onset ability of CaV1.1 to activate Ca release via RyR1. Previous work from our with a mean delay of 115 ms, presumably reflecting slow diffusion of FFN lab has shown that function (iii) likely depends on binding of the tandem while in amperometric detection catecholamine molecules are rapidly consumed SH3 domains to residues 745-765 of the CaV1.1 II-III loop, an interaction during the oxidation process. Supported by NIH grant R01GM121787. which is supported (Wong King Yuen et al., 2017) by crystallographic and calorimetric analysis of isolated segments of the two proteins. Here, by using 2597-Pos electrophysiology, and by testing for colocalization of fluorescently tagged NS510, a High Affinity Fluorescent Catecholamine Sensor for Monitoring fragments, we systematically tested the ability of discrete Stac3 domains to Norepinephrine Exocytosis 1 2 2 1 interact with intact CaV1.1 and its cytoplasmic domains in tsA201 cells. We Xin A. Liu , Le Zhang , Timothy Glass , Kevin D. Gillis . found that a mutation (P758L), recently reported to cause malignant hyperther- 1Dalton Cardiovas Res Ctr, Univ Missouri Columbia, Columbia, MO, USA, 2 mia susceptibility, abolishes the interaction of Stac3 with the CaV1.1 II-III loop. Chemistry, Univ Missouri Columbia, Columbia, MO, USA. We also found that the tandem SH3 domains (residues 243-360) co-localize NS510 is a fluorescent sensor with a high affinity for catecholamines based on a with the wild-type CaV1.1 II-III loop and promote insertion of full-length quinolone fluorophore incorporating a boronic acid recognition element and a CaV1.1 into the plasma membrane, where it produces gating charge movements turn-on response to norepinephrine. The excitation maximum of NS510 is near but not ionic currents. However, expression of Stac3 residues 1-242 together 488 nm. We are using adrenal chromaffin cells as a model cell to test NS510, with residues 243-360 restored ionic currents almost identical to those restored and sub-populations of cells enriched in epinephrine versus norepinephrine are by full-length Stac3, indicative of interaction site(s) between CaV1.1 and Stac3 isolated using gradient centrifugation. Fluorescence is partially quenched when other than the II-III loop and the SH3 tandem. Consistent with this possibility, the sensor interacts with epinephrine and dopamine whereas the fluorescence in- expression of only Stac3 residues 1-242, together with CaV1.1, was sufficient to creases upon binding with norepinephrine. Norepinephrine-enriched chromaffin restore Ca2þ currents almost similar in magnitude to those for full-length Stac3. cells loaded with NS510 exhibit distinct fluorescence punctae using confocal mi- croscopy and total internal reflection fluorescence (TIRF) microscopy, consistent Posters: Exocytosis and Endocytosis II with staining of norepinephrine-containing secretory granules. Amperometry us- ing underlying transparent electrodes in conjunction with TIRF microscopy dem- 2595-Pos onstrates that the sensor can be used to observe destaining of individual To Pinpoint the Location and the Orientation of Proteins Associated with chromaffin granules simultaneously with amperometric spikes indicating quantal Dense-Core Vesicles (DCVs) using CLEM exocytosis of catecholamine. To further validate the selective staining of Bijeta Prasai, Gideon Haber, Kem A. Sochacki, John A. Ciemniecki, norepinephrine-containing vesicles, we labelled fixed cells using an antibody Justin W. Taraska. against phenylethanolamine N-methyltransferase (PNMT), an enzyme that con- NHLBI NIH, Bethesda, MD, USA. verts norepinephrine to epinephrine, and thus is found in epinephrine-enriched,

BPJ 9463_9465 524a Wednesday, March 6, 2019 but not norepinephrine-enriched cell populations. NS510 labeling is higher in 2600-Pos norepinephrine-enriched cells as validated by PNMT staining. We conclude High throughput Drug Testing of Transmitter Release Events in Chro- that NS510 is the highest affinity and most selective fluorescent norepinephrine maffin Cells with Surface Modified CMOS Ic sensor currently available and can be used for measuring quantal exocytosis of Meng Huang1, Shailendra Rathore2, Manfred Lindau2. catecholamines in live-cell assays. Supported by NIH R01EB020415. 1Materials Science and Engineering, Cornell Univ, Ithaca, NY, USA, 2Applied & Engineering Physics, Cornell Univ, Ithaca, NY, USA. 2598-Pos Amperometry is an electrochemical method to measure the kinetics, quantal Conditional Knockout of the Serotonin Transporter (SERT) Demonstrates size and fusion pore properties of quantal release events. CMOS based electro- Its Role in Accumulating and Maintaining 5-HT Homeostasis in the Sym- chemical sensor array with surface-modified SU-8 microwell structures allow pathoadrenal System parallel recordings of multiple single cells, enhancing the recording efficiency 1 2 2 Rebecca L. Brindley , Mary Beth Bauer , L. Anne Walker , for amperometry. Many drugs, such as antidepressant drugs bupropion and cit- 3,4 4 5 Meagan A. Quinlan , Ana M.D. Carneiro , Ji-Ying Sze , alopram, have been studied in detail in terms of secretion. However, the effect Randy D. Blakely3,6, Kevin P.M. Currie1. 1 of the drugs on the kinetics of exocytosis at a single event level has never been Department of Biomedical Sciences, Cooper Medical School of Rowan m m 2 reported. In this work, the effects of bupropion (5 M) and citalopram (1 M) University, Camden, NJ, USA, Department of Anesthesiology, Vanderbilt m 3 and of the false fluorescent neurotransmitter FFN 511(7 M) on the kinetics and University School of Medicine, Nashville, TN, USA, Department of quantal sizes of exocytosis from single chromaffin cells have been investigated Biomedical Science, Charles E. Schmidt College of Medicine, Jupiter, FL, 4 using amperometry with these novel devices. FFN 511 does not affect the ki- USA, Department of Pharmacology, Vanderbilt University School of netics, quantal size, or the release frequencies of quantal release events. Bupro- Medicine, Nashville, TN, USA, 5Department of Molecular Pharmacology, 6 pion significantly increases the quantal size, the maximum peak current and the Albert Einstein College of Medicine, Bronx, NY, USA, Florida Atlantic foot amplitude due to its function as a vesicular monoamine transport enhancer. University Brain Institute, Jupiter, FL, USA. Larger vesicles (>0.14-0.20 pC) tend to have faster release after bupropion Adrenal chromaffin cells (ACCs) comprise the neuroendocrine arm of the sympa- treatment while smaller vesicles remain relatively unchanged. Citalopram thetic nervous system and secrete catecholamines to coordinate the physiological does not change the amperometry parameters much, but increases the release response to stress. Although SERT is prominently expressed in ACCs, its role frequency. Follow-up capacitance measurements revealed that this effect of cit- there has remained unclear. Our data show that SERT interacts with 5-HT1A recep- alopram is due to an increase of the readily releasable pool. CMOS based elec- tors to modulate catecholamine exocytosis from isolated ACCs and we propose trochemical sensor arrays are thus a suitable tool for high-throughput testing of that adrenal SERT coordinates accumulation of 5-HT for reuse in the autocrine drugs to determine their potential activity to modulate transmitter release. control of catecholamine secretion. Consistent with this, constitutive knockout Conflict of interest: M.L. is a partner of the ExoCytronics, which will develop of SERT (SERT/ mice) augments stress-evoked epinephrine secretion in vivo and commercialize the microchip array platform technology presented here. . However, the global nature of the knockout makes it difficult to determine Supported by NIH SBIR grant R43MH109212 awarded to ExoCytronics. if SERT might be acting in the CNS and / or ACCs. Furthermore, lack of SERT in platelets disrupts delivery of gut-derived 5-HT to the adrenal gland (and other tis- 2601-Pos sues) via the bloodstream. To address these confounding factors, we generated a Chromogranin A, the Major Lumenal Protein in Chromaffin Granules, novel transgenic mouse model with selective excision of SERT in the peripheral Controls Fusion Pore Expansion sympathetic nervous system (SERTDTH) by crossing floxed SERT mice with tyro- Prabhodh S. Abbineni1, Mary A. Bittner1, Daniel Axelrod2, sine hydroxylase Cre driver mice. SERT expression, assessed by western blot, was Ronald W. Holz1. abolished in the adrenal gland but not perturbed in the CNS of SERTDTH mice. 1Department of Pharmacology, University of Michigan, Ann Arbor, MI, SERT-mediated [3H] 5-HT uptake was unaltered in midbrain, hindbrain, and spi- USA, 2Department of Pharmacology and LSA Biophysics, University of nal cord synaptosomes, confirming transporter function was intact in the CNS. Michigan, Ann Arbor, MI, USA. Endogenous midbrain and whole blood 5-HT homeostasis was unperturbed in Lumenal contents of secretory granules are released into the extracellular envi- SERTDTH mice, contrasting with the adrenal gland, which showed a z 50 % ronment upon fusion of the granule and plasma membrane. Small molecules reduction of 5-HT content but with no change in catecholamine content. This can be discharged though the narrow fusion pore that forms immediately upon novel model confirms that SERT expressed in adrenal chromaffin cells is essential fusion, but protein discharge requires expansion of the initial fusion pore. We for maintaining 5-HT homeostasis and provides a powerful tool to help dissect the recently found that fusion pore expansion is slowed by tissue plasminogen acti- role of adrenal SERT in the sympathetic stress response. vator (tPA), a protein contained within the lumen of chromaffin granules. We further examined the influence of lumenal proteins on fusion pore expansion in 2599-Pos chromaffin cells using a combination of polarization and total internal reflection The Serotonin Transporter Modulates the Quantal Size of Vesicular fluorescence microscopy. We found that expression of Chromogranin A (CgA- Release Events in Adrenal Chromaffin Cells EGFP), the major lumenal protein in chromaffin granules, restricted fusion Rebecca L. Brindley, Kevin P. Currie. pore expansion such that fusion pore curvature durations often lasted longer Biomedical Sciences, Cooper Medical School of Rowan Universit, Camden, than 10 s. This was surprising because fusion pore curvature durations of granules NJ, USA. containing neuropeptide Y (NPY-EGFP) are significantly shorter (80 % lasting < The serotonin (5-HT) transporter (SERT) is an important regulator of 5-HT 1 s) than those that contain CgA-EGFP, despite CgA being a ubiquitous lumenal signaling in the CNS. SERT is also expressed in adrenal chromaffin cells protein. Immunocytochemistry revealed that overexpression of lumenal proteins, (ACCs), part of the sympathetic nervous system that coordinates the physiolog- including labeled NPY, led to downregulation of endogenous lumenal proteins ical response to stress through exocytosis of catecholamines and other transmit- including CgA. Fusion pore curvature durations in non-transfected cells were ters. Here, we used carbon fiber amperometry to show that SERT modulates longer than those associated with granules expressing exogenous NPY and catecholamine secretion from ACCs by at least two distinct mechanisms. First, shorter than those associated with granules expressing exogenous tPA, CgA, SERT accumulates small amounts of 5-HT into ACCs and modulates the ability or CgB. Expression of exogenous CgA with NPY-EGFP converted the fusion of 5-HT1A receptors to inhibit the number of vesicular fusion events in isolated pore from being transient to being longer-lived. These findings demonstrate ACCs. Additionally, SERT acts via a mechanism independent of 5-HT1A re- that several endogenous chromaffin granule lumenal proteins including CgA, ceptor activation to modulate the amount and kinetics of transmitter release the major lumenal protein in chromaffin granules, are regulators of fusion pore from individual vesicular fusion events. The half-width (duration) and charge expansion and that alteration of chromaffin granule contents affects fusion (amount of oxidizable transmitter released) of individual amperometric spikes / pore lifetimes. The rapid fusion pore expansion of NPY-EGFP-containing gran- (vesicular fusion events) was significantly smaller ( 35%) in SERT cells ules, rather than reflecting normal fusion pore dynamics as previously assumed, compared to wild-type. The same effect was recapitulated in wild-type cells is a consequence of the loss of endogenous CgA. by in vitro pharmacological block of SERT (48hrs culture with escitalopram) or by restricting the amount of extracellular 5-HT available for uptake (48hrs 2602-Pos culture dialyzed serum media). Adrenal 5-HT content is determined by SERT- Alpha to Beta Cells: A Pathway Towards a Diabetes Cure mediated uptake but only accounts for a small fraction (0.13%) of the total Michael R. DiGruccio, Dave W. Piston. adrenal gland monoamine content. Although 5-HT content was reduced in Cell Biology & Physiology, Washington University in St. Louis, University SERT / adrenal glands, the catecholamine content was unaltered making it City, MO, USA. unlikely that the reduced spike charge (35% decrease) is simply due to reduced Type 1 Diabetes (T1D) is insulin deficiency and hyperglucagonemia due to vesicular monoamine content. Ongoing work will dissect the underlying mech- auto immune destruction of the insulin-secreting beta cells. A viable strategy anisms by which adrenal SERT / 5-HT regulate catecholamine secretion and to treat T1D is to restore the beta cell mass. Here, our approach focuses hence the sympathoadrenal stress response. upon beta cell replacement from alpha cells, which are closely related to

BPJ 9463_9465 Wednesday, March 6, 2019 525a beta cells, but not destroyed in T1D. In rodent models containing specific fluo- 2605-Pos rescent genetic lineage labeling of alpha and beta cells, we identified a group of The Molecular Mechanism and Structural Analysis of Membrane Interac- cells at the islet periphery that appear to be beta cells that are transdifferentiated tion via Fera and C2 Domains in Ferlins Associated with Muscular Dystro- from alpha cells (AB). Preliminary live cell imaging and electrophysiology phy and Cancer studies suggest that these AB cells acquire a typical beta cell glucose response, Faraz M. Harsini1, Anthony A. Bui1,2, Michael Latham2,AnneM.Rice3, which suggests that beta cell regeneration from endogenous alpha cell popula- Mark A. White4, Mazdak Bradberry5, Edwin R. Chapman5, Sukanya Lakshmi1, tions may be a viable TID cure approach. In human islets, we also discovered Andrei Turtoi6, Isaac L. Scott1, Matthew Dominguez7, Elahe Masoumzadeh2, endogenous cells expressing both insulin and glucagon. The dynamics of these Jon J. McCord1,8, Jacob Gendelman9, Roger Bryan Sutton1,10. bi-hormonal cells in terms of glucose response and hormone (insulin and/or 1Cell Physiology and Molecular Biophysics, Texas Tech University Health glucagon) secretion are yet to be elucidated. We are performing imaging based Sciences Center, Lubbock, TX, USA, 2Chemistry & Biochemistry, Texas islet studies that utilize virally delivered far red fluorescent secretory pathway Tech University, Lubbock, TX, USA, 3Biophysics, Johns Hopkins fluorescent labels to determine the glucose response and inulin/ glucagon secre- University, Baltimore, MD, USA, 4Sealy Center for Structural Biology, tion dynamics of these bi hormonal cells. Our single cell islet secretion assays University of Texas Medical Branch, Galveston, TX, USA, 5Department of will also be used in combination with alpha and beta cell specific RCaMP1h Neuroscience, University of Wisconsin-Madison HHMI, Madison, WI, USA, and GCaMP6 Ca2þ response indicators in order to also understand how Ca2þ 6Metastasis Research Laboratory, University of Liege, Liege, Belgium, couples with secretion in these cells types. Findings from this study will 7Graduate School of Biomedical Sciences, Texas Tech University HSC, advance our understanding in these unique rodent and human cell types. The Lubbock, TX, USA, 8Mechanical Engineering, Texas Tech University, long term goal study goal is to target these unique cell populations for conver- Lubbock, TX, USA, 9Amherst College, Amherst, MA, USA, 10Center for sion into functional beta cells. Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX, USA. 2603-Pos Ferlins are a family of proteins involved in a variety of biological processes such as Detecting Early Risk of Type 2 Diabetes During an Oral Glucose Tolerance exocytosis, membrane repair mechanism, and other membrane fusion events. Dys- Test ferlin and myoferlin are two members of human Ferlins with the highest similarity, Joon Ha, Arthur Sherman. and are mostly expressed in skeletal muscle cells. Dysferlin is involved in mem- NIH, Bethesda, MD, USA. brane repair mechanism, and lack of functional dysferlin can cause Muscular Dys- Prediabetes is an intermediate state between normal glucose tolerance and dia- trophy. Although the role of myoferlin in membrane interaction has been proven, betes. 70% of individuals with prediabetes (PDM) will develop diabetes during its main function is not well understood. It has been shown however that myoferlin their lifetime. Finding early markers of prediabetes is essential to initiate ther- is overexpressed in several types of cancer including Triple-Negative Breast Can- apy to prevent or slow disease progression. We have previously shown that cer (TNBC), and TNBC patients with overexpressed myoferlin show a worse over- time to peak glucose during an oral glucose test (OGTT) is a risk factor for all survival. Dysferlin and myoferlin employ seven C2 domains to interact with the þ PDM (Chung et al. Clinical Endocrinology, 2017). Our mathematical model membrane in a Ca2 dependent manner. C2A domain is believed to function as the þ (Ha et al. Endo, 2016) predicts that one-hour glucose and time to peak of insulin main Ca2 sensor, playing an essential and indispensable role in their function. In þ during OGTTs are also early markers for prediabetes. In particular, one-hour this study, we solved the structure of Ca2 bound myoferlin C2A using X-ray crys- glucose passes the threshold of prediabetes earlier than two-hour glucose, tallography and compared that to dysferlin C2A structure which we previously þ which is the current standard criterion. Time to peak of both glucose and insulin solved. We also determined their specificities for Ca2 and different lipid compo- increases before two-hour glucose passes the threshold for PDM. Moreover, the sitions. In addition, we identified residues that are critical in membrane interaction mathematical model predicts that weak early-phase insulin secretion, which is via these domains. Finally, we present the first structure of the FerA domain solved common among East Asians and cystic fibrosis patients, contributes to earlier by 3D NMR and provide evidences of its interaction with the membrane using abnormality of one-hour glucose. To test our predictions, a linear mixed effect confocal microscopy, electron microscopy, and a co-sedimentation assay. Study- model (LME) was applied to analyze a longitudinal data set of Pima Indians. ing FerA pathogenic mutations by Circular Dichroism and Differential Scanning The LME analysis confirmed that time to peak of glucose and insulin and Calorimetry allowed us to have a better understanding of the pathogenicity of the one-hour glucose pass their thresholds before two-hour glucose. The results FerA mutations. Outcome of this study will further advance our understanding of recommend that time to peak glucose and insulin as well as one-hour glucose the function of these proteins and their relevance to human diseases. should be considered as new criteria for prediabetes. 2606-Pos Calcium Dependence, Kinetics, and Pore Dynamics of Physiological 2604-Pos Vesicle Fusion with Planar Supported Bilayers Extracellular zinc Contributes to the Slow Polyspermy Block Alex J.B. Kreutzberger1, Volker Kiesling1, Binyong Liang1, Katherine L. Wozniak, Wesley A. Phelps, Miler T. Lee, Anne E. Carlson. Patrick Seelheim1, Arun Anantharam2, J. David Castle1, Lukas K. Tamm1. Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA. 1Center for Membrane and Cell Physiology, University of Virginia, Ensuring that an egg is fertilized by a single sperm is essential for progression Charlottesville, VA, USA, 2University of Michigan, Ann Arbor, MI, USA. through embryogenesis. Eggs therefore have multiple mechanisms to prevent Content-labeled dense core vesicles (DCVs) can be purified from PC12 cells. sperm entry into an already fertilized egg. In the slow polyspermy block, fertil- We previously reconstituted their fusion to planar supported bilayers in a single ization induces exocytosis of cortical granules, which are enriched with com- vesicle assay that enabled docking, fusion, and fusion pore lifetime to be distin- pounds that transform the extracellular matrix (ECM) surrounding eggs into guished. Munc18 and complexin-1 were used to arrest docked DCVs in a a barrier impenetrable by sperm. The underlying mechanisms that enable the primed state that could be triggeredto fuse with micro molar concentrations creation of this protective barrier are largely-unknown. We hypothesize that of calcium. Fusion pore lifetimes were modulated in this system by the pres- extracellular zinc may contribute to the slow block. It has recently been shown ence of lipids with intrinsic negative curvature in the planar supported bilayer. in mice, primates, and humans that cortical granules are enriched with zinc; Here, we demonstrate the extension of this assay to fusion of purified content- furthermore, extracellular zinc impairs sperm motility and binding to the labeled synaptic vesicles and insulin granules. The fusion kinetics of synaptic ECM of eggs. Using confocal microscopy with the zinc indicator FluoZin-3, vesicles were faster than those of DCVs while the fusion kinetics of insulin we demonstrate that fertilization and egg activation evoke zinc release from Xenopus laevis, Danio rerio granules were slower. The different endogenous synaptotagmin isoforms on eggs of the African clawed frog, and zebrafish, . the secretory vesicles confer distinct calcium responses to the fusion reaction. Using the appearance of cleavage furrows 90-120 minutes after sperm addition X. lae- The interplay between synaptotagmin and PI(4,5)P2 in the planar supported as an indicator of embryonic development, we show that insemination of bilayer directly modulates the properties of the fusion pores. vis eggs in extracellular ZnSO4 blocks development in a concentration- dependent manner (IC50 =205 3 mM). To hone-in on when extracellular 2607-Pos zinc interferes with development, we inseminated X. laevis eggs in 0 or 1 Synaptotagmin-7 Endows a Subpopulation of Chromaffin Granules with mM ZnSO4 for 30 minutes, then transferred these eggs to new solutions with Distinct Calcium Sensing and Fusion Properties or without ZnSO4. None of the eggs inseminated in zinc developed cleavage Mounir Bendahmane1, Alina Chapman-Morales1, Noah A. Schenk1, furrows; whereas, eggs inseminated without zinc, but transferred to a zinc- Zhang Shuang1, Paul M. Jenkins1, David R. Giovannucci2, containing solution, developed normally. The effect of zinc on the ECM of Arun Anantharam1. 1 2 the egg is irreversible, as pretreatment with 1 mM ZnSO4 prior to sperm addi- Pharmacology, The Univ Michigan, Ann Arbor, MI, USA, Neurosciences, tion was sufficient to prevent embryonic development. Overall, our data dem- The University of Toledo, Toledo, OH, USA. onstrates that fertilization-induced zinc release is conserved in vertebrates, and Syt-7 is one of two major Ca2þ-sensors for regulated release from adrenal chro- this zinc may contribute to the slow polyspermy block. maffin cells. Its unique biochemistry provides cells with a highly Ca2þ sensitive

BPJ 9463_9465 526a Wednesday, March 6, 2019 pool of dense core granules for exocytosis. Syt-7 also participates in limiting the C2AB domains in pore regulation. We found C2AB domains promote pore dila- rate at which fusion pores expand via mechanisms that are not fully clear. All tion in a calcium, SNARE, and PI(4,5)P2 dependent manner. Both C2 domains previous imaging studies of Syt-7’s actions during exocytosis in chromaffin cells and calcium-induced membrane-insertion of hydrophobic loops at the tips of the have relied on overexpression of WT or mutant Syt protein. Here, we imaged calcium-binding loops are important for pore dilation. exocytosis in mouse chromaffin cells without Syt-7 for the first time, so that properties which depend on the protein could be directly determined. Our data 2610-Pos show that cells lacking Syt-7 exhibit at least a five-fold difference in fusion ef- Domain Stability and Functional Analysis at the AD3 Locus of Synaptotag- ficacy compared to WT cells in response to elevated Kþ depolarization. min 1 C2 Domains 1,2 1 3 1 Lumenal granule cargos are also released at faster rates from Syt-7 knockout Anthony A. Bui , Faraz M. Harsini , Anne M. Rice , Souvic Karmakar , Kerry Fuson1,4, R. Bryan Sutton1,4. (KO) cells, consistent with data from overexpression studies. Next, we measured 1 2 the responses of WT and KO cells to endogenous secretogoguges of the Texas Tech Univ Hlth Science Ctr, Lubbock, TX, USA, Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, USA, 3Department of sympatho-adrenal synapse, acetylcholine (ACh) and pituitary adenylate 4 cyclase-activating peptide (PACAP). Compared to WT cells, ACh-evoked Biophysics, John Hopkins University, Baltimore, MD, USA, Center for release in KO cells is significantly impaired with respect to granule fusion ki- Membrane Protein Research, Texas Tech University Hlth Science Ctr, netics and fusion efficacy. PACAP-evoked release, on the hand, is only mildly Lubbock, TX, USA. Synaptotagmin I (SytI) is a member of a family of proteins that play a crucial role suppressed. Our data provide evidence for Syt-7’s role in two critical aspects 2þ of exocytosis – fusion and fusion pore expansion. Moreover, the separable re- in facilitating Ca -mediated vesicle fusion in neurotransmitter release. They are sponses of WT and KO cells to endogenous secretogogues suggest that Syt pro- comprised of two tandem C2 domains, C2A and C2B, which are tethered to the teins occupy distinct roles in the physiology of the chromaffin cell system. membrane by a single transmembrane helix. Generally, the C2 domain motif is found in a broad range of proteins that bridge the interaction between protein and 2608-Pos membrane. A single point mutation (Y311N) in a highly conserved region of the PIP2 Drives Calcium-Independent Activation of Tandem C2-Domain Cal- C2B domain of Drosophila SytI has the ability to cripple Ca2þ-mediated vesicle cium Sensors fusion, despite the protein’s ability to fold correctly and target the membrane. In Mazdak M. Bradberry, Huan Bao, Xiaochu Lou, Edwin R. Chapman. addition to this mutation, we introduce another point mutation (Y311F) to com- University of Wisconsin-Madison, Madison, WI, USA. bat the hindered thermodynamic stability of the domain from the original muta- Tandem C2 domain proteins in the synaptotagmin and Doc2 families regulate tion. We show that the AD3 locus appears to regulate a delicate balance between þ exocytosis via direct interactions with Ca2 and phospholipid bilayers. the structural stability of the general C2 fold and its Ca2þ sensitivity by acting as þ Synaptotagmin-1 (syt1), a fast-acting, low-affinity Ca2 sensor, penetrates a ‘‘brace’’ through the combination of steric bulk and hydrogen bonding poten- þ membranes upon binding Ca2 to trigger synchronous vesicle fusion. Doc2b tial. We aim to elucidate the importance of the AD3 locus of Synaptotagmin I þ is a slow-acting, high-affinity Ca2 sensor for spontaneous and asynchronous through the use of X-ray crystallography, isothermal titration calorimetry vesicle fusion, but whether it shares a common, penetration-dependent mecha- (ITC), and guanidine denaturation coupled to circular dichroism (CD). nism with syt1 is unknown. In the case of both tandem C2-domain proteins, however, Ca2þ is thought to be a fundamental requirement for the triggering 2611-Pos 2D of exocytosis. Recent work has challenged this model by showing that uncaging How Ca And Synaptotagmin Trigger Snare-Mediated Membrane of phosphatidylinositol(4,5)-bisphosphate (PIP2) can trigger rapid, syt1- Fusion dependent exocytosis in the absence of Ca2þ flux. Here, we reconcile these Volker Kiessling1, Alex J.B. Kreutzberger1, Binyong Liang1, 2 1 3 2 findings by showing that PIP2 can drive membrane penetration by syt1 and Sarah B. Nyenhuis , Patrick Seelheim , J. David Castle , David S. Cafiso , Doc2b in the absence of Ca2þ.Ca2þ-independent penetration of membranes Lukas K. Tamm1. 1 by Doc2b was even more robust than that of syt1, with physiologic levels of Dept Molec Physiol/Biol Phys, Univ Virginia, Charlottesville, VA, USA, 2 3 PIP2 driving substantial activation of this sensor. To better define the differ- Dept Chemistry, Univ Virginia, Charlottesville, VA, USA, Dept Cell Biol, ences between syt1 and Doc2b, we used the parallax method to measure the Univ Virginia, Charlottesville, VA, USA. membrane penetration depths of syt1 and Doc2b in the presence and absence It has long been known that synatpotagmin-1 is the sensor that triggers fast, 2þ 2þ of Ca and PIP2. We found that, in addition to stimulating Ca [-independent evoked release of neurotransmitter by the fusion of synaptic vesicles to the penetration, PIP2 exerted different effects on syt1 and Doc2b in the presence of presynaptic membrane in response to calcium. It is also well established Ca2þ. While both Ca2þ sensors underwent Ca2þ-dependent membrane penetra- that the SNAREs syntaxin-1a, SNAP-25, and synaptobrevin-2/VAMP-2 tion in the presence of phosphatidylserine (PS), Ca2þ stimulated only shallow form the core of the membrane fusion machinery that drives calcium- penetration by Doc2b unless PIP2 was also present. Together, these results triggered neuronal exocytosis. However, how synaptotagmin-1 and the fusion demonstrate key similarities among tandem C2 domain proteins while defining machinery are mechanistically coupled has been the subject of intense recent a biophysical regulatory mechanism specific to Doc2b. Our findings provide research. Among several proposed models, no satisfactory molecular mecha- evidence that tandem C2 domain Ca2þ sensors can be directly activated by nism has emerged and been rigorously proven experimentally. Here, we pro- PIP2, establishing a new role for this lipid in regulating, and under some cir- pose a mechanism where the lipid membrane is intimately involved in cumstances triggering, exocytosis. coupling calcium sensing to fusion. Using TIRF- (total internal reflection fluo- rescence) and sd FLIC (site-directed fluorescence interference contrast) mi- 2609-Pos croscopy, we demonstrate that fusion of PC12 cell-derived dense core Fusion Pore Dilation by Synaptotagmin-1 vesicles (DCVs, which serve as surrogates for synaptic vesicles) is strongly Zhenyong Wu1, Nadiv Dharan2, Sathish Thiyagarajan3, Ben O’Shaughnessy2, linked to the tilt angle of the cytoplasmic domain of the nascent SNARE Erdem Karatekin1. complex with respect to the plane of the target membrane. As the tilt angle 1 2 Dept Cell/Molec Physiol, Yale Univ, New Haven, CT, USA, Chem. Eng., increases, force is exerted on the SNARE transmembrane domains to drive 3 Columbia University, New York, NY, USA, Dept Physics, Columbia Univ, the merger of the two bilayers as the trans-SNARE complex completes folding New York, NY, USA. into the cis-SNARE complex. We show that the tilt angle (a) dramatically in- Neurotransmitter and hormone release involve calcium-triggered fusion of a creases upon calcium-mediated binding of synaptotagmin to membranes, (b) cargo-loaded vesicle with the plasma membrane. The initial connection between strongly depends on the surface electrostatics of the membrane, and (c) is the fusing membranes, called the fusion pore, can evolve in various ways, strictly coupled to lipid order of the target membrane. Thus, the membrane including rapid dilation to allow full cargo release, slow expansion, repeated and its properties play a central role in the calcium-dependent coupling of syn- opening-closing, and resealing. Pore dynamics determine kinetics of cargo aptotagmin to SNARE-mediated fusion. release and mode of vesicle recycling, but how these processes are controlled are poorly understood. Most previous reconstitutions could not monitor single 2612-Pos pores, limiting mechanistic insight they could provide into pore regulation. Structural Characterization of Full-Length Synaptotagmin-1 to Cis or We recently developed a nanodisc-based fusion assay that allow reconstitution Trans Membranes and monitoring of single pores with unprecedented detail. The method uses cells Sarah B. Nyenhuis, David S. Cafiso. expressing ‘‘flipped’’ neuronal SNAREs on their surfaces as fusion partners with Chemistry, University of Virginia, Charlottesville, VA, USA. nanodiscs reconstituted with the complementary SNAREs. The fusion pore that Neurotransmission involves Ca2þ dependent fusion of synaptic vesicles (SV) to forms between cell and nanodisc membranes generates a connection between the presynaptic plasma membrane (PM), thereby releasing neurotransmitter into the cell interior and exterior that can be probed by monitoring pore conductance the synaptic cleft. Soluble N-ethylmaleimide sensitive factor attachment protein under voltage-clamp. Previously, using this assay we had characterized fusion receptors (SNARES) drive membrane fusion, but SNAREs are not calcium pores induced by SNAREs alone. Here we studied the role of Synaptotagmin-1 sensitive. The Ca2þ sensor, Synaptotagmin-1 (Syt1), thus couples neuron

BPJ 9463_9465 Wednesday, March 6, 2019 527a depolarization to synchronous neurotransmitter release. Syt1 is a vesicular- vesicle fusion process by inhibiting the nerve-evoked fusion via PKA phosphor- tethered protein, with two homologous C2A and C2B domains attached through ylation and promoting it in its non-phosphorylated form. a juxtamembrane linker. Upon Ca2þ influx, loops on the two domains bind Ca2þ and insert into charged membrane. The C2B domain contains other regions 2615-Pos capable of membrane interaction, including the lysine rich polybasic face. Sequential Link of Kiss-And-Run Mechanism and Classical Exocytosis at Syt1 can interact with either bilayer surface, binding cis- to the SV membrane, or Hippocampal Synapses trans- to the PM. Cis-binding likely plays an inhibitory role by back-binding both Andreas W. Henkel. domains to the SV preventing membrane fusion. Trans-binding then permits Dept Physiology, Kuwait Univ, Safat, Kuwait. fusion, docking the SV to the PM surface. These membrane interactions are lipid The mechanisms behind highly dynamic variation of synaptic vesicle release have specific in order to drive membrane bridging. On the synaptic vesicle surface, only been shown to be linked to different vesicle pools, variable modes of mobilization, phosphatidylserine (PS) contributes negative charge, while on the PM both PS and calcium levels, protein phosphorylation and a variety of endocytosis mechanisms. This study presents a model that may explain seemingly contradicting exocytosis phosphatidylinositol-4,5-bisphosphate (PIP2) contribute to the charge density. Through various EPR techniques, we explored cis- and trans-binding under mechanism like ‘kiss-and-run’ vs full vesicle fusion. It was found that the onset of various physiologically relevant lipid, salt, and ionic compositions, to deter- FM1-43 release of from individual synapses can be delayed by up to a few seconds mine if Syt1 may act as a distance regulator between the SV and PM. A series after the start of stimulation. This phenomenon is linked to modified opening ki- of methods were developed to determine and differentiate membrane insertion netics of a fusion pore that connects the vesicle lumen to the extracellular space. of the domains in the full-length protein. With this, we characterized an ATP Some synapses were completely inactive during the first few seconds after start of the stimulation, despite immediate rise of calcium.A low concentration of staur- and PIP2 competition to the polybasic face which blocks or promotes trans- binding. We also characterized the juxtamembrane linker and the preferential osporine, which has been used to induce ‘kiss-and-run’ exocytosis, increased the proportion of delayed synapses. Vesicle fusion was never delayed after start of binding of C2A to PS and C2B’s to PIP2 in the presence of SV or PM mimicking membranes with and without Ca2þ. stimulation, when it was monitored with pH-sensitive synaptopHlourin, indicating an instantaneous formation of a fusion pore that allowed rapid equilibration of the 2613-Pos vesicle lumenal pH but prevented release of FM1-43. The delay of FM release Synaptic Vesicle Fusion and Docking during the First 14 Milliseconds could be explained by the transient formation of a fusion pore (‘‘kiss-and-run’’) After an Action Potential that expanded to full vesicle fusion after a few seconds. Our observations point Grant F. Kusick, Shigeki Watanabe. to a regulated exocytosis mechanism in hippocampal synapses that combines Department of Cell Biology, Johns Hopkins, Baltimore, MD, USA. ‘kiss-and-run’- and full vesicle fusion exocytosis in a sequential process. Synaptic vesicles fuse with the plasma membrane to release neurotransmitter both synchronously and asynchronously following an action potential. How many ves- 2616-Pos icles can fuse at a single active zone, and where these vesicles fuse within the Calcium Channels Gate Calcium-Independent But Voltage-Dependent active zone, is not well understood, particularly in the context of these two phases. Secretion in Mammalian Cells To capture synaptic vesicle exocytosis during synchronous and asynchronous Zhuan Zhou, Rong Huang, Yuan Wang, Jie Li, Xiaohan Jiang, Yinglin Li, release at cultured mouse hippocampal synapses, we induced single action poten- Feipeng Zhu, Changhe Wang, Zuying Chai. tials (AP) by electrical field stimulation then subjected neurons to high-pressure Inst Molec Med, Peking Univ, Beijing, China. freezing to examine their morphology by electron microscopy. During synchro- The somata of primary sensory neurons, including dorsal root ganglion (DRG) nous release (<5 ms after AP) multiple vesicles can fuse at a single active neurons, release neurotransmitters and neuropeptides. Following physiological 2þ zone; this multivesicular release is augmented by increasing the extracellular cal- action potentials, in addition to Ca -dependent secretion, we have discovered 2þ cium concentration. Synchronous fusions are distributed throughout the active and studied Ca -independent but voltage-dependent secretion (CiVDS) in zone, whereas asynchronous fusions (5-11 ms after AP) are biased toward the cen- somata of freshly isolated DRG neurons. Major open question of CiVDS is the mo- ter of the active zone. As exocytosis proceeds, new vesicles are recruited to lecular mechanism, including 3 components: fusion-pore machinery, voltage- the active zone and fully replenish the docked pool, but docking of these vesicles sensor and the linker of fusion-pore and voltage-sensor. Here we report, by using is transient and they either undock or fuse within 100 ms. These results demon- exocytosis assays of membrane capacitance and single vesicle imaging, CiVDS is strate that multivesicular release occurs even at low-release-probability synapses jointly contributed by (1) SNARE protein SNAP-25 for fusion pore, (2) voltage and suggest a spatial organization underlying synchronous and asynchronous gated Ca channel (VGCC) Cav2.2 for Ca sensor; (3) and the ‘‘synprint’’, which release. is Cav2.2 intracellular loop718-963aa, as the linker between fusion pore and voltage sensor; (4) CiVDS presents in both somata and axons. Following auto- 2614-Pos matic knockdown of CiVDS by 3d-culture of DRG neurons, CiVDS is rescued Synapsin: A Novel Insight for PKA Phospho-Domains in Inhibiting by overexpressing any component of SNAP-25 or Cav2.2. On other hand, CiVDS Release Probability is inhibited by blockers against SNAP-25, Cav2.2 and synprint. CiVDS is blocked Agustin Gonzalez-Ruiz1, Jose Guzman-Gutierrez1, by Cav2.2-RNAi-KD in DRG pre-transfected in vivo (Chai et al, Neuron,2017). Pedro Feliciano-Ramos1,2, Ramon A. Jorquera1. Importantly, our recent new evidences show that CiVDS presents and functions 1 2 Neuroscience, Univ Central Caribe, Bayamon, PR, USA, CBMM, PILM, also robustly in other essential cells, implicating that CiVDS may impact mamma- BCS, MIT, Cambridge, MA, USA. lian physiology and disease much more than previously thought. Synapsin (Syn), a family of phosphoproteins are found abundantly in neurons of both vertebrate and invertebrate systems. Syn is located in the nervous terminals 2617-Pos and regulates the availability of synaptic vesicles by reversible association with Sec1/Munc18-Family Proteins Catalyze Directional SNARE Assembly by the actin cytoskeleton and other vesicles via phosphorylation and dephosphoryla- Templating SNARE Folding and Association tion cycle. Syn deficiencies have been found in several mental disorders including Junyi Jiao1, Mengze He1, Sarah Port2, Baker Richard2, Yonggang Xu1, Bipolar and Maniac Spectrum Disorders, Epilepsy, and short-term memory defi- Hong Qu1, Yujian Xiong1, Yukun Wang1, Huaizhou Jin1, Travis Eisemann2, ciencies. Evidence indicates that the development of short-term memory requires Frederick M. Hughson2, Yongli Zhang1. the phosphorylation of Syn domains via Protein Kinase-Activated by cAMP 1Department of Cell Biology, Yale University, New Haven, CT, USA, (PKA) during nerve activity. However, the neurophysiological effect of the 2Department of Molecular Biology, Princeton University, Princeton, NJ, USA. PKA phosphorylations of Syn in synaptic transmission and plasticity is not clear. Sec1/Munc18-family (SM) proteins are required for SNARE-mediated mem- We determined the effects of PKA-phosphorylation domains of Syn in synaptic brane fusion, but their mechanism(s) of action remain controversial. Using function at Drosophila neuromuscular junction of third-instar larvae. For this, single-molecule force spectroscopy, we found that the SM protein Munc18-1 we scrutinize the synaptic transmission of wild-type, Syn KO and its neuronal res- catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, cues with normal Syn and with a PKA phospho-incompetent mutant. Transgenic and SNAP-25) into a four-helix bundle. Catalysis requires formation of an in- animals containing the UAS-Syn constructs were expressed under the control of termediate template complex in which Munc18-1 juxtaposes the N-terminal re- the neuronal driver elav-Gal-4. Synaptic transmission activity was measured by gions of the SNARE motifs of syntaxin and VAMP2, while keeping their C- electrophysiological recording of the postsynaptic compartment by two- terminal regions separated. Next, SNAP-25 binds the templated SNAREs to electrode voltage-clamp. Synaptic responses were evoked by paradigms of nerve form a partially-zippered SNARE complex. Finally, full zippering displaces stimulation controlled by a programmable stimulator. Our work in Drosophila in- Munc18-1. Munc18-1 mutations modulate the stability of the template complex dicates that Syn is required for normal synaptic transmission and short-term syn- in a manner consistent with their effects on membrane fusion, indicating that aptic memory by modulating nerve-evoked release probability at rest and during chaperoned SNARE assembly is essential for exocytosis. Two other SM pro- high demand, in agreement with the previous evidence in other model systems. teins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a tem- Interestingly, neuronal rescues indicate that Syn may operate at the last step of plate complex, suggesting that SM protein mechanism is conserved.

BPJ 9463_9465 528a Wednesday, March 6, 2019

2618-Pos SNAP25, the average FRET efficiency increases to 38%. Combining electro- A Mechanism for Exocytotic Arrest by the Complexin C-Terminus chemical imaging of fusion events with TIRF-FRET imaging, we detected a Mazen Makke. SNAP25 conformational change preceding fusion pore opening in SCORE2 over- Physiology, Uniklinikum des Saarlandes, Homburg, Germany. expressing SNAP25/ cells, confined to a 0.1 mm2 area surrounding the fusion Hindering premature vesicular fusion is key to build up a molecularly primed site. Calibration of single molecule fluorescence indicates an average number of ready releasable pool. Complexin-II (CpxII), a 16 KD cytosolic protein, inhibits 500 overexpressed SCORE2 copies in this area, corresponding to 25 endog- non-synchronous vesicle fusion at release sites, and by that accumulates a ready enous SNAP25 copies in wild type cells. We estimate that at SCORE2 density cor- releasable pool of primed vesicles that are released in synchrony upon immediate responding to endogenous SNAP25 density 6-7 (25%) SCORE2 molecules at elevation of intracellular calcium. Functionally, CpxII is equipped with four inde- the fusion site undergo a conformational change from low to high FRET before pendent yet synergetic domains. A central helix mediates the CpxII-SNARE (N- fusion pore formation. Supported by ERC grant ADG 322699, the Max- ethylmaleimide-sensitive factor (NSF) attachment protein receptors) complex Planck-Society and NIH grant R01GM121787. interaction, while an accessory helix is important for the stability of the central helix and for reducing premature fusion. The upstream N-terminal domain facil- 2621-Pos itates exocytosis, whereas the downstream C-terminal domain (CTD) inhibits pre- Fusion Pore Dynamics and Snare Complex Mobility mature fusion of vesicles. The molecular mechanisms how the CTD blocks Satyan Sharma, Manfred Lindau. asynchronous release are still unclear. With the help of high resolution membrane Dept Nanoscale Cell Bio, MPI for Biophysical Chemistry, Gottingen, Germany. capacitance measurements coupled with calcium imaging, carbon fiber amper- SNARE proteins are the minimal machinery required for the exocytosis. The zip- ometry, and biochemical assays, we show that infusion of CpxII far CTD -derived pering of SNARE protein synaptobrevin-2 (syb2) on vesicle with syntaxin-1 and peptide into mouse chromaffin cells is able to boost the synchronous release by SNAP-25, on plasma membrane is known to overcome the fusion barrier. To gain reducing the premature vesicle fusion. A similar phenotype is also observed by a mechanistic understanding on SNARE-mediated fusion molecular dynamics over-expressing the full length WT protein. Using in vitro binding assays, we simulations have been employed previously, albeit using small-sized vesicles (15-20 nm). As the curvature strongly influences the fusion propensity, we car- show that the CTD interacts with SNARE proteins. Moreover, we found that the CTD peptide lowers the rate of SDS resistant SNARE complex formation. ried out coarse-grained molecular dynamics simulations of fusion of a 40 nm We also show that the CTD shares a high degree of similarity to the SNARE pro- vesicle with a planar bilayer, both with multicomponent lipid composition and tein SNAP25-SN1 domain. Furthermore chimeras of CpxII:SNAP25-SN1 fully leaflet asymmetry. The vesicle was bridged by either four or six partially unzip- restore CpxII function, and even ‘‘superclamp’’ tonic secretion. Collectively, ped SANRE complexes to the bilayer. In all simulations, stalk was formed while these results provide evidence for a clamping mechanism in which the CTD of no fusion pore formation was observed. Instead, the SNARE complexes moved CpxII prevents the spontaneous assembly of SNARE complexes, enabling the radially away from each other forming an extended hemifusion diaphragm. On build-up of a ready releasable pool for synchronised Ca2þ-triggered exocytosis. the contrary, when the vesicle was replaced by a nanodisc in a similar setup with four SNARE complexes, rapid fusion pores were formed but they flickered 2619-Pos and failed to expand. This suggests that the confinement of the SNARE com- Nsf-Mediated Disassembly of On- and Off Pathway Snare Complexes and plexes is thus required for rapid fusion pore formation. To further validate the Inhibition by Complexin requirement of the confinement of SNARE for rapid fusion pore formation, sim- Ucheor B. Choi1, Minglei Zhao2, K. Ian White1, Axel Brunger1. ulations of the vesicle/bilayer system were redone, with the lateral mobility of 1Dept Molec Cell Physiol, Stanford Univ, Stanford, CA, USA, 2Department SNARE complexes now constrained using two sets of harmonic restraints. Inter- of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, estingly, in both the simulations rapid fusion pore formation was observed. In USA. parallel simulations, where the nascent pore from the nanodisc/bilayer system SNARE complex disassembly by the ATPase NSF is essential for neurotransmitter was inserted in vesicle/bilayer system and SNARE complexes unconstrained, a release and other membrane trafficking processes. We developed a single- rapid pore expansion was observed. Altogether, the results suggest that the re- molecule FRET assay to monitor repeated rounds of NSF-mediated disassembly striction of SNARE complex mobility is required for rapid fusion pore formation and reassembly of individual SNARE complexes. For ternary neuronal SNARE while the constraints must be released for its expansion. complexes, disassembly proceeds in a single step within 100 msec. We observed short- (<0.32 s) and long-lived (>=0.32 s) disassembled states. The longlived 2622-Pos states represent fully disassembled SNARE complex, while the short-lived states Three Stages of Neurotransmitter Release: Ca-Triggered Unclamping, correspond to failed disassembly or immediate reassembly. Either high ionic SNARE Ring Assembly and Snare-Mediated Membrane Fusion strength or decreased aSNAP concentration reduces the disassembly rate while Zachary A. McDargh, Anirban Polley, Ben O’Shaughnessy. increasing the frequency of short-lived states. NSF is also capable of disassembling Chemical Eng, Columbia Univ, New York, NY, USA. anti-parallel ternary SNARE complexes, implicating it in quality control. Finally, Cognition and complex behavior depend on neurotransmitter (NT) release at complexin-1 competes with aSNAP binding to the SNARE complex; addition of neuronal synapses, accomplished by a machinery that senses Ca when an action complexin-1 has an effect similar to that of decreasing the aSNAP concentration, potential arrives, fuses the vesicular and plasma membranes, and releases NTs possibly differentially regulating cis and trans SNARE complexes disassembly. through a fusion pore. Synaptotagmin (Syt) is known to be the Ca sensor, and SNARE proteins constitute the core fusion machinery, but how they and others 2620-Pos cooperate as a NT-releasing machine is poorly understood. This remarkable The Number of SNARE Complexes Changing Conformation during machinery must clamp fusion, and execute unclamping and membrane fusion Vesicle Fusion on sub millisecond timescales following Ca arrival. Ying Zhao1, Qinghua Fang1, Satyan Sharma1, Shrutee Jakhanwal2, Mathematical modeling can help identify the mechanisms, but their collective Reinhard Jahn2, Manfred Lindau1,3. character, and the millisecond timescales, are beyond present all-atom or 1Department of Nanoscale Cell Biology, MPI for Biophysical Chemistry, coarse-grained Martini methods. To overcome this obstacle, we radically Goettingen, Germany, 2Department of Neurobiology, MPI for Biophysical coarse-grain the molecules using a systematic procedure retaining principal Chemistry, Goettingen, Germany, 3Dept Appl/Eng Phys, Cornell Univ, biophysical properties. This framework enables us to computationally reconsti- Ithaca, NY, USA. tute the NT-releasing machinery. Formation of the SNARE (Soluble NSF Attachment Receptor) complex We tested the hypothesis that fusion is clamped by a ring-like oligomer of composed of the proteins synaptobrevin 2, syntaxin1 and SNAP25 is thought to vesicle-associated Syt molecules, whose subsequent disassembly following provide the force and energy to overcome the energy barrier for membrane fusion. Ca injection triggers fusion and NT release (Wang et al, 2014). In our simula- It is presently unclear how many of them actually participate in fusion pore for- tions, SNARE complexes (SNAREpins) are initially bound to the Syt ring mation. We generated, a SCORE2 (SNARE Complex REporter2) FRET probe consistent with the published Syt1/SNARE crystal structure (Zhou et al, by inserting the FRET donor mCerulean3 and acceptor Venus at the N-termini 2015). Following Ca injection, a reaction scheme described Ca binding/disso- of two SNAP25 SNARE motifs SN1 and SN2, respectively. SCORE2 overex- ciation to the Syt C2AB domain and Ca-dependent Syt-Syt bond breaking. pression fully rescued release in SNAP25/ mouse chromaffin cells without Importantly, MD simulations quantitatively showed that fusion is initially affecting frequency, size and kinetics of quantal release events. When SCORE2 clamped. Simulations revealed a 3-stage process following Ca injection. First, is highly overexpressed and therefore not in complex with other SNARE proteins, stochastic Syt ring disassembly released the SNAREpins (unclamping). Sec- acceptor photobleaching it indicates an average FRET efficiency of 21% in the ond, entropic forces spontaneously assembled the SNAREpins into a ring at footprint of resting cells. Coarse-grain molecular dynamic simulations show var- the fusion site. Third, entropic forces expanded the ring, pulling the membranes iable donor acceptor distances (4-7 nm) and k2 varied from 0.14 to 1, which do not together and driving their fusion. The predicted NT release times are quantita- relax within the fluorescence lifetime, indicating that the average FRET efficiency tively consistent with experimental measurements of EPSCs following action may result from a wide range. When syntaxin is present at 10 fold excess over potentials or following Ca uncaging.

BPJ 9463_9465 Wednesday, March 6, 2019 529a

Posters: Membrane Receptors and Signal 2625-Pos Structural Correlates of Agonist Binding to Neurotransmitter Binding Transduction II Sites Sushree Tripathy1, Stephen M. Muehlemann1, Wenjun Zheng2, 2623-Pos Anthony Auerbach3. Monomers of AMPA-Type Glutamate Receptor Subunits Diffuse in and 1Physics, Univ at Buffalo, Buffalo, NY, USA, 2Dept Physics, State Univ New Out of Spines; Unraveling by Single-Molecule Tracking York Buffalo, Buffalo, NY, USA, 3Dept Physiology and Biophysics, State Jyoji Morise1, Kenichi G.N. Suzuki2,3, Ayaka Kitagawa1, Univ New York Buffalo, Buffalo, NY, USA. Yoshihiko Wakazono4, Kogo Takamiya4, Taka A. Tsunoyama5, Endplate acetylcholine receptors toggle between resting (R) and active (R*) Hiromu Takematsu1, Akihiro Kusumi3,5, Shogo Oka1. conformations. The main connection between these states is through binding 1 Department of Biological Chemistry, Division of Human Health Sciences, agonist molecules. The activation pathway starts with an agonist (A) arriving 2 Graduate School of Medicine, Kyoto University, Kyoto, Japan, Center for at a binding site by diffusion, and continues when the agonist enters a cavity Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu lined with aromatic side chains. This process, R/AR (‘catch’), forms a low- 3 University, Gifu, Japan, Institute for Integrated Cell-Material Sciences affinity protein-ligand complex via a local conformation change. Thereafter, 4 (WPI-iCeMS), Kyoto University, Kyoto, Japan, Department of Integrative a global ‘gating’ conformation change forms a high-affinity, open-channel Physiology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan, complex (AR/AR*;‘hold’), by a process that requires another local rearrange- 5 Membrane Cooperativity Unit, Okinawa Institute of Science and ment. To further understand the ‘catch’ mechanism, we built homology models Technology, Onna-son, Japan. of R/R* (apo) and AR/AR* conformations based on crystal structures of Memory and learning require structural synaptic plasticity. Among the key AChBP/a4b2, and equilibrated the structures by MD simulations. The results players for synaptic plasticity are the alpha-amino-3-hydroxy-5-methyl-4- suggest that R is more flexible and has a larger binding pocket that is more- isoxazolepropionic acid (AMPA) subtype of ionotropic glutamate receptors covered by loop C, compared to AR. This is consistent with our hypothesis (AMPARs), which are concentrated in the synapse to mediate excitatory that loopC acts as a protective lid that is actively opened by the agonist in order transmission. AMPARs are composed of four subunits (GluA1-4), and work to gain entry into the aromatic pocket. Regarding ‘hold’, previous work showed as ion channels in the forms of homo- and hetero-tetramers. These tetramers that the low/high binding energy ratio is constant for a series of agonists are formed at their exit from the endoplasmic reticulum and it was widely structurally-related to ACh, and that this ratio is related to agonist efficiency believed that these tetramers are very stable entities. However, we considered (the fraction of binding energy converted to gating energy). Further, the effi- that the direct evidence for the existence of stable tetramers in the plasma ciency of ACh-like ligands (50%) is correlated with the ratio of the distances membrane (PM) is lacking, and that if the tetramers were very stable entities, between the quaternary nitrogen atom and the pocket center. We have extended modulating the tetramer compositions in the synapse, which is known to occur these studies to include azabicycloamine agonists (related structurally to epiba- rapidly after stimulation, would be difficult. We examined this issue using tidine) that have a lower efficiency (39%). Preliminary results suggest that for single-molecule imaging and tracking. We found that, at variance with the these ligands, too, the distance between the bridge nitrogen atom and the cavity general belief, many GluA1 and GluA2 molecules expressed in the PM of center determines affinity, efficacy and efficiency. We are able to calculate HEK293 cells exist as monomers, and form metastable homo- and hetero- complete concentration-response curves from measurements of these distances tetramers (100 and 200 ms, respectively), while they instantaneously in equilibrated structures. NS064969. fall apart into monomers, dimers, or trimers, which again form tetramers readily. In the dendritic-shaft PM of mouse hippocampal neurons, GluA1 2626-Pos and GluA2 also dynamically merged into greater oligomers on similar time Role of B-Glucan Structure in Dectin-1 Signaling and Multimerization in scales to those in the HEK293-PM, suggesting that they form only metastable Innate Fungal Immunity tetramers in neuronal PM. GluA1 and GluA2 monomers and dimers were Eduardo U. Anaya, Aaron K. Neumann. much more mobile than tetramers in the dendritic-shaft PM and entered the Pathology, Univ New Mexico, Albuquerque, NM, USA. b spines quite readily. These results suggest a novel mechanism for synaptic Dectin-1 is a C-type innate immunoreceptor that recognizes -(1,3)- plasticity: During synaptic stimulation, the AMPAR subunit numbers and glucan, a structural component of Candida species cell walls. Dectin-1 has eight compositions in the postsynaptic membranes could be regulated readily by re- alternative splice isoforms, including ‘‘A’’ and ‘‘B’’. Due to its (hem)ITAM cruiting the preferred subunits pooled in dendritic-shaft PM, mostly in the motif, it’s thought that the receptor needs to dimerize to undergo signal trans- forms of monomers and dimers. duction. However, this has not been explored in live cells at the molecular level or in relation to signaling outcomes. Furthermore, innate immunocytes express one or both isoforms, but neither their responsiveness to b-glucan nor the po- 2624-Pos tential for inter-isoform signaling cross-talk is well understood. This study fo- Binding Free Energy Calculations of NMDA Glutamate Receptors cuses on the impact of ligation by soluble b-glucans varying in size and Adithya Polasa, Dylan S. Ogden, Mahmoud Moradi. quaternary structure on the Ca2þ signaling responses and dimerization states Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. of the major Dectin-1 isoforms. Stimulation with highly structured b-glucans N-methyl-d-aspartate (NMDA) ionotropic glutamate receptors are universal in resulted in greater amplitude of calcium influx in cells expressing Dectin-1A, mammalian central neurons that are engaged with neuronal advancement, syn- relative to less structured glucans. Meanwhile, Dectin-1B expressing cells aptic versatility and memory. The integrity of mixture of pre- and post-synaptic had decreased signaling compared to Dectin-1A and were less responsive to action by NMDA receptors requires a coupling between receptor’s binding and trends in glucans’ triple helical content. We observed that, in cells co- ion channel opening. Experimental studies have shown efficient interactions of expressing both isoforms, Dectin-1B negatively regulated signaling from the various bioactive compounds for therapeutic applications. We have performed Dectin-1A isoform. Furthermore, we utilized Fluorescence-Lifetime Imaging all-atom molecular dynamics (MD) simulations of two different NMDA recep- Microscopy for Forster Resonance Energy Transfer (FLIM-FRET) to better tors. Unbiased MD simulations of apo as well as agonist- and antagonist-bound resolve the biophysical process of Dectin-1 aggregation and activation in living proteins were used to characterize the local conformational dynamics of cells. Our results indicate that highly structured glucans result in a significant NMDA glutamate receptor mostly within the ligand binding site, while non- increase in donor-acceptor FRET populations whereas less structured glucans equilibrium pulling simulations were used to study the coupling between the result in no increase in donor-acceptor FRET populations. Lastly, we see the local and the global conformational changes associated with the channel acti- formation of Dectin-1A/B homo- and heterodimers and higher order oligomers vation process. These pulling simulations were employed to trigger the opening through western blot crosslinking studies. These results advance our under- of the channel by imposing rotational changes on the orientation of transmem- standing of Dectin-1 signaling by providing a better understanding of the brane (TM) alpha helices. The agonist-bound simulation has shown a closing role of Dectin-1 isoforms in signaling processes during pathogen recognition mechanism similar to clam-shell in ligand binding domains (LBD) activating as well as the nature of ligand induced Dectin-1 multimerization required for interconnecting loops between LBD and TM alpha helices. This conforma- signaling. tional shift initiated the pulling of TM alpha helices leading to channel opening, whereas in antagonistic binding state no such mechanism was observed. In 2627-Pos addition, relative binding free energy calculations were conducted for different Importance of Ordered Environment in the Early Stage of Mast Cell agonist and antagonist ligands to observe differential binding modes of ligands Signaling Studied by Imaging Fluorescence Correlation Spectroscopy in NMDA receptor ligand-binding domains. These simulations provide a mo- Nirmalya Bag, David A. Holowka, Barbara A. Baird. lecular basis for understanding the chemomechanical couplings in NMDA Dept Chem & Chem Biol, Cornell Univ, Ithaca, NY, USA. glutamate receptors and also for designing specific inhibitors to target the Plasma membrane (PM) localized receptors and other signaling components NMDA receptors. need to assume a poised, ‘resting state’ organization to optimally elicit

BPJ 9466_9469 530a Wednesday, March 6, 2019 transmembrane signaling in response to extracellular stimuli. Inherent PM neous cellular activation outcomes are detected based on localization of organizational principles often include, but are not limited to, ordered NFAT (calcium) and ERK (MAPK) activity reporters. We examine the rela- proteo-lipid nanodomains and cortical actin cytoskeleton. Such structural fea- tive timing and activation thresholds for these two signaling branches tures underpin the steady-state environment of signaling components to pro- thought to diverge downstream of the triggered TCR. Coordinated activation vide an optimal platform for signal initiation. Therefore, it is necessary to of NFAT and ERK is required for cytokine production. We control the im- evaluate the ‘resting state’ organization of signaling components and its mod- pulses to introduce a variety of stimulatory and non-stimulatory conditions, ulation after stimulation for complete understanding of the PM-localized stage with densities spanning physiological conditions. Overall, the results reveal of the cell surface signaling. In this study, we used RBL mast cells as a gen- characteristic behaviors that map accurately to details of the input ligand. eral paradigm of ligand induced transmembrane signaling. Here, extracellular NFAT activates only when cells are exposed to agonist pMHC. Notably, triggering of immunoglobin E complexed to its receptor, FcεRI, (IgE-FcεRI) ERK is strongly potentiated by adhesion (via LFA-ICAM interac- by multivalent antigen leads to receptor clustering and subsequent phosphor- tions) and its activity exhibits different temporal fluctuation patterns in ylation by Lyn tyrosine kinase to initiate signaling. We first examined lateral response to pMHC. Specifically, ERK activation dynamics reflect the ability environment ‘sensed’ in the resting state by IgE-FcεRI and Lyn by monitoring of the T cell to identify the presence of even null pMHC ligands, which fail their respective lateral diffusion modes in RBL cells with multiplexed Imag- to induce any NFAT response. This multi-dimensional activity readout may ing Fluorescence Correlation Spectroscopy (Imaging FCS) and associated spot help reveal insights into how synthetic TCR activators, such as bispecific variation FCS (svFCS). The diffusion of both receptor and kinase deviate antibodies used for cancer immunotherapy, activate multiple pathways en from free diffusion due to interaction with different confinement sources. route to full T cell activation. Furthermore, we identified the simultaneous presence of lipid-dependent and actin-dependent confinement sources for the diffusion of Lyn kinase, 2630-Pos whereas diffusion of IgE-FcεRI was weakly dependent on cortical actin orga- Beyond the TCR, Antigen Discrimination in T Cells Continues in the nization. Importantly, our Imaging FCS results for a dozen different mem- LAT:GRB2:SOS Protein Condensate brane proteins indicate that 70% of the plasma membrane exhibits the Shalini T. Low-Nam1, Jenny JY Lin1, Darren B. McAffee1, diffusion properties of an ordered environment. We further elucidate the asso- Steven A. Alvarez2, Scott D. Hansen3, Kole T. Roybal4, Jay T. Groves1. ciation of Lyn kinase and stimulated IgE-FcεRI receptor with their lipid- 1Dept Chem, UC Berkeley, Berkeley, CA, USA, 2Materials Science and driven ordered environments, which effectively enhances their spatial prox- Engineering, UC Berkeley, Berkeley, CA, USA, 3Chemistry and imity that is essential for optimal phosphorylation of antigen-activated Biochemistry, Univ. of Oregon, Eugene, OR, USA, 4Microbiology and receptors. Immunology, UCSF, San Francisco, CA, USA. T cells activate in response to a stochastic series of single molecule binding 2628-Pos events between T cell receptors (TCRs) and peptide ligands presented on Nanoscale Organization and Mobility of Ligands Direct T Cell Activation major histocompatibility complexes (pMHCs) accumulated at a cell-cell Joschka Hellmeier1, Viktoria Motsch1, Rene Platzer2, Andreas Karner3, interface. Discrimination between ligands with subtly different affinities en- Johannes Preiner3, Gerhard J. Schuetz1, Johannes B. Huppa2, Eva Sevcsik1. sures selective and robust immunological responses upon TCR triggering. 1Institute of Applied Physics, TU Wien, Wien, Austria, 2Medical University Formation of protein condensates based on the scaffolding protein Linker of Vienna, Wien, Austria, 3Campus Linz, University of Applied Sciences for Activation of T cells (LAT) is a key intermediate step in TCR signaling. Upper Austria, Linz, Austria. LAT is multi-phosphorylated on tyrosine residues by the kinase, ZAP70, The interaction of T cells with antigen presenting cells (APC) plays a central which is activated by triggered TCRs. The phosphotyrosines recruit Grb2, role in the adaptive immune system. However, the molecular mechanisms by which, via interactions with the multivalent GEF, SOS, forms an extended which binding of the T cell receptor (TCR) to its ligand triggers a signal LAT:Grb2:SOS assembly that resembles a phase transition. We mapped within the cell are still debated. Here, we use DNA origami decorated with single pMHC:TCR binding events in T cells expressing fluorescent TCR ligands anchored to a planar glass-supported lipid bilayer to assess the protein-tagged LAT and show that LAT assemblies are discrete and have effects of local ligand density, arrangement and mobility on T cell activation. characteristic features that are independent of the originating binding event. Our experimental setup allows for controlling the nanoscale arrangement of Furthermore, LAT assemblies can become spatially uncoupled from the TCR ligands on the DNA origami scaffold as well as the re-organization of pMHC:TCR complex. These individual assemblies are capable of deforming ligand and TCR during T cell activation. Further, the spatial distribution of the plasma membrane and creating local domains that may participate in ligands can be tuned independently of ligand concentration. TCRb-reactive protein segregation or control reaction kinetics. The probability of LAT as- single-chain antibody fragment (scFv) and peptide-loaded major histocompat- sembly is a function of the pMHC affinity, suggesting a second layer of ki- ibility complex II (pMHC) were used as ligands and placed on the DNA netic proofreading that exists beyond the triggered TCR. We recently origami at engineered capture sites in different layouts and densities. Func- introduced a molecular impulse-response assay which maps pMHC:TCR tionalized DNA origami platforms were characterized using single molecule binding events to early T cell activation. Results here argue that LAT as- fluorescence microscopy and high-speed atomic force microscopy (HS- semblies may confer a molecular memory that connects a series of spatio- AFM). The activation of T cells interfaced with the APC-mimicking surfaces temporally correlated binding events to cellular decision-making. By was determined by measuring intracellular calcium levels; the effects of local extending the impulse-response assay, we show that activating cells experi- ligand density, nanoscale ligand arrangement, platform mobility as well as the ence a characteristic, fast rate of LAT assembly. Taken together, we present nature of the ligand were assessed. Further, the rearrangement of TCR and new molecular dimensions to T cell activation that could provide actionable ligand in the process of T cell activation was monitored by single molecule therapeutic insights. microscopy. 2631-Pos 2629-Pos A Membrane-Activated, Universal T-Cell Receptor Agonist Live and Simultaneous Readout of NFAT and ERK Activation in T Cells Kiera B. Wilhelm1, Michael P. Coyle1, Geoffrey P. O’Donoghue2, Reveals Multiple Dimensions of TCR Signaling Jenny JY Lin1, Shalini T. Low-Nam1, Jay T. Groves1. Jenny J.Y. Lin1, Shalini T. Low-Nam1, Steven A. Alvarez2, Jay T. Groves1. 1Chemistry, UC Berkeley, Berkeley, CA, USA, 2Chemistry, UCSF, San 1Dept Chemistry, UC Berkeley, Berkeley, CA, USA, 2Dept Mat Sci/Eng, UC Francisco, CA, USA. Berkeley, Berkeley, CA, USA. T cells confer adaptive immunity by sensitively and specifically recognizing T cell activation is key in initiating adaptive immunity and targeted control antigenic peptides presented on Major Histocompatibility Complex over this process is core to cancer immunotherapy. Among the myriad of (pMHCs) molecules with their T-cell receptors (TCRs). Each developing ligands encountered during antigen scanning, T cells are exquisitely sensi- T cell expresses a unique TCR that binds productively to only one or a tive to small numbers of agonist ligands to ensure robust responses. We few pMHCs. TCR diversity within an individual facilitates robust, specific recently introduced a supported-bilayer-based, single-cell impulse- immune responses, but complicates efforts to study T-cell activation response assay that maps the complete sequence of single molecule binding because the pMHCs that activate T cells are generally unknown. Antibodies events between agonist peptide ligands loaded on major histocompatibility are able to universally activate T cells; however they activate T cells by complexes (MHC) and T cell receptors (TCR) to early cellular activation crosslinking TCR and are active from solution, neither of which is true via calcium signaling. Individual pMHC:TCR binding events are detected for natural antigen pMHC. When tethered to a membrane surface, but not based on mobility changes using single molecule fluorescence imaging from solution, pMHC monomers can activate T cells at the single molecule and spatiotemporal features of each complex are recorded. Here, simulta- level without crosslinking. Antibodies are likely to effectively short-circuit

BPJ 9466_9469 Wednesday, March 6, 2019 531a the TCR signaling system, and thus may not represent natural antigen (AFM), rigidity-based mechanical properties of these three different cell pMHC activation. We have designed a monovalent, membrane-tethered uni- lines under six different substrate stiffnesses are measured and quantified. versal TCR agonist that can trigger early T-cell activation, introducing a Magnetic tweezers are used to apply force directly to integrins at focal ad- synthetic TCR agonist that most closely mimics pMHC. This agonist, an hesions. The correlation between the mechanical properties and forces on anti-TCRb Fab’ fragment, is coupled to a supported lipid bilayer via talin which are showed in our data reveal the different mechanisms adopted DNA complementation to mimic the physiological cell-cell interface. In by normal and cancerous human breast cells. Our results indicate that the our experimental platform, every Fab’-DNA:TCR complex is visualized response of cancer cells to matrix is significantly different from that of and mapped to cellular readouts for signal propagation and activation. We normal cells, which provide an insight about the properties of cancer cells demonstrate that Fab’-DNA triggers TCRs and stimulates translocation of during the metastasis. the transcription factor Nuclear Factor for the Activation of T Cells (NFAT) with a similar potency to strongly activating pMHCs. Other dimen- 2634-Pos sions of Fab’-DNA triggered T cells will also be discussed, including differ- Computational Model of RGD-Containing Computational Model of RGD- ences between distinct binding epitopes. Ultimately, a comprehensive Containing Peptides and Their Effects on Integrin Binding characterization of multiple distinct Fab’-DNA constructs may help inform Tamara C. Bidone1, Aravind Rammohan2, Matt McKenzie3, design of cancer immunotheraputics, such as bispecific antibodies, that are Gregory A. Voth1. structurally similar to this synthetic ligand. 1Univ Chicago, Chicago, IL, USA, 2Univ Chicago, Corning Incorporated, NY, USA, 3Corning Research and Development Corporation, New York, 2632-Pos NY, USA. Single pMHC:TCR Binding Events Precipitate LAT Assemblies Capable Peptides based on cell adhesion regions, including Arg-Gly-Asp (RGD) se- of Spatially Decoupling from the Originating Ligand quences, are widely used in the design of functionalized surfaces. They can Darren B. McAffee1, Shalini T. Low-Nam1, Jenny JY Lin1, have different geometrical and chemical properties, varying for example for Steven A. Alvarez1, Scott D. Hansen2, Jay T. Groves1. height, localization of the RGD sequence, and specificity. Moreover, they 1Univ Calif Berkeley, Berkeley, CA, USA, 2University of Oregon, Eugene, can be used at different degrees of surface coverage in order to control OR, USA. integrin-based cell adhesions. However, the exact interplay between T cells are activated by peptide-MHC complexes (pMHC) that bind T cell geometrical and chemical factors of RGD-containing peptides in integrin receptors (TCRs) on the cell surface. These binding events enable the phos- binding is not fully understood. Here, we present a computational model phorylation and oligomerization of multiple downstream proteins. LAT is a of adhesion formation on functionalized surfaces based on the spacing be- scaffold molecule that mediates a phospho-dependent multi-molecular as- tween RGD-containing peptides, localization of RGD sequence and surface sembly in T cells that governs the activity of many signaling pathways. rigidity. The binding energies for modeling specific and non-specific inter- The quantitative dependence of this protein condensation on TCR input is actions between integrins and RGD sequences are incorporated from molec- unknown. By tracking individual pMHC:TCR binding events we are able ular level simulations and affinity measurements. The properties of to map spatio-temporal features of binding to the assembly of fluorescently individual peptides, including density and position of RGD region, are sys- tagged LAT. Remarkably, a single binding event is sufficient to trigger the tematically varied in order to characterize their relative effects on integrin assembly of hundreds of LAT molecules. These assemblies are transient and binding. Our results show that within the range of experimentally- self-limiting. The extent and dynamics of the LAT assembly — which measured peptide densities, integrin binding is promoted, whereas at lower feature phases of growth, steady state, and decay — are independent of or higher surface coverages integrin binding is reduced. The key factor ap- the dwell time of the initiating ligand. Assemblies have similar features pears to be the excluded volume between RGD-containing peptides, that regardless of the peptide ligand used for stimulation. This suggests that controls the degree of exposure of RGD-containing binding regions. These once phospho-LAT undergoes a phase transition into a LAT assembly and other results will be discussed in the context of the design of function- then subsequent molecular events are uncorrelated with the assembly’s orig- alized surfaces. inating binding event. This would indicate a completion of kinetic proof- reading. Moreover, these assemblies can often travel hundreds of 2635-Pos nanometers away from the binding event. We find that antigen discrimina- Total Reconstitution of Receptor-Mediated Ras Activation by SOS in Vitro tion between peptides arises from different probabilities to trigger LAT as- Reveals Kinetic and Conformational Layers of Regulation in MAPK sembly formation. Longer dwelling peptides produce LAT assemblies with a Signaling higher success rate. Together, these results suggest that LAT assemblies Steven Alvarez1, William Y.C. Huang2, Hiu Yue Monatrice Lam2, contribute discrete units of signal that are triggered, but not enhanced, by Shalini T. Low-Nam2, Young Kwang Lee2, Jean K. Chung2, long dwelling binding events. Scott D. Hansen2, Yasushi Kondo3, Kabir H. Biswas4, John Kuriyan3, Jay T. Groves2. 2633-Pos 1Dept. of Materials Science and Engineering, University of California Mechanical Responses of Cancer Cells to Different Matrices Measured by Berkeley, Berkeley, CA, USA, 2Dept. of Chemistry, University of California AFM and FRET Berkeley, Berkeley, CA, USA, 3Dept. of Molecular and Cell Biology, Fang Tian1, Tsung-Cheng Lin2, Liang Wang3, Sidong Chen4, Jun Chu3, University of California Berkeley, Berkeley, CA, USA, 4Mechanobiology Ching-Hwa Kiang2, Hyokeun Park1. Institute, National University of Singapore, Singapore, Singapore. 1Physics, University of Science and Technology, Hong Kong, Activation of the guanine nucleotide exchange factor Son of Sevenless Hong Kong, 2Physics, Rice University, Houston, TX, USA, 3Physics, (SOS) is a key step in controlling Ras signaling. SOS has a multidomain ar- Shenzhen Institutes of Advanced Technology, Shenzhen, China, 4Life chitecture comprised of a catalytic subunit flanked by regulatory domains. It Science, Hong Kong University of Science and Technology, Hong Kong, is highly autoinhibited in solution and achieves full activation through spe- Hong Kong. cific interactions, such as lipid and allosteric Ras binding, at the cell mem- Integrin-based focal adhesions matured at the front of the migrating cells brane. SOS is capable of accessing a long-lived, highly processive state and dissembled at the retracting edge of cells. In the formation, maturation that can activate up to hundreds of Ras molecules. Thus, a detailed, molec- and disassembly of focal adhesions, talin play an import role as a mediator ular understanding of full SOS activation is essential. We describe the total between mechanosensitive membrane protein, integrin, and actin cytoskel- in vitro reconstitution of Ras activation by SOS on supported lipid bilayers. eton. It is known that talin undergo conformational changes when they Imaging of single molecule SOS recruitment and Ras activation, detected us- are stretched with higher force. Vinculin binding sites and actin binding do- ing the Ras binding domain (RBD) of Raf1, on micropatterned membranes mains is known to be exposed when talin are stretched. Matrix can impact enables correlating SOS membrane localization with Ras activation in hun- cellular functions, such as cell growth and migration. However, how the dreds of reactions simultaneously. SOS activates Ras with two distinct ki- physical properties of cancer cells change with the extracellular matrix re- netics: switch-like activation, referred to as burst activation, and gradual mains to be an open question. Here, we measured the rigidities of cancer activation, referred to as transient activation. We demonstrate that burst ac- cells at different matrix using AFM and force transduction of cancer cells tivity, in which maximal Ras activation is reached with a sudden, steep tran- using a new Forster resonance energy transfer (FRET) tension sensor. Mea- sition, is the result of long-dwelling, stably-recruited single molecules of surements the force changes on talin were conducted in normal cells SOS. Both burst and transient SOS activities are modulated by the density (MCF10A), non-invasive cancerous cell (MCF7), and invasive cancerous of active Ras on the membrane, suggesting that a structural rearrangement human breast cells (MDA-MB-231). Using atomic force microscopy at the membrane, rather than allosteric Ras binding, is responsible for the

BPJ 9466_9469 532a Wednesday, March 6, 2019 distinct kinetic states. Furthermore, regulatory domains of SOS suppress 2638-Pos transient activity while retaining the burst activity, arguing that burst activity Quantification of G1-Cyclin Dynamics in Yeast by Scanning Number and might be the signaling competent kinetic state in the cell context. Structural Brightness rearrangements at the membrane and release of autoinhibition likely repre- Savanna Dorsey1, Pooja Goswami1, Jing Cheng2, Yogitha Thattikota2, sent kinetic proofreading steps that modulate full SOS activation and, ulti- Sylvain Tollis2, Catherine A. Royer1, Mike Tyers2. mately, MAPK signal propagation. 1Biology, Rensselaer Polytechnic Institute, Troy, NY, USA, 2Universitede Montreal, Montreal, QC, Canada. 2636-Pos Cell size homeostasis reflects the coordination between cell growth and di- Cancer Cell Has Lowered Threshold of Signaling Transduction Excitable vision and is vital to all living organisms. This coordination is achieved pri- Network Controlled by PIP-RAS Interaction marily at the commitment to cell division at the G1/S phase transition, David Huiwang Zhan. termed Start in budding yeast. Cells pass Start upon activation of kinase Biological Chemistry Department and Cell Biology Department, Johns Cdc28 by the G1 cyclin, Cln3, which phosphorylates the repressor Whi5, Hopkins University School of Medicine, Baltimore, MD, USA. thereby activating expression of the downstream G1 cyclins CLN1/2 and The dynamic change of cell migratory mode and the travelling actin wave 200 other genes in the G1/S regulon by the SBF/MBF transcription factor at the ventral surface are two typical characteristics of epithelial cancer complexes. Recently, we demonstrated that SBF/MBF abundance, and cell. Many distinct pathways have been reported to be involved, but an consequently Cln1/2 levels, are differentially regulated by nutrients. How- overall model to explain the interaction between the signaling transduction ever, it remains unclear how dynamics of Cln1/2 levels in G1 phase affect system and the cytoskeleton motor system is lacking. We proved that the Start. Moreover, the mechanism by which Cln3 activates Start remains signaling transduction network in epithelial cancer cell was excitable by elusive due to its low expression level and extreme instability. We per- showing the existence of threshold and refractory period. The annihilation formed time-course Scanning Number and Brightness (sN&B) fluorescence of two meeting waves and the results of global and local EGF stimulations fluctuation microscopy experiments on live yeast expressing Cln1/2/3 GFP indicated that the wave was also excitable. We manipulated the signaling fusions at their endogenous loci. We quantified single-cell protein concen- transduction network aiming to change the cell migratory mode and/or trations and localization patterns of each G1 cyclin throughout G1 phase actin wave. By lowering PI(4,5)P2 or recruiting active Ras to the cell in rich medium and in response to nutrient shifts. In early G1 phase, membrane, we were able to detect oscillatory spreading of cell and stron- Cln1/2 were expressed at low levels primarily in the cytoplasm. Cln1 cyto- ger wave formation and propagation. The threshold was found to be low- plasmic levels were generally higher (5nM)inearlyG1comparedtoCln2 ered with decreasing PI(4,5)P2 or recruiting Ras. Further investigation levels (1 nM). Unexpectedly, we observed low levels of Cln3 throughout revealed that the activity of downstream PI(3,4,5)P3, mTorC2, ERK and G1 phase, with distribution between the nucleus (20 nM) and cytoplasm PI(3,4)P2 oscillated accordingly to oscillatory spreading. The mutual inhi- (5 nM) varying randomly with respect to Start onset. This result suggests bition of PI(4,5)P2 and Ras served as positive feedback while the PI(3,4) that some aspect of Cln3 dynamics beyond its mere accumulation governs P2 and ERK served as negative feedback, which maintained the excit- the timing of Start. These data have been used to constrain a dynamic math- ability of the signaling transduction system. Too much lowering of ematical model of Start. PI(4,5)P2 broke up the excitable system and stopped cell oscillatory 2639-Pos spreading. Since tumor normally has increased migration ability, we Rhoa Mediated Juxtacrine Regulation of Glucagon Secretion then asked whether the cancer cell has lowered threshold compared to Yong Hee Chung, David W. Piston. its non-tumor counterpart. By comparing the Twist ON/OFF primary Cell Biology & Physiology, Washington University in St. Louis, St. Louis, mammary epithelial cells, and non-tumor MCF-10A cell VS MYC/RAS MO, USA. transformed tumor MCF-10A cell, we were able to demonstrate that can- Glucagon is secreted from pancreatic islet alpha-cells in response to hypo- cer cells had lowered threshold. This work provides a new theory to under- glycemia. Proposed models for glucose regulation of glucagon secretion fall stand the cancer malignancy and potentially a new therapeutic angle for into three general types: direct regulation by glucose, paracrine regulation cancer treatment. by other cell types within the islets of Langerhans, and juxtacrine regulation by neighboring cells. Here we are focused on one pathway of juxtacrine 2637-Pos regulation, which occurs through interactions of ephrin-A5 ligands on the Dynamic Partitioning and Convection: A New Mechanism for the Self- surface of beta-cells and EphA4 receptors on the alpha-cells. In this model, Organization Pattern of the Excitable Cortical Waves? stimulation of EphA4 leads to dense F-actin network that inhibits the secre- 1,2 1 3 3 Tatsat Banerjee , Yuchuan Miao , Debojyoti Biswas , Pablo A. Iglesias , tion of glucagon granules. However, the molecular pathway downstream of Peter N. Devreotes1. 1 EphA4 is poorly understood. We present evidence of the role of RhoA, a Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Rho family GTPase, in regulating glucagon secretion by modulating F-actin MD, USA, 2Chemical and Biomolecular Engineering, Johns Hopkins 3 network formation. Pharmacologial inhibition of RhoA resulted in an in- University, Baltimore, MD, USA, Electrical and Computer Engineering, crease of glucagon secretion and correlated to a loss of F-actin network den- Johns Hopkins University, Baltimore, MD, USA. sity. Through the use of fluorescent biosensors, we were able to The spatially distributed components of signal transduction excitable quantitatively measure glucose-dependent changes in activities of RhoA network (STEN) and cytoskeletal excitable network (CEN) in a Dictyoste- and F-actin in vivo using confocal microscopy. Electrophysiological exper- lium discoideum cell generate autonomous waves in the substrate attached iments are underway to explore possible signaling from EphA4 activation on basal surface, which contribute to the crucial physiological processes like putative changes in membrane permeability that may also occur downstream phagocytosis, macropinocytosis, cytokinesis, and pseudopodia-based cell of RhoA. The results implicate RhoA in the juxtacrine regulation pathway as motility. One key event in this characteristic cortical wave formation is a regulator of F-actin. the symmetry breaking of membrane phospholipids and self-organization of STEN and CEN proteins to dynamically generate distinct regions in 2640-Pos the cell cortex which resembles to the typical front and back regions of a Protein Kinase a Dynamics are Altered at the Outer Mitochondrial Mem- randomly migrating cell. The similar pattern of organization was widely brane in Cardiac Sympathetic Neurons from Prehypertensive Rats observed in chemotactic migration and cytokinesis of different eukaryotes Dan Li, Adib Tarafdar, Kun Liu, David J. Paterson. cell types. It was widely thought in the literature that the basis of this University of Oxford, Oxford, United Kingdom. self-organization is the dynamic shuttling mediated recruitment and dissoci- Cardiac sympathetic neurons experience larger Ca2þ transients in the spon- ation of STEN and CEN proteins in specific membrane domains. We studied taneously hypertensive rat (SHR) relative to normotensive Wistar rats, re- the selective localization based shape dynamics of these traveling excitable sulting in enhanced sympathetic activity at the end organ level. waves in electrofused giant cells and identified a novel pattern of a number Impairments in cyclic nucleotide signalling contribute to this phenotype, of STEN and CEN components. Our data additionally suggests that besides and are thought to arise due to excess phosphodiesterase (PDE) 2A activity. conventional recruitment based mechanism, a gliding/convection based Recently, it has been suggested that PDE2A overactivity occurs focally, dynamic partitioning process may exist in the self-organization pattern around the outer mitochondrial membrane (OMM) and is responsible for which facilitate a number of membrane proteins to stay on the membrane, the impaired cAMP dynamics. However, the functional significance of to not shuttle in and out of cytoplasm (in the timescale of wave dynamics), cAMP impairment has not been determined. This study investigated whether and yet to be localized preferentially in particular domains on the cell Protein Kinase A (PKA) activity was altered at the OMM in sympathetic membrane. neurons from the SHR. Cardiac stellate ganglia were cultured from 4-5

BPJ 9466_9469 Wednesday, March 6, 2019 533a weeks old pre-hypertensive SHRs and Wistar rats. Two PKA biosensors ex- cence resonance energy transfer (BRET) as well as western blot assays indi- pressing cytoplasmic and the outer mitochondrial membrane (OMM) tar- cate the destabilization of oligomer by the mutation. Indeed, our MD geted AKAR4 were transferred to cultured sympathetic neurons. simulations demonstrated that the E102Q mutation disrupts the local Fluorescence resonance energy transfer technique was used to measure dy- hydrogen bond network and destabilizes the region connecting the C-termi- namic changes of PKA concentration. No significant difference in nal domain and the transmembrane helix (anchor region), as well as the Forskolin-evoked PKA activity was observed in the cytosol or at the conformation of the ligand binding site. Together, results herein provide first OMM between SHR and Wistar neurons. However, direct PKA activation account of molecular mechanism of s1R dysfunction by E102Q mutation using Sp-8-Br-cAMP led to significantly higher PKA activity being detected and characterize the importance of the anchor region in the receptor’s at the OMM in SHR. In the presence of Bay60-7550 (a PDE2A specific in- function. hibitor), greater Forskolin-evoked PKA activity was also observed in SHR neurons at the OMM. Evidence presented in this study demonstrates that there is impairment of PKA signalling at the OMM in sympathetic neurons 2643-Pos of the SHR. This might be caused by over-activity of PDE2A. Our findings Elucidation of Signaling Mechanism of ANP Receptor by X-Ray Crystal- suggest that impaired cAMP-PKA signalling at the OMM may have impor- lography tant functional consequences linked to enhanced sympathetic neurotransmis- Haruo Ogawa, Masami Kodama. sion. PDE2A may be a valuable therapeutic target for dysautonomia IQB, Univ Tokyo, Tokyo, Japan. associated in the treatment of hypertension. Atrial natriuretic peptide (ANP) plays a major role in the regulation of blood pressure and volume regulation. The actions of ANP are mediated 2641-Pos by a ANP receptor, a single span transmembrane receptor carrying intrinsic Role of Store Operated Calcium Channel Complex in the Inflammatory guanylate cyclase activity. The ANP receptor consists of a glycosylated Signaling in Peripheral Sensory Neurons extracellular ANP-binging domain and an intracellular domains including a guanylyl cyclase catalytic domain, and acts as a homodimer. The receptor Alexandra S. Hogea, Shihab Shah, Nikita Gamper. is a member of a family of GCase-coupled receptors that share a similar Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom. Inflammation underlies many acute and chronic pain conditions. Excitation overall molecular configuration and, presumably, a common signal trans- of peripheral afferents innervating the inflamed tissue is a primary event in duction mechanism. However, the mechanism remains largely unknown. generation of the inflammatory pain. Such excitation is produced by ‘inflam- Here, we have solved crystal structures of extracellular ANP-binging matory mediators’ - reactive compounds released by the damaged tissue and domain in complex with three ligands (ANP, ANP lacking its C-terminus region and Dendroaspis natriuretic peptide (DNP), isolated from the venom the immune cells at the inflammation site. Many of these inflammatory me- of the green Mamba snake Dendroaspis angusticeps). High resolution struc- diators (e.g. , histamine, prostaglandins, ATP etc.) are ligands of G protein coupled receptors (GPCR) which activate phospholipase C (PLC) ture allows us to build the bound ligands precisely, including water mole- and induce release of Ca2þ from the endoplasmic reticulum (ER). Ca2þ sig- cules. Plausible mechanisms of ligand recognition and transmembrane nals induced by these receptors in sensory neurons produce excitation and signal transduction mechanism, and insight from the structures will be trigger pain. A crucial mechanism maintaining the ER Ca2þ store refill discussed. and the GPCR-mediated Ca2þ signaling, is the calcium release-activated channel (CRAC). Here we characterized the expression of CRAC compo- nents within the rat dorsal root ganglion (DRG) neurons and explored the Posters: TRP Channels role of CRAC channel complex in the inflammatory GPCR signaling in 2644-Pos DRG. STIM1/2 and Orai1/3 were expressed within DRG neurons of Molecular Mechanism of Ligand-Induced TRPV2 Channel Activation different sensory modalities with STIM1 and Orai1 being the predominant Shasha Feng1, Huisun Lee1, Ruth Anne Pumroy2, Amrita Samanta2, subunits. STIM1 and Orai1 formed clusters upon ER store depletion, as Vera Moiseenkova-Bell2, Wonpil Im1. demonstrated by the proximity ligation assay (PLA). STIM2 was preferen- 1Department of Biological Sciences, Lehigh University, Bethlehem, PA, tially expressed in large-diameter (presumed mechanosensory) neurons. 2 þ USA, Department of Systems Pharmacology and Translational Store-operated Ca2 entry (SOCE) in DRG neurons was sensitive to Therapeutics, University Pennsylvania, Philadelphia, PA, USA. CRAC inhibitors YM58483 and Synta66. CRAC channel complex assembly The transient receptor potential vanilloid 2 (TRPV2) belongs to the transient relies on the close proximity of the plasma membrane and endoplasmic re- receptor potential vanilloid (TRPV) subfamily, which consists of six members ticulum. We show that junctional proteins junctophilin-3 and junctophilin-4 (TRPV1-6). While TRPV2 shares 50% sequence identity with TRPV1, the are expressed in DRG. Knocking-down JPH4 disrupted the Orai1-STIM1 most extensively studied heat and pain sensor, it differs from TRPV1 in its clustering and inhibited SOCE. Moreover, JPH4 knockdown impaired the þ overall cellular function. TRPV2 is sensitive to a variety of stimuli, including ability of DRG neurons to generate multiple Ca2 transients in response ligand binding, mechanical stress, and pH, and has been proposed to play a crit- to inflammatory mediators (bradykinin and ATP). Taken together these find- ical role in neuronal development, cardiac function, immunity, and cancer. ings reveal the importance of CRAC channel complex in inflammatory Despite its diverse functions and ubiquitous expression profile, little is known signaling in sensory neurons. about TRPV2 at the molecular level.In our study, we used molecular dynamics simulations to study TRPV2 gating by endogenous and exogenous modulators. 2642-Pos Our simulations probed the effect of modulators interacting with several critical Mechanistic Characterization of the E102Q Mutation in the Sigma 1 Re- regions of TRPV2. We also examined the ligand binding of a potent TRPV2 ceptor agonist and the conformational changes of TRPV2 upon ligand interaction. Hideaki Yano, Ara M. Abramyan, Sett Naing, Leanne Liu, Lei Shi. Simulation offers a unique approach to shed light into structural mechanisms National Institute on Drug Abuse National Institutes of Health, Baltimore, of TRPV2 modulation by endogenous factors, as well as channel activation MD, USA. by ligands. The sigma 1 receptor (s1R) is a unique endoplasmic reticulum membrane pro- tein, sharing no similarity with other mammalian proteins and mainly func- tioning as a molecular chaperone. s1R ligands have shown therapeutic 2645-Pos potentials in the treatments of cancer, neurological disorders, and substance Short- and Long-Distance Allosteric Coupling in Capsaicin-Induced use disorders. The E102Q mutation of s1R has been found to elicit familial TRPV1 Activation cases of amyotrophic lateral sclerosis (ALS). Despite recent reports on its Simon Vu1, Bo Hyun Lee2, Xian Xiao3, Fan Yang4, Jie Zheng1. downstream signaling consequences, mechanistic details of the functional 1University of California, Davis, Davis, CA, USA, 2University of impact of the E102Q mutation at molecular level is not clear. Washington, Seattle, WA, USA, 3Westlake University, Hangzhou, China, Here we investigated the mechanism of the E102Q mutation with a spectrum 4Zhejiang University, Hangzhou, China. of biochemical, biophysical and pharmacological assays. In combination Capsaicin binding opens TRPV1 through allosteric coupling; however, the with in silico molecular dynamics (MD) simulations based on the s1R structural basis for the coupling process remains largely unknown. Binding crystal structures, we reveal the perturbation of this mutation on the oligo- of capsaicin is mediated partly by two hydrogen bonds: one between the hy- merization of s1R as well as the impact on s1R ligand binding. Specif- droxyl group on the capsaicin head and that of T551 on S4, another between ically, the radioligand binding assayshowedthatthemutationdecreased the amide group of capsaicin neck and the carboxyl group of E571 on S4-S5 the affinity as well as expression levels of s1R. Results from biolumines- linker. The formation of these H-bonds is thought to bring the S4-S5 linker

BPJ 9466_9469 534a Wednesday, March 6, 2019 away from the pore, allowing S6 (the lower gate) to open. Indeed, the 2648-Pos capsaicin-bound cryo-EM structure indicates movements of both the S4- Regulation of PKD2L1 by Calcium Effectors S5 linker and S6. However, we recently found that capsaicin binding also Amitabha Mukhopadhyay, Leo Ng, Thuy Vien, Paul DeCaen. induces outer pore movement, which opens the selectivity filter (the upper Feinberg School of medicine, Pharmacology, Northwestern University, gate) to yield an open pore. How this is achieved, and the relationship be- Chicago, IL, USA. tween the two gates, remain unclear. To understand how capsaicin binding Architecture of a tissue is determined by the function and positioning of cilia. controls both the lower and upper gates, we carried out a detailed kinetic Cilia is a membrane bound compartment, projected like antenna from the pe- analysis that took advantage of the knowledge of structural details underly- riphery of cell, acting as a sensor for external stimuli. Ciliary malfunction leads ing the capsaicin activation process. This analysis revealed both short and to defects in tissue organization, developmental anomalies and sensory recep- long-distance structural coupling. tion, which are collectively called as ciliopathies. One of the most common and life-threatening ciliopathy is autosomal dominant polycystic kidney disease (ADPKD) – where fluid-filled cysts develop and gradually lead to kidney fail- 2646-Pos ure. Mutation in Polycystin ion channels (TRPP family) are known to be the Structural Insights on TRPV5 Gating by Endogenous Modulators primary cause of ADPKD, but we lack a clear understanding of how the Poly- 1 1 2 3 Taylor Hughes , Ruth Pumroy , Aysenur Yazici , Marina Kasimova , cystin channels function. We demonstrate a novel mechanism of regulation of 1 4 1 Edwin Carl Fluck , Kevin Huynh , Amrita Samanta ,SudheerKumar PKD2L1 (or Polycystin-L, or TRPP2, formerly TRPP3) by calcium effectors 1 4 3 2 Molugu , Hong Zhou , Vincenzo Carnevale , Tibor Rohacs , which control channel activity. Vera Moiseenkova-Bell1. 1University of Pennsylvania, Philadelphia, PA, USA, 2Rutgers University, 2649-Pos Newark, NJ, USA, 3Temple University, Philadelphia, PA, USA, CaMKII Regulates TRPC6 Mediated Stress Stimulated Contractility in 4California NanoSystems Institute, University of California, Los Angeles, Muscular Dystrophy CA, USA. Brian L. Lin1, Sumita Mishra1, Grace E. Kim1, Suraj Kanann1, TRPV5 is a transient receptor potential channel involved in calcium reab- Jinying Yang1, Chulan Kwon1, Mark Anderson1, Steven S. Pullen2, sorption. Here we present the interaction of two endogenous modulators David A. Kass1. with TRPV5. Both phosphatidylinositol 4,5-bisphosphate (PI(4,5)P )and 2 1Dept Cardiology, Johns Hopkins University, Baltimore, MD, USA, calmodulin (CaM) have been shown to directly bind to TRPV5 and 2External Innovation, Boehringer-Ingelheim, Ridgefield, CT, USA. activate or inactivate the channel, respectively. Using cryo-electron micro- Duchenne muscular dystrophy (DMD) is an X-linked muscular dystrophy, in scopy (cryo-EM), we determined TRPV5 structures in the presence of which patients lose the ability to walk in their teens and die from heart failure. dioctanoyl PI(4,5)P and CaM. The PI(4,5)P structurerevealsabinding 2 2 DMD patients lack the dystrophin protein that is responsible for cellular stabil- site between the N-linker, S4-S5 linker and S6 helix of TRPV5. These þ þ ity, but the secondary insult of Ca2 overload is caused by upregulation of Ca2 interactions with PI(4,5)P induce conformational rearrangements in the þ 2 channels. We identified a mechanosensitive Ca2 channel known as Transient lower gate, opening the channel. The CaM structure reveals two TRPV5 Receptor Potential Canonical 6 (TRPC6) as a major contributor to the DMD C-terminal peptides anchoring a single CaM molecule and that calcium þ pathology. TRPC6 can be activated upon cellular stretch, mediating Ca2 inhibition is mediated through a cation-p interaction between Lys116 influx that results in an increase in myocyte force generation, known as on the C-lobe of calcium-activated CaM and Trp583 at the intracellular stress-stimulated contractility (SSC). SSC is a cardiac phenomenon that con- gate of TRPV5. Overall, this investigation provides insight into the endog- tributes to rapid cardiac responses but is exaggerated in disease. Recently, a enous modulation of TRPV5, which has the potential to guide drug TRPC6 phosphorylation site at threonine 487 was discovered to be phosphor- discovery. þ ylated by Ca2 /calmodulin-dependent kinase II (CaMKII), though the rele- vance of CaMKII activation of TRPC6 in cardiac physiology is unknown. 2647-Pos The membrane instability and hyperactivated CaMKII activity in DMD are The Role of Calmodulin in Regulating Calcium-Permeable PKD2L1 Chan- likely to exacerbate TRPC6-mediated SSC and pathology in DMD. Therefore, nel Activity we hypothesized 1) CaMKII is necessary to activate TRPC6-mediated SSC, Eunice Y. Park, Misun Kwak, Youngjoo Baik, Insuk So. and 2) this pathological pathway can be interrupted by inhibition of TRPC6. Dept Physiol, Seoul Natl Univ Col Med, Seoul, Republic of Korea. Using a custom force-length microscope, we attached, stretched, and stimu- Polycystic kidney disease 2-like-1 (PKD2L1), or polycystin-L or TRPP2, lated single cardiomyocytes in the presence of CaMKII and TRPC6 inhibitors. formerly TRPP3, is a transient receptor potential (TRP) superfamily member. Drugs that targeted either CaMKII or TRPC6 blunted SSC in WT cardiomyo- It is a calcium-permeable non-selective cation channel that regulates intracel- cytes, reflecting blunted SSC in cardiomyocytes from TRPC6 knock-out mice / lular calcium concentration and thereby calcium signaling. Calmidazolium (TRPC6 ) and CaMKII-inhibited (AC3-I) transgenic mice. In a severe (CMZ), which is well-known as a calmodulin (CaM) inhibitor, is an activator dystrophin-utrophin-deficient double-knockout (DKO) mouse model of of PKD2L1 channel, but the activating mechanism remains unclear. The pur- DMD, TRPC6 and CaMKII expression and activity are elevated. Inhibition pose of this study is to clarify whether CaM takes part in the regulation of of TRPC6 rescued function and dramatically increased lifespan. Together, PKD2L1 channel, and if so, how. our results introduce the role of CaMKII in cardiac mechanosensitivity by regu- To clarify the interaction between PKD2L1 channel and CaM, we co- lating TRPC6, and demonstrate the therapeutic potential of interrupting expressed the proteins in HEK293 cells and observed the changes in CaMKII-TRPC6-SSC pathway. PKD2L1 current amplitudes. When co-expressed with CaM(WT), the current amplitudes of PKD2L1 were significantly reduced while co- 2650-Pos expression with CaM(DN), the double-negative mutant, barely altered the Understanding Protein-Lipid Interactions of TRP Channels of the Polycys- current amplitudes of the channel. To further examine whether the binding tin Family, through MD Simulations and Structural Studies of CaM (either Ca2þ-CaM or apo-CaM) affects the channel currents, we Qinrui Wang1,2, George Hedger1, Prafulla Aryal3, Mariana Grieben2, generated the truncated CaM constructs which are CaMdeltaC and CaMdel- Ashley C.W. Pike2, Jiye Shi4, Elisabeth P. Carpenter2, Mark S.P. Sansom1. taN. Interestingly, CaMdeltaC significantly reduced the currents of PKD2L1 1Department of Biochemistry, University of Oxford, Oxford, United while CaMdeltaN did not alter the current amplitudes. This result suggests Kingdom, 2Structural Genomics Consortium, University of Oxford, Oxford, that the N-lobe of CaM carries more crucial role in regulating PKD2L1 and United Kingdom, 3Department of Physiology and Biophysics, Univ guides us into our next question on the different functions of two lobes of Colorado, Aurora, CO, USA, 4Department of Chemistry, UCB Pharma, CaM. Slough, United Kingdom. We also identified the predicted CaM binding site, and generated dele- Polycystin-2 (PC2) is a TRP channel protein encoded by Polycystic kidney dis- tion and truncation mutants. The mutants showed significant reduction ease-2 gene. Mutations in PC2 are associated with autosomal dominant polycy- in currents losing PKD2L1 I-V curve, suggesting that the C-terminal stic kidney disease (ADPKD), which is one of the most prevalent genetic region from 590 to 600 is crucial for maintaining functionality of disorders in human, estimated to occur in 1:800 live births. The recently solved PKD2L1 channel, but the correlation of CaM-binding has to be further veri- cryo-EM structures of human PC2 show that the overall architecture of the PC2 fiedwithCo-IP. homo-tetramer has the same fold as other TRP channels. The interaction of in- This research project was supported by BK21-plus education program of the tegral proteins with their lipid environment is crucial for the modulation of pro- MOE (Ministry of Education) by the National Research Foundation of Korea, tein stability and function, and may be investigated via molecular dynamics and NRF grant funded by the Korea government (2018R1A4A1023822). simulations. Using multi-scale molecular dynamics simulations, we identified

BPJ 9466_9469 Wednesday, March 6, 2019 535a a hydrophobic pocket in PC2 formed between S3, S4 transmembrane helices function mutations in TRPV3 result in spontaneous dermatitis and pruritus and S4-S5 linker, which showed a preference for phosphatidylinositol bisphos- in humans and rodents. TRPV3 also plays important roles in hair morpho- phate (PIP2) binding. This has been further tested using a lipid binding assay, genesis and maintenance of epidermal barrier function. Thus, understanding suggesting that detergent-solubilised PC2 can bind a range of phosphatidylino- the mechanism of regulation of TRPV3 has important implications in skin sitol phosphates but not other simple anionic (phosphatidylserine, phosphatidic health. Previously, it has been shown that protons exert both stimulatory acid) or zwitterionic (phosphatidylethanolamine, phosphatidylcholine) lipids. and inhibitory actions on TRPV3. While the large part of the stimulatory Simulations of other members of the TRP channel family suggest this lipid- action may be attributed to the direct modification by protons of the binding site may be shared amongst a number of TRP channels. Simulations TRPV3 ligand, 2-aminoethoxydiphenyl borate (2APB), used in these and cryo-EM data also suggest the presence of a second lipid-binding site on studies, the mechanism of proton inhibition of TRPV3 remains unknown. PC2, which may be occupied by cholesterol. Here, we examined the extracellular and intracellular effects of low pH on TRPV3 expressed in HEK 293 cells using whole-cell and inside-out 2651-Pos patch clamp techniques. Lowering extracellular pH from 7.2 to 6.5 and Essential Residues Required for the Opening of a Polycystin TRP Channel 5.5 caused a transient and persistent inhibition of TRPV3 whole-cell cur- Leo CT Ng, Thuy N. Vien, Amitabha Mukhopadhyay, Paul G. DeCaen. rents evoked by 2APB and , respectively. The inhibition was abol- Pharmacology, Northwestern University, Chicago, IL, USA. ished not only by the mutation of Asp 641 to Asn at the selectivity filter of Polycystins (PKD2 and homologs) belong in the family of the Transient Recep- the channel, but also two glutamate and one aspartate residues at its C-ter- tor Potential (TRP) nonselective cation channels. Their biophysics and cellular minus. However, only with the D641N mutation, did intracellular acidifica- functions are still poorly understood, except for their association with auto- tion become stimulatory in the absence of any other TRPV3 ligand, somal dominant polycystic kidney disease (ADPKD), a common monogenetic suggesting Asp 641 to be the key residue for proton inhibition. Our study disorder characterized by proliferative cysts which leads to renal failure in reveals a critical residue involved in TRPV3 inhibition by low pH and adulthood. TRP channels are known to be stimulated by a wide range of several protonatable residues at the C-terminus of TRPV3 that regulate external stimuli, where some classes are thermo-sensitive, ligand-gated, while the overall functionality of the channel. others are regulated by osmotic stress or extracellular pH. Despite the diversity, TRP channels are not traditionally considered as voltage-gated, or being acti- 2654-Pos vated in response to changes in the membrane potential. Here, we examined Conserved Allosteric Pathways for Activation of TRPV3 Revealed through the property of PKD2-L1 (also called TRPP3)- a member of the polycystin Engineering Vanilloid-Sensitivity sub-class of TRP channels. Endogenously, PKD2-L1 forms heteromeric chan- Feng Zhang1,2, Kenton Swartz1, Andres Jara-Oseguera1. nels with other polycystins in the primary cilia, but can also form a homomeric 1Molecular Physiol/Biophys, NINDS NIH, Bethesda, MD, USA, channel when expressed heterologously. We investigated on the structural 2Biochemistry, University of Utah, Salt Lake City, UT, USA. movements which control voltage-dependent and polymodal gating of the ho- The Transient Receptor Potential Vanilloid 1 (TRPV) cation channel is acti- momeric form of this channel. Through analysis of new functional and struc- vated by an array of stimuli, including heat and vanilloid compounds. tural data sets, we have defined a series of interactions which are absolutely TRPV2, TRPV3 and TRPV4, close homologues of TRPV1, are also acti- essential for channel opening. vated by heat, but sensitivity to vanilloids and many other agonists is not conserved among TRPV subfamily members. We recently discovered that 2652-Pos four mutations in TRPV2 are sufficient to render the channel sensitive to Force Transduction in the NOMPC Mechanosensitive Channel the TRPV1-specific vanilloid agonist (RTx). Here we show Sara Capponi1,2, David Argudo1,2, Neville Bethel1,2, Michael Grabe1,2. that mutation of six residues in TRPV3 corresponding to the vanilloid site 1Cardiovascular Res Instit, UCSF, San Francisco, CA, USA, 2Pharmaceutical in TRPV1 is sufficient to engineer RTx binding. However, robust activation Chemistry, University of California in San Francisco, San Francisco, CA, of TRPV3 by RTx requires further facilitation of channel opening, either by USA. introducing individual mutations in the pore, applying RTx at temperatures The recent structure of the NOMPC mechanosensitive channel, responsible > 30C, or co-stimulating TRPV3 channels with the non-selective TRPV for hearing and touch in fly, provides a great opportunity to understand how agonist 2-aminoethyl diphenylborinate (2-APB). Our results demonstrate external mechanical forces are converted into electrical impulses through that the energetics of channel activation can determine the apparent sensi- the opening of ion channels in sensory cells. NOMPC is a homo-tetramer tivity to a specific stimulus and suggest that allosteric pathways for activa- composed of a transmembrane domain containing the ion channel pore tion are conserved in the TRPV family. Furthermore, we found that and a cytoplasmic domain made up of 4 helical chains of 150 A˚ length. mutations in the pore that enable TRPV3 activation by RTx have distinct The chains made of 29 Ankyrin repeats come into contact with each other effects on the heat- and 2-APB sensitivity of the channel, indicating that at two points and associate with microtubules. The transmission of external different regions in the pore can become selectively engaged in the activa- forces responsible for NOMPC gating is believed to occur by tethering the tion by specific stimuli. Ankyrin chains to microtubules. Previous work has demonstrated that iso- lated Ankyrin chains behave as biological springs under extension. Here, 2655-Pos we combine full-atom molecular dynamics simulations, normal mode anal- Selectivity and Characterization of the Permeant Ion Effect in the Rapid- ysis (NMA), and continuum mechanics to characterize the material proper- transitions on the Pore of TRPV1 Channel ties of the chains under extreme compression and extension. The NMA Miriam Garcia Avila, Javier Tello Marmolejo, Gisela E. Rangel-Yescas, reveals that the lowest energy modes correspond to 4-fold symmetric Leon D. Islas. compression/extension along the long-axis of the channel parallel to the Departamento de Fisiologı´a, Facultad de Medicina, UNAM, Mexico City, membrane normal, while higher energy modes correspond to symmetric dis- Mexico. placements of the TRP domain in the channel. The finite element model re- The transient receptor potential vanilloid 1 (TRPV1) channel can be acti- veals that the Ankyrin chains behave as a soft spring with a linear restoring vated by stimuli such as capsaicin, pH <5.4, heat and voltage. Its structure force of 4 pN/nm for deflections %15 A˚ , and as a non-linear spring for is similar to voltage-dependent potassium channels, but in contrast the larger deformations. Detailed force-balance analysis on the chains during mechanism voltage detection has remained unknown.TRPV1 is classified compression reveal that they exert a clockwise twisting moment on the as a non-selective cation channel with more permeability to divalent ions TRP domain when viewed from the cytoplasm, which is solely a conse- as calcium. We characterized the selectivity of TRPV1 through inside-out quence of the bundle geometry. This rotation of the TRP domain is consis- patch clamp recordings in cells HEK293. We find a selectivity sequence þ z þ > þ z þ > þ > þ tent with the gating mechanism suggested for the closely related TRPV1 ion for monovalent ions: Cs NH4 Rb K Na Li . Addition- channel based on open and closed structure. ally, we find that, in single-channels recordings, there are rapid fluctuations on the current that occur during the open state of the channel. These 2653-Pos fluctuations are observed as an asymmetric tail in the all-points current Mechanism of Proton Inhibition of TRPV3 amplitude histograms. This asymmetry increases at positive voltages and Haiyuan Wang, Qiaochu Wang, Jinbin Tian, Michael X. Zhu. varies depending on the permeant ion. These fluctuations can be explained Department of Integrative Biology and Pharmacology, McGovern Medical by rapid transition between the open state and several closed states. The School The University of Texas Health Science Center at Houston, Houston, lifetime of the open state is related to the type of ion that occupies the TX, USA. channel pore. Amplitude histograms are analyzed with through simulation Transient Receptor Potential Vanilloid 3 (TRPV3) forms Ca2þ-permeable of histograms resulting from several models considering one open nonselective cation channels highly expressed in keratinocytes. Gain-of- state and two or more closed states with appropriate transition rate

BPJ 9466_9469 536a Wednesday, March 6, 2019 constants. These experiments indicate that the selectivity filter undergoes The transient receptor potential vanilloid receptor 1 (TRPV1) is a nonselective rapid transitions that might be relatedtogatingandmightreflect cation channel, member of the transient receptor potential superfamily of ion the movement of a selectivity filter gate. seems to occur in the selectivity channels. TRPV1 is expressed in peripheral sensory neurons, where it trans- filter. Supported by grants DGAPA-PAPIIT: IN203318 and CONACYT: duces noxious stimuli into electrical signals, and is responsible for body tem- 252644. perature regulation, heat sensation, noxious and chronic pain. TRPV1 is activated by a variety of stimuli, including ligand binding at the vanilloid 2656-Pos site. We previously discovered that rotation of a conserved residue on the S6 Agonist-Dependent Plasticity in the TRPC3 Selectivity Filter segment from the S4-S5 linker towards the pore is also implicated in TRPV1 1 1 2 Oleksandra Tiapko , Sanja Curcic , Gema Guedes de la Cruz , activation. This movement is correlated with dehydration of peripheral cavities 2 1 Toma Glasnov , Klaus Groschner . (PCs) located between S6 and the S4-S5 linker, and with hydration of the pore. 1Gottfried-Schatz-Research-Center - Biophysics, Med Univ Graz, Graz, 2 However, the structural details for this mechanism remain to be elucidated. Austria, Department of Chemistry, Univ of Graz, Graz, Austria. Here we explore the hypothesis that annular lipids mediate the allosteric Transient receptor potential canonical 3 (TRPC3) forms non-selective cation coupling between the vanilloid site and the PCs, ultimately leading to channels that are opened in response to phospholipase C stimulation. TRPV1 activation. Our multi-microseconds molecular dynamics simulations This physiologic mode of activation involves a direct interaction of diacyl- and free energy calculations of several TRPV1 systems (i.e. TRPV1 bound glycerols with residues within a lateral fenestration of the pore domain to vanilloid ligands and/or to the spider venom toxin, Dk/Tx) revealed that behind the selectivity filter. Benzimidazols (GSK1702934A derivatives) annular lipids have a peculiar ‘‘buried’’ conformation upon concomitant bind- activate TRPC3 by a mechanism bypassing the phospholipase cascade. ing of vanilloids and Dk/Tx. Interestingly, lipids in this conformation project We set out to explore the impact of diacylglycerol interactions within the the hydrophobic tails into the PCs, thereby triggering their dehydration and pore domain on the ion selectivity of the channel by using optical probes the consequent opening of the channel. In conclusion, our results elucidate for lipid-mediated and lipid-independent activation of TRPC3. Fast and the molecular underpinnings of TRPV1 allosteric regulation by annular lipids, temporally precise activation of recombinant TRPC3 channels by a photo- and sheds light on the mechanism by which the binding of vanilloid ligands is switchable di-arachidonyl-mimicking glycerol (OptoDArG) and a newly coupled to gating motion. developed, photochromic benzimidazol (OptoBI-1) enabled reliable deter- mination of Ca2þ/monovalent permeability ratios at essentially low current densities. These experiments revealed a significant agonist dependence of 2659-Pos Conformational Ensemble of the Human TRPV3 Ion Channel the TRPC3 selectivity profile. The permeability ratio PCa2þ/PCsþ was close to 3 for TRPC3 pores opened via phospholipase C stimulation or photoiso- Lejla Zubcevic1, Mark A. Herzik Jr.2, Mengyu Wu2, William F. Borschel1, merization of OptoDArG, but 0.8 for channels activated by photoisomeriza- Marscha Hirschi1, Albert Song2, Gabriel C. Lander2, Seok-Yong Lee1. 1 2 tion of OptoBI-1. These differences were eliminated by a mutation of Dept Biochemistry, Duke University, Durham, NC, USA, The Scripps the glycine residue critically involved in DAG-TRPC3 interaction Research Institute, La Jolla, CA, USA. (G652A). We suggest DAG association with the pore domain of TRPC3 Transient receptor potential vanilloid channel 3 (TRPV3), a member of channels determines the architecture of the selectivity filter and thereby the thermosensitive TRP (thermoTRPV) channels, is activated by warm Ca2þ permeation. temperatures and serves as a key regulator of normal skin physiology through the release of pro-inflammatory messengers. TRPV3 sensitizes 2657-Pos upon repeated stimulation, yet a lack of structural information about the Temperature-Dependent Heat Capacity (DCP(T)) Modification of the channel precludes a molecular-level understanding of TRPV3 sensitization Thermodynamic Framework for Thermotrp Channels Eliminates Pre- and gating. Here, we present the cryo-electron microscopy structures of dicted Dual Thermosensitivity apo and sensitized human TRPV3, as well as several structures of TRPV3 Frank Yeh, Richard Aldrich. in the presence of the common thermoTRPV agonist 2-aminoethoxydi- Institute for Neuroscience, University of Texas at Austin, Austin, TX, USA. phenyl borate (2-APB). Our results show a-to-p-helix transitions in the Many candidate protein domains have been proposed as the thermosensor in S6 during sensitization, and suggest a critical role for the S4-S5 linker p-he- temperature sensitive TRP channels (thermoTRPs). In contrast, Clapham & lix during ligand gating. Miller, 2011 proposed that the whole protein, rather than a localized area of the channel, could be responsible for thermosensation (C-M framework). The proposed thermosensitivity is caused by the heat capacity difference 2660-Pos Phosphatidylinositol Inhibits TRPV1 via its Vanilloid Binding Site (DCp) between the open and closed states. This theory makes a surprising Aysenur Torun Yazici1, Eleonora Gianti2, Marina A. Kasimova3, prediction that thermoTRPs should have two thermosensitive regions, 4 5 responding to both hot and cold temperatures. In the original formulation, Vincenzo Carnevale , Tibor Rohacs . 1Rutgers Univ, Newark, NJ, USA, 2Dept Chem/ICMS, Temple Univ, DCp was assumed to be temperature-independent, an assumption that could Philadelphia, PA, USA, 3SciLife Lab, Solna, Sweden, 4Temple Univ, be reasonably relaxed. Using the Po vs. temperature data of Jara-Oseguera 5 and Swartz (Jara-Oseguera et al., 2016), we used the C-M framework Philadelphia, PA, USA, Dept Pharmacology, Rutgers New Jersey Med Sch, Newark, NJ, USA. to backcalculate the DCp per degree temperature. The estimated DCp Regulation of the heat- and capsaicin-activated Transient Receptor Po- showed clear temperature dependence that warranted modeling. Here, we tential Vanilloid 1 (TRPV1) channels by phosphoinositides is complex chose to use a Taylor series expansion of DCp as a function of temperature and controversial. In excised inside-out patches phosphatidylinositol (DCp(T)). Each of the DCp(T) functions were then used to calculate Po over temperature and were compared to the original P vs. temperature data. 4,5-bisphosphate (PIP2) and its precursor phosphatidylinositol 4-phosphate o (PIP) reproducibly potentiate TRPV1 activity. In a recent cryoEM structure The second and the third order DCp(T) approximations were the best fits to the original data. Over the temperature range of 273oCto233oC, a phosphoinositide was detected in the vanilloid binding site in the absence of agonists. Phosphoinositides were proposed to negatively regulate the third order equation predicted only one thermosensitive temperature TRPV1 as competitive inhibitors of vanilloids, but the resolution of the region. The modified DCp(T) C-M framework eliminates the necessity to experimentally determine the dual thermosensitive temperature regions structure was sufficient to determine the exact nature of the lipid. Here in order to prove the C-M framework. As a key component of protein we used computational modeling, site directed mutagenesis and patch heat capacity is solvation, further work on the effects of solvent clamp electrophysiology to identify the phosphoinositide in the vanilloid binding site. We find that PIP2 diffuses away from the vanilloid binding and lipid composition could help validate the modified DCp(T) C-M framework. site in Molecular Dynamics simulations, but phosphatidylinositol, the precursor of PIP and PIP2, remains there. We also find that mutating two 2658-Pos residues predicted to interact with phosphatidylinositol, but not with Activation of TRPV1 by Lipids: Can Lipid Tails Bridge the Gap between capsaicin shifts the dose-response of capsaicin to the left. We also the Vanilloid Binding Site and the Peripheral Cavities? show that in excised inside-out patch clamp experiments phosphatidylinosi- Eleonora Gianti1, Michael L. Klein2, Tibor Rohacs3, Vincenzo Carnevale4. tol inhibited TRPV1 activity induced by PIP2 at low capsaicin concentra- 1Dept Chem/ICMS, Temple University, Philadelphia, PA, USA, 2Coll Sci tions. We conclude that phosphatidylinositol binds to the Vanilloid &Tech, Temple Univ, Philadelphia, PA, USA, 3Dept Pharmacology, Rutgers binding site of TRPV1 in resting conditions, and this may serve to set New Jersey Med Sch, Newark, NJ, USA, 4Temple Univ, Philadelphia, PA, the basal sensitivity of the channel to vanilloids as well as heat and low USA. pH.

BPJ 9466_9469 Wednesday, March 6, 2019 537a

2661-Pos pulmonary arterial hypertension (PAH). Therefore, it is important to An Ancient TRPM2 Ortholog is a True Channel-Enzyme, But its Catalytic understand how TRPC4 channels are regulated. Using HEK293 cells that Activity is Uncoupled from Pore Gating stably co-expressing m opioid receptor and TRPC4b, we examined intracel- 2þ 2þ Balazs Toth, Iordan Iordanov, Laszlo Csanady. lular Ca concentration ([Ca ]i) changes evoked by the TRPC4/5 agonist, Department of Medical Biochemistry, Semmelweis University, Budapest, Englerin A, and the m agonist, DAMGO. While the EC50 of Englerin Hungary. A increased by 10 folds when the cell confluence increased from 80% 2þ Human TRPM2 (hTRPM2) is a non-selective Ca permeable cation channel, to 100%, the EC50 of DAMGO did not change with the increase in cell den- expressed abundantly in brain, immune cells, and pancreatic b-cells. Under sity. By Ca2þ imaging, we observed that Englerin A only elicited Ca2þ re- physiological conditions it plays a role in glucose-induced insulin secretion, sponses in cells located at the boundary of a cell cluster, but not those in the and in the immune response. Besides, TRPM2 enhances the sensitivity of cells middle. However, DAMGO induced Ca2þ responses in all cells. These data toward oxidative stress induced damage, e.g., facilitating neuronal cell death suggest that cell-to-cell contact inhibits TRPC4 activation by Englerin A but during brain ischemia/reperfusion. not Gi/o proteins stimulated by DAMGO via the coexpressed m receptors. Nematostella vectensis TRPM2 (nvTRPM2) is an ancestor of hTRPM2. Both This differential effect was not altered by the co-expression of TRPC1, 2þ 2þ are activated by intracellular Ca and ADP-ribose (ADPR), and contain a which only decreased the amplitude the [Ca ]i elevation evoked by C-terminal NUDT9-H domain, which shows high with Englerin A and DAMGO. The differential effects of cell-to-cell contact the mitochondrial ADPR-hydrolase enzyme NUDT9. We have recently shown onTRPC4activationbyEnglerinAandGi/o signaling may suggest a unique that the NUDT9-H domain of hTRPM2 has lost its ADPRase activity due to mechanism of regulation relevant to TRPC4 in the endothelium, where mutations of the conserved ‘‘Nudix box’’ motif.Interestingly, in nvTRPM2 different triggers and/or signaling pathways may be required to activate the Nudix-box motif is still canonical. Correspondingly, we found that purified channels located within and outside the tight junctions to regulate nvTRPM2 protein, or its isolated NUDT9-H domain, shows robust ADPRase permeation. activity. We next addressed whether nvTRPM2 enzymatic activity plays any role in channel gating. Enzymatic activity of isolated nvNUDT9-H could be reduced or abolished by 2664-Pos point mutations, as well as by Mg2þ removal. On the other hand, in inside-out Receptor Mediated Activation of TRPC3 Channel When at ER PM patch clamp experiments the kinetics of channel gating of full-length Junctions nvTRPM2 remained unaffected by these manipulations: none of them changed Haiping Liu. the apparent affinity of the currents towards ADPR, or the current relaxation National Institutes of Health, Bethesda, MD, USA. time constant after sudden ADPR removal. We also investigated the effect of TRPC3, a member of the Transient Receptor Potential (TRP) superfamily, functions as non-selective cation channel permeable to Ca2þ and mediates the non-hydrolyzable ADPR analogue AMPCPR on nvTRPM2 channel gating. 2þ Compared to ADPR, AMPCPR showed a lower apparent affinity, and caused part of the receptor-stimulated Ca signals. In polarized cells, TRPC3 is shorter channel openings, suggesting that it fits less well into the binding confinedtotheapicalpolewhereitinteracts with the IP3 receptors. How- pocket. ever, the exact microdomain localization of TRPC3 (and any of the other These results together suggest that nvTRPM2 is a true channel-enzyme, but its plasma membrane TRP channels) and how localization of the channels at enzymatic activity is not coupled to channel gating. the ER/PM junctions affect channel functions are not clear. An essential tether for functional ER/PM junctions is Extended Synaptotagmin 1 (Esyt1), while STIM1 increases the number of ER/PM junctions while regu- 2662-Pos lating TRPC3 activity. We show here that knockdown of Esyt1 reduced Mining the Drosophila Gustatory Receptor Family for New Thermosensi- TRPC3 current, while overexpression of Esyt1 increased the carbachol- tive Proteins activated TRPC3 current. Mutation analysis showed that L241S mutation Marzie Amirshenava, Benton R. Berigan, Aditi Mishra, Benjamin C. Zars, increased, while the I807S mutation eliminated TRPC3 interaction with Taylor G. Hallman, Troy Zars, Lorin S. Milescu, Mirela Milescu. STIM1. Measurement of dose dependence for activation by carbachol Univ Missouri, Columbia, MO, USA. showed that the L241S mutation increased the maximal current at all con- Gustatory receptors (Gr) are a family of transmembrane proteins that have been centrations while increasing apparent affinity for carbachol. The I807S mu- extensively studied in the context of insect taste and odor sensory systems. tation has the opposite effect. Notably, knockout of STIM1 reduced the Drosophila Gr28b(D) is the first gustatory receptor shown to be involved in current of WT-TRPC3 and TRPC3(L241S) but not that of TRPC3(I807S) thermosensation. In a previous study, we showed that Gr28b(D) exhibits a to equalize their currents and the dose response to that observed with non-inactivating, temperature dependent, cation non-specific current, when ex- TRPC3(I807S) in WT and STIM1/ cells. Knockdown of Esyt1 markedly pressed in Xenopus laevis oocytes. More recently, we explored the temperature inhibited TRPC3(L241S) current with relatively smaller effect on the sensitivity of Gr28b(D) orthologs from five Drosophila species (D. yakuba, TRPC3(I807S) current while reducing the affinity for stimulation of WT- simulans, pseudoobscura, willistoni, and mojavensis) that inhabit different en- TRPC3 and TRPC3(L241S), but not of TRPC3(I807S), by carbachol. Our vironments. When expressed in Xenopus oocytes, Gr28b(D) orthologs exhibit findings so far reveal profound role of the ER/PM junctions on receptor- various temperature sensitivities and current densities. Additionally, when ex- mediated activation of TRPC3, and likely other TRP channel. Additional pressed in Drosophila motor neurons, along with a genetically-encoded cal- properties of TRPC3 when at and when outside the junctions are being cium sensor (GCaMP6f), Gr28b(D) orthologs exhibit unique changes in the examining. firing pattern of individual cells, in a temperature-dependent fashion. Finally, flies over-expressing GR28b(D) orthologs exhibit different temperature- dependent behavior. By comparing their primary structure and through muta- 2665-Pos genesis experiments, we identified functional domains within the Gr proteins Investigation of TRPM4 in Prostate Cancer Cells with Novel Small Mole- and obtained mutants with various properties. These results identify novel ther- cule Inhibitors 1 1 1  2 mosensitive gustatory receptors in Drosophila and expand the pool of potential Anna Borgstro¨m , Barbara Hauert , Sven Kappel ,Clemence Delalande , 2 1 candidates for thermogenetics tools. They also provide clues for further struc- Jean-Louis Reymond , Christine Peinelt . 1NCCR TransCure, Inst. of Biochemistry and Molecular Medicine, ture/function studies aimed at elucidating the molecular biophysics of 2 thermosensation. University of Bern, Bern, Switzerland, NCCR TransCure, Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland. Transient receptor potential melastatin 4 (TRPM4) is a monovalent cation 2663-Pos channel, mainly conducting Naþ and Kþ. TRPM4 is widely expressed, Differential Effects of Cell-To-Cell Contact on TRPC4 Channel Activation although its expression is most prominent in prostate and colon. TRPM4 by Englerin a and Gi/o-Coupled Receptor Agonist protein and RNA expression levels are increased in prostate cancer Lin Gao1, Qiaochu Wang2, Michael X. Zhu2. compared to normal prostate tissue and TRPM4 was recently identified as 1Beijing University of Chinese Medicine, Beijing, China, 2Department of a cancer driver gene in androgen-insensitive prostate cancer. TRPM4 is acti- Integrative Biology and Pharmacology, McGovern Medical School, The vated by Ca2þ and plays an important role in intracellular Ca2þ signaling University of Texas, Houston, TX, USA. through the initiation of an important negative feedback mechanism of The endothelium forms a semipermeable barrier lining the entire vascula- store-operated calcium entry (SOCE). As a universal second messenger cal- ture. Transient Receptor Potential Canonical 4 (TRPC4) channels have cium is regulating multiple biological processes. Therefore, it is important been shown to regulate endothelial permeability and be involved in that calcium homeostasis is tightly regulated. However, in several cancers,

BPJ 9466_9469 538a Wednesday, March 6, 2019

SOCE and its signaling components are dysregulated, contributing to myocytes, even in 72 hours of culture time. Loss of TRPV2 from the hearts of several cancer hallmarks such as increased proliferation and enhanced juvenile mice resulted in disassembly of the intercalated disc structure, even in migration. Sodium influx via TRPM4 depolarizes the membrane and by adult age. The TRPV2-deficient adult mice showed cardiac dysfunction with that reduces the driving force for further calcium influx. We hypothesize conduction defects. These observations suggest that TRPV2 is critical for the that TRPM4, as negative regulator of SOCE, plays a role in migration developments of the intercalated disc of myocytes. and proliferation of prostate cancer cells. We recently successfully evaluated a new TRPM4 blocker, 4-Chloro-2-(2-(2- 2668-Pos chlorophenoxy) acetamido) benzoic acid (CBA), as a potent inhibitor of Biophysical Properties of the Electropermeabilization-Induced Membrane TRPM4 current. CBA blocks endogenous TRPM4 currents in LNCaP cells Conductance in Patch Clamped Adrenal Chromaffin Cells in a low micromolar range. Furthermore, LNCaP cells treated with CBA Lisha Yang, Sophia Pierce, Gale L. Craviso, Normand Leblanc. showed decreased viability. Additionally, two additional small molecule inhib- Pharmacology, Univ Nevada Reno, Reno, NV, USA. itors were tested on the same cell line whereof one showed strong negative ef- Our group recently showed that a single 5 ns high intensity electric pulse (NEP) fect on the viability of LNCaP cells. Furthermore, DU145 cells treated with elicited membrane permeabilization in whole-cell voltage clamped bovine ad- CBA demonstrated slight decrease in migration compared to vehicle treated renal chromaffin cells, inducing an instantaneous inward conductance at the þ cells. Stable TRPM4-K.O. cells were used to further study the role of holding potential of 70 mV. When Na was completely replaced in the bath solution with n-methyl-d-glucamine, the NEP-induced current was signif- TRPM4 in prostate cancer migration and proliferation. In line with our previous þ data using the blockers, DU145 TRPM4-K.O. cells displayed decreased migra- icantly reduced suggesting that the current was partially carried by Na . Later, tion and proliferation. a slow voltage ramp protocol from 100 mV to 80 mV was used to inactivate Naþ channel, whereas Kþ and Ca2þ channels were blocked by cesium, tetrae- 2666-Pos thylammonium and nitrendipine. With this ramp protocol, the fact that the TRPC4 Channels are a Key Player in Hippocampal Neuronal Develop- NEP-induced conductance showed a consistent reversal potential around 10 mV revealed the properties of the current that resembled protein ion channel- ment 3þ Jaepyo Jeon, Michael Xi Zhu. like activity. We further exposed cells to SKF-96365 and La , both com- Univ Texas Health Science Center at Houston, Houston, TX, USA. pounds considered to be able to alter the activity of TRP channels. The results Transient receptor potential canonical 4 (TRPC4) forms Ca2þ-permeable showed 33% and 59 % inhibition effect on the NEP-induced current. Therefore, nonselective cation channels with many biological, including neuronal, func- one possibility of the NEP-induced conductance is a TRP-like channel that is tions. We report here that TRPC4 plays a critical role in dendrite development activated via some sort of elctromechnical coupling triggered as a consequence of hippocampal neurons. Hippocampal neurons of young (P21) TRPC4 of the NEP. It is known that many TRP channels are localized in caveolae, a knockout (TRPC4/) mice exhibited a decreased number of apical dendritic subset of membrane lipid rafts involved in compartmentalized signal transduc- b branches as compared to the wild type mice. This difference was abolished tion. Methy- -cyclodextrin, as cholesterol depletion drug which disrupts cav- in older (P60) mice, indicating a delayed dendrite development in the absence eolae integrity, produced a strong inhibitory effect on the NEP-induced of TRPC4. In primary cultures of hippocampal neurons, dendritic arborization current suggesting the hypothesis that caveolae disruption altered TRP traf- proceeded more slowly between days in vitro (DIV) 7 and 16 in TRPC4/ than ficking and/or compartmentalization leading to a diminished ability of NEP TRPC4þ/þ neurons. More importantly, unlike wild type neurons, the to activate these channels, although this observation cannot rule out the possi- TRPC4/ neurons failed to show an increase in dendritic arborization in bility that membrane cholesterol depletion altered the biophysical properties of response to brain-derived neurotrophic factor (BDNF) or a low concentration the membrane in a way that would prevent or limit the formation of lipid- (5 mM) of glutamate. Examination of intracellular Ca2þ concentration changes containing electronanopores. near plasma membrane in dendritic regions using GCaMP6-CAAX revealed 2669-Pos that both Gq- and Gi-coupled metabotropic glutamate receptors are involved PHB Regulates Trafficking of TRPM8 to the Plasma Membrane in generating the sustained Ca2þ signal in response to glutamate, which is / Lusine Demirkhanyan, David Goa, Eleonora Zakharian. largely missing in TRPC4 neurons. These glutamate receptors are also crit- Cancer Biology and Pharmacology, University of Illinois College of ical for TRPC4-mediated dendritic arborization. Using long-term (12 hrs) time- Medicine, Peoria, IL, USA. lapse live cell imaging of GFP-CAAX-expressing neurons in an environmental TRPM8 is a Ca2þ-permeable ion channel that mediates testosterone signaling m control chamber, we observed that in response to 5 M glutamate, dendrites of in prostate epithelial cells. Previously, we demonstrated that TRPM8 is post- TRPC4þ/þ neurons extended continuously for about 6 hrs and then stabilized, / translationally modified by poly-R-3-hydroxybutyrate (PHB). Here, we tested whereas that of TRPC4 neurons stopped the extension after 2 hrs and often whether removal of PHB from TRPM8 affects channel trafficking to the plasma retracted. These results suggest that downstream from metabotropic glutamate membrane (PM). Using confocal microscopy, we found that PHB is highly co- receptor activation, TRPC4 channels play an important role in dendritic arbor- localized with TRPM8 in the heterologous expression system and human pros- ization of hippocampal neurons during early development. tate tissues. In cells transfected with PHB hydrolyzing enzyme PHB- depolymerase (PhaZ7) TRPM8 was no longer detected on the PM and showed 2667-Pos an internalized pattern. The response of TRPM8 treated with PhaZ7 or PhaZ7- TRPV2 is Crucial for the Development of Intercalated Discs in Mouse S136A, the mutant lacking enzymatic activity, to cold (15C) or Hearts demonstrated decreased TRPM8 activity with PhaZ7 treatment, but not the 1 1 2 2 Yuki Katanosaka , Makoto Shibuya , Yoshihiro Ujihara , Satoshi Mohri , inactive form of the enzyme. Western Blot analysis of PhaZ7-treated Keiji Naruse1. 1 2 TRPM8, following biotinylation of cell surface proteins, showed decreased Dept Cardiovas/Physiol, Okayama Univ, Okayama, Japan, Dept TRPM8 expression on the PM upon PHB hydrolysis. Removal of PHB moieties Physiology, Kawasaki Med Sch, Kurashiki, Japan. from TRPM8 not only affected the channel trafficking to the PM but also TRP vanilloid family type 2 channel (TRPV2) is a key molecule in stretch- 2þ directly impacted its function. As a result, testosterone-induced TRPM8 activ- induced Ca response of cardiomyocytes, and is highly localized mammalian ity was completely abolished in planar lipid bilayers after the PhaZ7 treatment. intercalated discs, which is cyclically stretched within working hearts. Elimina- In line with our earlier work, this inhibition was achieved from both sides of the tion of TRPV2 from hearts of adult mice led to a rapid and severe decline in channel but required more prolonged incubation at the inner side, further sug- cardiac pump function through the disorganization of intercalated discs. gesting the prevalence of PHB-content on the internal side of the channel. Gene expression of intercalated disc proteins was also acutely downregulated Overall, these findings support the importance of PHBylation for TRPM8 traf- by TRPV2-elimination. Here, we show the involvement of TRPV2 in the devel- ficking to the PM and its function. Enzymatic cleavage of PHB could be opment of intercalated disc. In neonatal cardiomyocytes, the hypotonicity- and 2þ responsible for downregulation of TRPM8 activity and its enhanced degrada- stretch- induced Ca responses were observed within 12 hours of culture after tion in prostate cancer and present a possible mechanism of prostate cancer enzymatic isolation. These responses were decreased by tranilast, an inhibitor development. of TRPV2, suggesting involvement of TRPV2 channel. These myocytes show the robust expression of TRPV2, without any expression of 43 and N- 2670-Pos cadherin. After 48 hours, we can observe the localization of connexin 43 and N- Spectroscopic Studies of Purified Rat TRPV1 cadherin in the longitudinal cell-edges interface forming intercalated disc. In Gilbert Q. Martinez, Marium M. Raza, Sharona E. Gordon. þ contrast, TRPV2-deficient myocytes showed no Ca2 response to the hypoto- Dept Physiol/Biophys, Univ Washington, Seattle, WA, USA. nicity- and stretch-stimulation at 12 hours after isolation. These myocytes Transient receptor potential vanilloid-1 (TRPV1) ion channels are polymo- formed no intercalated disc-like connection between neighbouring cells. In dal signal integrators of noxious stimuli including heat, vanilloids such as addition, sarcomeric actin and synchronous beating were not observed in these capsaicin, peptide toxins, acid, and inflammatory mediators. It is unknown

BPJ 9466_9469 Wednesday, March 6, 2019 539a whether activation of TRPV1 by different stimuli is achieved through the 2673-Pos same structural mechanism or if different stimuli activate the channel Conformational Dynamics of the Intrinsic Ligand in the CNBHD of the through different structural mechanisms. Clinical trials using TRPV1 Voltage-Gated Potassium Channel hERG antagonists resulted in patients exhibiting hyperthermia, suggesting that Sara J. Codding. TRPV1 plays a role in maintaining body temperature and highlighting Physiology, Univ Maryland Baltimore, Baltimore, MD, USA. the need to ensure that therapeutics targeting the channel do not disrupt ther- Human ether-a´-go-go related gene (hERG) voltage-activated potassium mal homeostasis. Hence, knowledge of different structural mechanisms for channels are critical for cardiac excitability. Characteristic slow closing channel activation would aid in design of therapeutic agents targeting (deactivation) in hERG is regulated by a direct interaction between the N- TRPV1. To address this, we have expressed a series of functional single- terminal Per-Arnt-Sim (PAS) domain and the C-terminal cyclic nucleotide cysteine rat TRPV1 channels for spectroscopic analysis, including electron binding homology domain (CNBHD). An intrinsic ligand in hERG is paramagnetic resonance, double electron-electron resonance, and Fo¨rster located at the PAS-CNBHD interface, but its mechanism is not well under- resonance energy transfer spectroscopy. By probing several structural re- stood. Our recent work with co-expressed channel fragments in which the gions within TRPV1 we can determine which regions of the channels hERG PAS domain fused to CFP was expressed in trans with the ‘core’ move during activation and whether those are the same for different noxious of the hERG channel (i.e. a channel lacking the PAS domain that is fused stimuli. to Citrine at the C-terminus, hERG DPAS-Citrine) showed that deactivation gating was disrupted (was made faster) by intrinsic ligand mutations and that the activation time course of the channel was perturbed (was 2671-Pos made faster), indicating that the intrinsic ligand is necessary for the Directly Activates TRPV2 functional and structural interaction of the PAS domain and the CNBHD. Aaron Gochman, Andres Jara-Oseguera, Kenton Swartz. To better understand the dynamics for intrinsic ligand regulation of National Institutes of Health, Bethesda, MD, USA. hERG gating, we incorporated the fluorescent non-canonical amino acid TRPV2 is an enigmatic vanilloid receptor that was discovered not long after ANAP using amber codon suppression at the intrinsic ligand, and engi- its closest homologue, TRPV1. While TRPV1 directly modulates the sen- neered dihistidines that were structurally adjacent to this ANAP, to allow sory response to many pain-producing environmental stimuli and inflamma- 1 us to probe a change (1) using an environmentally-dependent spectral shift tory mediators , TRPV2 is not activated by any of these stimuli despite of ANAP and/or (2) using transition metal FRET. This technique will deter- having significant sequence homology2. In fact, TRPV2 does not have a in vivo mine how the intrinsic ligand at the PAS-CNBHD interface may undergo documented endogenous ligand or physiological function .Ouraim conformational changes with voltage to regulate channel deactivation was to study TRPV2 activation by cannabidiol (CBD), a phyto- gating. cannabinoid compound, which has been shown to activate TRPV2 in þ Ca2 imaging experiments3. CBD recently received FDA approval as a 2674-Pos treatment for epilepsy and has long been used as an analgesic but has an un- Differential Sensitivity of Cardiac Ion Channels to Polyunsaturated Fatty known molecular mechanism of action in vivo. In whole-cell patch clamp Acid Analogues experiments, we discovered that TRPV2 directly activates the channel but Briana Bohannon1, Sara I. Liin2, Peter Larsson3. with slower activation and deactivation kinetics relative to non-specific 1Physiology and Biophysics, Univ Miami, Miami, FL, USA, 2Clin TRP channel agonist 2-APB and is a low efficacy agonist despite CBD hav- Experimental Medicine, Linko¨ping University, Linkoping, Sweden, 3Dept ing higher affinity for TRPV2 than 2-APB3. We are currently using single Physiol/Biophys, Univ Miami, Miami, FL, USA. channel recording techniques to further study the kinetic basis of CBD acti- Ion channels are important regulators of cellular excitability in tissues such vation of TRPV2 and are exploring whether CBD can activate other related as the heart and brain. In the heart, the ventricular action potential depends TRP channels. on the activity of several different ion channels including Naþ channels, Ca2þ channels, and Kþ channels. When any of these currents are disrupted by mutation, patients can develop Long QT Syndrome (LQTS). LQTS Posters: Voltage-gated K Channels II predisposes the individual to ventricular fibrillation that can lead to sudden cardiac death. Polyunsaturated fatty acids (PUFAs) have emerged as thera- 2672-Pos peutic candidates for LQTS because they are known to modulate the activity Determining Functional KCNQ1/KCNE1 Subunit Interactions in the of different ion channels. We have demonstrated that PUFAs can regulate the KCNQ1/KCNE1 Channel activity of the cardiac IKs channel (Kv7.1/KCNE1), by shifting the voltage- Xiaoan Wu1, Marta E. Perez1, Kevin J. Sampson2, Robert S. Kass2, dependence of the channel to more negative voltages. It is not known, how- Peter H. Larsson1. 1 2 ever, whether PUFAs influence specific ion channels or whether the PUFAs Dept Physiol/Biophys, Univ Miami, Miami, FL, USA, Dept Pharmacology, nonspecifically modify multiple ion channels in the heart. To understand the Columbia Univ, New York, NY, USA. selectivity of different PUFAs, we applied PUFAs and modified PUFAs to KCNQ1 subunits and KCNE1 subunits together form the cardiac voltage- the cardiac Nav1.5 channel, the Cav1.2 channel, and the IKs channel. Here gated potassium channels that regulate the repolarization process of the ac- we demonstrate that PUFA analogues range from highly specific for single tion potential. How the KCNE1 subunit interacts with the KCNQ1 subunit ion channels to nonspecifically activating all three cardiac ion channels remains unclear though different hypotheses have been proposed. For (Nav1.5, Cav1.2, and IKs). We have found that PUFAs containing a glycine instance, both the S4 and S6 segments of the KCNQ1 channel have been head group have specific effects on IKs channels, whereas PUFAs shown to directly interact with KCNE1. Recently, more attention has been with a taurine head group are more selective for Nav1.5 and Cav1.2 chan- paid to the extracellular end of the S1 segment, a part of voltage sensor nels. Our data suggest that modified PUFAs could be tailored towards spe- domain (VSD) of the KCNQ1. In this region, there is a short stretch of cific forms of LQTS based on the channel carrying the disease causing residues (positions 140-147) that (when mutated) are associated with mutation. cardiac arrhythmia. For example, S140G and V141M, two gain-of- function mutations that cause short QT syndrome, greatly slow the 2675-Pos KCNQ1/KCNE1 channel deactivation and hence increase the repolarizing Re-Education of Tumor Associated Macrophages by Trabectedin Kþ current in the action potential. Our previous study has proposed a kinetic Diego A. Peraza1, Ana B. Garcia-Redondo2, Adrian Povo-Retana1, model wherein S140G and V141M affect the VSD-pore coupling and slow Sara Arias1, Ana M. Briones2, Lisardo Bosca´1, Carlos M. Galmarini3, pore closing in the KCNQ1/KCNE1 channel. To identify the functional in- Carmen Valenzuela1. teractions between the extracellular ends of KCNQ1 and KCNE1, I will use 1Inst Investigaciones Biomedicas CSIC-UAM, Madrid, Spain, 2School of thermodynamic mutant cycle analysis (MCA), which is a powerful approach Medicine UAM, Madrid, Spain, 3PharmaMar, S.A., Colmenar Viejo, Madrid, to measure the coupling of two residues in proteins. If the two residues are Spain. independent of each other, the sum of the free-energy changes of the two Immune cells have an important role in the tumor-microenvironment. Mac- single mutant will be equal to the free-energy change of the double mutant. rophages may tune the immune response toward inflammatory or tolerance Conversely, if the two residues are interacting with each other, the sum pathways. Tumor associated macrophages (TAM) have immunosuppressive of the free-energy changes of the two single mutant will be different from functions and they are considered a therapeutic target in cancer. The aim of the free-energy change of the double mutant. This difference is known as this study was to evaluate the effects of trabectedin, a new class of antitumor the coupling energy, suggesting the strength of interactions between two agent, on the tumor-microenvironment through the study of electrophysio- residues. logical and molecular phenotype of macrophages. Experiments were

BPJ 9466_9469 540a Wednesday, March 6, 2019 performed using the whole-cell configuration of the patch-clamp technique We have developed a series of novel imidazolidinedione derivatives, that spe- in resident peritoneal mouse macrophages under different types of polariza- cifically modulate Kv3.1 and Kv3.2 channels. These compounds act by tion. Trabectedin decreased macrophage viability and increased ROS pro- increasing the open probability of the channels at threshold voltages duction. Trabectedin does not directly interact with KV1.5 and KV1.3 Using HEK and CHO cells stably transfected with Kv3.1 and Kv3.2, respec- channels, but treatment (16 h) of macrophages with sub-cytotoxic concen- tively, we here investigate the effects of temperature on the ability of the com- trations (0.1-5 nM) increased their KV current in a concentration- pounds to shift the voltage of activation of Kv3.1 and Kv3.2 currents towards dependent manner due to an upregulation of KV1.3 channels. In vitro gener- negative potentials. ated TAM (TAMiv), by a co-culture of ID8 cells and macrophages, exhibited Modulation of Kv3 currents represents a novel tool for manipulation of a M2 phenotype. TAMiv generated a small KV current, similarly to M2 neuronal excitability. Understanding the effects of these compounds under polarized macrophages, and expressed high levels of M2 markers. In this physiological conditions and temperatures will be important for translating study, we demonstrated that TAMiv polarization could be re-educated by us- their in vitro pharmacological effects into in vivo models, and predicting the ing sub-cytotoxic concentration of trabectedin. TAMiv treated with sub- concentration of compound that may be required to treat human disorders cytotoxic concentration of trabectedin exhibited an upregulation of KV1.3 such as schizophrenia and hearing loss. channels and their M2 phenotype changed towards M1 pro-inflammatory one. Funded by PharmaMar, CSIC 201820E104, CIBERCV and 2678-Pos SAF2016-75021-R. EPR Spectroscopic Studies of the Voltage Sensor Domain (Q1-VSD) of Hu- man KCNQ1 Potassium Ion Channel in Lipid Bilayers Gunjan Dixit. 2676-Pos Cell Molecular and Structural Biology, Miami University Ohio, Oxford, OH, Two-Pronged Aromatic Arginine-Mimics as Hv1 Proton Channel Inhibi- USA. tors KCNQ1 is a voltage gated potassium ion channel expressed in several tis- Chang Zhao1, Liang Hong1, Jason D. Galpin2, Christopher A. Ahern2, 1 sues of the body and modulated by another membrane protein KCNE1 Francesco Tombola . (E1). Q1 is involved in the repolarization phase of cardiac action poten- 1Physiology and Biophysics, University of California Irvine, Irvine, CA, 2 tial. Q1/E1 interaction slows down the activation kinetics required for USA, Molecular Physiology and Biophysics, University of Iowa, Iowa city, proper channel and heart function. Mutations in Q1 can cause Long-QT IA, USA. syndrome resulting in atrial fibrillation, sudden infant death syndrome, The voltage-gated proton channel Hv1 regulates cellular pH homeostasis cardiac arrhythmias and congenital deafness. Q1-VSD is important as and the production of reactive oxygen species by the NADPH oxidase. the channel is activated by a change in voltage, which is sensed by the The channel is made of two identical subunits, which gate cooperatively. VSD. Around 40% of the >200 reported disease-related mutations arise Each subunit contains a voltage-sensing domain (VSD) that conducts pro- from the amino acid substitutions in the VSD, making structural and func- tons and is inhibited by the arginine mimic 2-guanidinobenzimidazole tional studies of Q1-VSD important. The structure of a membrane protein (2GBI). 2GBI operates from the intracellular side and gains access to its varies from micelles to lipid bilayer environment. Site-directed spin label- binding site when the channel is open. The blocker must leave the binding ing (SDSL) EPR is a powerful structural biology technique to study the site for the channel gate to close. We previously found that mutation F150A structural dynamics and topology of membrane proteins in lipid bilayers. strongly increases the binding affinity. We now show that this increase in In this study, the structural dynamics and topology of several residues of affinity is due to a rearrangement of the binding site allowed by the smaller Q1-VSD in the POPC/POPG vesicles were studied using CW-EPR residue at position 150 and use this information to design new arginine line shape analysis and CW power saturation techniques. Spin labeled mu- mimics with improved affinity for the non-rearranged binding site of the tants F130C and F232C were found to be located inside the lipid bilayer, wild type channel. The new compounds consist of two ‘‘prongs’’, an amino- while Q147C and F222C were found to be solvent accessible. The study imidazole ring and a fluorinated aromatic group connected by extended provides novel insights to the structural details of Q1-VSD reconstituted linkers. We find that Hv1 inhibition by the new compounds presents fast in POPC/POPG vesicles for more advanced structural and functional and slow binding modalities, which are affected by distinct types of pertur- studies. bations of the binding site. The conservative amino acid substitution D112E in the Hv1 selectivity filter abolishes the fast component, leaving the slow 2679-Pos component unaltered, whereas the F150A substitution affects primarily the Effect of Lipophilic Molecules on Epilepsy-Causing Mutations of Neuronal slow component. Based on kinetic analysis of the inhibition process, we pro- KCNQ Channels pose that the new arginine mimics are able to bind the open channel like Ludwig Andersson1, Marta E. Perez2, Sara I. Liin3, Fredrik Elinder3, 2GBI. However, unlike 2GBI, the new compounds become ‘‘trapped’’ inside Peter H. Larsson2, Rene Barro-Soria4. the VSD when the gate closes, and they can slowly reach the binding site in 1Linko¨ping Univ, Linkoping, Sweden, 2Dept Physiol/Biophys, Univ Miami, the trapped conformation even from the closed state. These pharmacological Miami, FL, USA, 3Clin Experimental Medicine, Linko¨ping Univiversity, properties are desirable features for the treatment of diseases associated with Linkoping, Sweden, 4Dept of Med, Univ Miami, Miami, FL, USA. Hv1 gain of function. One of the major factors known to cause neuronal hyperexcitability is mal- function of the potassium ion channels underlying the M-current -formed by 2677-Pos KCNQ2 and KCNQ3-. The slow kinetics of activation and deactivation of Pharmacological Modulation of Kv3 Potassium Currents KCNQ2/KCNQ3 channel ensures that the M-current regulates the mem- Nadia Pilati1, Michele Speggiorin1, Giuseppe Alvaro2, Charles H. Large2. brane potential and impede repetitive neuronal firing. Inherited mutations 1Autifony srl, Padova, Italy, 2Autifony Therapeutics Limited, Stevenage, in KCNQ2 and KCNQ3 channels are linked with a wide spectrum of United Kingdom. early-onset epileptic disorders ranging from benign familial neonatal sei- Voltage-gated potassium channels (Kv) are a family of transmembrane ion zures to severe epileptic encephalopathies. Here, we investigate the molec- channels which are voltage-sensitive and selective for potassium ions. Among ular mechanisms by which epilepsy-associated mutations in the voltage Kv channels, the Kv3 channels have unique biophysical properties that enable sensor (S4) of KCNQ2/KCNQ3 cause channel malfunction. Because poly- neurons to fire brief action potentials at high frequencies. unsaturated fatty acids (PUFAs) have been shown to alleviate the symptoms Kv3 channels are typically expressed in rapidly spiking neurons, such as of intractable epileptic seizures, we also investigate the mechanisms by cortical GABAergic interneurons and in certain neurons within the auditory which these compounds reverse channel malfunction and therefore improve brainstem, where rapid and accurate firing is critical to network synchronisation neuronal function. We use voltage clamp fluorometry (VCF), to measure and information transfer, respectively. simultaneously voltage sensor movement and gate opening during channel One feature that distinguishes Kv3 channels from other voltage-dependent po- activation in these mutations. We show that that epilepsy-causing mutations tassium channels is their very rapid activation and deactivation kinetics in in KCNQ channels affect voltage sensor movement and that PUFA variants response to transient depolarization. Moreover, Kv3 channels tend to activate can restore normal voltage dependence of voltage sensor movement in at more positive membrane potentials (above 10mV) where they contribute mutated KCNQ channels. Using optogenetic, we also show that PUFA var- to the initial repolarization phase of the action potential. Until now, modula- iants reduce activity in iPSC-derived cortical neurons bearing epilepsy- tion of Kv3 current in neurons has been obtained by genetic knockdown of the associated mutations in KCNQ channels. Our results suggest that, com- channels, use of non-specific potassium channel blockers, such as tetraethy- pounds that left-shift the voltage dependence of S4 activation in loss-of- lammonium (TEA), or by injecting artificial Kv3 conductances using dynamic function mutations would promote gate opening and have therapeutic poten- clamp. tial.

BPJ 9466_9469 Wednesday, March 6, 2019 541a

2680-Pos The voltage-gated potassium channel Kv1.3 contributes to peripheral insulin PKC Activation Decreases Kv1.5 Protein Expression through Accelerating sensitivity. KO mice for Kv1.3 are resistant to diet-induced weight gain. Endocytic Channel Degradation Therefore, this protein is being considered as a pharmacological target for Tingzhong Wang, Yuan Du, Jun Guo, Wentao Li, Tonghua Yang, obesity and associated type II diabetes. However, the expression of Kv1.3 Shetuan Zhang. in adipocytes generates controversy. Here, we found that Kv1.3 is present Department of Biomedical and Molecular Sciences, Kingston, ON, Canada. in white adipose tissue from humans and rodents. In addition, Kv1.1, The voltage-gated potassium channel Kv1.5 is primarily expressed in the atria Kv1.2, Kv1.4 and especially Kv1.5, from the same family are also þ and conducts the ultra-rapidly delayed rectifier K current (IKur), which is present. Although elevated insulin levels and adipogenesis remodeled the important for atrial repolarization and closely related to atrial fibrillation. Kv phenotype, which could trigger to numerous hybrid complexes, Kv1.3 Elevated stress is associated with several cardiovascular outcomes, including noticeably participated in the insulin-dependent regulation of glucose trans- atrial fibrillation in which Kv1.5 is thought to play a role. Since protein kinase port in differentiated adipocytes. Adipogenesis increased the expression of C (PKC) activation is associated with stress, we examined the effects of PKC Kv1.3 into caveolae by molecular interactions with caveolin 1. In a caveolin activation on Kv1.5 channels. Previous studies have shown that PKC regulates 1-deficient 3T3-L1 adipocyte cell line, we verified that the localization of Kv1.5 channel when co-expressed with Kvb1.2 or Kvb1.3. In the present Kv1.3 in caveolar lipid raft structures is necessary for proper insulin study, using Western blot analysis and whole-cell patch clamp recording in signaling. Insulin-dependent phosphorylation of the Kv1.3 occurs at the human embryonic kidney cells stably expressing Kv1.5 alone, we found beginning of insulin-mediated signaling. However, when Kv1.3 localized that PKC activation by phorbol 12-myristate 13-acetate (PMA) at 10 nM out of these lipid microdomains, reduced phosphorylation was demon- nearly abolished Kv1.5 channel expression and current within 3 hours. Inhibi- strated. Our data contribute to the knowledge of the putative role of tion of PKC by Bisindolylmaleimide I completely prevented PMA-induced Kv1.3 in adipocyte physiology. Kv1.5 channel reduction. Co-immunoprecipitation experiments showed that Supported by the Ministerio de Ciencia, Innovacio´n y Universidades, Spain PMA treatment enhanced ubiquitination of Kv1.5 channels; proteasome inhib- (BFU2014-54928-R and BFU2017-87104-R) and Fondo Europeo de Desar- itors MG-132 or lactacystin partially prevented the PMA-induced Kv1.5 chan- rollo Regional (FEDER). nel reduction. We also found that transfection of dominant-negative Rab5 partly impeded PMA-induced Kv1.5 channel reduction. Furthermore, trunca- 2683-Pos tion of the N-terminus of Kv1.5 up to residue 209 (DN209) abolished PMA- Development of BK Channel Agonists and Antagonists that Target a mediated reduction in expression and current of the channel. Intriguingly, Common Recognition Area in the Accessory Beta1 Subunit deletion of a distinct motif in the N-terminus for non-receptor tyrosine kinase Anna N. Bukiya1, Guruprasad Kuntamallappanavar1, Abby L. Parrill2, Src also abolished PMA-mediated reduction in Kv1.5 expression and current. Alex M. Dopico1. 1Dept Pharmacology, Univ Tennessee Hlth Sci Ctr, Memphis, TN, USA, We propose that interplay between PKC and Src mediates degradation of 2 mature Kv1.5 channels partially through ubiquitination and Rab5-mediated Dept Chemistry, Univ Memphis, Memphis, TN, USA. endocytosis. These findings extend our understanding of the regulation of Calcium-/voltage-gated, large conductance potassium channels (BKs) are Kv1.5 channel by PKC activation. (Supported by the Canadian Institutes of ubiquitously expressed. Smooth muscle (SM) BKs, which contain accessory Health Research). beta1-subunits, regulate SM contractility. Upon activation, BKs generate out- ward Kþ currents that counteract depolarization-induced Ca2þ entry and limit 2681-Pos contraction. Given the rather specific expression of beta1-subunits in SM, Identifying Common Structural Features for Electromechanical Coupling their pharmacological targeting constitutes an attractive tool for selective between Domain-Swapped and Non-Domain Swapped Potassium Channels modulation of SM BKs. We have previously identified a steroid-sensing Ana I. Ferna´ndez-Marin˜o, Kenton J. Swartz. site in BK beta1 that included Thr169 and Leu172,173. Herein, we advance Mol. Physiol. & Biophysics, NINDS-NIH, Bethesda, MD, USA. non-steroidal analogs (NSTAs) that interact with this site but exert opposite Voltage-gated potassium channels play a pivotal role in physiological pro- effects on BK current and organ function. Based on a pharmacophore that cesses such as neuronal excitability and cardiac function. The crystal struc- described endogenous ligands for the BK beta1 steroid site (McMillan tures of the Kv1.2 and Kv1.2/2.1 chimera established a ‘‘domain-swapped et al., 2014), we performed chemical library search to yield NSTAs. Hits architecture’’ due to the structure of the S4-S5 linker, a rigid alpha helix were tested on BK currents using robotic patch-clamp. While some agents that positions the voltage-sensing S4 helix of one subunit near the S5 helix activated BK (decreased V0.5; ‘‘agonists’’), others did not decrease or even of the neighboring subunit. In the tetrameric channel, the S4-S5 linker helices increased V0.5 (collectively termed ‘‘antagonists’’). Pharmacophore model are positioned parallel to the membrane, wrapping the pore domain like a refinement based on functional data led to development of two distinct phar- ‘‘cuff ’’so that movements of the S4 helix can be coupled to the pore. Similar macophores: BK beta1 agonists hydrogen-bond with Thr169 and hydrophobi- domain-swapped architecture architectures are also seen in Nav and Cav chan- cally interact with Leu172,173. In contrast, antagonists lack the former feature nel structures. However, experiments in the ‘‘Ether-a-go-go’’(EAG) and in the while still forming hydrophobic interactions with Leu172,173. Both pharma- ‘‘human EAG-Related-Gene’’ (hERG), showed that the voltage sensor re- cophores were used to search 400,000 structures in the ZINC database. The mained coupled to the pore in constructs lacking a covalent link between agonist pharmacophore matched 84 and the antagonist pharmacophore the VSD and the pore, challenging the view that a structured S4-S5 linker is matched 131 chemically diverse compounds, with zero compounds matching necessary for the coupling between the two. Interestingly, the cryoEM struc- both pharmacophores. Thus, our models have exquisite selectivity. Two an- tures of the EAG channel family (EAG, CNG, HCN1 and hERG) revealed that tagonists that did not modify BK current effectively blunted BK activation these channels have a ‘‘non-domain swapped’’ architecture, where the S4-S5 and resulting dilation of rat cerebral arteries evoked by the beta1 Thr169- linkers are short loops instead of rigid helices positioning the S4 helix close to targeting lithocholic acid. Further optimization of BK beta1-targeting NSTA the S5 of the same subunit. These unexpected findings raise the question of agonist-antagonist pairs will lead to the development of novel pharmaceuticals how the coupling between the voltage sensor domain and the pore occur in with potential to combat prevalent disorders where SM function is disrupted. this Kv family. In the present work, we successfully engineered functional chi- R01 HL104631; R37 AA11560 (AMD). meras between Shaker Kþ channel (domain swapped channel) and hERG (non-domain swapped), transplanting parts of the hERG pore into the Shaker 2684-Pos KCNMB1 voltage sensor scaffold. We are using electrophysiological and pharmacolog- Proteomics Analysis Points at Novel Cellular Partners for the ical approaches to characterize the properties of the chimeras and elucidate Protein Product Kelsey North1, David Kakhniashvili2, Alex M. Dopico1, Anna N. Bukiya1. which common features are shared between these two families of channels 1 with different architecture. Dept Pharmacology, Univ Tennessee Hlth Sci Ctr, Memphis, TN, USA, 2Proteomics Core Lab, Univ Tennessee Hlth Sci Ctr, Memphis, TN, USA. 2682-Pos The protein product of the KCNMB1 gene (‘‘KCNMB1’’) has been charac- Caveolar Kv1.3 Targeting Participates in the Adipocyte Physiology terized as a regulatory subunit of the voltage-/calcium-gated potassium Mireia Perez-Verdaguer1, Jesusa Capera Aragones2, channel of large conductance (BK). In smooth muscle, KCNMB1 is highly Maria Ortego-Dominguez3, Joanna Bielanska4,Nu´ria Comes1, abundant and enhances calcium sensitivity of plasmalemma BK, which con- Rafael J. Montoro5, Marta Camps4, Antonio Felipe4. tributes to regulation of myocyte contractility. Observations from our lab 1Univ Barcelona, Barcelona, Spain, 2Biochemical and Molecula, Univ De and others document that up to 90% of KCNMB1 has an intracellular loca- Barcelona, Barcelona, Spain, 3Dpto. de Fisiologı´a Medica y Biofı´sica, tion. We hypothesized that KCNMB1 has intracellular protein partner(s). Universidad de Sevilla, Sevilla, Spain, 4Biochemistry and Mol Biomed, Univ We used murine (C57BL6/J) aorta lysates and immunoprecipitation to Barcelona, Barcelona, Spain, 5Univ Sevilla, Sevilla, Spain. isolate KCNMB1-containing protein complexes for the following protein

BPJ 9466_9469 542a Wednesday, March 6, 2019 identification. To identify background, non-specific proteins, control immu- mine the trajectory of KCNQ1 VSD activation motion. We further demonstrate noprecipitation was performed using the same antibody pre-incubated with the physiological relevance of this intermediate-state conductance as regulated immunogenic peptide (aa 90-103 of human KCNMB1). Efficiency of immu- by different auxiliary-subunits. Together, these findings provide structure- nogenic peptide was validated by Western blotting of eluate, which showed function insights into the elusive intermediate VSD conformation and VSD mo- shading of a KCNMB1 (24 kDa) band. Experimental and control immuno- tion during voltage-dependent activation. Additional studies into the distinction precipitations were performed in five replicates. Eluates were analyzed by between the KCNQ1 intermediate-VSD and activated-VSD states may lead to mass spectroscopy for protein identification. Proteins identified with high novel discoveries regarding KCNQ1 physiology, electromechanical coupling, confidence were annotated using the Mus musculus protein subset in Uniprot and disease pathogenesis. database. The following proteins were present in all eluates from the exper- imental group while absent in eluates from the control group: trifunctional 2687-Pos enzyme beta subunit (encoded by Hadhb, Q99JY0), murinogloblulin-1 Dynamics of the PAS Domain and Cyclic Nucleotide-Binding Homology (P28665), MKIAA0866 (Q69ZX3) and transgelin-2 (Q9WVA4). We per- Domain Interaction Probed with a Fluorescent Noncanonical Amino formed immunofluorescence co-staining of mouse cerebral arteries against Acid (L-ANAP) in HERG Potassium Channels KCNMB1 and Hadhb protein product. The latter locates in the mitochondria Ashley A. Johnson, Matt C. Trudeau. where participates in fatty acid beta-oxidation. Co-staining against Dept Physiology, Univ Maryland, Baltimore, MD, USA. KCNMB1 with conventional mitochondrial marker COX-IV was used as The voltage-gated potassium channel hERG plays a critical role in cardiac control. Immunofluorescence Pearson’s coefficient of KCNMB1:Hadhb repolarization and is characterized by unusually slow deactivation kinetics. (r=0.25) was significantly higher when compared to the KCNMB1:COX- Slow deactivation in hERG channels is regulated by a direct interaction be- IV (r=0.08) pair (p<0.05). Studies on the functional consequences of tween the N-terminal PAS domain and the C-terminal CNBHD. The PAS- KCNMB1:Hadhb co-localization are underway. Our data may lead to recon- CNBHD interaction is sensitive to point mutations at the domain interface sidering of the current view on KCNMB1 function, which is limited to regu- in hERG but less is known about potential dynamic rearrangements of the lation of BK expression and gating mechanisms. PAS domain relative to the CNBHD and the rest of the channel. Here, we report that hERG channels formed from PAS-CFP domains and DPAS- 2685-Pos Citrine channels showed FRET (Fo¨rster resonance energy transfer) that is Using Click Chemistry and Voltage Clamp Fluorimetry to Study Struc- sensitive to potassium-induced cell depolarization, consistent with a PAS- tural Dynamics of Membrane Proteins CNBHD rearrangement. To examine this in more detail, we took advantage Kanchan Gupta, Gilman E.S. Toombes, Kenton J. Swartz. of a fluorescent noncanonical amino acid (L-ANAP) and a metal ion bound MPBU, NIH NINDS, Bethesda, MD, USA. to a dihistidine motif that can be used to monitor small structural rearrange- The conformational dynamics of membrane proteins has been extensively ments within ion channels using patch-clamp fluorometry (PCF) and transi- investigated using cysteine mutagenesis to probe accessibility with thiol- tion metal FRET. We found that amber stop codon-containing mutants reactive compounds and/or introduce fluorophores, spin labels, etc. Here N33X, E50X, and C64X in the PAS domain incorporate L-ANAPs using we explored a complementary approach using an unnatural amino acid con- amber codon suppression as shown by robust currents measured with two- taining an azide group that can selectively react with alkynes through click electrode voltage-clamp. Likewise, the addition of dihistidine mutations in chemistry. We found that the azide-containing amino acid can be efficiently the CNBHD (N33X,E788H-L790H, E50X,H851-S855H and C64X,V796H- introduced into Shaker, a voltage-activated potassium (Kv) channel and al- I798H) also result in functional channels. The voltage dependence of these lowed us to efficiently tag the protein with alkyne derivatized biotin. The channels was comparable to control, indicating incorporation of L-ANAP azide containing amino acid could also be labeled with alkyne-conjugated flu- and introduction of histidines did not measurably alter channel gating. We orophores to track the conformational transitions of the voltage-sensor acti- then performed PCF recordings of excised patches with L-ANAP incorpo- vation and deactivation, and the resulting fluorescence voltage (F-V) rated hERG channels and found that hERG channels are stable in excised relationships were very similar to those obtained with fluorophores attached patches and that hERG E50X,H851-S855H channels showed robust L- to introduced cysteine residues via thiol-based chemistry. Our results estab- ANAP and Citrine fluorescence. The above approaches will determine how lish the potential of fluorescent labeling through click chemistry to probe the PAS-CNBHD interaction dynamically regulates conformational changes the structural rearrangements of membrane proteins, which when combined and ionic current in hERG. with cysteine-based labeling should allow two-color labeling of membrane protein assemblies for studying both intramolecular and intermolecular struc- 2688-Pos tural dynamics. CP1 Is a Potent IKs Channel Activator Which Acts by Substituting Phos- phatidylinositol 4,5 Bisphosphate 2686-Pos Yongfeng Liu1, Xianjin Xu2, Moawiah M. Naffaa3, Hongwu Liang1, Structure of the Intermediate State of the Human KCNQ1 Channel Guohui Zhang1, Panpan Hou1, Hongzhan Wang4, Junyuan Gao4, Jingyi Shi1, Voltage-Sensor Domain Ira Cohen4, Xiaoqin Zou2, Jianmin Cui1. Keenan C. Taylor1, Po wei Kang2, Panpan Hou2, Nien-Du Yang2, 1Department of Biomedical Engineering, Washington University in Saint Georg Kuenze3, Jingyi Shi2, Jarrod A. Smith1, Kelli McFarland White2, Louis, Saint Louis, MO, USA, 2Department of Physics and Astronomy, Hui Huang1, Dungeng Peng1, Alfred L. George Jr.4, Jens Meiler3, University of Missouri, Columbia, MO, USA, 3Biomedical Eng, Washington Robert L. McFeeters5, Jianmin Cui2, Charles R. Sanders1. Univ St Louis, St Louis, MO, USA, 4Department of Physiology and 1Department of Biochemistry, Vanderbilt University, Nashville, TN, USA, Biophysics, Stony Brook University, New York, NY, USA. 2Department of Biomedical Engineering, Washington University in St. Louis, KCNQ1 voltage-gated potassium channel assembles with its KCNE1 auxil- 3 St. Louis, MO, USA, Department of Chemistry and Pharmacology, iary subunit to form the slow delayed rectifier (IKs) channel, which acceler- Vanderbilt University, Nashville, TN, USA, 4Department of Pharmacology, ates the repolarization of action potentials in cardiac myocytes. Loss-of- Northwestern University Feinberg School of Medicine, Chicago, IL, USA, function mutations of KCNQ1 gene have been associated with long QT 5Department of Chemistry, University of Alabama in Huntsville, Huntsville, (LQT) syndrome and lead to an elevated risk of fatal cardiac arrhythmias. AL, USA. The activation of IKs channel depends on its physiological activator, phos- The KCNQ1 voltage-gated potassium channel resembles many voltage-gated phatidylinositol 4,5-bisphosphate (PIP2). In this study, we docked the previ- potassium channels in that its voltage-sensor domain (VSD) transitions be- ously identified PIP2 binding sites in a homology model of KCNQ1 and tween three stable conformations during voltage-dependent activation: resting, developed an in-silico screening to the Available Chemical Database. One þ intermediate, and activated. However, KCNQ1 uniquely conducts K flux of the hit compounds, CP1, potently increases the amplitude of IKs current when its VSDs adopt the intermediate conformation. This unique conductance (EC50=7.2 mM) heterogenously expressed in Xenopus oocytes and makes contributes to KCNQ1’s ability to conduct diverse physiological currents the channel constitutively open. Long intervals between voltage clamp including the cardiac IKs current and an epithelial leak current. However, pulses revealed that 2 mM CP1 slowed the deactivation of IKs channel by experimental-structure insights into this conductance so far remain elusive. 13-fold. The half-point for activation was 40 mV more negative than Here, we present the three-dimensional atomic structure of the human wild-type channels. Further studies in guinea pig ventricular myocytes KCNQ1 VSD in the intermediate state. We functionally map major stabilizing showed that 0.2 mM CP1 apparently shortens the prolonged action potential interactions in both the KCNQ1 intermediate-VSD and activated-VSD states. duration. In addition, CP1 is capable of rescuing the loss of IKs current These results provide an experimental-structure glimpse of VSD movements caused by PIP2 depletion, suggesting that CP1 substitutes for PIP2 in acti- during voltage-dependent activation and define major interactions that deter- vating IKs channels. Our results indicate that CP1 is a novel IKs channel

BPJ 9466_9469 Wednesday, March 6, 2019 543a opener and may provide a novel therapy to treat congential and acquired voltages at which BK channels were open nearly 100% of the time, the LQT syndromes. IC50 did not significantly increase or decrease as a function of voltage, sug- gesting that loperamide does not inhibit as an open-channel blocker. Single- 2689-Pos channel recordings revealed that loperamide does not reduce unitary current Single Channel Studies of the Cation Permeation Pathway of the Shaker amplitudes, and does not inhibit BK channels at voltages where voltage Kv Isolated Voltage-Sensing Domain (iVSD) sensors were at rest (between 140 and 160 mV; Po = 0.0035 5 0.0002 Juan Zhao, Rikard Blunck. and Po = 0.0033 5 0.0001 with 0 and 30 mM loperamide, respectively). þ Dept Physics, Univ Montreal, Laval, QC, Canada. Loperamide inhibited BK channels in the nominal absence of Ca2 , in chan- In the absence of a pore domain, the isolated voltage-sensing domain (iVSD) of nels co-expressed with the g1 subunit (LRRC26), suggesting that inhibition þ Shaker Kv channel forms a cation-selective ion channel with a strong prefer- does not strictly depend on the presence of Ca2 . Together, these data are ence for protons. iVSDs produce hyperpolarization-activated ionic currents, consistent with the idea that loperamide inhibits BK channels either through but the location of the gating pore in the iVSD is unknown. Identifying the mo- directly inhibiting activation of the BK voltage sensor, or by inhibiting allo- lecular determinants underlying iVSD gating would help in the understanding steric coupling between voltage sensor activation and pore opening. This work of VSD relaxation since iVSD gating and relaxation have been shown to be has been supported by NIH grant R01 GM126581 and AHA Grant-in-Aid to correlated. We studied the single channel behavior of iVSD ionic currents; B.S.R. the single channel recordings displayed two conformational sublevels, which can be effectively inhibited by the intracellular guanidine derivative 2GBI in 2692-Pos inside-out patch configuration, or by the extracellular ZnCl2 in outside-out Modulation of Kv1.3 by the Gut Peptide Glucagon-Like Peptide 1 patch configuration. We also characterized the effects of a series of residue sub- Daniel R. Landi Conde, Genevieve A. Bell, Debra A. Fadool. stitutions along the suspected gating pore of the Shaker channel on the IVSD Biological Sciences, Florida State University, Tallahassee, FL, USA. single channels. Our study supports the notion that the cation permeation of The voltage-dependent potassium channel Kv1.3 is a mammalian homolog Shaker IVSD follows the pathway through the gating pore like in HV1 channels of the Shaker subfamily that is implicated in processes related to immunity, and u currents. glucose metabolism, energy homeostasis, and olfaction, in addition to action potential (AP) excitability. It has been previously demonstrated that Kv1.3 2690-Pos is modulated by important metabolic factors and hormones including insu- Decoupling between Voltage Sensor Movement and Pore Opening of Kv2.1 lin, glucose, and glucagon-like peptide 1 (GLP-1). Although GLP-1 is an in- Channels cretin hormone secreted by L-cells of the intestine following a meal, we Matthew J. Marquis, Rebecka J. Sepela, Jon T. Sack. discovered GLP-1 signaling in the olfactory bulb wherein the peptide en- Dept. of Physiology and Membrane Biology, University of California, Davis, hances AP firing of the major output neurons in a Kv1.3-dependent manner. CA, USA. Herein, we co-expressed Kv1.3 and the receptor for GLP-1 (GLP-1-R) in a The relationship between membrane potential and the conductance of Kv2- heterologous expression system (HEK 293) to biophysically characterize the type voltage gated Kþ channels is modulated by cells. To begin studies into molecular mechanism of modulation. Recording macrocurrents from cell- the mechanistic underpinnings of coupling between voltage sensing and Kþ attached patches in voltage-clamped cells, we found that Kv1.3þGLP-1-R conductance, we are developing transition state theory kinetic models of co-expression did not alter basal biophysical properties of the channel, but coupling between voltage sensor movement and pore opening. We have con- bath application of the peptide decreased the peak outward current by strained these gating models with voltage-clamp recordings of the dynamics 50% over a 25-minute recording interval and elicited a 6 mV hyperpolariz- of rat Kv2.1 channels expressed in CHO-K1 cells. Consistent with prior re- ing shift in voltage at half-activation (V1/2). Using Quantity One Software, ports, our models suggest that Kv2.1 gating consists of fast voltage sensor predicted Ser phosphorylation sites were mapped to S105, S470, S472, and movement, and a slower pore opening step that has little inherent voltage S502 in the N- and C-terminal aspects of the channel. A channel was con- dependence. We found that the activation kinetics of Kv2.1 conductance structed with all four phosphorylation sites removed (S to A point muta- are influenced by the probability of voltage sensor activation, and a tions), named delta4SerKv1.3. Cells co-expressed with delta4SerKv1.3þGLP- concerted pore opening step is rate limiting. The degree of sigmoid delay 1-R were resistant to current suppression and shift in V1/2 in response to of activation kinetics in response to voltage steps predicts the kinetics of GLP-1 peptide. We are using a channel immunoprecipitation strategy fol- a fast component of gating current. A relatively slow pore closing step lowed by Western Blot to determine if delta4SerKv1.3þGLP-1R transfected with little voltage dependence leads to voltage insensitive deactivation ki- conditions fail to exhibit Kv1.3 phosphorylation when stimulated with netics over a range of membrane potentials. In response to sufficiently nega- GLP-1. These results highlight a phosphorylation-dependent modulation tive membrane potentials, deactivation voltage dependence steepens. This of Kv1.3 by GLP-1 signaling that could regulate metabolism in the olfactory change of voltage dependence requires an alternate route of exit from the bulb. open state. Modeling predicts that open channels subject to sufficiently Funded by the FSU LAC Scholarship and NIH grant DCR01013080 from the negative voltages enter a short-lived state where voltage sensors have deac- NIDCD. tivated before the concerted pore closing conformational change occurs. This transient decoupling of voltage sensor movement from pore closing al- 2693-Pos lowed estimation of the coupling energy between voltage sensor and pore Conserved Residues at the Interface between the S4 and S5 Segments are conformational changes in Kv2.1 channels. Critical for Normal Gating of HCN Channels Rosamary Ramentol, Marta E. Perez, H. Peter Larsson. 2691-Pos Department of Physiology and Biophysics, University of Miami, Miami, FL, Mechanism of BK Channel Inhibition by the Opioid Agonist Loperamide USA. Alexandre G. Vouga1, Michael E. Rockman1, Marlene A. Jacobson2, Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have Brad S. Rothberg1. the unique characteristic of being activated at negative membrane potentials, 1Dept Med Genetics Mol Biochem, Temple Univ Sch Med, Philadelphia, PA, but the mechanism of HCN reverse voltage-dependence is not known. HCN USA, 2Moulder Center for Drug Discovery Research, Temple Univ School of channels share a similar tetrameric architecture with depolarization- Pharmacy, Philadelphia, PA, USA. activated Kv channels, having six transmembrane domains, where S1-S4 Large conductance Ca2þ-activated Kþ (BK) channels are expressed in a vari- form the voltage-sensing domain (VSD) and S5-S6 form the pore domain ety of tissues and control a range of physiological functions, including action (PD). As opposed to Kv channels, HCN channels have a non-domain swap- potential duration in some neurons and contractility in smooth muscle. Phar- ped architecture with a short S4-S5 linker that covalently binds the VSD to macological BK channel modulators may be promising therapeutic options in the PD. The non-domain swapped architecture implies that the S4 from one human disease; however, the current library of effective BK-selective modu- subunit directly interacts with the S5 from the same subunit. As well as in lators is limited. Using a high-throughput screening approach, we observed depolarization-activated channels, S4 is the positively charged voltage that the diphenylpropylamine m-opioid agonist, loperamide, could inhibit sensor of HCN channels and moves across the membrane in response to BK channel activity. Here we analyze the molecular mechanism of this inhi- changes in the membrane potential. It has been shown before that cutting bition using patch-clamp electrophysiology. In excised inside-out patch re- the S4-S5 linker of HCN channels and other non-domain swapped channels cordings (Ca2þ =88mM at the cytosolic side), loperamide reversibly does not impair their typical gating. Therefore, the coupling between the inhibited BK current in a dose-dependent manner when applied to the cyto- VSD and the PD in HCN channels may involve residues located at the inter- solic face of BK channels, with an IC50 of 1.0 5 0.2 mM. Over a range of face between the S4 and S5 segments. The interface between S4 and S5 of

BPJ 9466_9469 544a Wednesday, March 6, 2019

HCN channels possesses several residues that are absolutely conserved in Shijiazhuang, China, 3Medicinal Chemistry, Shanghai Zhimeng BioPharma the HCN family and that therefore, are potentially important for their unique Ltd, Shanghai, China. gating. We individually mutated several of these conserved residues at the Voltage-gated Kv7/KCNQ/M-potassium channels play an essential role in S4-S5 interface in the sea urchin HCN (spHCN) channel and expressed control of membrane potential and neuronal excitability. Activation of them in Xenopus laevis oocytes to measure the effects of each mutation us- neuronal Kv7/KCNQ/M-current represents an attractive therapeutic strategy ing Voltage Clamp Fluorometry. Our results show that some residues at the for treatment of hyperexcitability-related neuropsychiatric disorders such as S4-S5 interface are critical for normal S4 movement, while others are epilepsy, pain and schizophrenia. In this study, we synthesized and charac- important to stabilize the gate or are involved in the coupling between S4 terized a novel series of 2,6-dimethyl-4-(piperidin-yl)phenyl)-amide deriva- and the gate. tives that exhibit selective and potent activation of neuronal Kv7/KCNQ/M channels. Whole-cell patch clamp recordings of HEK293 cells expressing 2694-Pos Kv7.2/Kv7.3 channels show that a representative compound SCR2682 Endocannabinoids Facilitate the Opening of the M-Channel selectively activates the channel current in dose-dependent manner with Johan E. Larsson, Liin Sara. an EC50 of 9.8 5 0.4 nM, which is about 100-fold potent than antiepileptic Linko¨ping Univ, Linko¨ping, Sweden. drug retigabine approved by FDA for partial epilepsy. Compound SCR2682 The M-channel is a neuronal ion channel complex that is important for sta- shifts the voltage-dependent activation of Kv7.2/7.3 current towards more bilizing neurons at subthreshold potential, limiting continuous neuronal negative membrane potential about 37 mV (V1/2). SCR2682 also activates firing. The M-channel belongs to the KV7 channel family and is formed native M current of rat hippocampal or cortical neurons, causing a marked mainly by KV7.2/KV7.3 heteromultimers. The physiological importance of hyperpolarization and potent inhibition of evoked action potentials. In rat the M-channel is evident by channel mutations that have been linked to an epileptic models, intraperitoneal and intragastric administrations of inherited form of juvenile epilepsy. We have previously described that poly- SCR2682 results in a dose-dependent inhibition of seizures. Taken together, unsaturated fatty acids activate the M-channel and thereby dampen neuronal our findings demonstrate that a novel SCR2682 selectively and potently ac- excitability. Several members within the large family of endocannabinoids tivates neuronal Kv7 channels and reverses epileptic activity in rats. Thus, are structurally related to polyunsaturated fatty acids. We therefore hypothe- SCR2682 may warrant further evaluation for clinical development of antiep- size that endocannabinoids may modulate the activity of the M-channel. To ileptic therapy. test that hypothesis, we studied the effect of different endocannabinoids on the human KV7.2/KV7.3 channel expressed in Xenopus oocytes, using two- electrode voltage clamp. We found that specific compounds belonging to Posters: Bacterial Mechanics, Cytoskeleton, and the endocannabinoid family activate the M-channel by shifting the voltage Motility dependence of channel opening towards negative voltages and increasing the maximal conductance. In particular, N-Arachidonoyl L-serine (ARA-S) 2697-Pos had a large activating effect, with significant effects from 0.3 mM. In contrast, A Comprehensive View of Type IV Pilus Retraction Forces across the some of the most commonly known endocannabinoids, e.g. 2-Arachidonoyl- Bacterial Domain glycerol (2-AG) and N-arachidonoylethanolamine (anandamide), did not acti- Nicolas Biais. vate the M-channel. From a systematic comparison of compounds with City Univ New York, Brooklyn, NY, USA. distinct chemical properties, we conclude that a negative charge of the Type IV pili are long flexible polymeric appendages emanating from many bac- head group is crucial for the activating effect, whereas the specific length teria. They are playing key roles in many bacterial functions: DNA uptake, and number of double bonds of the acyl tail is less important. Altogether, motility, colonization, infection, biofilm formation, aggregation. A central these findings suggest that specific members within the family of endocanna- feature of these appendages is their ability to undergo cycles of elongation binoids may signal non-canonically via the M-channel. Understanding which and retraction enabling them to exert physical forces on their surroundings. endocannabinoids target the M-channel is of importance for interpretation of In order to gain insight in the differences and commonalities of the biophysical endocannabinoid effects on neuronal excitability and may be utilized for characteristics of those retraction forces across evolution, we have measured, future development of pharmaceutical agents. combining different techniques, the retraction forces of type IV pili in a wide range of bacterial genus (Neisseria, Escherichia, Vibrio, Synechocystis, Strep- 2695-Pos tococcus). Of particular interest is the role that the so-called retraction ATPase A Structural Model of Fast Inactivation in Shaker KV Channels (pilT) plays in the biophysical characteristics of the retraction forces. The direct Miguel Holmgren1, Ariela Vergara-Jaque2, Horacio Poblete2, comparison of the retraction forces in different mutants of those very different Francisco Palma1, Adam S. Lowet1, Angel de la Cruz Ladrau1, genus will help ascertain the common biophysical features of the Type IV pilus Alexander Sukharev1, Jeffrey Comer3. machinery across bacteria. 1NINDS/NIH, Bethesda, MD, USA, 2Center for Bioinformatics and Molecular Simulation, Talca, Chile, 3Department of Anatomy and 2698-Pos Physiology, Institute of Computational Comparative Medicine, Manhattan, Mechanical Forces are a Reactivity Switch for an Adhesin Thioester Bond KS, USA. Daniel J. Echelman1, Alvaro Alonso2, Shubhasis Haldar3, Rafael Tapia- After opening, the Shaker voltage-gated potassium channel rapidly inactivates Rojo2, Edward C. Eckels2, Julio M. Fernandez2. when one of its four N-termini enters and occludes the channel pore. While it is 1Columbia University Vagelos College of Physicians and Surgeons, New known that the tip of the N-terminus reaches deep into the central cavity, the York, NY, USA, 2Columbia University, New York, NY, USA, 3Ashoka conformation of this region during inactivation and the nature of its interactions University, Sonipat, India. with the rest of the channel have remained unclear. Here, we use molecular dy- Bacteria must recognize and bind to target ligands against large mechanical namics simulations coupled with electrophysiology experiments to reveal the perturbations, such as coughing. Towards this end, Gram-positive bacteria atomic-scale mechanisms of inactivation. We find that the first six amino acids have evolved a subset of adhesins with an internal Cys-Gln thioester bond of the N-terminus spontaneously enter the central cavity in an extended confor- capable of forming stable covalent crosslinks to host ligands. Initial work has mation, establishing hydrophobic contacts with residues lining the pore. A sec- suggested that covalent binding via this conserved thioester bond is force- ond portion of the N-terminus, consisting of a long 24 amino acid a-helix, dependent. Herein, we applied Magnetic Tweezers single-molecule force spec- forms numerous polar contacts with residues in the intracellular entryway of troscopy to resolve the binding mechanics of the Streptococcus pyogenes the T1 domain. Double-mutant cycle analysis revealed a strong relationship be- Spy0125 adhesin thioester bond under force. We find that nucleophilic thioester tween predicted interatomic distances and empirically observed thermody- cleavage and the thioester reformation are in a stable equilibrium in the absence namic coupling, establishing a plausible model of the transition to the of force. However, both processes of cleavage and reformation are negatively inactivated state. force-dependent, inhibited by loads above 30 pN and 7 pN, respectively. When a ligand-bound Spy0125 protein refolds at low force, the Cys-Gln thio- 2696-Pos ester spontaneously reforms to substitute out the ligand, which remarkably oc- Electrophysiological and Pharmacological Characterization of a Novel and curs without exogenous enzymes or cofactors such as ATP, and on timescales Potent Neuronal Kv7 Opener SCR2682 for Anti-Epilepsy of less than 5 seconds. Thus, mechanical force switches ligand binding between Yani Liu1, Fan Zhang2, Bo Liang3, huangming Chen3, Hailin Zhang2, reversible covalent binding at low force and an inert state at high force. More- KeWei Wang1. over, we report that the reversible binding can be inhibited by small molecule 1Department of Pharmacology, Qingdao University School of Pharmacy, suicide substrates that target the cysteine thiol, establishing a novel target for Qingdao, China, 2Department of Pharmacology, Hebei Medical University, mechanically active antibiotics.

BPJ 9466_9469 Wednesday, March 6, 2019 545a

2699-Pos pected energy that would come from hydrolysis of six ATP molecules, indi- Constrictive Force as the Key Symmetry-Breaking Factor for Bacterial cating that 12 ATP molecules are required for a single rotation, and Cell Division suggesting a model for the mechanism of archaellar motor rotation. Lam T. Nguyen. [1] Y. Kinosita, N. Uchida, D. Nakane, T. Nishizaka, Nature Microbiology 1, Dept Biol/Bioeng, Caltech, Pasadena, CA, USA. 16148 (2016). To divide, Gram-negative bacterial cells must remodel their peptidoglycan cell [2] J. Yajima, K. Mizutani, T. Nishizaka, Nature Structural & Molecular wall to a smaller and smaller radius at the division site, but how this process Biology 15, 1119-1121 (2008). occurs remains debated. While the tubulin homolog FtsZ is thought to generate a constrictive force, it has also been proposed that cell wall remodeling alone is 2702-Pos sufficient to drive membrane constriction, possibly via a make-before-break Rapid Light-Triggered Spatial Reorganization of Proteins in Living mechanism in which new hoops of cell wall are made inside the existing hoops Bacteria Cells (make) before bonds in the existing wall are cleaved (break). Previously, we Ryan J. McQuillen, Jie Xiao. constructed software, REMODELER 1, to simulate cell wall remodeling in Biophyiscs & Biophysical Chemistry, Johns Hopkins University School of rod-shaped bacteria during growth. Here, we used this software as the basis Medicine, Baltimore, MD, USA. for an expanded simulation system, REMODELER 2, which we used to explore In gram-negative bacteria, the cytoplasm is surrounded by two lipid bilayers that different mechanistic models of cell wall division. We found that simply orga- encapsulate the peptidoglycan cell wall. Due to a lack of membrane-enclosed or- nizing the cell wall synthesis complexes at the midcell was not sufficient to ganelles within the cytoplasm, the proper formation and spatiotemporal regula- cause wall invagination, even with the implementation of a make-before- tion of subcellular protein structures and scaffolds are critical to the cell’s ability break mechanism. Applying a constrictive force at the midcell could drive di- survive and function. Here we demonstrate, for the first time, that the CRY2/ Arabidopsis thaliana vision if the force was sufficiently large to initially constrict the midcell into a CIB1 system from can be used to induce the fast reorgani- compressed state before new hoops of relaxed cell wall were incorporated be- zation of proteins inside living bacterialcells. We further show that this system Escherichia coli tween existing hoops. Adding a make-before-break mechanism could drive di- can be used to rapidly inhibit cytokinesis in cells. vision with a smaller constrictive force sufficient to bring the midcell 2703-Pos peptidoglycan into a relaxed, but not necessarily compressed, state. MinC-MinD Copolymers Capture FtsZ Filaments to Facilitate the Regula- tion of Z-Ring Localization 2700-Pos Yaodong Chen1, Ping Wang2, Na Wang1, Xueqin Ma1, Li Bian1. Tracking the Movement of a Single Prokaryotic Cell in Extreme Environ- 1Coll Life Sci, NorthWest Univ, Xian, China, 2Department of Anesthesia, mental Conditions Duke University, Durham, NC, USA. Masayoshi Nishiyama1, Yoshiyuki Arai2. 1 2 Self-organized MinDE proteins act as a dynamic reaction-diffusion device Department of Physics, Kindai Univ, Higashiosaka, Japan, Osaka Univ., leading to their oscillation in vivo and facilitate to localize the bacterial contrac- Suita, Japan. tile ring (Z ring) at the center of the cell. The previous model is set up by a feed- Many bacterial species move toward favorable habitats. The flagellum is one of back loop of the ATPase MinD and its activator MinE. MinC, an inhibitor of the most important machines required for the motility in solution and is FtsZ polymerization, binds MinD and is carried as a passenger. However, conserved across a wide range of bacteria. The motility machinery is thought MinC in vivo is around 0.7 mM, which is 6-8 times less than FtsZ and MinD; to function efficiently with a similar mechanism in a variety of environmental it contradicts the previous results that only excess MinC could affect FtsZ poly- conditions, as many cells with similar machineries have been isolated from merization significantly. The recent results suggested that MinC and MinD harsh environments. To understand the common mechanism and its diversity, could assemble into copolymers with 1:1 stoichiometry. However, the proposal microscopic examination of bacterial movements is a crucial step. Here, we was challenged since the previous study showed that the assembly of MinC- describe two methods to characterize the swimming motility of cells in extreme MinD required a high concentration of proteins and had a long lag time. We environmental conditions. The developed microscopy system is optimized both discovered new features of the coassembly of MinC-MinD from Pseudomonas for the best image formation and for the stability to hydrostatic pressure up to aeruginosa. MinD alone dominates two properties of coassembly: critical con- 150 MPa [1]. The temperature and oxygen concentration can also be manipu- centration and lag time. If MinD is above 4 mM, it will copolymerize any low lated. By using the system, we successfully monitored the swimming motility concentration MinC. The long lag time is also due to slow MinD dimerization: of hyperthermophilic bacterium, Aquifex aeolicus at 85C and extremely low premixing MinD with ATP eliminated it all. It fits in vivo data well. Also, concentrations of O2 ( 1 %) [2]. In addition, we report a method to track MinCD copolymers could bind FtsZ filaments and form huge bundles quickly the movement of particles using an ImageJ plugin (file name: Simple_PTA.jar) and be disassembled by MinE protein. Based on these results, we propose a new [3]. This enables us to characterize the swimming motility of the selected cells. ‘‘capture’’ model. Most MinC in vivo co-assembles with MinD into short co- Cell motility could be characterized by analyzing the fraction and speed of the polymers quickly, and they will capture FtsZ filaments diffused away from swimming cells under each condition. This analysis could be extended to other the center. These tight bindings will let MinC shorten FtsZ filaments. The motile systems of bacteria (or archaea) such as swarming, gliding, twitching, or bound FtsZ filaments will be released after GTP is used up. It can explain floating. why so low concentration MinC can regulate Z-ring formation. [1] Nishiyama M. 2017. Biophys Chem. 231: 71-78. [2] Takekawa N. et al. 2015. Sci Rep. 5: 12711. 2704-Pos [3] Nishiyama M. and Y. Arai. 2017. Methods Mol Biol. 1593: 175-184. Dynamics of FtsI, an Essential Bacterial Cell Wall Synthesis Protein Joshua McCausland1, Jie Xiao2. 2701-Pos 1Biophysics and Biophysical Chemistry, Johns Hopkins University School of Insights into the Mechanism of Archaellar Motor Rotation from Observa- Medicine, Baltimore, MD, USA, 2Johns Hopkins Sch Med, Baltimore, MD, tion of Unexpectedly High Torque USA. Takayuki Nishizaka. Bacterial cell division is complicated, requiring the orchestration of over thirty Dept. Physics, Gakushuin Univ., Tokyo, Japan. different proteins at midcell. One essential member of the septal cell wall pepti- It is unknown how the archaellum—the rotary propeller used by Archaea for doglycan synthesis machinery, FtsI (PBP3), cross-links newly-made glycan motility—works. The archaellum is a helical filament that generates thrust strands and helps drive membrane invagination, exhibiting directional move- when rotated by a membrane-embedded, ATP-driven archaellar motor. The ment coupled to the treadmilling tubulin homologue FtsZ. However, how only energy-transducing archaellar motor protein is the hexameric ATPase FtsZ regulates the spatiotemporal dynamics of FtsI indirectly through a FlaI that powers both assembly and rotation, yet the mechanism of torque gen- protein-protein interaction relay in not well understood. Namely, whether or eration by FlaI is unknown. We previously characterized motor function in the not FtsI’s enzymatic activity contributes to its directional movement at the model organism Halobacterium salinarum using advanced fluorescent micro- septum and how FtsI communicates with the Z-ring and other cell wall remod- scopy [1], but challenges manipulating motor load prevented us from measuring elers to advance division remain unknown. We have constructed functional torque. To gain insights into the molecular mechanism underlying how FlaI fluorescent fusions of FtsI in the E. coli chromosome and have determined drives rotation, here we describe determination of motor torque through impo- that the directional FtsI molecules are likely the active subpopulation. Through sition of various loads on archaella using markers of different sizes, trajectory single molecule tracking and super-resolution microscopy, we tracked FtsI in quantification using three-dimensional tracking [2], and high-speed recording. Escherichia coli to determine its dynamics with point mutants that perturb its We show that rotation slows as the viscous drag of markers increases, and torque interaction with divisome partners or that reduce its enzymatic activity. We remains constant at 160 pN,nm, independent of rotation speeds between 0.5 and are currently testing FtsI interaction mutants between its critical partners to 30 Hz. Notably, the estimated work done in a single rotation is twice the ex- parse out the communication relay of the divisome.

BPJ 9466_9469 546a Wednesday, March 6, 2019

Posters: Cell Mechanics, Mechanosensing, and rent in vitro tests focusing mainly on cytotoxicity, many important biochemical and biophysical characteristics are not being explored. Such biochemical and Motility III biophysical cues play an integral role in regulating cell morphogenesis, move- ment, and metabolism during fetal development. In this study, we investigated 2705-Pos the biophysical and biochemical effects of rotenone (an active ingredient of in- Modelling Collective Gradient Sensing with Leader and Follower Cells secticides) on human neural stem cells (NSCs). NSCs were exposed to a wide Austin Hopkins, Brian A. Camley. range of rotenone concentrations and using dose-response curves, toxic concen- Dept. Physics & Astronomy, Johns Hopkins University, Baltimore, MD, tration (IC50 = 0.24 mM) was flagged. IC50 and lower concentrations were tested USA. for biophysical assessment of rotenone-exposed NSCs. Using a MFP-3D-BIO Cells in clusters can cooperate to improve sensing chemical gradients. Recent AFM and customized culture platform, the biomechanical characteristics were experiments suggest that in some cell clusters there are cells which try to sense quantified from force-deflection curves obtained at various locations on live and follow a chemical gradient (leader cells) and other cells that follow their NSCs cultured under exposure conditions (n > 50 cells/ dose/ time). Cytoskel- neighbors (follower cells). Single cell experiments have indicated that a cell eton marker (actin) expression was assessed to correlate to changes in biome- can more effectively follow steeper chemical gradients, and information theory chanics. Results showed a negative dose-dependent effect on cell viability (p approaches have shown that a steeper chemical gradient can reduce the error in < 0.001) and a significant dose-time dependent reorganization of cytoskeleton a cell’s measurement of the gradient’s direction. In light of these results, we (p < 0.05). NSC stiffness varied from 450 Pa to 5 kPa in the presence and absence have developed models of cluster chemotaxis that include leader cells and fol- of rotenone, respectively. The changes in membrane tension suggests a strong lower cells. In the models, the accuracy of the leader cells depends on gradient correlation between dosage and duration of rotenone exposure (p < 0.01). Adhe- steepness, number of chemical receptors on each cell, and the dissociation con- sive forces between AFM probe and NSCs were strongly dependent on exposure stant of the ligand-receptor interaction. We simulate cluster chemotaxis in an duration and dosage of rotenone (p < 0.01), demonstrating the alterations of cell environment with a transition between high and low chemoattractant. In a steep membrane with rotenone exposure. gradient, a cell cluster with leaders and followers can be guided by leaders near the transition, which have more information about the gradient’s direction. If 2708-Pos the follower cells perfectly follow the cluster’s direction, a few leaders alone Transition between Swimming and Crawling: A Model of Eukaryotic Cell can successfully guide the cluster. However, as the followers become noisy, Motility the number of leaders for optimal chemotaxis increases. At a fixed follower Melissa H. Mai1, Brian A. Camley2. noise level, the optimal number of leaders changes as a function of the width 1Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA, of the gradient transition. As the width increases, the optimal number of leaders 2Department of Physics & Astronomy, Johns Hopkins University, Baltimore, initially increases since more cells are within the transition region and have suf- MD, USA. ficient information to effectively lead. However, as the gradient widens further, Eukaryotic cells can migrate spontaneously or in response to external stimuli it becomes too shallow for even some cells within the transition region to accu- such as chemical gradients and may travel either individually or collectively. rately sense, and the optimal number of leaders decreases. Eukaryotic cell motility is crucial during development, wound healing, the im- mune response, and cancer metastasis. In liquids, eukaryotic cells can swim by 2706-Pos pushing on the surrounding fluid, but they more often crawl along the extracel- Dynamic Crosslinking of the Actin Cytoskeleton Governs Intracellular lular matrix. We are interested in studying the relationships between hydrody- Mechanics namics and adhesion that regulate a cell’s transition between swimming and Loı¨c Chaubet, Hossein K. Heris, Allen J. Ehrlicher, Adam G. Hendricks. crawling. Therefore, we develop a simple model of a cell capable of both kinds Bioengineering, McGill University, Montreal, QC, Canada. of motion based on the three-sphere swimmer proposed by Najafi and Golesta- A dynamic network of actin filaments and crosslinkers is essential for maintain- nian. The model includes additional adhesive friction terms, geometry moti- ing the cell’s structural integrity and organization. Crosslinking proteins tran- vated by cells crawling on fibers, substrate surface hydrodynamic effects, siently link actin filaments to control the viscoelasticity of the cytoplasm. At and parameters from experimental data. Hydrodynamic effects diminish in long time scales, crosslinkers unbind to allow filaments to slide past one the high adhesion limit, and the crawling cell can achieve significant center- another resulting in a more viscous network. At time scales faster than the un- of-mass velocity compared to its swimming counterpart. The crawling cell binding rate, filaments remain crosslinked resulting in a more elastic network. maintains the same net displacement per cycle regardless of its sequence of mo- While the effect of crosslinking dynamics on viscoelasticity has been examined tions, including reciprocal cycles that would be unproductive in the frictionless in purified systems, their impact on the mechanics of living cells is largely un- limit, which constrains cell motion by the Scallop Theorem of low-Reynolds explored. Here, to probe the frequency-dependent viscoelastic properties of the number hydrodynamics. The center-of-mass velocity of the cell plateaus at a cytoplasm, we used an optical trap to apply multi-harmonic sinusoidal oscilla- maximum value as adhesion increases until the frictional drag exceeds the tions to 500 nm beads. Using in vivo calibration of the optical trap, we show largest force the cell can generate, after which velocity decreases with that the mechanics of fibroblast cells are best described by a model that assumes increasing adhesion. This recapitulates the common experimental observation viscoelasticity is governed by the unbinding kinetics of a single dominant that cell speed is not monotonic with adhesion strength. crosslinker, as opposed to a power law model which describes a system with multiple crosslinkers with different unbinding rates. We next modulated the dy- namics of one important actin crosslinker, a-actinin 4 (ACTN4), by introducing 2709-Pos a point mutation (K255E) associated with kidney disease. ACTN4 exhibits Cell Response to Liquid Crystal Order catch-bond behaviour, while the binding of the mutant is not tension- Kirsten D. Endresen, Francesca Serra, Michael A. Lepori. dependent. In normal conditions, we observe no significant difference between Physics and Astronomy, Johns Hopkins University, Baltimore, MD, USA. wild type (WT)- and K255E-expressing cells. However, when intracellular ten- Liquid crystal (LC) elastomers are an interesting platform to investigate the sion is reduced using Blebbistatin, WT ACTN4-expressing cells relax faster response of cells to mechanically anisotropic substrates. LC elastomers are compared to untreated cells. K255E-expressing cells display similar relaxation formed by liquid crystalline molecules, which spontaneously align in a uniform regardless of intracellular tension. These results indicate that viscoelastic prop- direction imposed by the surface anchoring. For this reason, they combine rubber erties are governed by the tension-dependent binding kinetics of actin cross- elasticity with the anisotropic properties of LCs. For example, uniformly align- linkers, and provide a mechanism for the cell to tune its mechanical ing the LCs in the horizontal plane yields an elastomer which is stiffer along the properties through modulating crosslinking dynamics. axis of alignment and softer in the perpendicular direction. In this study, we investigate the response of 3T6 fibroblasts growing on LC elastomers. Goals 2707-Pos of this research are to know if cell shape and motility can be tuned by substrate Correlating Biochemical Impact of Environmental Toxicants on Human anisotropy, i.e. if cells will contract or elongate in a preferred direction based on Neural Stem Cells to Biophysical Changes cues from the anisotropic elasticity of the substrate on which they are grown. On Gautam Mahajan, Moo-Yeal Lee, Chandrasekhar R. Kothapalli. LC elastomers with imposed uniform planar alignment, 3T6 cells demonstrate a Chemical and Biomedical Engineering, Cleveland State University, preferred elongation parallel to the LC alignment, corresponding to the stiffer Cleveland, OH, USA. axis of the material. By computing image intensity gradients to probe the anisot- Today one out of six children are diagnosed with developmental disorders and ropy of cells, we obtain an order parameter, analogous to the order parameter of there is a critical need to screen compounds such as environmental toxicants nematic LCs, and we compare the results for cells on uniformly aligned elasto- and pharmaceutical drugs for their potential developmental toxicity. It is widely mers with that for cells grown on isotropic substrates. These values vary with the accepted that developing central nervous system is highly susceptible to damage confluence of the cells. We also show how cells can follow the LC elastomer by exposure to toxicants due to the still immature blood-brain barrier. With cur- intrinsic order on more complexly patterned substrates.

BPJ 9470_9474 Wednesday, March 6, 2019 547a

2710-Pos 3Dept Biochem, Kyoto Univ, Kyoto, Japan, 4Grad Sch Pharm Sci, Kyoto ECM-Substrate Interfacial Forces Dictate Cell Coalescence on Viscoelastic Univ, Kyoto, Japan, 5College of Optometry, Columbus, OH, USA, 6Dept Substrates Surgery, Ohio State Univ, Columbus, OH, USA. Divyanshu Mishra1,2, S.U. Guo1,2, Paul Matsudaira1,3. Simple corneal wounds usually heal quickly; however, diabetic patients often 1Mechanobiology Institute, National University of Singapore, Singapore, have more fragile corneas and experience delayed and painful wound healing. Singapore, 2Center for BioImaging Sciences, National University The present study is designed to quantify the healing capacity of mouse and hu- of Singapore, Singapore, Singapore, 3Center for BioImaging Sciences, man corneal epithelial cells (CEC) in normal and diabetic conditions and Department of Biological Sciences, National University of Singapore, explore potential cellular and molecular mechanisms. Singapore, Singapore. Primary murine CEC, derived from wild type and diabetic (db/db) mice, and Basement membrane is composed of ECM proteins that have viscoelastic prop- primary human CEC were used in this study. Human CEC were grown in erties. When the viscoelasticity is mimicked in vitro, epithelial cells coalesce the presence or absence of high glucose (30 mM) to mimic diabetic conditions. by ‘‘dragging’’ the ECM protein through the PDMS substrate. This mechano- Scratch test and MTT assays were performed to evaluate migration and prolif- sensing of viscoelasticity is achieved through the translocation of vinculin from eration, respectively. Live cell imaging was used to measure reactive oxygen the focal adhesions to the cell-cell junctions and is sensitive to the level of vin- species (ROS); N-acetyl cysteine was used as an antioxidant. Transepithelial culin in the cell. Apart from the composition of cell-matrix and cell-cell adhe- electrical resistance (TEER) and zonula occludens-1 expression were used sion complexes within the cell, we find that other biophysical and biochemical to determine tight junction integrity. Stratified human CEC were used to cues from environment affect the cell response on a viscoelastic substrate. By confirmed changes in barrier formation. Western blot analysis evaluated varying the interfacial force between ECM protein, fibronectin, and the PDMS expression of Akt and ERK. substrate through physisorption or covalent linkage, we found that increasing Cellular proliferation and migration were slower for the diabetic CEC than the adhesion force hinders the coalescence of cells on a viscoelastic substrate those of normal CEC from both mice and humans. Increased ROS production as if on a soft-elastic substrate, suggesting the role of ECM-substrate interac- was observed in CEC grown under diabetic conditions. Antioxidant treat- tion for in vitro models. Also, stronger cell-cell adhesions cause coalescence ment significantly decreased ROS production and increased wound healing when coated with either fibronectin or collagen-1 alone but not when coated in diabetic CEC. Barrier function was significantly reduced in both diabetic with Matrigel, consisting of collagen-IV, laminin, and other ECM proteins. mouse and human CEC, while antioxidant treatment mitigated these effects. To gain further insights into this phenomenon, we are using quantitative Western blot analysis indicated that Akt signaling was impaired in diabetic super-resolution microscopy to investigate how the difference in ECM CEC. anchoring is sensed by the cell at the molecular level and traction force micro- Our study demonstrates that under diabetic conditions, CEC have delayed scopy to quantify the ECM remodeling and substrate deformation. These re- wound healing capacity and impaired tight junction formation in both mice sults on the viscoelastic substrate would provide new insights into in vivo and humans. Increased ROS production and reduced Akt signaling may basement membrane and cell-cell dynamics in general, and help to have better contribute this outcome, implicating these as potential targets for improving in vitro model, mimicking in vivo ECM arrangement and viscoelasticity, where diabetic wound healing. these are crucial. 2713-Pos 2711-Pos The Role of CLP36 in Pancreatic Cancer Cells during Migration and in IPMK Loss Inhibits Cellular Motility and Contractility Cell Shape Morphology Abinash Padhi1, Becky Tu-Sekine2, Matthew Apperson1, Sunghee Jin2, Eleana Parajon, Dustin G Thomas, Eric S. Schiffhauer, Amrinder S. Nain1, Sangwon F. Kim2. Douglas N. Robinson. 1Mechanical Engineering, Virginia Tech, Blacksburg, VA, USA, 2Medicine, Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD, USA. Division of Endocrinology, Diabetes and Metabolism, Johns Hopkins Principles that govern cell shape change ultimately drive tissue development University, Baltimore, MD, USA. and regeneration. By deciphering these principles, we become better Cellular adhesion is dependent on the type of adhesion receptors displayed on poised to understand diseases such as cancer. Due to the overexpression of the cell surface, and activation of adhesion receptors initiates downstream adaptor protein CLP36 and its binding partner, a-actinin-4, in other cancers, signaling cascades that are context dependent. Cellular expression of adhesion we hypothesized that these proteins might contribute to migration, cell receptors is modulated by a complex array of extracellular and intracellular sig- morphology, and mechanoresponsiveness in pancreatic cancer cells. In these nals, and we have found that Inositol Phosphate Multikinase (IPMK) is a tran- cells, migration and invasion were impaired by clp36 knockdown. Immuno- scriptional regulator of integrin b1 protein in murine embryonic fibroblast fluorescence studies revealed that CLP36 primarily resides at the cell cortex (MEF) cells. Further, we show that although loss of IPMK dramatically reduces and is significantly enriched in actin protrusions. We are now exploring how levels of both active and inactive integrin b1 protein, activation of the focal CLP36 may contribute to a-actinin-4’s role in cell mechanics and mechanor- adhesion proteins FAK and Src are increased while total protein levels are un- esponsiveness. Overall, our results already suggest CLP36 is an important changed, suggesting that IPMK is a negative regulator of focal adhesion com- regulator of pancreatic cancer cell migration and may contribute to cancer plex activity. Importantly, IPMK regulation of integrin gene expression is progression. activity dependent. Using native extracellular matrix mimicking fibers as substrate, generated using STEP (Spinneret-based Tunable Engineered 2714-Pos Parameters) technique, we show that loss of this enzyme inhibits migration Physical Model for Cell Migration Guided by Elastic Properties of the (0.68 mm/min in Wild Type cells vs 0.35 mm/min in IPMK Knock-out cells) Substrate due to decreased cell-matrix adhesion and reduced cellular contractility. Using Susana Ma´rquez, Rodrigo Soto, Miguel Concha, German Reig. nanonet force microscopy (NFM), we calculated the forces of migrating MEF Universidad de Chile, Santiago, Chile. WT cells to be 226.8 nN which was significantly higher than those of the IPMK During annual fish epiboly, between the egg yolk and the enveloping layer cells KO cells (115.6 nN). Attempts to rescue IPMK pharmacologically resulted in (EVL),there is a group of deep cells that move in contact with EVL layer. It is increased contractility (154.3 nN) but not to the same degree as that of WT experimentally observed that these cells migrate toward the EVL borders and cells. To further elucidate the impact of IPMK on adhesion strength, we move freely on them. Apparently,the phenomenon is due to elastic properties stretched the cells suspended between fibers until their adhesion bonds failed of the EVLs: either a difference in the Young modulus on the border or a stress and the cells detached from the adhered fibers. WT cells demonstrated higher gradient inside the cell. In this work,a model is developed to elucidate how this failure force (45.4 nN) as opposed to KO cells (36.2 nN). Based on these find- kind of migration works and what are sensing the cells. It considers a cell ings, we hypothesize that IPMK modulates cell migration and contractility by modelled as a circle with four contacts on a thin elastic membrane,representing regulating gene expression of cell-matrix molecules. the EVL layer. Each of the four contacts simulates a random protrusion which elongates their end out of the center of the cell,attach rigidly to the substrate, 2712-Pos and contracts without gliding. We study the elastic response of the substrate Under Diabetic Conditions Reactive Oxygen Species Inhibits Corneal and its implications in the cell dynamics when it has different stiffness and Epithelial Cell Migration and Tight Junction Formation via Akt Signaling when it is under internal tensions. In the first case, the substrate has a stiffer Qiwei Jiang1, Denis Kaili1, Jonaye Freeman1, Bingchuan Geng1, Tao Tan1, strip which simulates the EVL border which in theory has a higher Young Yanhong Luo2, Jianfeng He2, Miyuki Takeshima3, Hiroshi Takeshima4, modulus. In this case,the results do not show the expected durotaxis effect. Heather Chandler5, Hua Zhu6. In fact, the cell shows a random walk movement added to a slight repulsion 1Davis Heart and Lung Research Institute, Columbus, OH, USA. generated by the border so that a stiffer strip on a thin plate is not enough to 2Children’s Hospital Chongqing Medical University, Chongqing, China, reproduce the phenomenon. In the second case,when the substrate is under

BPJ 9470_9474 548a Wednesday, March 6, 2019 internal applied tensions,the results show that in a substrate with circular geo- layers, we developed a multicellular computational model of cell-cell and cell- metry,if the contractive circumferential stress component increases with the substrate forces. Thermodynamically-constrained cells migrate by generating radial distance,the cell migrates and reproduce experimental observations. traction forces on a finite element substrate to minimize the total energy of the We conclude that cell migration could be due to differences in internal tensions system. Junctional forces at cell-cell contacts balance traction forces thereby instead of thickness or stiffness differences. producing a stable epithelial monolayer. As a result, cell traction forces and me- chanical stress in the substrate are largest at the edge of the epithelial monolayer 2715-Pos when compared to the center. However, cell-cell junctional forces balance sub- Thermotaxis Involves Spontaneous Backward Swimming in Chlamy- strate forces and remain isotropic across the entire monolayer. In the absence of domonas cell-cell adhesions and junctional forces, similar to a mesenchymal cell, no simu- Masaya Sekiguchi, Shigetoshi Kameda, Satoshi Kurosawa, Megumi Yoshida, lated epithelial monolayer forms. These findings suggest that force transmission Kenjiro Yoshimura. across cell-cell adhesions organize otherwise migratory cells into a stable epithe- Dept Machin Cont Sys, Shibaura Inst Tech, Saitama-shi, Japan. lial monolayer. Future work will investigate the role of anisotropic mechanical Many microorganisms migrate away from harmful temperature and accumulate stress in the cellular microenvironment as a mechanism of inducing the EMT Chla- in an environment favorable for growth. In this study, we examined how program, and therefore regulating cellular mobility in tissue morphogenesis mydomonas reinhardtii cells perform this behavior, thermotaxis. Cell suspen- and disease. sion was placed in a trough with a linear temperature gradient of 5C. pf17 Immotile mutant cells, , did not accumulate at either end indicating that 2718-Pos convection is negligible. When a 5C gradient from 10Cto15C, from Nanomechanical Properties as a Biomarker to Differentiate State of Cell 15 Cto20C, from 20 Cto25C, from 25 Cto30C, was generated, cells using AFM accumulated toward the lower temperature in each temperature gradient. The Jyoti Wala. tendency of migrating toward lower temperature did not depend on cultivation Sch of Med Sci & Tech, Indian Inst of Tech Kharagpur, Kharagpur, India. ppr2 temperature (15 C, 20 C, and 25 C). Thermotaxis was not observed in the The underlying goal of biomechanics experiment is to construct a realistic ppr3 and mutants, which do not swim backward because of a defect in gener- description of cell’s mechanical properties that can be used to provide new per- ating calcium current in flagella. By contrast, thermotaxis was observed in the spectives in disease. Nanomechanical properties of cells such as elastic modulus, ptx1 mutant, which does not show phototaxis because of impaired control of the adhesion force and stiffness have a great impact on cellular processes like balance of the motility of two flagella. The frequency of spontaneous backward morphogenesis, mechanotransduction, focal adhesion, motility, metastatis and swimming decreased with temperature, suggesting that frequent occurrence of several disease treatments. Cell elasticity, adhesion and stiffness are an integra- spontaneous backward swimming randomize the swimming direction at higher tive parameter to summarize the biophysical and biochemical outcome of many temperatures. When we applied reagents to change intracellular reduction- known and unknown biological process that could serve as a biomarker. Thus to oxidation (redox) conditions, thermotaxis was enhanced, suppressed, or study these nanomechanical changes, Atomic Force Microscopy (AFM) is the reversed, depending on the redox conditions and cultivation temperature. The most powerful tool to monitor dynamic changes in cell structure and mechanics above result suggests that spontaneous backward swimming generated by of untreated and treated cancerous cell. In this study, MCF-7 and MDA-MB 231 Chlamydomonas membrane excitation mediates thermotaxis in . breast cancer cells were cultured on Polydimethylsiloxane (PDMS) substrates of 2716-Pos altered elasticity from 0.5 MPa - 3 MPa to understand the cell adaptation to sub- 4-Hydroxyacetophenone Modulates the Cytoskeleton through Nonmuscle strate stiffness and modulation of cell internal stiffness involving rearrange- Myosin-2C to Reduce Metastasis ments of cytoskeleton and actin structures. Both breast cancer cells were Darren Bryan1, Melinda Stack1, Katarzyna Krysztofiak2, Urszula Cichon2, treated with 50 nM doxorubicin anticancer drug to examine effect of drug on Dustin Thomas3, Alexandra Surcel3, Eric Schiffhauer3, Douglas Robinson3, cell’s biomechanics. It was observed Young’s modulus and stiffness of cells Ronald S. Rock2, Ralph Weichselbaum1. are decreasing towards lower stiffness of PDMS substrate for untreated and 1Univ. Chicago, Chicago, IL, USA, 2Biochemistry & Molecular Biol, Univ. treated cells. Young’s modulus increased approximately 23 % and 38 % and Chicago, Chicago, IL, USA, 3Johns Hopkins, Baltimore, MD, USA. the cell adhesion force reduced 21 % and 50 % respectively for MDA-MB Metastases are the cause of the vast majority of cancer deaths. In the metastatic 231 and MCF-7 cells after drug treatment. Thus, results demonstrate that a process, cells migrate to the vasculature, intravasate, extravasate, and establish greater understanding of the mechanics of untreated & treated breast cancer cells new metastatic colonies. This pattern of spread requires the cancer cells to deformability and its interactions with the extracellular environments offer enor- change shape and to navigate tissue barriers. Approaches that block this me- mous potential for new developments in disease diagnostics, therapeutics and chanical program represent new therapeutic avenues. We show that 4-hydrox- drug treatment in several complex processes. yacetophenone (4-HAP) inhibits colon cancer cell adhesion, invasion, and migration in vitro, and reduces the metastatic burden in an in vivo model of co- 2719-Pos lon cancer metastasis to the liver. Treatment with 4-HAP activates nonmuscle Cellular Traction Forces and Locations of Adhesion Site Regulate Cell myosin-2C, increasing cortical tension and generating contractile, mixed- Functions polarity arrangements of actin. These mechanical alterations overcome the cells Jyoti Wala, Soumen Das. ability to polarize, disseminate, and invade. Our results identify nonmuscle Sch of Med Sci & Tech, Indian Inst of Tech Kharagpur, Kharagpur, India. myosin-2C (MYH14) for the first time as a therapeutic target. Targeting acto- Biomechanical characterization of cells is becoming one of the non-invasive myosin contractility is a promising approach to address metastatic disease, techniques to monitor the different physical phenomenon of cells including cell one that may be readily combined with other therapeutic strategies. growth, proliferation, migration and adhesion on micropatterned substrates. The present study demonstrates micropillars patterned-Polydimethylsiloxane 2717-Pos (PDMS) substrate to investigate the biophysico-mechanical properties of normal, Junctional Forces Maintain Isometric Tension of the Epithelial Monolayer cancer and drug-treated cancer cells. The compositions of PDMS were varied to Lewis E. Scott, Christopher A. Lemmon, Seth H. Weinberg. explore the effect of substrate rigidity providing important signals to change in Department of Biomedical Engineering, Virginia Commonwealth University, cell-cell and cell-substrate interactions on micropillar substrates. Further, micro- Richmond, VA, USA. pillars of different PDMS compositions were fabricated through lithography fol- Mechanical stress of the cellular microenvironment mediates tissue homeostasis lowed by softlithography technique. Subsequently, normal HaCaT cells, through mechanically-sensitive gene networks that regulate cellular adhesions. untreated and doxorubicin-treated MDA-MB 231 & MCF-7 breast cancer cells Cells respond to mechanical stress by undergoing the phenotypic transition, were cultured on without any protein coated micropillar substrates for 24 h that epithelial-mesenchymal transition (EMT), to maintain normal tissue homeosta- individually act as a force sensor. Deflection of pillar allows the direct visualiza- sis. Epithelial cells downregulate cell-cell adhesions to gain migratory function, tion and quantification of cell traction forces applied to the pillar using python im- indicative of the mesenchymal phenotype, during development to form bodily age processing algorithm developed by us. Results revealed cell focal adhesion structures. However, this process is dysregulated in diseased tissues, such as site on micropillar varied among HaCaT, untreated and treated MDA-MB 231 in fibrosis and cancer progression. Hence, adhesion switches constitute an under- & MCF-7 cancerous cells. It was observed HaCaT cells were adhered at (20- lying mechanism of EMT-linked cellular patterning observed in developmental, 26)% of pillars height, however, both untreated cancer cells adhered above physiological, and aberrant processes. Previous work has demonstrated an upre- 68% but after drug treatment cancer cells adhesion site shifted at (35-55)% of gulation of EMT markers in the boundary population of a spatially confined pillar height from bottom. Therefore, changes in the deflection magnitude and epithelial monolayer relative to the confluent population, possibly due to dispro- traction force with cell types are attributed to the alterations in their different loca- portionate cell-cell contacts and anisotropic tension. To further understand the tion of cell adhesion site that constitute different mechanical properties of cells. role of intercellular tension in EMT-driven spatial patterning of epithelial mono- Thus, traction force exerted by various cell lines due to their different biophysico-

BPJ 9470_9474 Wednesday, March 6, 2019 549a mechanical properties exploring new insights and advances in cell mechanics and 2723-Pos differentiation of normal and cancer cells as well as drug effect on cancer cells and Biomechanical and Structural Investigation of Peripheral Nervous System in cancer therapeutics. Microenvironment During Development Gonzalo Rosso1, Jochen Guck1,2. 1 2720-Pos Biotechnology Center, Center for Molecular and Cellular Bioengineering, 2 Changes in Mechanical Property of Human Dermal Fibroblast Induced by Technische Universit€at Dresden, Dresden, Germany, Max-Planck-Institute Electric Field Stimulation for the Science of Light, Erlangen, Germany. Se Jik Han1, Kyung Sook Kim2, Sangwoo Kwon3. Peripheral nervous system (PNS) nerves are constantly exposed to different 1Department of Bioengineering Kyung Hee University, Hoeki-dong, Seoul, types of mechanical stresses such as shear, tension, and compression associated Republic of Korea, 2Kyung Hee Univ, Seoul, Republic of Korea, 3Kyunghee with limb movements during regular daily activities. Moreover, impaired biome- Univ., Dongdaemun-gu, Republic of Korea. chanics of peripheral nerves (PNs) is linked to neuropathies with the liability to Cellular elasticity is a unique mechanical property of single cell, which is ex- mechanical stresses paralleled by significant impairment of PNS physiological pressed by the complex interaction of cytoskeleton, membrane, and other fac- functions. On the other hand, recent investigations indicate that Schwann cells tors. This cellular elasticity is closely related to various functions and (SCs) are highly mechanosensitive and able to respond to a heterogeneous me- biological processes in the cell. Therefore, many researchers have been study- chanical microenvironment during PNS development and myelination. Further- ing cellular elasticity in various ways. In particular, studies on the changes of more, the correlation between tissue biomechanics and structure is relevant to cellular elasticity by external stimuli are actively performed in the fields of understand the response of SCs and neurons to physical stress and has clinical stem cells, cell death, and drug delivery. In this study, the changes of cellular implications for PNS regeneration and repair. However, currently there is a elasticity were induced by electric field stimulation (EFs). Continuous EF (50 lack of data on local biomechanical properties of PNS tissue during development mV/mm) was applied to human dermal fibroblasts (HDFs) for 2 h and the and myelination. Here, we used atomic force microscopy to investigate ex vivo changes in elastic property was measured with an atomic force microscope the apparent elastic modulus of living PNS tissue of young, juvenile and adult (AFM). The Young’s modulus of treated cells was increased by 2 4 times mice, and to correlate the biomechanical data with the underlying histological compared to the control cells and the changes depends on the exposure time to microstructure. We found an elastic modulus of 433.9 5 8.5 Pa, 128.7 5 4.5 EF. To understand the changes, the changes of F-actin and cellular calcium Pa and 270.4 5 10.9 Pa for young, juvenile and adult nerves, respectively. His- ion (Ca2þ) by EF were analyzed. F-actin changes were consistent with tological analysis revealed that the cell body density, the endoneurial collagen IV changes in cellular elasticity. Concentration of Ca2þ increased up to 6.9 times fibrillar content, and the density of myelinated axons contribute most to the by EF. The changes of F-actin and Ca2þ showed the similar dependency on biomechanical properties of early developing nerves. Conversely, the increase the EFs time. in myelin thickness and axon caliber may contribute to nerve biomechanics in the adulthood stages. These data provide comprehensive biomechanical and 2721-Pos structural information on the developing PNS tissue microenvironment, and In Vivo Tension Sensors Deliver Novel Insight into Mechanics of Zebrafish they may help to understand the SC and neuron mechanosensitivity mechanisms. Gastrulation Finally, local nerve tissue elasticity provides valuable mechanical information Bernhard Wallmeyer, Arne Hofemeier, Timo Betz. for the design of bioengineering nerve scaffolds to aid nerve regeneration and Institute for Cell Biology, Munster,€ Germany. repair. Mechanical tension has recently been recognized as a key element to under- 2724-Pos stand many biological processes such as collective cell migration during can- Regulation of Epithelial Mesenchymal Transition under Compliant Poly- cer invasion and embryogenesis. However, direct experimental access to in vivo dimethylsiloxane Substrate determine tension in a non-destructive way remains a major challenge. Mousumi Mandal1, Monika Rajput1, Anji Anura1, Tanmaya Pathak2, Here, we present a novel experimental approach that allows direct measure- 1 in vivo Jyotirmoy Chatterjee . ment of stress inside tissue. By injecting fluorescent polyacrylamide 1School of Medical Science and Technology, Indian Institute of Technology (PAA) beads of known elasticity in the tissue, we can generate time resolved Kharagpur, Kharagpur, India, 2Department of Chemistry, Indian Institute of 3D images of the beads that are used to quantify the bead deformation. Solv- Technology Kharagpur, Kharagpur, India. ing the inverse elastic problem yields an approximation of the stress field in- Epithelial mesenchymal transition (EMT) is a cellular trans-differentiation phe- side the tissue. PAA beads are injected into zebrafish embryos to investigate nomenon, where polarized epithelial cells convert to motile and invasive mesen- the role of tissue stress for the collective cell migration during embryogenesis. chymal cell through remodeling of cytoskeletal protein, cell-cell and cell-matrix At the same time positions of nuclei are detected and tracked over time using interaction. The EMT program is crucial in embryogenesis, wound healing, state of the art tracking techniques. Combining tissue flow, mechanical ten- fibrosis and cancer metastasis. Molecular mechanism lead to EMT are well- sion and cell density allows to directly differentiate between pure mechanical studied whereas influence of matrix mechanical property has not been studied control of cell motion, for example via hydrodynamic or elastic effects, and yet. In this study, HaCaT (epithelial normal) and AW13516 (epithelial cancer) active cell motion. cells were cultured on two different PDMS substrate viz, 10:1 and 40:1 having Young’s Modulus 1.26 5 0.047 MPa and 795 5 15 kPa respectively with treat- 2722-Pos ment of EMT inducing transforming growth factor beta - 1 (TGFb-1) upto a Force-Dependent Allosteric Enhancement of e-Catenin Binding to F- 72 hours. HaCaT and AW13516 cells were observed very compact on 10:1 Actin by Vinculin and 40:1 PDMS substrate at 0 hr, whereas after 72 hrs PDMS (10:1) showed Nicolas A. Bax1, Derek L. Huang2, Sabine Pokutta1, Alexander R. Dunn3, more elongated morphology and loose cell-cell interaction. The proliferation William I. Weis1. rate was decreased on both substrates validated by Ki67 expression. Addition- 1Structural Biology, Stanford University, Stanford, CA, USA, 2Biophysics, 3 ally, expression of E-cadherin was analyzed where 40:1 PDMS substrate Stanford University, Stanford, CA, USA, Chemical Engineering, Stanford induced membranous expression indicated strong cell-cell interaction in com- University, Stanford, CA, USA. parison to 10:1. Interestingly, mesenchymal markers, vimentin and fibronectin The linkage between cadherin-based transmembrane adhesions and the acto- were seen decreased on 40:1 PDMS substrate with increased expression of myosin cytoskeleton is a fundamental feature of metazoan tissues. Mechanical mechanosensitive vinculin aid the formation of E-cadherin based junctional tension on the minimal cadherin-catenin complex increases the actin-binding complex which elucidated the inhibition of EMT progression. This study thus lifetime of alpha-catenin (termed catch-bond behavior) and reveals a cryptic indicated that by modulating the mechanical property of the substrate, the a binding site in E-catenin for the actin-binding protein vinculin. While vinculin EMT process can be inhibited which itself is revolutionary in the field of cancer is thought to reinforce the cadherin-catenin/actin linkage, the mechanistic de- diagnosis and therapy. tails of this process remain poorly understood. Although the actin-binding ac- tivity of vinculin likely contributes to junctional strengthening, we find that that 2725-Pos complex formation with an aE-catenin binding domain from vinculin led to Novel Method of Determining Cellular Traction Forces during EMT longer-lived actin binding, but in a manner that was enhanced under force. Brian P. Griffin, Christopher A. Lemmon. We use computational modeling to probe how force-dependent strengthening Biomedical Engineering, Virginia Commonwealth University, Richmond, of individual bonds affects resilience of the adhesion to fluctuating loads. VA, USA. Our results demonstrate a form of force-dependent allosteric regulation that In both physiological settings and pathological settings, epithelial cells, which may enhance the ability of cells to form robust connections and respond to me- line organs and tissues, change their phenotype from one that primarily serves a chanical cues at cell-cell contacts. barrier function to one which facilitates new tissue growth. This change, known

BPJ 9470_9474 550a Wednesday, March 6, 2019 as epithelial-mesenchymal transition (EMT), allows cells to facilitate cell non-tumorigenic MCF10A cells. Our findings suggest that the breast cancer migration, extracellular matrix assembly, and tissue patterning. The role of me- cells with the more invasive phenotype exhibit a highly significant increase chanical properties in EMT behavior is not well understood. Cellular mechan- in glycolysis when probed on denser substrates than do the less and non- ical properties affect cell migration, proliferation, signaling, and aggressive cells. Additionally, these invasive cells have a decreased membrane differentiation. Despite wide recognition of the importance of mechanobiology fluidity on glass as compared to the less and non-aggressive cells. The results of in cellular behavior, shortcomings in techniques to measure cellular forces these studies motivate an investigation of a specific therapeutic target of meta- generated by epithelial cells are difficult to accurately measure due to the bolic disease by exploiting the more invasive cell’s ability to sense a density low magnitude of cell forces and the lack of cytoskeletal rigidity in these cells. change in the surrounding ECM and shift to a glycolytic metabolic state. Additionally, EMT is regulated by two distinct sets of mechanical forces: This work was supported in part by NIH P41GM103540 and the National Sci- forces between cells and the underlying matrix, and forces between neigh- ence Foundation Graduate Research Fellowship award DGE-1839285. boring cells. We have developed a system that can quantify both of these values, and determine how their interactions drive EMT. 2728-Pos Our data shows that cell-matrix forces increase as the distance from the cell Cell Cycle Synchronization for the Study of DNA Wrapped Single Walled centroid increases, as has previously been modeled computationally by our lab. Carbon Nanotube Influence on Neural Stem Cells We also observe that epithelial cell colonies distribute forces as a single large Swetha Chandrasekar. aggregate, instead of as individual cells. This suggests that cellular forces are Bioengineering, Lehigh University, Bethlehem, PA, USA. linked between cells via cell-cell adhesions, resulting in all force summing to- Nanomaterials have been employed as imaging agents and drug delivery vehicles wards a collective cell centroid. Most notably, EMT stimuli such as TGF-beta for disease therapy across the medical field. Carbon nanotubes in particular are of produce significant increases in cellular traction forces, as well as a loss of this the appropriate size and can be modified with functional groups to allow transport collective force pattern. This finding supports the hypothesis that mesenchymal through the blood brain barrier and aid in delivery of therapeutics to the central cells are able to apply greater local cell-matrix forces. Together, these results pro- nervous system. In this study, we aim to examine the micro-level interactions be- mote a greater connection between the process of EMT and cellular forces which tween nano-molar concentrations of (GT)20 DNA-wrapped single walled carbon could provide future insight into understanding and treating diseases. nanotubes (SWNT, roughly 1 nm dia, 500 nm length) and C17.2 neural progenitor cells. Though commonly used concentrations are often in the mmolar range, expo- 2726-Pos sure to even nanomolar concentrations up to 72 hours yields evidence of cytotox- Study of Cancer Cell Mechanics by Traction Force Microscopy icity and reorganization of cellular sub structure. Low concentrations of SWNT Yuwen Mei, Justin Raupp, Takeshi Sakamoto. exposure have even shown to augment cell division and differentiation. However, Physics, Wayne State University, Detroit, MI, USA. the mechanisms of these irregularities in cell structure and fate are widely un- Pancreatic cancer is the fifth most common cancer in the United States. Despite the known. To understand the implications of these materials, it is imperative to advancements in modern medicine, five-year survival rate of such cancer is gener- differentiate the potential micro-correlations caused by the presence of the nano- ally less than 10%. To better understand the underlying cause, mechanobiology of materials from the natural intracellular dynamics of a proliferating stem cells. the PANC-1 cell line is ought to be investigated. Mechanical cellular interactions Chemical arrest agents were used to optimize cell cycle synchronization (G2/ heavily influence major cellular processes such as metastasis, embryogenesis, M, early G1) and ultimately induce phase-dependent normalized cytoskeletal ar- angiogenesis, etc. Most of these physiological processes are in direct relations rangements upon which further interactions with nanomaterials can be assessed. to cell migration. Such biological function is responsible for positive responses Population cell distributions were discerned via flow cytometry. To connect cell in one’s body in aid of healing of wounds, or infamously, invasion of cancer cells cycle organization with uptake, localization and correlation of nanotubes within through the connective tissues. To better understand these major physiological intracellular compartments, hyperspectral imaging was performed via confocal phenomena, it’s critical to understand how these contractile motions are generated micro-Raman spectroscopy. Focus on the internal endocytic pathways and con- and quantification of traction forces is necessary. To measure these forces, resent nections to cell cycle regulation will lead to future studies investigating the inter- studies showed that fluorescent beads were embedded in the substrate as markers actions of SWNTs with intracellular components. to tract the forces being applied, named Traction Force Microscopy (TFM). How- 2729-Pos ever, such setup has no control over marker positions and often introduce back- Mechanobiological Control of the Immune Response ground noise, e.g. loss of spatial resolution. In addition, beads usually have no Huw Colin-York1, Yousef Javanmardi2, Emad Moeendarbary2, direct contact with the cells, meaning traction force has higher degree of estimation Christian Eggeling1, Marco Fritzsche1. as it’s relied heavily on substrate properties to extrapolate forces. Thus, we have 1MRC Weatherall Institute of Molecular Medicine, University of Oxford, improved our method of force detection by employing fluorescent superparamag- Oxford, United Kingdom, 2UCL, London, United Kingdom. netic beads. Under the influence of the external magnetic field, superparamagnetic Cytoskeletal actin dynamics are essential for T-cell activation, a key step in the beads formed a single layer along the surface of the substrate. Such configuration adaptive immune response. The intimate cell-cell contact that forms between the minimized background noise, hence spatial resolution is improved. Since the mag- T-cell and antigen presenting cell during activation, known as the immunolog- netic beads are located at the surface, marker positions are directly influenced by ical synapse, has been shown to be a dynamic, physical structure where complex traction forces, improving the accuracy of measurements. Employing traction mechanical forces are generated. Using a range of biophysical techniques, we force microscopy, cellular motions of PANC-1 cells were captured sequentially, show evidence that the binding kinetics of the antigen engaging the T-cell recep- and the mechanical forces were analyzed. tor controls the nanoscale actin organization and mechanics of the immune syn- apse. By stimulating T-cells expressing a specific T-cell receptor by a range of 2727-Pos antigens, force measurements revealed that the peak force experienced by the T- A Non-Invasive Metabolic Investigation of Breast Cancer Invasion cell receptor during activation was independent of the kinetics of the stimulating 1,2 2,3 1,2 Austin E.Y.T. Lefebvre , Freddie A. Adame , Michelle A. Digman . antigen. Conversely, quantification of the actin retrograde flow velocity at the 1Biomedical Engineering, Univ CA Irvine, Irvine, CA, USA, 2Laboratory for 3 synapse revealed a striking dependence on the antigen kinetics. Taken together, Fluorescence Dynamics, Irvine, CA, USA, Biological Sciences, UC Irvine, these findings suggest that the dynamics of the actin cytoskeleton actively Irvine, CA, USA. adjusted to normalize the force experienced by the T-cell receptor in an antigen Over 90% of cancer related deaths are the result of complications arising due to specific manner. Consequently, tuning actin dynamics in response to antigen ki- cancer metastasis. The reconfiguration of an invasive cancer cell’s metabolic netics may thus be a mechanism that allows T cells to tune their activation signature due to its mechanosensing ability is well acknowledged yet remains response by adjusting the length- and time-scale of T-cell receptor signaling. poorly understood. By probing the metabolic response of cells of differing in- vasivity using non-invasive imaging techniques, novel insights into the mech- 2730-Pos anisms of metastatic disease may be elicited.In this work, we used fluorescence Probing Fluctuations and Avalanches in the Cytoskeleton with Active lifetime imaging microscopy (FLIM) together with phasor analysis, to deter- Micropost Arrays mine the free to bound NADH ratio present within the different cells. This Yu Shi1, Daniel H. Reich1, Christopher L. Porter2, John C. Crocker2. free to bound NADH ratio analysis can determine whether a cell is relying rela- 1Physics and Astronomy, Johns Hopkins University, Baltimore, MD, USA, tively more on glycolytic metabolism or on oxidative phosphorylation. Addi- 2Department of Chemical and Biomolecular Engineering, University of tionally, we used spectral imaging with phasor analysis on LAURDAN Pennsylvania, Philadelphia, PA, USA. stained cells, allowing us to determine the cell’s membrane fluidity under vary- The cellular actomyosin network plays an important role in a wide range of cell ing conditions. We probed the metabolic and fluidity profiles of three breast behavior, including motility, morphology and mechanotransduction. Although cells of varying invasivity. These cells include highly invasive, triple- the building blocks of actomyosin networks are well understood at the molec- negative MDA-MB-231 cells, non-invasive, tumorigenic MCF-7 cells, and ular level, the picture connecting these microscale units to cell-scale behavior

BPJ 9470_9474 Wednesday, March 6, 2019 551a remains incomplete. Here we present results using active micropost array de- ment of APC on actin dynamics. We find that IQGAP alone binds tightly to tectors (AMPADS) to characterize the dynamical fluctuations and local actin filament sides (dwell time > 15 min) and potently bundles F-actin. rheology of cellular actomyosin networks in detail. AMPADS are poly(dime- Further, IQGAP interacts transiently with barbed ends (dwell time 30 sec), thylsiloxane) (PDMS) micropillar arrays with embedded magnetic nanowires blocking growth, thus defining it as a transient capper. As such, IQGAP atten- that enable mechanical actuation of an adherent cell. We classified the cell- uates actin assembly induced by APC-C and Dia1. Consistent with these in vi- associated microposts based on their traction force, and identified two distinct tro observations, RNAi silencing of IQGAP in HeLa cells leads to a marked populations, one containing posts coupled to stress fibers and the other containg increase in APC-dependent F-actin accumulation at the leading edge. Thus, IQ- posts coupled to the actomyosin cortex. Both groups show weak power law GAP restricts APC/Dia1-mediated actin assembly, possibly in order to make rheology, but we find that the fluctuations of stress fiber-associated posts are actin assembly signal-responsive. Analysis of the N-terminal armadillo more active and highly anisotropic in comparison to the cortically-associated (ARM) domain of APC revealed three novel functions: (1) APC-ARM inhibits posts. Notably, the fluctuations of both populations are highly heterogenous APC-C-mediated actin assembly in trans, suggesting that APC is autoregu- and resemble a Levy process. Specifically, the mean-squared displacements lated; (2) APC-ARM inhibits IQGAP’s barbed end capping effects, i.e., pro- of both types of posts display broadly distributed amplitudes and super- moting actin assembly; (3) APC-ARM enhances Dia1’s actin assembly- diffusive behavior. We find that the amplitude distribution is fat-tailed, and promoting activities. Together, these observations suggest a complex interplay is dominated by a corresponding distribution of intermittent, large step-like dis- among these three large, multi-domain proteins, and provide evidence of their placements, resembling avalanches and earthquakes in physical systems. Our mutual activation to promote actin assembly. regular array of detectors also allows us to determine the spatial extent and average symmetry of the largest rearrangements in the cortex, which are 2733-Pos both spatially and temporally complex, resembling those in plastic solids. Experimentally Varying the Number of Super-Repeats in the Neb Gene of the These results suggest that the dynamics of the cellular actomyosin network re- Mouse - Assessing the Role of Nebulin in Thin Filament Length Regulation sembles what is seen in soft matter systems such as sandpiles and foams, where Balazs Kiss, Paola Tonino, Justin Kolb, John E. Smith, Henk L. Granzier. constituent components self-organize into marginally stable, plastic networks, Dept Cell & Molec Med, Univ Arizona, Tucson, AZ, USA. with significant implications for future models of the cortex. Nebulin is a giant, modular actin binding protein that spans along the thin fila- ment in skeletal muscle. Nebulin consists of numerous tandem copies of 38.5 nm 2731-Pos ‘super-repeats’ that correspond to the arrangement of troponin and tropomyosin Point Mutation of the Ice-Binding Site in Antifreeze Protein Modify the on the thin filament. Besides being known as a thin filament stabilizer, nebulin Cold Tolerance in Caenorhabditis Elegans has been speculated to act as a thin filament ruler in skeletal muscle, yet little is 1 1 2,3 Masahiro Kuramochi , Chiaki Takanashi , Akari Yamauchi , known about nebulin’s direct influence on actin length or its mechanism of ac- Motomichi Doi4, Kazuhiro Mio5, Sakae Tsuda2,3, Yuji C. Sasaki1. 1 2 3 tion. Here we developed two novel mouse models in which nebulin super- Univ Tokyo, Kashiwa, Japan, AIST, Sapporo, Japan, Hokkaido Univ, NebDSR9-11 NebdupSR9-11 4 5 repeats 9-11 are deleted ( ) or duplicated ( ), respectively. Sapporo, Japan, AIST, Tsukuba, Japan, AIST-UTOKYO Operando Oil, Mice of either model are viable and develop without muscle weight deficits. Kashiwa, Japan. Super-resolution microscopy (SR-SIM) and immunoelectron microscopy on Antifreeze proteins (AFPs) inhibit the ice-crystal growth from the surface- extensor digitorum longus (EDL) muscle revealed that the N-terminus of nebu- bound waters that are spatially organized to bind to an ice crystal. The surface lin, together with tropomodulin (Tmod) localizes at the thin filament pointed structure complementarity between AFP and the ice plane has been evaluated end. Furthermore, nebulin and thin filaments were found to be shorter by based on structural docking studies. The ice-binding properties are closely 115 nm in NebDSR9-11 and longer by 115 nm in NebdupSR9-11 EDL. Com- related to the freezing point depression activity of the AFP solution, which is pound heterozygous animals (NebDSR9-11,dupSR9-11) express both the shorter evaluated as the difference between the melting and freezing temperatures and the longer nebulin close to a ratio of 1:1 and the difference in their lengths (thermal hysteresis: TH). Although AFPs are thought to facilitate the survival is 230 nm. Tmod localization suggests a bimodal thin filament length distribu- of organisms living in cold environments, it is poorly understood that whether tion in NebDSR9-11,dupSR9-11 EDL muscle fibers according to the shorter and the exogenous expression of AFPs can improve the cold tolerance of animals. longer nebulin. Functional studies on skinned EDL fibers revealed leftward shift Moreover, it is also unclear whether TH activity is correlated with the survival of the descending limb of the force-sarcomere length relationship in NebDSR9-11 ability of animals during cold shock. Here, we examined the functions of AFPs and rightward shift in NebdupSR9-11 consistent with their altered thin filament in vivo Caenorhab- through behavioral and cellular observations of transgenic lengths. We conclude that nebulin rules the thin filaments in EDL muscle ditis elegans expressing AFPs. We showed that the fungal AnpAFP improves with each of nebulin’s super-repeats being responsible for a quantal 38 nm the survival rate of worms and protects their cells from lethal cold shocks. thin filament length. Studies on other muscle types are currently ongoing. The AnpAFP T156Y mutant exhibiting low TH resulted the reduced cold toler- ance at the freezing temperature. The fungal TisAFP8 exhibiting high TH ac- 2734-Pos tivity was observed to dramatically improve the survival rate and strongly Elucidating Steps in Actin Polymerization and Nucleation using a Coarse- protect muscle cells. Structural modification of the ice-binding site is crucial Grained Model of Multiprotein Complex Formation to regulate the ice-binding strength of AFPs. These results suggest that AFPs Brandon G. Horan1, Dimitrios Vavylonis1, Jeetain Mittal2. play a crucial role in the animal body, improving cold tolerance through the 1Dept Physics, Lehigh Univ, Bethlehem, PA, USA, 2Dept Chem Eng, Lehigh ice-binding mechanism. In this presentation, we will discuss the relationship Univ, Bethlehem, PA, USA. between the ice-binding abilities of AFPs and their in vivo roles in cold toler- Nucleation and polymerization of actin monomers into filaments is crucial for ance and cellular protection in a living animal. many cellular processes including motility, cytokinesis, and muscle contrac- tion. To understand how intermolecular actin-actin contacts are established, Posters: Actin Structure, Dynamics, and we utilize molecular dynamics simulations with a coarse-grained protein model Associated Proteins having each residue represented by a single interaction site located at the C- alpha atom. We approximate each protein or protein assembly as a rigid unit 2732-Pos (lacking intramolecular conformational fluctuations) to identify the equilibrium Novel Actin Regulatory Activities of the IQGAP-APC-Dia1 Complex Re- structural ensemble of interprotein complexes formed. Nonbonded residue- vealed by Single-Molecule Imaging residue interactions are obtained from the Kim-Hummer model. We find the Gregory Hoeprich, Maria Angeles Juanes, Bruce Goode. incoming subunit binds at both barbed and pointed ends of a short actin filament Dept Biol, Brandeis Univ, Waltham, MA, USA. (using Oda et al. F-actin model). Reduced temperature was adjusted to match Cell motility and morphogenesis depend on the precise spatiotemporal control experimentally known binding affinity to the barbed end. At each end we of actin assembly at the cell periphery. IQGAP is a downstream effector of find two distinct bound states, with one closely resembling the expected struc- Cdc42 and Rac1 and a key factor linking signaling to cytoskeletal rearrange- ture in the Oda et al. model. We discuss possible kinetic importance of the other ments at the leading edge. IQGAP binds to F-actin and scaffolds several actin state in addition of a new actin subunit to the filament. Additionally, we find the regulatory proteins, including Adenomatous polyposis coli (APC) and the for- incoming subunit binds near the barbed end in a configuration similar to an anti- min Dia1. However, the specific mechanisms by which IQGAP regulates actin parallel dimer—another structure found to be involved in actin polymerization. dynamics remain poorly understood. Our previous work showed that a large C- We examine the difference in barbed end binding affinity depending on terminal domain of APC (APC-C) nucleates actin polymerization and works whether the incoming subunit is G-actin or F-actin, and whether the D-loop collaboratively with Dia1 to promote actin assembly by a ‘Rocket Launcher’ and residues near the N- and C- termini are held rigid or flexible. Our model mechanism (Breitsprecher et al., 2012). Here, we used single molecule imaging finds contacts between residues of the incoming subunit and filament consistent to characterize the effects of IQGAP and an N-terminal IQGAP-binding frag- with expectation from models based on crystal structures and find additional

BPJ 9470_9474 552a Wednesday, March 6, 2019 contacts not present in the native bound complex may also be important. We sensing, are elongated thanks to actin filaments bundled in parallel. Actin fila- also discuss the ability of this model to describe mechanistic details of actin ments within filopodia are elongated at their barbed ends by formins linked to filament nucleation pathways and polymerization. the cell membrane. Key characteristics of formins are its processivity, which refers to the duration of time that formins remain attached to actin barbed 2735-Pos ends and its ability to accelerate elongation rate of filaments. These formin- A New Twist on the Mechanism of Mutation-Induced Tropomyosin elongated actin filaments are cross-linked the bundling protein fascin, stiff- Dysfunction ening structures and allowing filopodia to emerge from the cell surface. How 1 2 3 Michael J. Rynkiewicz , Jeffrey R. Moore , Stuart G. Campbell , formin/fascin machineries collaborate is not known. William Lehman4. in vitro 1 2 To tackle these questions, we use an microfluidics-based approach to Dept Physiol/Biophys, Boston Univ, Boston, MA, USA, Department of probe the activity of mDia1 formins and human fascins in a large range of Biological Sciences, University of Massachusetts-Lowell, Lowell, MA, USA, 3 4 biochemical conditions: first on single filaments, then scaling up to bundles Dept Biomed Eng, Yale University, New Haven, CT, USA, Dept Physiol/ of multiple filaments. We have experimented with different formin anchoring Biophys, Boston Univ Sch Med, Boston, MA, USA. methods, ranging from setups that limit formin movement, to anchoring to lipid Tropomyosin, a well-known regulator of muscle contraction, participates in a bilayers to allow free diffusion and rotation of formins, a more physiological macromolecular scale steric-mechanism to control myosin cross-bridge - actin in- situation. We observe that formin elongation rate is slowed down and its proc- teractions and consequently contraction. Here, head-to-tail linked tropomyosin ca- essivity is strongly diminished by fascin bundling if formin rotation is hindered. bles lying along thinfilaments move azimuthallytoopenand close myosin-binding 2þ Conversely, when formins are allowed to freely rotate processivity are compa- sites on actin in response to changing Ca -levels and the actions of troponin and rable to those of single filaments. Our results offer insight into the conditions myosin. Our recent analysis of molecular dynamics simulations on tropomyosin where formins activity is modulated by actin bundling. suggests that previously unrecognized twist-variance of tropomyosin is critical for thin filament function. It is likely that tropomyosin twist-dysfunction, quite 2738-Pos distinct from frequently invoked flexural anomalies, can result from mutations Impact of a Beta-Iii-Spectrin Mutation on the Structure and Function of in tropomyosin and lead to faulty contractile function, presaging human disease. Dystrophin ABD1 Such twist-dysfunction may decrease or enhance apparent affinity of tropomyosin Adam W. Avery1, Michael E. Fealey2, Thomas S. Hays1, David D. Thomas2. mutants to F-actin, yielding functional changes, which over time can progress to 1Department of Genetics Cell Biology and Development, University of overt myopathy. Indeed, in many cases disease-associated tropomyosin mutants, Minnesota, Minneapolis, MN, USA, 2Department of Biochemistry, Molecular for example E40K tropomyosin, cannot directly affect actin-tropomyosin binding, Biology & Biophysics, University of Minnesota, Minneapolis, MN, USA. since they occur in tropomyosin b-zones suspended over subdomain 2 of actin, and We have used circular dichroism, co-sedimentation and pulsed EPR to assess thus lie far from the actin surface. In fact, MD indicates that E40K acts indirectly to the structural and functional impact of a disease-causing mutation belonging affect tropomyosin twist-variance, which then propagates over much of the tropo- to beta-III-spectrin within dystrophin’s first actin-binding domain. Beta-III- myosin coiled-coil and thereby would compromise interactions with actin subunits spectrin and dystrophin have several commonalities including a structural pref- along thin filaments. Unlike the previously examined D137L mutation that causes erence for ‘‘closed’’ conformations of their calponin-homology domains as under-twisting of tropomyosin, E40K causes over-twisting and likely reposition- well as an apparent hydrophobic driving force for occupation of such closed ing of tropomyosin on F-actin. In this context, we now have also begun to system- conformations. Most of the known disease-causing mutations in dystrophin atically examine twist-variance parameters of a number of representative ABD1, however, cause misfolding with subsequent targeting of the protein tropomyosin mutants that lead to HCM and DCM and which define unique do- to the proteasome, so it is difficult to use mutations to gain further insights mains of the tropomyosin coiled-coil structure. Our analysis of coiled-coil twisting into its structural mechanism for regulating actin binding. Here, we explore and untwisting provides a new paradigm for understanding the action of the the possibility of a beta-III-spectrin missense mutation site that is strongly plethora of tropomyosin mutants both in muscle and in non-muscle cells. conserved in dystrophin’s sequence as a potential tool to probe structure- function relationships of the dystrophin ABD1 that may parallel its close evolu- 2736-Pos tionary relative. Quantitative Mass Imaging of Actin Nucleation Nikolas Hundt, Gavin Young, Daniel Cole, Max Hantke, Philipp Kukura. Department of Chemistry, University of Oxford, Oxford, United Kingdom. Posters: Membrane Pumps, Transporters, and Actin filaments are one of the major components of the cytoskeleton and mediate a Exchangers II vast number of cellular processes that regulate shape and motility, such as cell migration, cell adhesion and muscle contraction. The mechanism of actin polymer- 2739-Pos ization has been studied for more than 50 years and a basic mechanism has been Structural Determinants of the hASBT-Ligands Interactions formulated where actin subunits form a stable nucleus which is extended into a bi- Gamsj€ager Viktoria, Claire Colas, Gerhard F. Ecker. polar filament. This model was largely inferred from the observation of time- Pharmaceutical Chemistry, Vienna University, Vienna, Austria. dependent increases in scattering or fluorescence of a solution of polymerizing The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) actin filaments or by following macroscopic growth of individual filaments using is a membrane protein that is responsible for the uptake of bile acids across the fluorescence microscopy. None of these methods, however, provides a means of enterocytes apical membrane. hASBT is a key drug target for the treatment of quantifying actin oligomers formed during the nucleation process directly, which hypercholesterolemia, as inhibitors blocking bile acids uptake would increase would be very useful to verify the existing model and for investigating how actin the de novo synthesis of bile acids and thus, the consumption of cholesterol. nucleation is mediated by actin-binding proteins. Our lab has recently developed Additionally, hASBT is an interesting target for prodrugs, as its high specificity mass photometry based on interferometric scattering microscopy, which enables and capacity could considerably improve the bioavailability of bile acids derived direct visualization and size quantification of polydisperse biomolecular distribu- drugs. The study presented here describes the interactions of this transporter with tions in a solution environment. The latest improvements in detection technology its ligands using computational methods. We have first built homology models of and data analysis enable mass resolution to 13 kDa (mass peak FWHM). As a hASBT in distinct conformations of transport cycle using prokaryotic trans- result, we can now generate mass spectra of actin solutions above the critical nucle- porters as templates. The models have then been used for the docking of known ation concentration allowing us to identify and quantify the oligomers present ligands. Our results improve our understanding on how substrate specificity is shortly after initiating polymerization. Since we can quantify the amount of each determined in hASBT, providing guiding rules for the development of new com- individual oligomeric species and its evolution with time, we are able to directly pounds targeting this pharmacologically important transporter. compare and validate predictions of oligomeric distributions obtained from kinetic simulations. We find direct evidence for a nucleation mechanism of actin, demon- 2740-Pos strating a general methodology for studying nucleation processes in biology. Substrate Binding and Conformational Changes of the Bile Acid Sym- porter ASBTNM 2737-Pos Fiona B. Naughton1, Patrick Becker2, Deborah Brotherton2, Mechanosensitivity of Actin Bundles Alexander D. Cameron2, Oliver Beckstein1,3. Jahnavi Chikireddy. 1Department of Physics, Arizona State University, Tempe, AZ, USA, 2School Institute Jacques Monod, Paris, France. of Life Sciences, University of Warwick, Coventry, United Kingdom, Many essential cellular processes including cell division, migration and 3Center for Biological Physics, Arizona State University, Tempe, AZ, USA. morphogenesis involve dynamical actin networks, which consists of cross Bile acids are synthesised from cholesterol in the body to aid with digestion, but linked actin filaments regulated by actin binding proteins. Filopodia, dynamic are largely recycled via reabsorption in the intestine through transporters such finger-like structures emerging at the cell periphery and important for cell as the apical sodium-dependant bile acid transporter (ASBT). These

BPJ 9470_9474 Wednesday, March 6, 2019 553a transporters are thus attractive drug targets for the treatment of hypercholester- thiocyanate, and perchlorate with varying affinities. The mechanism and res- olaemia and type II diabetes, and are additionally of interest as vehicles for drug idues responsible for substrate selectivity and inhibitor sensitivity are poorly delivery to the cell following oral administration. ASBT uses the sodium understood. of NIS proteins from many species across gradient to drive movement of bile acids across the cell membrane, operating evolutionary history and environments with varying iodide availability pro- through an alternating access mechanism. Although structures of bacterial ho- vided unique clues to NIS transport dynamics. Comparison of a selevted mologues of ASBT have been determined in both inward and outward facing nine species of NIS (D. rerio, X. laevis, M. musculus, R. norvegicus, S. scrofa, conformations many details remain unclear, including the binding of the bile C. lupus familiaris, B. acutorostrata scammoni, P. anubis, and H. sapiens) acid substrate and the nature of the conformational transition. We have used with radioisotope uptake assays and competitive substrate uptake assays as- computational and experimental methods to examine details of ASBT function sessing the impact of sixteen substrates and inhibitors on I-125 uptake re- on an atomistic scale. Homology modelling and docking were used to generate vealed differences in substrate selectivity and transport kinetics despite the apo and substrate-bound structures of the ASBT homologue from Neisseria high level of sequence and tertiary structure conservation. B. acutorostrata meningitidis (ASBTNM) in both the inward and outward facing conformations. scammoni (minke whale) NIS exhibited the highest selectivity for iodide, Molecular dynamics simulations were performed to investigate substrate bind- whereas iodide uptake by D. rerio (zebrafish) NIS was the most severely in- ing and compared to structural results from X-ray crystallography. Structural hibited by all compounds tested. Subsequent molecular modeling of the hu- transition pathways were explored with fully atomistic dynamic importance man, minke whale, and zebrafish isoforms revealed a high degree of sampling (DIMS) simulations. Through our work, we characterise substrate conservation in the putative ion binding pocket, which suggests the residues binding and potential transition pathways between the inward and outward fac- responsible for substrate selectivity lie elsewhere in the protein. Mutagenesis ing structures of ASBTNM as an initial step towards an understanding of the studies of variable residues provide insight into the regions responsible for molecular mechanism of sodium-driven symport in ASBT. substrate selectivity. Further examination of interspecies differences will improve understanding of the substrate transport mechanism and guide future 2741-Pos models. Functional Characterizations of Purified Ctr Copper Transporter Proteins Reveal a Novel Mechanism of Ion Selectivity and Transport 2744-Pos Kehan Chen, Yaping Pan, Ming Zhou. Perchlorate Binding to a Cryptic Allosteric Site Changes the Mechanism of Biochemistry, Baylor Coll of Med, Houston, TX, USA. Iodide Transport by the NaD/I Symporter (NIS) Copper (Cu) is a cofactor in a number of oxidoreductases and is an essential Alejandro Llorente Esteban1, Andrea Reyna-Neyra1, Rian W. Manville2, nutrient for human. The CTR copper transporters are the major protein for selec- Geoffrey W. Abbott2, Nancy Carrasco1, Leon M. Amzel3. tive Cu uptake. Previous functional studies of CTR have relied on cell-based as- 1Department of Cellular and Molecular Physiology, Yale School of says, and are limited by complications caused by uncertainty in the amount of Medicine, New Haven, CT, USA, 2Department of Physiology and Biophysics, surface expressed CTR protein and by potential interference from other endoge- University of California at Irvine, Irvine, CA, USA, 3Dept Biophys/Biophys nous transporters and channels. We have expressed and purified CTR proteins of Chem, Johns Hopkins Univ Med School, Baltimore, MD, USA. twoeukaryotic species, and measured ion binding and transport. Using isothermal The sodium/iodide symporter (NIS), which mediates the first step in thyroid titration calorimetry, we found that CTRs have 10 fold higher affinity for the hormone biosynthesis, has an unusual property for a transporter: it transports monovalent transition metal ion Agþ than the divalent transition metal ions þ þ þ different anions with different stoichiometries. Although NIS transports its such as Co2 ,Ni2 .and Zn2 . Using a liposome-based flux assay, we found þ þ physiological substrate, I , using two Na ions per I transported, it translo- that CTRs transport Ag at least 10 fold faster than the divalent cations. These cates some oxyanion substrates [including ReO4 , TcO4 , and the environ- results are consistent with previous observations and provide quantification for mental pollutant perchlorate (ClO4 )] with a 1:1 stoichiometry. There are ion binding and transport. We then examined the role of the two highly conserved two possible explanations for these different stoichiometries: 1) the larger an- methionine residues and found opposing effects on ion binding and transport. ions binding to their transport site inhibit Naþ binding to one of the two Naþ These results provided hints of a novel mechanism of ion selectivity and transport, sites; 2) the oxyanions also bind to a non-transporting site (NTS) that either and inspired further experiments directly test the mechanism. overlaps or allosterically interferes with binding of Naþ to one of its sites. 2742-Pos We show experimentally that NIS has an NTS, and when the oxyanions bind to this site, they prevent Naþ from binding to one of its two sites. For example, Structural Basis of Ion Selectivity and Permeation in the High-Affinity remarkably, low concentrations of ClO4 or ReO4 change the stoichiometry Copper Transporter Ctr1 þ þ Peng Yuan. of I transport from electrogenic (2 Na :1 I ) to electroneutral (1 Na :1 I). Furthermore, the dependence of the transport kinetics of the oxyanions Dept Cell Bio/Physiol, Washington Univ in St Louis, St Louis, MO, USA. þ Copper (Cu) is an essential trace element for growth and development and on the Na concentration at low anion concentrations is sigmoidal, indicating abnormal Cu levels are associated with anemia, metabolic disease and cancer. a 2:1 stoichiometry. At higher anion concentrations, by contrast, when the þ NTS is occupied, a 1:1 stoichiometry is observed. Consistently with these ob- Evolutionarily conserved from fungi to humans, the high-affinity Cu transporter Ctr1 is crucial for both dietary Cu uptake and peripheral distribution, yet the servations, ClO4 elicits currents at low but not at high concentrations. Global mechanisms for selective permeation of potentially toxic Cuþ ions across cell fitting of transport data to a statistical thermodynamics model provides values membranes are unknown. Here we present X-ray crystal structures of Ctr1 in for the affinities and the interaction constants that are in agreement with a great þ þ þ many experimental kinetics results. Taken together, our results reveal that both Cu -free and Cu -bound states, revealing a homo-trimeric Cu -selective ion channel-like architecture. Two layers of methionine triads form a selectivity ClO4 inhibits NIS-mediated I transport not only because it is a substrate filter, coordinating two bound Cuþ ions close to the extracellular entrance. Below of NIS, but also because it profoundly alters the mechanism of NIS. Funding: the filter, polar amino acids line the pore and are poised to facilitate and regulate GM114250. Cuþ movement. The Ctr1 C-terminus may operate as a dual-functional þ þ 2745-Pos intracellular gate, both controlling Cu conduction and mediating Cu transfer D D to intracellular chaperones. These structures, together with Ctr1 functional char- Atypical RCK Domain Present in a Two Component K /H Antiporter acterization, provide the first high-resolution picture to understand Cuþ transport Tatiana Cereija, Joao Pedro, Leitao Guerra, Joao H. Morais Cabral. across cellular membranes and suggest new therapeutic opportunities for inter- I3S, Porto, Portugal. vention in diseases characterized by inappropriate Cu accumulation. Potassium is the major intracellular cation in both bacterial and eukaryotic cells. The concentration of potassium inside the cells is tightly regulated by a number of 2743-Pos influx and efflux systems. KhtU is a member of the monovalent cation/proton Species Differences in Monovalent Anion Substrate Selectivity in the antiporter-2 family of transporters and its activity is modulated by an ancillary Sodium Iodide Symporter (NIS) protein (KhtT) with sequence similarity to RCK domains. Functional analysis þ þ Susanna C. Concilio1, Hristina Zhekova2, Sergei Noskov2, of KhtTU using everted vesicles revealed its K /H antiport activity and its pref- þ þ Stephen J. Russell3. erence for K over Na . We now show the structure of the RCK KhtT in complex 1Department of Molecular Medicine, Mayo Clinic Graduate School of with cyclic-di-AMP solved by X-ray crystallography at 1.8 A˚ resolution. While Biomedical Sciences, Rochester, MN, USA, 2Centre for Molecular N-terminal domain has no sequence homology to any other structurally character- Simulation, University of Calgary, Calgary, AB, Canada, 3Department of ized proteins, the C-domain displays similar fold to other C-lobes of RCK do- Molecular Medicine, Mayo Clinic, Rochester, MN, USA. mains and binds cyclic-di-AMP. In agreement, the antiport activity of KhtTU The sodium iodide symporter (NIS) transports iodide, which is necessary for in the everted vesicles was highly increased in the present of cyclic-di-AMP, indi- thyroxine production. NIS can also transport other naturally occurring and cating that this molecule acts as an activator of KhtTU transporter by interacting synthetic monovalent anions such as nitrate, tetrafluoroborate, pertechnetate, with the RCK domain. Also, the KhtT structure and the conservation degree of its

BPJ 9470_9474 554a Wednesday, March 6, 2019 residues allow inference of the molecular determinants of KhtT and KhtU inter- pathological conditions, NCX can reverse direction and contribute to cell dam- action. Altogether, the results contribute for a better understanding of the mech- age by Ca2þ overloading. These critical functions have fueled the debate over anisms of regulation of KhtTU, clarify the contribution of cyclic-di-AMP and of the pharmacological potential of NCX since many years. Inhibition of the RCK domains in the modulation of Kþ transport. reversed mode of NCX is thought to be beneficial in ischemia/reperfusion injury. Moreover, inhibition of NCX has been proposed to exhibit an anti- 2746-Pos arrhythmic effect and implications for safety screening are discussed. Quantitative Simulations of Alternating Access in Sodium-Solute Sym- Human iPSC derived cardiomyocytes (hiPSC-CMs) are an evolving model sys- porters tem in cardiac research. We investigated NCX function in hiPSC-CMs. By 1 1 2 1 Paola Bisignano , Sara Capponi , John M. Rosenberg , Michael Grabe . developing a suitable sensor based method (SSM based electrophysiology) 1Department of Pharmaceutical Chemistry, Cardiovascular Research 2 we recorded specific NCX current responses of high amplitude. These currents Institute, UCSF, San Francisco, CA, USA, Dept Biol Sci, Univ Pittsburgh, showed similar calcium affinity compared to NCX1 expressed in HEK cells and Allison Park, PA, USA. were sensitive to nickel, KB-R7943 and SEA0400. The beating behavior of Sodium-solute symporters (SSS) exploit the sodium ion concentration gradients hiPSC-CMs was further investigated using an impedance based system. We to transports solute across the plasma membrane. Through a series of conforma- observed a significant increase in beating rate as a long term effect when inhib- tional changes, collectively described as alternating access, transporters expose iting NCX with SEA0400. their substrate cavity from one side to the other of the membrane, and eventually These findings clearly demonstrate the presence of functional NCX in hiPSC- release their cargo. How substrates and energizing ions coordinate to drive this CMs which until now has been poorly investigated. The observed increase in processes remains elusive and is likely very different from one structural family beating rate after inhibition of NCX is to some extent in disagreement with to the next. I will use the Weighted Ensemble enhanced sampling technique other studies. However, previous studies have employed different methods us- together with a novel progress coordinate based on structural similarity to simu- ing non-human systems and usually investigate short term effects of up to one late alternating access of the bacterial sodium galactose transporter (vSGLT) hour. Further investigation is necessary, but at this time we can state that we inward-facing out- starting from the apo and sugar bound states using the have found an intriguing new combination of tools which will help to generate ward-facing structure of the closely related sialic acid transporter (SiaT) as a new insights into the physiology and pharmacology of NCX. target. Thanks to these two structures we can explore the biophysics of the trans- port cycle at the atomic level to answer to the following questions: 1) what is the 2749-Pos order of binding?, and 2) how does sodium and substrate coupling drive confor- Unravelling the Topological Organization of the a2-Repeat of the mational change? It is very likely that information gained through these studies Mammalian Sodium-Calcium Exchanger will shed light on the transport cycle of mammalian homologues (hSGLT1/2) Namuna Panday, Kyle J. Scranton, Shuzhen Zhang, Scott John, and other members in the Leucine Transporter superfamily. Michela Ottolia. 2747-Pos Anesthesiology, UCLA, Los Angeles, CA, USA. The Naþ-Ca2þ Exchanger (NCX) is a plasma membrane protein that exchanges Using Phylogeny to Decipher Electrogenicity in Cation/Proton Antiporters þ 2þ Gal Masrati1, Manish Dwivedi2, Abraham Rimon2, Yael Gluck-Margolin2, three Na for one Ca . Transport function is associated with two regions of Amit Kessel1, Haim Ashkenazy3, Itay Mayrose3, Etana Padan2, Nir Ben-Tal1. NCX that have similar sequence and are conserved among the exchanger family: a 1Department of Biochemistry and Molecular Biology, Tel Aviv University, the -repeats. The recently resolved crystal structure of archaebacterial Tel Aviv, Israel, 2Department of Biological Chemistry, The Hebrew NCX_Mj has raised an important question in the existing topological model a University of Jerusalem, Jerusalem, Israel, 3Department of Cell Research and of the mammalian -repeats. Biochemical and electrophysiology studies have a Immunology, Tel Aviv University, Tel Aviv, Israel. shown that the -repeats include two re-entrant loops, which have not been With more than 100,000 entries in UniProt, cation/proton antiporters (CPAs) observed in the archaebacterial homolog. We have applied cysteine-scanning a constitute one of the largest superfamilies of transporters. They control pH ho- mutagenesis to investigate whether the residues within the 2-repeat of the meostasis and salt concentrations alongside other essential biological processes, mammalian NCX form a re-entrant loop or face the extracellular environment and their malfunction is associated with a growing number of pathologies as seen in the archaebacterial NCX_Mj. The sulfhydryl reagent, MTSET, was ranging from hypertension to autism. Whereas some CPAs are electroneutral, applied intracellularly to determine the cytoplasmic accessibility of cysteine exchanging one proton per one cation (mostly Naþ or Kþ), others are electro- introduced at positions 835, 836, 838, 842, 854-859, 862, 863, 865, 867 and genic, exchanging two protons per one cation. CPAs are also commonly divided 875. Mutation at 835 slightly decreased the affinity of NCX to cytoplasmic 5 5 into two phylogenetic groups, CPA1 and CPA2, which have been suggested to Na (mM, WT=20 0.8 n=5, V835C 29 1.6, n=6) and its activity was blocked correlate with the two electrogenicity phenotypes. However, such a correlation by intracellular MTSET application only when the transport sites were facing has not been established, and the molecular mechanism of CPAs electrogenicity the cytoplasm (inward state). This residue is part of TMS8 in NCX_Mj. Cyste- is still unclear.Reconstructing the superfamily’s phylogeny can elucidate the ines at positions 854-859, 862, 863 and 875 showed no activity change upon molecular determinants of the different electrogenicity phenotypes and intracellular MTSET application. This is in contrast to previous reports showing contribute towards defining the superfamily’s subdivision into different clades. that residues 855 and 858 face the cytoplasm and, suggests that this region Here, we present the most comprehensive phylogenetic analysis of CPAs to maybe extracellular - as seen in the NCX_Mj crystal structure. Mutation 5 date, encompassing 6,537 different CPAs that collectively capture the superfam- E863C had increased sensitivity for intracellular Na (14 0.4 mM, n=8). Ex- ily’s immense diversity. We identify a well-defined sequence motif of eight changers F865C and V867C were inactive, suggesting an important role of amino acids that distinguishes CPA1s from CPA2s, and that appears to deter- this region in NCX function. We are currently screening further positions to mine electrogenicity. On the basis of this phylogenetic analysis, we propose a assess the topological organization of the a2-repeat of the mammalian NCX. new approach to classifying CPAs and show that, in contrast to previous sugges- tions, the CPA1/CPA2 division only partially correlates with the different 2750-Pos D D electrogenicity phenotypes of CPAs. Supporting our analysis, we rationally- Molecular Basis for Ion Recognition and Transport in a Na /Ca2 designed, on the basis of phylogeny, a triple mutant of the electrogenic anti- Exchanger porter EcNhaA, converting its phenotype to electroneutral. To that end, we Fabrizio Marinelli, Emel Ficici, Jose’ D. Faraldo-Go´mez. introduced two compensatory mutations to an EcNhaA mutant that was origi- TMB, NHLBI NIH, Bethesda, MD, USA. þ þ þ nally designed to be electroneutral, but became inactive. Finally, we introduce Na /Ca2 exchangers are membrane antiporters playing a crucial role in Ca2 CAPTCHER (http://bental.tau.ac.il/CAPTCHER), a searchable database that signaling. They function by an alternating access mechanism in which the maps a query CPA sequence onto the phylogenetic tree, identifies its CPA motif outward-to-inward protein conformational change is coupled to the transport þ þ and offers a prediction of its phenotypical characteristics. of 3Na ions in exchange for 1 Ca2 ion through the cell membrane. Previous structural and computational studies on the outward-facing prokaryotic 2748-Pos exchanger, NCX_Mj, revealed the mechanism of protein binding-site occlusion NaD/Ca2D Exchanger in Human iPSC Derived Cardiomyocytes: Func- arising from the binding of either 3 Naþ ions or 1 Ca2þ ion. Such occlusion pro- tional Evidence and Relevance for Beating Behavior cess and thereby the transport itself, becomes energetically unfeasible in pres- Maria Barthmes, Krisztina Juhasz, Andre Bazzone, Ulrich Thomas, ence of partial ion occupancies, proton instead of Naþ binding or when the Sonja Stoelzle-Feix, Andrea Bruggemann, Michael George, Niels Fertig. binding site is unoccupied. This finding rationalizes the physiological require- Nanion Technologies, Munich, Germany. ment for a selective and strictly-coupled ion exchange mechanism. This notwith- The Naþ/Ca2þ-exchanger (NCX) plays an important role in cellular calcium standing, a thorough assessment of this process requires that a similar occlusion homeostasis. Under physiological conditions NCX removes Ca2þ from the mechanism occurs also for the yet unknown inward-facing conformation of the cell lumen of excitable cells and is involved in regulatory processes. Under Naþ/Ca2þ exchanger. Using extensive enhanced sampling simulations and

BPJ 9470_9474 Wednesday, March 6, 2019 555a repeat-swap homology modelling we identified a unique inward facing structure Lysophospholipid transporter (LplT) is a member of the major facilitator super- for NCX_Mj. The reliability of such model is assessed against recent hydrogen- family (MFS), found in many Gram-negative bacteria. LplT catalyzes flipping of deuterium exchange experiments and mutational analysis. Based on this inward lysophospholipids (LPLs) across the bacterial inner membrane, playing an impor- facing model, we calculated the conformational free energy landscapes for tant role in bacterial membrane homeostasis. We found that LplT promotes both different ion occupancy states giving an explicit evidence for a quasi- uptake of exogenous LPLs and intramembranous LPL flipping across the bilayer. symmetric binding-site occlusion mechanism between inward and outward fac- To gain mechanistic insight into this novel dual-LPL flipping activity, we imple- ing states of the protein. Our study also provides insights into the molecular mented a combination of computational approaches and LPL transport analyses driving forces by which only specific ion occupancy states trigger the occlusion to study substrate binding and translocation of LplT. Our data suggest that LPLs process, revealing the prominent role in this regard of ion-protein and water are translocated in an elongated cavity exhibiting an extremely asymmetric polar- mediated interactions. Overall, our work set the molecular basis for ion recogni- ity. Two D(E)N motifs form a head group binding site, in which the carboxylate tion and associated coupled transport in Naþ/Ca2þ exchangers. group of D30 is important for LPL head group recognition. Mutation of residues in the head group binding site disrupts both LPL uptake and flipping activities. 2751-Pos However, alteration of hydrophobic residues on the interface between the N- CLC Conformational Landscape as Studied by SMFRET and C- domains impairs LPL flipping specifically, resulting in accumulation of 1,2 2 3 Ayush Krishnamoorti , Ricky C. Cheng , Vladmir Berka , LPLs in the membrane, whereas their activities of LPL uptake still remain active. 2 Merritt Maduke . These results suggest a dual-substrate accessing mechanism, in which LplT re- 1The Kincaid School, Houston, TX, USA, 2Dept Molec/Cell Physiol, Stanford 3 cruits LPLs to the substrate binding site via two routes, either from its extracel- University, Stanford, CA, USA, Biochemistry and Molecular Biology, lular entry or through a membrane-embedded groove between transmembrane University of Texas Health Science Center at Houston, Houston, TX, USA. þ helices, and then moves them towards the inner leaflet of the membrane. This CLC secondary active transporters catalyze the exchange of Cl for H ions LPL flipping mechanism is likely conserved in many bacterial species and our across the cellular membrane. The available CLC crystal structures all share findings illustrate how LplT adjusts the MFS translocation pathway to perform the same global protein conformational fold, which suggests that the transport its versatile lipid homeostatic functions. mechanism may involve only movements of a conserved glutamate residue that has been observed to adopt different side-chain rotamers. However, NMR 2754-Pos and DEER measurements have revealed additional motions throughout the Uptake Dynamics in the Lactose Permease (LacY) Membrane Protein CLC protein, including in a region 20 A˚ away from this glutamate site. Transporter Cross-linking to restrict movement at this latter region inhibits transport, thus Dari Kimanius1, Erik R. Lindahl2, Magnus Andersson3. connecting the protein conformational dynamics to its function. To investigate 1Stockholm University, Stockholm, Sweden, 2Dept Biochemistry & Bioph, further the CLC conformational landscape, we used single-molecule FRET Stockholm Univ, Solna, Sweden, 3Umea˚ University, Umea˚, Sweden. (smFRET) to investigate substrate (Hþ) induced protein conformational The sugar-transporter Lactose permease (LacY) of Escherichia coli has changes. These smFRET studies show that there are Hþ-dependent conforma- become a prototype to understand the underlying molecular details of mem- tional changes at both the extracellular and intracellular sides of the protein. brane transport. Crystal structures have trapped the protein in sugar-bound The relevance of these changes to the Hþ transport mechanism is supported states facing the periplasm, but with significant differences in the extent of by their absence in a mutant that transports Cl but not Hþ. To better understand the structural opening. Therefore, the molecular details of sugar uptake remain these changes, triangulation experiments are being designed and performed to elusive. In this work, we have used extended simulations and metadynamics obtain distance constraints at both the extracellular and intracellular sides. sampling to explore a putative sugar-uptake pathway and associated free en- ergy landscape. We found an entrance at helix-pair 2 and 11, which involved 2752-Pos lipid head groups and residues Gln 241 and Gln 359. Furthermore, the Modelling of Ion Binding and Selectivity in SLC4 Transporters protein displayed high flexibility on the periplasmic side of Phe 27, which is 1 1 1 2 Hristina R. Zhekova , Mirna Damergi , Sergei Yu Noskov , Jiansen Jiang , located at the narrowest section of the pathway. Interactions to Phe 27 enabled 3,4 5 5,6 Z. Hong Zhou , Alexander Pushkin , Ira Kurtz . passage into the binding site, which was associated with a 2454 kJ/mol bind- 1Dept. Biol. Sci., University of Calgary, Calgary, AB, Canada, 2NHLBI, 3 ing free energy in excellent agreement with an independent binding free energy NIH, Bethesda, MD, USA, Dept. Microbiol. Immunol. Mol. Genet., UCLA, calculation (24510 kJ/mol) and experimental data (23 kJ/mol). Two free Los Angeles, CA, USA, 4California NanoSystems Institute, UCLA, Los 5 6 energy minima corresponding to the two possible binding poses of the Angeles, CA, USA, Dept. Medicine, UCLA, Los Angeles, CA, USA, Brain. lactose analog b-D-galactopyranosyl-1-thio-b-D-galactopyranoside (TDG) Res. Inst., UCLA, Los Angeles, CA, USA. 2 þ þ were aligned with the crystal structure-binding pocket. This work outlines The SLC4 family of transporters mediate HCO3 ,CO3 ,Cl ,Na and H the chemical environment of a putative periplasmic sugar pathway and paves transport and play important roles in acid-base chemistry, blood pressure regu- way for understanding substrate affinity and specificity in LacY and sugar lation, and maintenance of cell function and growth. To better understand the transport in general. mechanisms of SLC4 transporter function at the atomic level we studied (published in Scientific Reports, 25 September 2018, 8, 14324). AE1 (SLC4A1), NBCe1 (SLC4A4) and NDCBE (SLC4A8) which mediate þ 2 electroneutral Cl /HCO3 exchange, electrogenic Na -CO3 cotransport 2755-Pos þ 2 and electroneutral Na -coupled Cl /CO3 exchange, respectively. The Direct Protein-Lipid Interactions Shape the Conformational Landscape of outward-facing (OF) structures of AE1 and NBCe1 have been recently resolved Secondary Transporters by X-ray diffraction and cryoelectron microscopy to 3.5 and 3.9 A˚ , respec- Chloe Martens1, Mrinal Shekhar2, Antoni Borysik1, Andy Lau1, tively. Putative ion coordination sites in these transporters were predicted using Eamonn Reading3, Emad Tajkhorshid2, Paula Booth1, Argyris Politis1. the available structures and functional mutagenesis experiments. However, the 1Chemistry Dept, King’s Coll London, London, United Kingdom, 2Dept positions and orientation of the ions in these sites is yet to be determined. In the Biochemistry, Univ Illinois Urbana Champaign, Urbana, IL, USA, 3King’s present study, we used computational modelling techniques including all atom Coll London, London, United Kingdom. molecular dynamics (MD) simulations with discreet protonation states of rele- A growing number of studies report lipids interacting with membrane proteins vant titratable residues to assess the ion-protein interactions in the putative ion to modulate their folding, stability and function, yet the molecular details un- coordination sites in the OF states of AE1, NBCe1, and NDCBE. Selected mu- derpinning such processes remain elusive. This is even more pertinent for trans- tants of NBCe1 that cause renal tubular acidosis were also studied. Since the porters, which despite being an important class of drug targets lack atomic structure of NDCBE is not available, we have constructed a homology characterization at molecular level. Here, we combine hydrogen-deuterium ex- model based on the OF AE1 and NBCe1 templates. The role of specific residues change mass spectrometry (HDX-MS) with molecular dynamics (MD) simula- in the ion coordination sites and ion permeation pores of the three transporters tions to understand how lipids regulate the conformational dynamics of was elucidated and their effect on ion selectivity was determined. secondary transporters at the molecular level. Using the homologous trans- porters XylE, LacY and GlpT from Escherichia coli as model systems, we 2753-Pos reveal that specific protein-lipid interactions modulate the conformational equi- Dual-Substrate Accessing Mechanism of an MFS Transporter for Lyso- librium between outward- and inward- facing conformations. We first identify phospholipid Flipping across the Cell Membrane the structural elements involved in the conformational transition between 1 1 2 2 Lei Zheng , Yibin Lin , R.N.V. Krishna Deepak , Hao Fan . different states by combining hydrogen deuterium exchange mass spectrometry 1Dept Biochemistry and Molecular Biology, Univ Texas Hlth Sci Ctr, 2 (HDX-MS) with systematic mutagenesis. We then use tunable nanodiscs and Houston, TX, USA, Bioinformatics Institute (BII), Agency for Science, MD simulations to uncover how specific lipids directly modulate such transi- Technology and Research, Singapore, Singapore. tion. We found that this modulation arises from interactions between the

BPJ 9470_9474 556a Wednesday, March 6, 2019 phopspholipid headgroup and a specific network of residues shown through our zation domain, following an elevator-like motion, involving both translational work to control conformation. This network is highly conserved across homol- and orientational components. In this study we have performed extensive all- ogous transporters and previous biochemical characterization demonstrated atom simulations of recently crystalized human EAAT1 in cholesterol-rich that its integrity is an absolute requirement for function. Together, our data pro- and cholesterol-free lipid-bilayers. Our results suggest that compared to the vides novel insights into the role of lipids in molecular mechanisms and lay the modeled POPC membranes, the cholesterol-rich bilayer modifies the orienta- foundation for a comprehensive model of secondary transport in lipid bilayers. tional dynamics of EAAT1, which we attribute to the increased lateral pressure of the lipid bilayer. Distribution of lipids around the protein shows cholesterol 2756-Pos binding to the interface between the trimerization and transport domains of Exploring Dynamic Transitions in an Arginine Transporter EAAT1. A combination of equilibrium simulations, string calculations and Zhiyi Wu, Simon Newstead, Philip C. Biggin. 1D-BEUS free energy calculations demonstrate that cholesterol has a high af- Dept Biochemistry, Univ Oxford, Oxford, United Kingdom. finity towards the interface and alters the OF-IF transitions in EAAT1. Remark- Amino acids serve as modulators of important cellular pathways, ranging from ably, the same interface forms the binding pocket for an allosteric inhibitor regulating metabolism and inflammation to neuronal signalling pathways. The UCPH, thus we suggest cholesterol might play an important role in governing amino acid arginine is a precursor of nitric oxide synthesis and an important regu- the dynamics and kinetics of EAAT1. lator of the mTORC1 pathway. The level of intracellular arginine is regulated by the solute carrier (SLC) 7 family of cationic amino acid transporters (CATs). 2759-Pos Although no structure of a human CAT exists, the structure of a bacterial homolog, Mechanism and Potential Sites of Potassium Interaction with the Gluta- GkApcT, has been recently solved in an inward occluded state (Jungnickel, Parker mate Transporter EAAC1 et al. 2018). One of the questions arising from the structure is how the protein cou- Jiali Wang, Christof T. Grewer. ples the movement of protons to the transport of arginine. Our working hypothesis Chemistry, Binghamton University, Binghamton, NY, USA. is that release of arginine is facilitated by deprotonation of a glutamate residue, Plasma membrane glutamate transporters are expressed in the brain in neuronal located at the bottom of the binding pocket and which acts as the intracellular and glia cells. Glutamate transporter malfunction is connected with Alzheimers’ gate, allowing the protein to transition from an inward-occluded to an inward- disease, stroke and other neuro-degenerative diseases. Glutamate transport open conformation. To explore this hypothesis, we have used molecular dynamics across the membrane is coupled to co-transport of three sodium ions, one proton and umbrella sampling toshowthatthe transition frominward-occludedtoinward- and counter-transport of one potassium ion. While potassium-induced relocation open is more energetically favourable when this key glutamate is deprotonated. is one of the major properties of glutamate transporters, resulting in Kþ-induced We also used absolute free energy calculations to show that the binding of arginine glutamate efflux under conditions of energy deprivation, the mechanisms and in the binding pocket is more unfavourable in the inward-open state compared than site(s) of interaction with Kþ are not well understood. Here, we combine electro- in the inward-occluded state, supporting the view that deprotonation of this gluta- physiology and molecular dynamics (MD) simulations to study the potential po- mate is vital to the correct function of the transporter. tassium binding site(s) of the glutamate transporter subtype EAAC1. Site- directed mutagenesis was used to create EAAC1 variants with mutations to pre- 2757-Pos dicted Kþ binding site, and electrogenic properties were compared to the wild- Transport Activity and Novel Inhibitors of Human GLUT9 Characterized type transporter. In MD simulations, potassium was found to spontaneously bind by Molecular Modeling and Electrophysiology to positions near residues Y98A, D443A, D365A, remaining in the binding site Jinping Zhang, Yanyu Chen, Ting Wu, Qunsheng Lan, Ze-an Zhao, for more than 100 ns. Consistently, we found that potassium induced-relocation Ying Cao, Pingzheng Zhou, Jianxin Pang. together with glutamate translocation were fully blocked in the D443A and Southern Medical University, Guangzhou, China. D365A mutant transporters. Our results provide new information on potential hGLUT9(SLC2A9) is a voltage sensitive urate transporter and has been sug- Kþ binding sites, which can be further tested in future studies. gested as a promising therapeutic target for hyperuricemia and gout. In the pre- This work was supported by the National Science Foundation Grant 1515028 sent study, whole-cell patch clamp technique in CHO cells was successfully awarded to C.G. established to evaluate hGLUT9 activity. The 3D structure of hGLUT9 was constructed by homology modeling taking hGLUT1/3/5 as the templates and 2760-Pos several key residues were proposed to play key roles in uric acid transport in Voltage Dependent Inhibitor Binding to Plasma-Membrane Glutamate hGLUT9. The transporting activity of uric acid mediated by mutants Transporters (L303A, F397A, W430A and N433A) were significantly decreased compared Laura J. Zielewicz, Jiali Wang, Elias Ndaru, Christof T. Grewer. to wild type in CHO cells. Further, two potent inhibitors (3170-M and 171#) Department of Chemistry, Binghamton University, Binghamton, NY, USA. were discovered and demonstrated potent inhibitory effect in hGLUT9. Finally, Plasma-membrane glutamate transporters of the excitatory amino acid transporter in vivo studies revealed that both 3170-M and 171# could effectively decrease (EAAT) family are important for maintaining a low glutamate concentration in the serum uric acid level in hyperuricemia mice induced by potassium oxonate extracellular space of the mammalian brain. Glutamate is believed to be trans- without affecting liver and kidney functions. Taken together, our study indi- ported in its negatively-charged form, energetically driven by the co-transport cates that L303, F397, W430 and N433 play important roles in the binding of three sodium ions. At least two of these sodium ions are bound within the dielec- of uric acid with hGLUT9. 3170-M and 171# present new inhibitors for the pre- tric of the membrane, resulting in voltage dependent association and dissociation vention and treatment of hyperuricemia and gout. kinetics. It was speculated that glutamate binding is also electrogenic because the binding site of the transported substrate, glutamate, is located somewhat close to 2758-Pos the center of the membrane. Furthermore, it was hypothesized that competitive in- Modulation of Orientational Dynamics of Excitatory Amino Acid hibitor binding is voltage dependent for the same reason. Here, we rapidly applied Transporter-1 by Cholesterol a low-affinity competitive inhibitor, kainate, to the glutamate transporter subtype Shashank Pant1, Emad Tajkhorshid2. 1 EAAT2, resulting in outward transient current caused by movement of net nega- Center for Biophysics and Quantitative Biology, Department of tive charge of the inhibitor into the low dielectric of the protein/membrane. Biochemistry, Beckman Institute, University of Illinois, Urbana, IL, USA, 2 Consistently, inhibitor dissociation kinetics are voltage dependent, accelerating Biochemistry, University of Illinois, Urbana, IL, USA. with increasingly negative transmembrane potential. In contrast, binding kinetics Glutamate is the most important excitatory neurotransmitter in the human of a high-affinity, slow binding inhibitor, TFB-TBOA, showed the opposite brain. Its concentration controls synaptic transmission, thus maintaining low voltage dependence compared to dissociation. Consistent with previous studies, concentration of glutamate in the synapse is of critical importance. The synap- our results show that the substrate and inhibitor binding site is located within tic concentration of glutamate in mammalian brain is regulated by excitatory the membrane environment with low dielectric constant. amino acid transporter-1 (EAAT1), which harvests the energy of sodium, pro- This work was supported by the National Science Foundation Grant 1515028 ton, and potassium gradients to uptake glutamate from the synapse. The trans- awarded to C.G. port mechanism involves outward-facing (OF) to inward-facing (IF) conformational changes of EAAT1, where glutamate is transported from the 2761-Pos extracellular to the intracellular region. EAAT1 is reported to partition into Evolution of Ion Specificity in Glutamate Transporters cholesterol-rich regions of the membrane, suggesting that cholesterol might Krishna Reddy, Olga Boudker. play an important role in its function. Each monomer of EAAT1 consists of Physiology and Biophysics, Weill Cornell Medical College, New York, NY, USA. two distinct domains, the trimerization domain and the transport domain. A family of secondary active glutamate transporters imports glutamate into The interconversion between the two can be described as nearly a rigid body the cell through coupling to electrochemical gradients. In mammals, these movement of the transport domain in lipid bilayer with respect to the trimeri- proteins clear the synaptic cleft of excess neurotransmitter, while in

BPJ 9470_9474 Wednesday, March 6, 2019 557a prokaryotes they serve in nutrient uptake. In prokaryotes, aspartate/glutamate 2764-Pos transporters appear to couple transport to either exclusively protons or so- Ibogaine Binds Human Serotonin Transporter in Multiple Functional dium. While the mechanism of sodium-coupling is well-established for an States 1,2 1,2 3 3 archaeal aspartate transporter homolog (GltPh), the basis of proton-coupling Zhiyu Zhao , Po-Chao Wen , Jonathan Coleman , Dongxue Yang , is unknown. Based on evolutionary and structural analysis, we have designed Eric Gouaux3, Emad Tajkhorshid1,2. 1 two point mutations in GltPh that introduce increased transport rates in the Center for Biophysics and Quantitative Biology, Department of presence of a proton gradient. The mutant protein still requires the presence Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA, of sodium, indicating that the mutant protein may be an intermediate between 2NIH Center for Macromolecular Modeling and Bioinformatics, Beckman sodium and proton-coupled transporters. Upon aspartate binding, the mutant Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA, protein does not appear to bind or release protons, suggesting that pH- 3Vollum Institute, Oregon Health & Science University, Portland, OR, USA. dependent transport affects the transition rate between inward and outward- Serotonin transporter (SERT) belongs to the family of neurotransmitter:sodium facing states. Interestingly, at high sodium concentrations, the mutant protein symporters (NSSs), which harness the transmembrane electrochemical gradient binds aspartate in a more complex binding mode than wild-type GltPh, poten- of ions for the uphill transport of their substrates. The hallucinogen ibogaine is tially due to a change in distribution of structural states. Further investigation a unique SERT inhibitor that behaves differently from many antidepressants into the dynamics of the proton-coupled GltPh mutant may reveal the general characterized in recent crystal structures, all of which lock SERT in an mechanism of proton-coupled transport in prokaryotic aspartate/glutamate outward-open conformation. We have obtained cryo-electron micrographs of transporters. the ibogaine-bound SERT in outward-open, occluded, and inward-open states, showing that they all bear ibogaine within the central binding site. However, 2762-Pos the exact binding pose of ibogaine in these structures remained ambiguous Millisecond Time Resolution by HS-AFM Line Scanning of Fast GltPh Dy- due to limited resolution. To reveal the atomic details of ibogaine binding, namics we developed a computational approach to systematically search for the Tina R. Matin1, George R. Heath1, Gerard Huysmans2, Olga Boudker3, most probable binding poses, followed by extensive molecular dynamics sim- Simon Scheuring1. 1 ulations to substantiate stable ibogaine-bound states. The systematic search Anesthesiology, Physiology and Biophysics, Weill Cornell Medicine, New included all possible translations, rotations, and rotamer configurations of ibo- York, NY, USA, 2Physiology and Biophysics, Weill Cornell Medicine, New 3 gaine within the binding pocket, and scored by a combination of a force-field York, NY, USA, Physiology and Biophysics, Howard Hughes Medical based energy function and the cross-correlation to the electron density. The Institute, Weill Cornell Medicine, New York, NY, USA. optimal binding poses in all three states share a salt bridge between an aspartate Mammalian glutamate transporter proteins play the crucial role in and the charged ammonium group of the ligand, a hallmark of substrate binding removing excitatory neurotransmitters from the synaptic cleft. Malfunction conserved among NSSs. In the inward-open state, the binding site is capped of Glutamate transporters is identified in various neurological disorders from the extracellular side by an aromatic sidechain and the bound ibogaine such as cerebral stroke, epilepsy, Alzheimer’s disease, dementia, Hunting- is shifted slightly from the central binding site toward the cytoplasm. In all ton’s disease, amyotrophic lateral sclerosis (ALS) and malignant glioma. states, ibogaine fluctuates within the central binding site but remains bound During the past decade, our understanding of the structure and function within the binding pocket. The consistent ibogaine-binding modes character- of these proteins have evolved profoundly from studying a prokaryotic ized here indicate that ibogaine blocks serotonin access to SERT due to its glutamate transporter homolog (GltPh). Here, we report the development binding to the central binding site in all functional states. Arguably, the and use of novel high-speed atomic force microscopy (HS-AFM) based apparent noncompetitive inhibition of ibogaine might arise from the conforma- techniques that reach millisecond time resolution, and explored previously tional preference of the ibogaine-bound SERT under physiological conditions. undetectable short-lived transport states in unlabeled membrane reconsti- tuted GltPh. We find that GltPh exhibits much faster dynamics than previ- 2765-Pos ously thought. Bridging the Gap between Functional and Structural Data Verena Burtscher1, Matej Hotka1, Thomas Stockner1, 2763-Pos Jan-Philipp Machtens2,3, Walter Sandtner1. Mechanistic Characterization of the Allosteric Communications between 1Pharmacology, Medical University of Vienna, Vienna, Austria, the Central Binding Site and the Extracellular Vestibule of the Serotonin 2Neurophysiology, Hannover Medical School, Hannover, Germany, Transporter 3Cellular Biophysics, Forschungszentrum Juelich, Juelich, Germany. Ara M. Abramyan1, Per Plenge2, Pia Weikop2, Ulrik Gether2, The voltage at the outer and inner membrane-solution interface deviates from Benny Bang-Andersen3, Lei Shi1, Claus J. Løland2. the voltage at the bulk solution. This is due to fixed charges at the membrane 1National Institute on Drug Abuse, National Institutes of Health, Baltimore, 2 3 surface. Charged residues of proteins that are exposed to the aqueous solution MD, USA, University of Copenhagen, Copenhagen, Denmark, Lundbeck contribute to these charges. The voltage that drops across the membrane is Research, Copenhagen, Denmark. therefore set by the difference of the inner and outer membrane surface poten- The serotonin transporter (SERT) is a primary target for antidepressants, e.g. tial and not by the bulk potential. By utilizing capacitance measurements, we S-citalopram and imipramine to alleviate symptoms of depression and anx- have recently shown that ligand binding to membrane proteins changes the sur- iety, and functions by exploiting pre-existing ion gradients of Naþ,Cl,and þ face potential and that this is seen by a change in apparent capacitance. It is K to translocate serotonin from the synaptic cleft into the presynaptic conceivable that also rearrangement of solvent-accessible charged residues, neuron. SERT crystal structures reveal that two S-citalopram molecules as can occur in conformational transitions, change the surface potential. We bind in both the central binding (S1) site and a site in the extracellular ves- tested this hypothesis by incorporating structural information of the serotonin tibule (S2 site). We investigated the allosteric communications between the transporter (SERT) into a kinetic model of the transport cycle. For this, we S1 and S2 sites by carrying out extensive molecular dynamics (MD) simu- counted the number of water-accessible charges of SERT for different confor- lations of hSERT in complex with various combinations of S1- and S2- mational states. Using a kinetic model, we then were able to calculate the time bound ligands. The comparative analysis of the simulation results in combi- dependent change of the inner and outer net surface charge densities during the in vitro nation with binding assays and mutagenesis reveal that the binding transport cycle. Subsequently, this allowed us to determine the associated of S-citalopram or imipramine to the S1 site is allosterically coupled to the capacitance changes. Comparison with experimental data revealed that the S-citalopram binding to S2 through the Thr497-Phe335 motif. The different capacitance signal estimated from structural data is similar in size and shape. configurations of this motif in turn affect the propensity of Glu494 in the S2 Thus, the complex capacitance signal of the SERT transport cycle derives site to form a salt bridge with the S2-bound S-citalopram. In addition, our mainly from changes in surface potential due to conformational transitions. MD simulation results indicate that Lu AF60097, the first high-affinity We further propose that capacitance measurements in combination with kinetic S2-ligand, allosterically couples the ligand binding to S1 through a similar models might serve as a useful tool to link functional and structural mechanism. In the presence of S1-bound imipramine but not S-citalopram, information. our combined in silico and in vitro analysis show that Lu AF60097 occupies a dynamically formed sub-pocket in the extracellular vestibule. We propose 2766-Pos that the occupation of this cavity is responsible for the significantly Identifying Structural Determinants of High-Potency MDPV Binding at improved affinity of Lu AF60097 in the presence of S1-bound imipramine. the Human Dopamine Transporter S1 Binding Site Together we reveal a coupling mechanism between the S1 and S2 sites Tyler W.E. Steele, Brian Ruiz, Zachary Spires, Jose M. Eltit. that will facilitate rational development of high-affinity SERT allosteric Dept Physiology and Biophysics, Virginia Commonwealth University, inhibitors. Richmond, VA, USA.

BPJ 9470_9474 558a Wednesday, March 6, 2019

Methylenedioxypyrovalerone (MDPV) is a prominent component of and the positions of all tracks are combined computationally to create a com- designer ‘‘bath salts’’, an illicit family of drugs, that is a potent inhibitor posite map of nuclear positions in the developing embryo. Currently, we (IC50 5-50nM, assay dependent) of the human dopamine transporter have incorporated 162 nuclei, including 22 seam, 20 intestinal, 22 neurons, 4 (hDAT). In contrast with cocaine, MDPV is specific for hDAT, and has little neuroblast, 90 muscles, 3 glia, and 1 hypodermal syncytium nuclei into our affinity at the human serotonin transporter (hSERT). Despite information model, imaging with 3x coverage for improved accuracy. We are also in the gleaned from crystal structures of hSERT and the D. melanogaster dopa- process of refining a convolutional, neural network capable of segmenting mine transporter (dDAT), the structural determinants of high affinity nuclei, which we anticipate will greatly speed up cell tracking. We are also MDPV-binding at hDAT are unknown. Here we sought to identify the exploring multi-color approaches in which several fluorophores, each driven structural features of hDAT in the S1 binding site that contribute to the by a unique promoter, are expressed in the same embryo to provide overlapping high-affinity binding of MDPV. Assessments of transporter function labeling of nuclei. Such approaches should also speed completion of the atlas. were carried out using a transporter-induced Ca2þ fluorescence assay. In This 4D atlas can help scientists to efficiently study C. elegans neuro- and glio- contrast to cells expressing hDAT, cells expressing dDAT showed weak af- genesis, e.g. how its neurites and axons are guided during development, and to finity to MDPV. Using this information several residues near to the S1 bind- identify neuronal patterns of brain activity. ing site of dDAT were mutated to cognate human residues, and these ‘‘humanized’’ dDAT versions where permanently expressed in mammalian 2769-Pos cells. Using this strategy, we identify residues near the S1 binding site along Analysis of Phosphoinositide-Dependence of Action Potential Firing in helices 8 and 3 that are essential for high affinity binding of MDPV at Sympathetic Neurons by Electrophysiological Recordings and Mathemat- hDAT. ical Modeling Martin Kruse1, Rayne Whitten2. Posters: Computational Neuroscience 1Department of Biology and Program in Neuroscience, Bates College, Lewiston, ME, USA, 2Program in Neuroscience, Bates College, Lewiston, 2767-Pos ME, USA. The Missed Role of Cytoskeletal Filaments in Information Processing Superior cervical ganglion (SCG) neurons play an important role for the inner- Christian C. Hunley. vation of the pineal gland. Due to their essential role in controlling the circa- Physics, University of Texas at San Antonio, San Antonio, TX, USA. dian rhythm, we analyzed action potential firing and its dependence on the rare Recently, substantial experimental evidence on Cytoskeleton filaments have phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in SCG neu- demonstrated that the concurrent propagation of both electrical signals along rons isolated from adult male rats by a combination of electrophysiological, cytoskeleton filaments and electrochemical currents along the axonal mem- biochemical, and mathematical approaches. Previously, we found action po- brane are highly possible. This discovery opened an unexplored frontier in tential firing to be controlled by KCNQ2/3 and Maxi-K potassium channels, understanding brain information processing, neural plasticity and changes and a decrease in their activities by more than 80% was necessary to evoke ac- in the cytoskeleton. However, the molecular mechanisms governing the bio- tion potential firing. Physiologically, action potential firing in SCG neurons is nanowire properties in cytoskeleton filaments under physiological and path- induced by activation of muscarinic acetylcholine receptors, which triggers ological conditions are still poorly understood. In this presentation, we activation of phospholipase C and hydrolysis of PI(4,5)P2. While KCNQ2/3 introduce an innovative multi-scale approach able to account for the atom- channel activity has been shown to be strongly dependent on PI(4,5)P2, a regu- istic details of a protein molecular structure, its biological environment, lation of Maxi-K channel activity by phosphoinositides is under debate and has and their impact on electrical impulses propagating along wild type Actin fil- been speculated to be dependent on the subunit composition of Maxi-K chan- aments. Our results for in-vitro and intracellular conditions show a significant nels. To analyze which Maxi-K subunits are expressed in SCGs, we isolated influence of the voltage stimulus and biological environment on the electrical total RNA from SCGs and performed RT-PCR with oligonucleotides for a b impulse shape, attenuation and propagation velocity. The filament is shown both - and -subunits of Maxi-K channels. Our results showed expression a a to sustain the ionic wave propagation at almost constant velocity for the of only the Maxi-K -subunit. Expression of Maxi-K -subunits in HEK293 in-vitro condition, whereas the intracellular condition displays a remarkable cells together with either muscarinic acetylcholine receptors or a voltage- deceleration. As a unique feature, this multi-scale theory is able to account sensitive lipid phosphatase to transiently deplete PI(4,5)P2 showed no decrease for molecular structure conformation (mutation) and biological environment in Maxi-K channel activity upon depletion of PI(4,5)P2. We incorporated our changes (pH and ionic concentrations) often present in pathological condi- electrophysiological and biochemical results into a model of phosphoinositide tions. Thus, it may be potentially useful in providing a molecular understand- metabolism of SCG neurons previously published by our group to develop a ing for how and why age and inheritance conditions induce dysfunction and mathematical description of phosphoinositide metabolism and PI(4,5)P2- malformation in cytoskeleton filaments associated with a variety of neurolog- dependent ion channel activity in SCG neurons. In conclusion, our model al- ical diseases. It is also applicable to other conducting biopolymers with rele- lows for the first time a simulation of phosphoinositide metabolism and its vance in biomedicine and biophysics and their potential nanotechnological regulation of action potential firing of SCG neurons. applications. 2770-Pos 2768-Pos Effect of Columnar Neural Grouping on Network Synchronization Generating a 4D Atlas of Nuclear Positions in Embryonic Caenorhabditis Joseph S. Tumulty, Luis Cruz. elegans Physics, Drexel University, Philadelphia, PA, USA. Ryan Christensen1, Alexandra Bokinsky2, Anthony Santella3, Mark Moyle4, Connectivity in the brain has long been explored on varying scales: from Min Guo1, Andrew Lauziere1, Evan Ardiel1, Harshad D. Vishwasrao1, connectivity of large regions down to groups of only a few neurons. In Brandon Harvey1, Michael Levin1, Nensi Karaj1, William Mohler5, this work we explore how a connectivity scheme inspired by columnar or- Daniel Daniel Colo´n-Ramos4, Zhirong Bao3, Hari Shroff1. ganization in the neocortex effects the synchronization of a system of neu- 1NIBIB, National Institutes of Health, Bethesda, MD, USA, 2CIT, National rons. Neurons are grouped based on (x,y) positions in a grid to form Institutes of Health, Bethesda, MD, USA, 3Developmental Biology Program, columnar groups, and a control system is grouped independent of position. Sloan-Kettering Institute, New York, NY, USA, 4Department of Cell These neurons are connected with a bias towards connections within their Biology, Yale University, New Haven, CT, USA, 5Department of Genetics groups, based on bias parameter, d.Thisschemeleadstoasmall-world and Developmental Biology, University of Connecticut, Farmington, CT, network containing highly connected groups as well as inter-group connec- USA. tions. Systems were created with purely excitatory neurons as well as sys- Prior studies in Caenorhabditis elegans have identified the invariant cell line- tems of 80% excitatory neurons and 20% inhibitory neurons. Simulations age in the developing embryo; imaging and computational approaches harness are run with external stimulation to each Hodgkin-Huxley type neuron, this information so that cell identities can be efficiently tracked during the first and distance-dependent delays are included in the propagation of each ac- half of embryogenesis. This ability makes C. elegans an excellent system for tion potential. We find that initially the connectivity must reach a critical studying developmental questions during embryogenesis. However, rapid em- point in order for the system to synchronize. Above that point, the randomly bryo movement and morphological changes hinder cell identification and grouped control system becomes less synchronous (measured by SPIKE- tracking during the second half of C. elegans embryogenesis. To tackle these distance) than the system grouped in a columnar structure. This difference challenges, we are using light sheet microscopy and custom software to create is significant in a particular region of our parameter phase space at very an atlas of cell positions in the embryo, from the two-cell stage until hatching. low signal propagation speeds. Additional work has been done to further We use a sparse-labeling approach, where mCherry marks unique subsets of characterize the dynamics of these systems including the frequency of nuclei in each imaged strain. Then, cell positions are tracked in each strain, global spiking events.

BPJ 9470_9474 Wednesday, March 6, 2019 559a

Posters: Computational Methods and are affected in the USA alone, and patients exhibit varying symptoms and re- actions to treatments, so that diagnosis and finding an effective treatment can be Bioinformatics II challenging. The causes are not well understood, but both genetic and environ- mental factors are involved. NGS (Next Generation Sequencing) and GWAS 2771-Pos (Genome-Wide Association Studies) studies published by several groups QM/MM Study on Cleavage Mechanism Catalyzed by Zika Virus NS2B/ have identified dozens of alleles that vary between patients and healthy individ- NS3 Serine Protease uals and might be involved in genetic predisposition to IBDs. Over 100 of these Bodee Nutho1, Adrian Mulholland2, Thanyada Rungrotmongkol3. IBD-associated SNPs result in single amino-acid substitutions in protein se- 1Program in Biotechnology, Chulalongkorn University, Bangkok, Thailand, quences (SAPs, missense mutations), and it is not always clear how these mu- 2School of Chemistry, University of Bristol, Bristol, United Kingdom, tations lead to genetic predisposition or if some might be due to sequencing 3Biochemistry, Chulalongkorn University, Bangkok, Thailand. ‘‘noise’’. For each SAP, we performed an in silico analysis on the type of amino Zika virus (ZIKV), a mosquito-borne flavivirus, was originally isolated from acid substitution and the location on the protein’s three-dimensional structures. sentinel rhesus monkey in the Zika Forest of Uganda in 1947. The virus is trans- The results of the analysis identified amino acid substitutions that are likely to mitted to humans by Aedes mosquitoes. The recent evidences have revealed that affect protein function, structure, and/or stability, and therefore might be this virus is linked to serious pathological disorders including microcephaly in involved in genetic predisposition to IBDs. The results of analyses like this newborns and Guillain-Barre syndrome in adults. Up to now, there is no one can suggest which amino acid substitutions may be of highest priority currently available vaccine or therapeutic drug for preventing or controlling for in vitro analysis of the purified mutant proteins to aid in further understand- ZIKV infection. One of the attractive drug-targets for ZIKV treatment is the ing of the molecular mechanisms of disease and aid in diagnosis and future NS2B/NS3 serine protease that is crucial for viral polyprotein during infection. development of therapeutics to alleviate the symptoms of IBD. Herein, we have used a hybrid Quantum Mechanics and Molecular Mechanics (QM/MM) umbrella sampling simulation at the PM6/ff14SB level of theory to 2774-Pos investigate the acylation process of the reaction catalyzed by the ZIKV protease. Computational Analysis of Spectroscopical Properties and Binding Affin- The obtained results reveal that proton transfer from S135 to H51 and nucleo- ities of Oxyluciferin Analogs in Firefly Luciferase Protein philic attack on the substrate by S135 occur in a concerted manner. The rate- Vardhan Satalkar1, Xiaoliang Pan1, Enrico Bennasi2, Yihan Shao1. 1 limiting step is the formation of tetrahedral intermediate with an energy barrier Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA, 2 of 10.0 kcal,mol1. In addition, single-point energy QM/MM calculations on School of Science and Technology, Nazarbayev University, Astana, the BH&HLYP-D3/6-31G(d)/ff144SB optimized geometries were carried out at Kazakhstan. the SCS-(L)MP2/(aug)-cc-pVTZ/ff14SB and L-CCSD(T)/(aug)-cc-pVTZ/ Design of novel luciferin substrates as near-infrared bioluminescence probes to ff14SB to correct the potential energy surfaces. The average values of computed improve deep-tissue imaging is an active field of research. Several effective an- activation energies at the SCS-LMP2/(aug)-cc-pVTZ/ff14SB of 17.8 5 1.2 alogs of oxyluciferin, such as AkaLumine (bioluminescence wavelength = kcall,mol1 and L-CCSD(T)/(aug)-cc-pVTZ/ff14SB of 16.3 5 1.4 kcal,mol1 677nm), cyclic alkylamino luciferin (CycLuc1) (bioluminescence wavelength = are in good agreement with the experimental data. Therefore, the ability of the 604 nm) have indeed resulted in longer bioluminescence wavelengths as QM/MM presented here could be informative and useful for further designing compared to standard D-luciferin (bioluminescence wavelength = 562nm). of NS2B/NS3 inhibitors based on transition state analogues. Additionally, lower substrate concentration, bioavailability of substrate in the protein binding pocket and higher quantum yield of bioluminescence are also 2772-Pos important criterions for the design of ideal luciferin substrate. Network-Based Modeling of Amyloid Fibril Formation In order to investigate the effect of substitutions on the oxyluciferin, we have de- Gianmarc Grazioli1, Yue Yu2, Megha H. Unhelkar3, Rachel W. Martin4, signed a series of luciferin substrates with various functional groups on both 6- Carter T. Butts5. hydroxy-benzothiazole and 4(5H)-thiazolone heterocyclic rings. Through per- 1California Institute for Telecommunications and Information Technology forming TDDFT calculations in both gas phase and condensed-phase (using im- (Calit2) & Dept. of Chemistry, Univ Calif Irvine, Irvine, CA, USA, plicit solvent model), we identified several promising combinations of electron 2California Institute for Telecommunications and Information Technology donating groups and electron withdrawing groups, as emitted light from these (Calit2), Univ Calif Irvine, Irvine, CA, USA, 3Dept. of Chemistry, Univ Calif analogs is predicted to be significantly red-shifted compared to standard oxylu- Irvine, Irvine, CA, USA, 4Dept Chemistry, Univ Calif Irvine, Irvine, CA, ciferin. In order to explain shifts in the computed emission wavelengths of oxy- USA, 5Calit2 & Dept. Computer Science & Depts. Sociology and Statistics, luciferin analogs, energy decomposition analysis was performed to study the Univ Calif Irvine, Irvine, CA, USA. interaction between molecular orbitals from both oxyluciferin and substituents. Amyloid fibrils are locally ordered protein aggregates that self-assemble under a In order to design effective bioluminescent probes, effect of microenvironment variety of physiological and in vitro conditions. Their formation is of funda- will be considered along with substrate modifications. QM/MM calculations on mental interest due to its central role in Alzheimer’s disease, Type II diabetes, luciferin analogs and various mutant luciferase systems can provide useful in- and other human diseases. As the number of known amyloid fibril structures sights to explain substrate-protein interactions responsible for modulation of has grown, the need has arisen for a nomenclature for describing and classifying bioluminescence wavelength. Herein, we also report relative binding free en- fibril types, as well as a theoretical description of the physics that gives rise to the ergies of newly designed luciferin analogs with native firefly luciferase. self-assembly of these structures. We have developed a systematic nomenclature and coarse-graining methodology for describing the topology of fibrils and other 2775-Pos protein aggregates, along with a computational methodology for simulating In Silico Experiments as a Method to Compare Transportes Mechanism protein aggregation on the scale of thousands of monomers interacting on the Yuly E. Sa´nchez, Julian Aguilar. timescale of hours. Our coarse-graining methodology has mathematical under- Physics, Universidad Nacional de Colombia, Bogota´, Colombia. pinnings in both graph theory and statistical mechanics, providing the foundation Sodium-mediated solute transporters (SLC5 Family) translocate their sub- upon which we have built software capable of both quantitatively mapping fibril strates using sodium gradient as driving force. These transporters are found topology from atomistic structures, and simulating their formation from a set of in diverse fields of life, and have the unique ability to harness the energy stored free monomers via either thermodynamic sampling using Markov chain Monte in ionic gradients to accumulate small molecules essential for life, as well as Carlo (MCMC) sampling, or time-dependent sampling based in generalized Ar- expelling toxins and other harmful compounds. Unfortunately, there is no pre- rhenius kinetics. Our simulation strategy is capable of recapitulating the forma- cise experimental structural information on many of the sodium-dependent tion of all currently known amyloid fibril topologies found in the Protein Data transporters. Therefore, taking advantage of the versatility of the experiments Bank, as well as the formation kinetics of fibrils and oligomers. in Silico, it is possible to develop computational studies that aim to reveal and examine regions and / or individual amino acids in members of this family 2773-Pos that may be important for the binding or translocation of substrates and Na þ, In Silico Analysis of Amino Acid Substitutions Resulting from SNPs Asso- as well as elucidating the effect of amino acid substitutions on affinity, changes ciated with Inflammatory Bowel Disease in stoichiometry, and the activity of study proteins, which may provide guide- Constance Jeffery1, Chang Chen2. lines for the study of future therapeutic targets. In the present study, the changes 1Dept Biol Sci, Univ Illinois Chicago, Chicago, IL, USA, 2Dept of the global movements of the native form of three proteins of the SLC5 family Bioengineering, Univ Illinois Chicago, Chicago, IL, USA. in the presence and absence of the substrates translocating the transporter were Inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative compared from computational point of view, applying Normal Modes Analysis colitis, comprise a group of immune disorders with symptoms that can have sig- using the model of Anisotropic Network (ANM), which has proved to be an nificant negative impact on a patient’s quality of life. Over one million people useful computational method for predicting membrane protein dynamics and

BPJ 9470_9474 560a Wednesday, March 6, 2019 has been shown to be consistent with experimental data in terms of the forms than utilizing sequence or complete structural similarity since interface mim- and mechanisms of membrane proteins movements. icry is more common. We applied our interface-based approach to H. pylori, dominant species in gastric microbiome that greatly increases gastric cancer 2776-Pos risk in order to understand how they modulate host immunity and lead to tumor- Comparative Study of Flagellar and Cytoplasmic Dyneins igenesis. We found that its proteins interfere with the functioning of host Nayere Tajielyato, Joshua Alper, Emil Alexov. apoptosis pathway, cytokine and chemokine pathways, and also cell-cell Clemson University, Clemson, SC, USA. adhesions. Dyneins are minus-end microtubule motor proteins which can be grouped into two groups, cytoplasmic and flagellar dyneins. The cytoplasmic dynein is proc- 2779-Pos essive, i.e. ‘‘walks’’ along the microtubule; while flagellar dynein is involved in Computational Modelling of Triadin’s Contribution to Sudden Cardiac oscillatory beating of cilia and flagella. These functional differences led us to Death in Catecholaminergic Polymorphic Ventricular Tachycardia-5 study the similarity and dissimilarity of these two dynein proteins in binding (CPVT-5) to the microtubule. Here, we compare the binding of flagellar and cytoplasmic Laura Coonfield1, Aman Ullah2, W. Jonathan Lederer3, M. Saleet Jafri2. microtubule binding domain (MTBDs) to microtubule emphasizing on intrinsi- 1Drexel University, Philadelphia, PA, USA, 2Krasnow Institute, George a b cally disordered regions (IDRs) at the C-terminal of - and -tubulins. We Mason University, Fairfax, VA, USA, 3Center for Biomedical Engineering demonstrate that the interactions between cytoplasmic and flagellar MTBD and Technology, University of Maryland School of Medicine, Baltimore, with E-hooks are different, in terms of residues involvement and number of MD, USA. contacts at various MTBD-microtubule distances. Furthermore, the computa- Catecholaminergic ventricular tachycardia (CPVT) is a rare disorder typically tional modeling indicates that there is a correlation between number of contacts first appearing in young adults characterized by arrhythmia in response to between MTBD and E-hooks and the RMSF (conformational changes) of the beta-adrenergic stimulation and risk of sudden cardiac death. In this study, corresponding MTBD. we are leveraged the results of experimentally-derived data to develop a 2777-Pos computational model to simulate the pathophysiology behind CPVT-5, a vari- Computational Design of Druglike Allosteric Inhibitors of AXL and MET ation of CPVT caused by a mutation in the protein triadin. We adapted the Receptor Tyrosine Kinases model of cardiac excitation-contraction coupling developed by Wagner et Circ. Res. D.S. Dalafave, K. Sureshkumar. al. ( 111(4):402-414) according to our findings from the literature Physics, The Coll New Jersey, Ewing, NJ, USA. about the mutated form of triadin. To simulate the triadin mutation, the num- We report on the computational design of drug-like small molecules that could ber of calcium release units was reduced by 50%, calsequestrin was reduced allosterically bind both the AXL and MET kinases. Such binding would inhibit by 50%, and the transfer rate was increased up to 20%. To simulate beta the kinases and potentially promote apoptosis in cancer cells. AXL and MET adrenergic stimulation, L-type calcium current was increased by 40%, are receptor tyrosine kinases (RTKs) that play important roles in healthy cells. SERCA was increased by 50%, and RyR2 channel open probability was However, both AXL and MET are overexpressed in many cancers where they increased by 50%. The model was paced at 1-5 Hz for up to 40 seconds. have been linked to drug resistance and metastasis. Several inhibitors of AXL We successfully observed an arrhythmic pattern using our model. The transfer and MET are in various stages of clinical trials. However, generally, they bind rate between the network and junctional sarcoplasmic reticulum appears to be to the active site of the kinases. The structure of the active site is similar in a critical factor, as arrhythmia appears to occur at some point between a 0 to many kinases, making them difficult to target selectively. Molecules that 10% increase in the transfer rate. Overlapping action potential tracings were broadly inhibit several kinases may damage healthy tissue and lead to undesir- observed, which indicates an atypical arrhythmia. We successfully modeled able side effects in patients. Efforts in drug discovery have been shifting toward CPVT-5 using computational modeling. For future tests, we will examine inhibiting the allosteric sites of kinases. Allosteric sites differ among kinases, the data on a shorter time scale and determine more precisely which factors allowing for selective targeting. The small molecules designed here showed are responsible for the arrhythmia. These findings suggest possible areas for comparable or better drug-like properties than the known AXL and MET further investigation for basic science research to better understand the path- active-site inhibitors. Also, they had no implied toxic risks, showed good ophysiology of CPVT-5. bioavailability, and had promising overall drug potential. The molecules bonded AXL and MET allosterically with high binding affinities. In the pro- 2780-Pos cess, the kinase’s active site conformation changed, preventing the ATP bind- Integrated Computational Model of Lung Tissue Bioenergetics 1 2 3 4 ing. Our findings, together with previous experimental investigations of active- Xiao Zhang , Ranjan K. Dash , Anne Clough , Dexuan Xie , 5 1 site AXL and MET inhibitors, strongly imply that the molecules designed here Elizabeth Jacobs , Said Audi . 1Marquette Univ, Milwaukee, WI, USA, 2Dept BME, Med Coll Wisconsin, may be effective allosteric inhibitors of both kinases and may lead to the devel- 3 opment of novel, more effective cancer treatments. Milwaukee, WI, USA, Department of Mathematics, Statistics, and Computer Science, Marquette Univ, Milwaukee, WI, USA, 4Department of 2778-Pos Mathematical Sciences, University of Wisconsin-Milwaukee, Milwaukee, Prediction of Novel Host-Pathogen Interactions for Helicobacter Pylori WI, USA, 5Division of Pulmonary and Critical Care Medicine, Zablocki V.A. Through Interface Mimicry and their Implications to Gastric Cancer Medical Center, Milwaukee, WI, USA. Emine Guven Maiorov, Chung-Jung Tsai, Buyong Ma, Ruth Nussinov. Altered lung tissue bioenergetics plays a key role in the pathogenesis of lung Cancer and Inflammation Program, Leidos Biomedical Research Inc. diseases. A wealth of information exists regarding the bioenergetics processes Frederick, Frederick, MD, USA. in mitochondria isolated from rat lungs and from intact rat lungs under physi- About 20% of the cancer incidences worldwide have been estimated to be asso- ological and pathophysiological conditions. However, the interdependence of ciated with infections. There is a strong correlation of some pathogens with bioenergetics processes makes it difficult to quantify the impact of a change various cancer types, such as Helicobacter pylori with gastric cancer. However, in a single or multiple process(es) on overall lung tissue bioenergetics. Inte- the molecular mechanisms how they trigger cancer in the host is generally un- grated computational modeling provides a mechanistic and quantitative frame- known/incomplete. Pathogens interact with the host mainly through proteins. work for the bioenergetics data at different levels of biological organization. To subvert host defense, they mimic host proteins at different levels: sequence, Thus, the objective of this study was to develop and validate an integrated structure, motif and interface -binding surface-. Interface mimicry seems to be computational model of lung tissue bioenergetics. The model expands our the most common type. This similarity in interfaces permits pathogenic protein recently developed rat lung mitochondrial bioenergetics model by accounting to compete with host proteins to bind to a target protein, alter physiological for glucose uptake, glycolysis, and the pentose phosphate pathway. For mito- signaling and cause persistent infections, as well as cancer. Detection of chondrial region, kinetic parameters were fixed at those estimated in our recent host-pathogen interactions (HPIs) and mapping the re-wired HPI network - isolated rat lung mitochondria bioenergetics model. For cytosolic region, along with its structural details - is critical for in-depth understanding of the un- intrinsic parameters such as binding constants were determined based on pre- derlying pathogenesis mechanisms of infections, pathogen-triggered cancers, viously published enzyme kinetics data, whereas extrinsic parameters such as and developing efficient therapeutics. HPI data including structural details is maximal reaction and transport velocities were estimated by fitting the model far from complete and experimental characterization of the large-scale inter- solutions to published data from isolated rat lungs. The model was then vali- species interactions is challenging. Thus, computational tools are becoming dated by assessing its ability to predict existing experimental data not used increasingly important in enriching the HPI data, uncovering their complex for parameter estimation, including the relationships between lung nucleotides (bound) structures, and complementing the experiments. Here, we developed content, lung lactate production rate, and energy charge under different exper- the first computational approach to identify novel HPIs that utilizes solely inter- imental conditions. The model was used to gain novel insights on how lung tis- face mimicry. Employing interface mimicry is promising to identify more HPIs sue glycolytic rate is regulated by exogenous glucose and lactate, and assess

BPJ 9470_9474 Wednesday, March 6, 2019 561a differences in the bioenergetics of isolated mitochondria and those of mito- ment. Our work models the crowded nature of a cell through including chondria in intact lungs. The model provides a mechanistic and quantitative randomly-placed low-dielectric cavities in the surrounding environment. We framework for integrating available lung tissue bioenergetics data, and for also incorporate snapshots from molecular dynamics simulations in order to ac- testing novel hypotheses regarding the role of different bioenergetics processes count for how optimal binding properties depend on conformational changes in lung tissue bioenergetics. over time. Preliminary findings suggest that optimization of specific residues within static, crowded environments yields more favorable interactions than 2781-Pos optimization modeled in dilute solution. As a whole, this study investigates Cancer-Ml: Modeling Fitness of Unregulated RAS Mutants using Compu- the implications of how conformational dynamics and the crowded nature of tational Mutagenesis and Machine Learning the biological environment affects optimal binding properties, providing Majid Masso, Arnav Bansal, Akhil Gajjala, Preethi Prem, Iosif I. Vaisman. insight for future molecular design applications. School of Systems Biology, George Mason Univ, Manassas, VA, USA. 1. Lee, L.-P., and B. Tidor. 2001. Barstar is electrostatically optimized for tight RAS protein plays a pivotal role as an oncogene by participating in a wide binding to barnase. Nat. Struct. Mol. Biol. 8(1):73. range of signaling events including those linked to uncontrollable cell growth and cell division. Using artificial intelligence methods and computational muta- 2784-Pos genesis, we developed models for predicting functional effects of all single Roles of Nuclear Confinement, Excluded Volume, and Persistence on TAD missense mutations based on experimental data. The computational mutagen- Formations, Chromosome Territories, and Chromatin-Nuclear Envelope esis technique empirically quantifies structural changes to RAS upon mutation, Interactions while experimental fitness measurements correlate with mutant functional Samira Mali1, Alan Perez-Rathke1, Qiu Sun2, Gamze Gursoy3, Jie Liang1. changes relative to wild-type RAS. We implemented Random Forest classifica- 1University of Illinois at Chicago, Chicago, IL, USA, 2Shanghai Jiao Tong tion and Tree regression machine learning algorithms to train predictive models University, Shanghai, China, 3Yale University, New Haven, CT, USA. of RAS mutant functional changes, by representing the mutants using their Understanding genome organization is essential for gaining insight into respective sets of structural change attributes. Model performance was evalu- cellular function. Cis chromosome looping interactions, trans chromosome- ated using leave-one-out and tenfold cross-validation using the Weka suite of chromosome interactions, and chromosome-nuclear envelope interactions machine learning tools. To assess statistical significance, control experiments dictate the overall 3D nuclear architecture. Specifically, looping interactions were similarly performed on datasets with randomly shuffled functional effects. among different loci of a chromosome lead to formation of topologically asso- Classification models displayed a balanced accuracy rate (BAR) as high as ciating domains (TADs), whose disruptions are often associated with cellular 82%, with a corresponding Matthew’s correlation coefficient (MCC) of 0.65, malfunction; chromosome-chromosome interactions give rise to well-defined and an area under the ROC curve (AUC) of 0.91. Similarly, Tree regression chromosomal territories, which play important roles in gene regulation; r models reflected a Pearson’s correlation coefficient ( ) reaching 0.79, with a chromatin-nuclear envelope interactions position specific genes to the nuclear corresponding root mean squared error (RMSE) of 0.19. On the other hand, lamina and regulate their expression. While the importance of these structural all control datasets yielded performance values consistent with random guess- organizations is well-recognized, the origins of their physical formations are ing. The models we developed for predicting the mutational effects can be used less clear. Here we examine the roles of nuclear confinement, excluded volume, to better understand mechanisms of RAS protein involvement in oncogenesis and persistence length in determining these interactions. We generate large en- and provide useful insights to design future diagnostic and treatment modalities sembles (each of 10^4 chromatin chains of up to 2,000 structural units) through for cancer. deep sampling using fractal Monte Carlo. We characterize how the key factors 2782-Pos of nuclear confinement, excluded volume, and chromatin stiffness influences Anomalous Heat Dissipation of a Brownian Motor these interactions. Specifically, we discuss how TADs and chromosome Karina Mazzitello1, Yi Jiang2, Miguel Arizmendi1, Jose Iguain1, territories can arise at appropriate nuclear confinement and chromosome Fereydoon Family3. persistence length, and how biologically important chromosome-lamina inter- 1Physics, Universidad Nacional de Mar del Plata, Mar del Plata, Argentina, actions can be identified. We discuss our results in the context of several Hi-C 2Mathematics and Statistics, Georgia State University, Atlanta, GA, USA, studies. 3Physics, Emory University, Atlanta, GA, USA. Brownian motors behave completely different than macroscopic Carnot en- 2785-Pos gines because they are kept away from equilibrium by external fluctuations New Method to Determine the Effect of Dimerization on Protein Flexibility or temperature gradients. Although the studies on the Thermodynamics of these from Molecular Dynamics Simulation using Structural Hierarchy 1 2 1 motors have been mainly focused on efficiency, high efficiency may be Arghya Chakravorty , Jonathan Higham , Emil Alexov , 3,4 achieved with considerable heat dissipation losses. In this work the heat dissi- Richard H. Henchman . 1Department of Physics and Astronomy, Clemson University, Clemson, SC, pation from the motor to two thermal baths is numerically obtained. The pro- 2 cess of heat transport is related to the effective temperature through the USA, School of Human Genetics Unit and Institute of Genetics & Molecular Medicine, Edinburgh, United Kingdom, 3Manchester Institute of generalization of the fluctuation-dissipation theorem for systems far from equi- 4 librium. The effective temperature, defined as the comparison between induced Biotechnology, Manchester, United Kingdom, School of Chemistry, The and spontaneous fluctuations, can be greater than both reservoir temperatures or University of Manchester, Manchester, United Kingdom. can be between them. We found that the heat transport in Brownian motors is Macromolecular binding involves two major terms: enthalpy and entropy. Most different from Fourier’s Law and is instead proportional to the nth power of the efforts are focused on evaluating the enthalpy component because it can be difference between the effective temperature of the motor and the temperature computed simply from the atomic positions, which makes it convenient to of the respective bath, where the power n depends only on the temperature of compute interactions among binding partners or with the solvent phase. None- the bath. In this way an expression of anomalous heat transport is obtained as a theless, for numerous cases, the entropy component can be equally or even non-equilibrium measure. more important than the enthalpy. However, computing entropy for large bio- molecular systems is not trivial because the large number of atoms involved can 2783-Pos make it very time-intensive. In this work, we present a simple and fast method The Effect of the Cellular Environment and Conformational Dynamics on to compute the entropy of solvated protein-dimer systems directly from molec- Optimal Electrostatic Interactions Within the Barstar-Barnase Complex: ular dynamics simulations which builds on an existing theory of entropy of liq- A Computational Approach uids and isolated, flexible molecules. The total entropy is split into two terms: Alyssa J. Kranc1, Mala L. Radhakrishnan2. vibrational and topographical. The vibrational term quantifies the population of 1Wellesley College, Wellesley, MA, USA, 2Chemistry, Wellesley Coll, energy states accessible in a single multi-dimensional energy well in phase Wellesley, MA, USA. space and the topographical term depicts the effective number of these energy Understanding the driving factors that govern molecular recognition is critical wells. The vibrational term is derived using a hierarchical approach whereby when studying the efficacy of cellular processes and drug therapies. In partic- covariance matrices of forces and torques are evaluated at different length- ular, we are interested in the barstar-barnase system, a ribonuclease and inhib- scales associated with the macromolecular structure - whole-molecule, residue itor found in bacillius amyloquefacien, because this well-studied complex has a and united-atom levels. The topographical term is derived hierarchically from high binding affinity mediated predominantly by electrostatic interactions. As the distribution of dihedrals and molecular contacts to account for the confor- has been done previously1, we use a continuum electrostatics framework to mations of side chains and orientations of the residues in the complex. In total- optimize the charge distribution over specific barstar residues to maximize ity, the theory is scalable across the hierarchical levels and, therefore, averts the electrostatic affinity. However, our goal here is to understand how the optimal need to deal with large number of atoms simultaneously. The theory is tested on charge distributions depend on the crowded nature of the system’s environ- a large library of proteins to demonstrate its application in computing the

BPJ 9470_9474 562a Wednesday, March 6, 2019 entropic changes that occur due binding and is benchmarked against the con- GS-align for alignment of two glycan structures and their similarity measure- ventional Quasiharmonic method. ment. A PDB survey study of protein-glycan interactions in the PDB is also pre- sented for modeling of protein-glycan interactions and prediction of glycan 2786-Pos binding proteins. Computational Predictions of Drug-Protein Binding Kinetics with a Hybrid Molecular Dynamics, Brownian Dynamics, and Milestoning 2789-Pos Approach Physical Binding of the Smoke Carcinogen NNK Diazonium Ion Benjamin R. Jagger, Christopher T. Lee, J. Andrew McCammon, to the Human Tumor Suppressor Gene Tp53 Exon 5 Rommie E. Amaro. Christos Deligkaris. Univ Calif San Diego, La Jolla, CA, USA. Geology & Physics, University of Southern Indiana, Evansville, IN, USA. Molecular binding events are a fundamental aspect of cellular processes and are Among all cancer types, lung cancer results in the greatest number of cancer therefore of particular interest for the development of novel therapeutics. Simu- deaths and smoking tobacco is its main cause. The molecule N-nitrosamine lation based approaches that can estimate binding and unbinding kinetics are 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the most abundant desirable, as they can provide critical detail on the molecular level. We present lung carcinogen present in tobacco smoke and classified as a Group 1 carcin- recent developments to our hybrid molecular dynamics, Brownian dynamics, ogen according to the International Agency for Research on Cancer. NNK and milestoning software for estimating protein-ligand association and dissoci- can be metabolized from Cytochrome P450 enzymes to reactive intermediates, ation rates, called SEEKR. This approach reduces the simulation time required such as the NNK diazonium ion, which can damage DNA. NNK-DNA adducts to observe binding and unbinding events and also benefits from an embarrass- have been detected in tobacco users and are considered mutagenic. In order to ingly parallel implementation. We describe the effectiveness of the approach both (i) understand and (ii) prevent the process of NNK carcinogenesis, we for rank-ordering ligands by dissociation rates and binding free energies. We need to elucidate the role that the physical NNK diazonium ion-DNA interac- also describe improvements focused on making such calculations feasible for tions play. We used AutoDock’s physics-based free energy function and search larger systems of pharmaceutical relevance. This includes implementation algorithm (LGA) to predict the binding modes of NNK upon physical binding changes to reduce simulation cost and on-the-fly convergence monitoring. to TP53 exon 5. The free energy function was used to estimate the magnitude of the DNA-carcinogen physical interaction at a specific geometry of the carcin- 2787-Pos ogen, whereas the LGA was used to find the carcinogen geometry that mini- Toward Reducing HERG Affinities for Dat Inhibitors with a Combined mized the free energy. The free energy function includes the following Machine Learning and Molecular Modeling Approach terms: van der Waals, hydrogen bonding, electrostatic, desolvation and 1 2 1 3 Andrew D. Fant , Soren Wacker , Joslyn Jung , Jiqing Guo , torsional. We found that the NNK diazonium ion binds predominantly to the 1 3 1 2 Ara M. Abramyan , Henry J. Duff , Amy H. Newman , Sergei Y. Noskov , minor groove in various modes: type I sites have no NNK-TP53 hydrogen Lei Shi1. 1 bonds, type II-V sites include hydrogen bonds with NNK participating as the National Institute on Drug Abuse, National Institutes of Health, Baltimore, acceptor and Guanine’s exocyclic amino group as the donor. We will present MD, USA, 2Department of Biological Sciences, University of Calgary, 3 these and additional results and discuss their implication for TP53 damage Calgary, AB, Canada, Department of Cardiac Sciences, University of and mutations. Calgary, Calgary, AB, Canada. The human Ether-a`-go-go-Related (hERG) potassium channel plays a critical 2790-Pos role in repolarization of cardiac action potentials. hERG blockade by drugs Transient Anomalous Subdiffusion of DNA-Binding Species in the Nu- may result in abnormal heart rhythms such as the potentially fatal Torsades cleus: the Final Model de Pointes. Experimental methods to identify compounds with high hERG af- Michael J. Saxton. finities are time-consuming and costly. Compulsory hERG screening by the Dept Biochem/Molec Med, Univ Calif Davis, Davis, CA, USA. FDA has resulted in an increase of publicly available hERG binding data. Single-particle tracking experiments have measured the distribution of dwell Utilizing machine learning algorithms, we constructed structure activity rela- times of DNA-binding species - including CRISPR-Cas9, TetR, and LacI - tionship regression models to predict hERG binding affinity. The models diffusing in living cells. The observed truncated power law distribution has were trained on molecules with known hERG affinities from the ChEMBL two major implications. First, it is inconsistent with a Gaussian distribution database. We filtered out erroneous points among the low affinity data that of binding energies. Second, it leads to transient anomalous subdiffusion. were found to affect prediction accuracy. We compared the performance of Monte Carlo simulations are used to characterize the time-dependent diffusion the eXtreme gradient boosting (XGBoost) and the random forest (RF) algo- coefficient D(t) and to relate the time dependence to the dwell time distribution. rithms against several test sets and found that XGBoost slightly outperformed The time dependence is described in terms of the anomalous diffusion expo- RF. The XGBoost models were then used to predict the hERG affinities of a nent, the limits D(0) and D(8), and the crossover time between anomalous sub- series of dopamine transporter (DAT) inhibitors. The predictions showed diffusion at short times and normal diffusion at long times. The trap good correlation with experimentally measured affinities. In addition, we im- concentration has a major effect on the anomalous diffusion exponent, but plemented a machine-learning based classifier to identify compounds which much of this effect is captured in D(0) and D(N), which are easily calculated appear to have extremely low affinity for hERG and are therefore unlikely to from the truncated power-law parameters, and are important quantities to mea- be well-predicted by the consensus regression model. sure in experiments. We examine how the underlying dwell time distribution is In coordination with ligand-based approaches, we have used a protein broadened by the statistics of escape to give the ideal observed distribution of structure-based method to characterize binding modes of these ligands against dwell times. The actual observed distribution may be modified by experimental both our hDAT models and the recent cryo-EM structure of hERG, and to iden- constraints. The simplest interpretation of the model is that the dwell times are tify clues to improve hDAT affinities while reducing hERG affinities. Together, actual binding times to DNA. One alternative is that the dwell times are the pe- we established a combined ligand-based and protein structure-based computa- riods of 1D diffusion on DNA in the standard combination of 1D and 3D search. tional framework, which is integrated with experimental approaches, to predict Both binding and obstruction affect diffusion. In the absence of a consensus hERG affinities. We specifically applied it to facilitate the rational optimization model of chromatin geometry, obstruction effects ought to be characterized of lead compounds targeting DAT for the treatment of psychostimulant use by experiment as well as by modeling. Suggested controls for obstruction are disorders. GFP as a calibration standard among laboratories and cell types, and the DNA-binding species with the binding site unobtrusively inactivated. (Sup- 2788-Pos ported in part by NIH grant GM038133) Glycan Structure Modeling and Simulation Wonpil Im. 2791-Pos BioS and BioE, Lehigh Univ, Bethlehem, PA, USA. Dynamical Network Analysis of Protein:RNA Complexes Made Easy I would like to share our ongoing efforts toward computational (structural) gly- Marcelo Cardoso dos Reis Melo1, Rafael C. Bernardi2, cobiology in terms of various tools that we have developed and are available in Zaida Luthey-Schulten3. GlycanStructure.ORG (http://www.glycanstructure.org)and in CHARMM- 1Ctr Biophys/Comp Bio, Univ Illinois Urbana Champaign, Urbana, IL, USA, GUI (http://www.charmm-gui.org): Glycan Reader for automatic detection 2Beckman, Univ Illinois Urbana Champaign, Urbana, IL, USA, 3Dept and annotation of carbohydrates and their glycosidic linkages and chemical Chemistry, Univ Illinois Urbana Champaign, Urbana, IL, USA. modifications in PDB files, Glycan Fragment DB for carbohydrate fragment Molecular interactions are essential for regulation of cellular processes, from structures in the PDB, torsion angle distributions of specific glycosidic linkages the formation of multi-protein complexes, to binding of ligands in active sites, of searched structures, and their clustered structures, Glycan Modeler for and allosteric activation of enzymes. Identifying the essential residues and mo- modeling of N-/O-glycan or glycan-only structures from its sequence, and lecular features that regulate such interactions is paramount for understanding

BPJ 9470_9474 Wednesday, March 6, 2019 563a the biochemical process in question, allowing for suppression of a reaction ulations of nucleosomes. For every simulation we present Amber formatted through drug interventions, or optimization of a chemical process using bio- topology and coordinate input files, NAMD formatted output, log and trajectory engineered molecules. In order to identify important residues and information files, and RMSD and DNA helical parameter analysis data. Closely related sim- pathways within molecular complexes, the Dynamical Network Analysis ulations are grouped together. A summary analysis is provided for each group, method was developed and has since been broadly applied in the literature. and a meta analysis of all simulations is provided. All data can be navigated in a In this work we provide an evolution of the method, application and interface, file browser or iBIOMES Lite web browser format or downloaded with com- with all data processing and analysis, including visualization, conducted mand line tools. Results indicate that the workflow and simulation protocol through a Jupyter notebook. It provides an optimized and parallel generalized developed are robust, that DNA sequence can affect the structure and dynamics correlation implementation that is linear with respect to the number of nodes in of nucleosomal DNA at some locations but not others, and that sequence dif- the system, automatic detection of solvent and ion residues stably bound to the ferences can be observed in 10’s of nanoseconds. Our TMB-iBIOMES server target molecule, and subsequent community clustering, calculation of between- is: http://dna.engr.latech.edu/ibiomes. ness of contacts, and determination optimal paths for the entire system.The new implementation was applied to the leucine tRNA synthetase complexed with its 2794-Pos cognate tRNA and adenylate. Our analysis was capable of identifying experi- Implementation of the FDA CiPA Qnet Model for Drug Safety Screening mentally verified identity elements throughout the tRNA:protein interface, as Which Increases Efficiency 25 Fold Leigh Korbel1, Glenna Bett2, Randall Rasmusson2. well as essential catalytic residues in the active site. The hypothesis that a mag- 1 2 nesium ion is required for the charging of the tRNA was investigated, and we Cytocybernetics, Buffalo, NY, USA, Dept Physiol/Biophys, State Univ observed significant destabilization of communities and contacts in the active New York Buffalo, Buffalo, NY, USA. In silico site, indicating the ion does not create a favorable environment for the reaction. computer modeling to assess drug proarrhythmia risk is a key compo- in vitro This enhanced and updated methodology will provide the community with an nent of the Comprehensive Proarrhythmia Assay (CiPA) initiative. The intuitive and interactive interface, that can be easily applied to large macro- recently-published qNet metric shows promise for correctly determining drug- molecular complexes. induced Torsades-de-Pointes (TdP) risk (high/intermediate/low) of drugs using an in silico model of the human ventricular action potential (AP). The model 2792-Pos combines simulations of multiple ionic channels (including O’Hara Rudy dy- Megadock-Web: An Integrated Database of High-Throughput Structure- namic (ORd) drug-binding hERG model) and in vitro pharmacology data. Us- Based Protein-Protein Interaction Predictions ing this model, TdP risk can be determined via qNet for the 12 CiPA training Masahito Ohue1, Takanori Hayashi1, Yuri Matsuzaki2, and 16 validation compounds. The FDA made an open source R script of this Keisuke Yanagisawa1, Yutaka Akiyama1. model available (github). This program can be used to calculate both the qNet 1School of Computing, Tokyo Institute of Technology, Tokyo, Japan, metric and its uncertainty quantification. Simulating the AP and determining 2ACLS, Tokyo Institute of Technology, Tokyo, Japan. the uncertainty quantification is a computationally-intensive task, not well- Protein-protein interactions (PPIs) play several roles in living cells, and compu- suited for R. We translated the FDA R script to C and parallelized the code us- tational PPI prediction is a major focus of many researchers. The three- ing both OpenMP (shared memory parallelism) and MPI (distributed memory dimensional (3D) structure and binding surface are important for the design parallelism). The serial C code produces the same results as the R script, but of PPI inhibitors. Therefore, rigid body protein-protein docking calculations with a five-fold increase in efficiency. Running the OpenMP C code on a PC for two protein structures are expected to allow elucidation of PPIs different with an eight core processor results in 25-fold increase in efficiency vs. R from known complexes in terms of 3D structures because known PPI informa- script. The MPI version of the code can be run on a high performance tion is not explicitly required. We have developed rapid PPI prediction software computing resource and results in an even greater increase, depending on the based on protein-protein docking, called MEGADOCK. In order to fully utilize number of processors employed. Using the R script to generate a simulation the benefits of computational PPI predictions, it is necessary to construct a of the effect of a single drug on the AP using 500 samples takes 16.5 days using comprehensive database to gather prediction results and their predicted 3D R script. With the parallel C code, it takes just 15.3 Hours. This reduction in complex structures and to make them easily accessible. Although several data- computational time required will significantly reduce the time taken to calcu- bases exist that provide predicted PPIs, the previous databases do not contain a late the arrhythmogenic profile of any compound. sufficient number of entries for the purpose of discovering novel PPIs. In this study, we constructed an integrated database of MEGADOCK PPI predictions, 2795-Pos named MEGADOCK-Web. MEGADOCK-Web provides more than 10 times TCPRO: An In-Silico Risk Assessment Tool for Biotherapeutic Protein the number of PPI predictions than previous databases and enables users to Immunogenicity conduct PPI predictions that cannot be found in conventional PPI prediction da- Osman N. Yogurtcu, Zuben E. Sauna, Joseph R. McGill, tabases. In MEGADOCK-Web, there are 7,528 protein chains and 28,331,628 Million A. Tegenge, Hong Yang. predicted PPIs from all possible combinations of those proteins. Each protein CBER, FDA, White Oak, MD, USA. structure is annotated with PDB ID, chain ID, UniProt AC, related KEGG As immune responses to biotherapeutic proteins can impact drug efficacy pathway IDs, and known PPI pairs. Additionally, MEGADOCK-Web provides and safety, immunogenicity assessments are required prior to licensure of four powerful functions: 1) searching precalculated PPI predictions, 2) the product. There is an unmet need for non-clinical assessments of immu- providing annotations for each predicted protein pair with an experimentally nogenicity so that risks can be identified at early stages in the drug develop- known PPI, 3) visualizing candidates that may interact with the query protein ment process. Most immune responses to biotherapeutic proteins involve the on biochemical pathways, and 4) visualizing predicted complex structures development of anti-drug antibodies (ADAs) and the response is T-cell through a 3D molecular viewer. MEGADOCK-Web is freely available for dependent. Ex-vivo assays using blood cells from patients or normal donors use at http://www.bi.cs.titech.ac.jp/megadock-web/. that monitor T-cell proliferation are routinely used to assess immunogenicity risk. Such assays can be expensive and time-consuming. In addition, T-cell 2793-Pos proliferation requires presentation of the immunogenic epitope by major his- TMB-iBIOMES: A Database of All Atom Simulation and Analysis for tocompatibility complex class II (MHCII) proteins on antigen presenting Nucleosomes cells. The MHC proteins are the most diverse in the . Ran Sun, Zilong Li, Thomas Connor Bishop. Thus, obtaining donor cells from subjects that reflect the distribution of Louisiana Tech, Ruston, LA, USA. the different MHCII proteins in the human population can be challenging. Nucleosomes are the fundamental building blocks of chromatin, the biomate- Moreover, the frequencies of MHCII proteins are very different in human rial that houses the genome in all higher organisms. A nucleosome consist of subpopulations and understanding the potential immunogenicity risk in 145-147 base pairs of double stranded DNA wrapped 1.7 times around eight different sub-populations would thus require the generation of multiple data- histones. There are approximately 100 atomic resolution structures of the sets and complex subject recruitment. We develop TCPro, an in-silico tool nucleosome available from the protein data bank. Collectively they explore his- that predicts T-cell proliferation for cells obtained from an individual sub- tone mutations, species variations, binding of drugs and ionic effects but only a ject. By using TCPro, we can generate in-silico pools of subjects based on few sequences of DNA. Given a four-letter code (A, C, G, T) there are on the MHCII frequencies and estimate immunogenicity risk for different popula- order of 4147 1088 possible sequences of DNA that can form a nucleosome. tions. The tool provides a rapid and inexpensive initial screen for a new bio- Exhaustive studies are not possible. Here we introduce a database containing therapeutic under development. Also, it can be used to determine the over 20 microseconds of all atom molecular dynamics simulations for over potential immunogenicity risk of new sequences introduced while bioengi- 500 different realizations of the nucleosome. Our iBIOMES-Lite based data- neering proteins. *This abstract reflects the views of the authors and should base serves as a reference for comparative studies and future on-demand sim- not be construed to represent FDA’s views or policies.

BPJ 9470_9474 564a Wednesday, March 6, 2019

Posters: Optical Spectroscopy: CD, UV-VIS, properties of the cortical parts of femur bone tissues using Fourier transform infrared microspectroscopy (FTIRM) and Atomic force microscopy (AFM). Vibrational, Fluorescence Carbamazepine was administered by intraperitoneally (50 mg/kg/day) to 6- month male Wistar rats for 5 weeks in drug-treated group. According to 2796-Pos the FTIR studies, the mineral to matrix ratio was significantly decreased, car- A Revised Coarse-Grained Model of Circular Dichroism of Proteins bonate/phosphate ratio was increased, collagen crosslinks were decreased and Mauricio D. Carbajal-Tinoco1, Carmen Giovana Granados-Ramı´rez2. mineral crystallinity was significantly increased in drug-treated group 1 2 Dept Physics, Cinvestav IPN, Mexico City, Mexico, Universidad Distrital compared to the control. Raw bone cross-sections from cortical part of femur Francisco Jose de Caldas, Bogota´, Colombia. tissues were used for AFM studies. Root mean square (RMS) and arithmetic Circular dichroism (CD) spectroscopy is a powerful technique to study the average height (Ra) give information about surface roughness of the samples. secondary structure of certain biomolecules such as proteins. Numerous ap- In the current study, RMS and Ra values of drug-treated healthy group were proaches are based on the analysis of CD spectra to elucidate the percentage significantly lower than the values of control group, implying a decreased of structural components. Some of the most successful methods of this type, bone surface roughness in Carbamazepine-treated group. AFM and FTIRM e.g. BeStSel, take into account the morphological and spectral diversity of studies revealed that Carbamazepine treatment decreased the bone strength the distinct secondary structures. Here, we present a different kind of in healthy rats. This study will provide new strategies to prevent the side- approach that is based on the classical theory of optical activity. From the effects of anti-epileptic drugs on bone metabolism in epilepsy and other analysis of synchrotron radiation CD spectra, we estimate an effective com- neurological disease treatments. plex polarizability (ECP) per residue of each type of secondary structure, which can be defined by a similar classification to the one proposed at 2799-Pos BeStSel. The inputs of our model are the ECPs and the protein structure ob- Environment-Dependent Photophysics of an Asymmetrical Cyanine tained from the PDB, and both allow us to calculate the corresponding CD Nikita Kumari, Marcia Levitus. spectrum. The approximated spectra are in good agreement with their exper- School of Molecular Sciences and The Biodesign Institute, Arizona State imental counterparts. University, Tempe, AZ, USA. Thus, this model could be used to describe conformational changes of a given Cyanine dyes are amongst the most commonly used fluorescent probes in the protein. biophysical research. The photophysics of these dyes and their interaction with DNA vary significantly based on the hydrophobic properties, structure 2797-Pos etc. This study investigates fluorescent properties of a relatively new class of Spectral Assignment of Lysozyme Collective Vibrations asymmetrical red cyanine dyes from Dyomics (Dy630, Dy632), which have Yanting Deng1, Jeffrey Mckinney1, Tod Romo2, Alan Grossfield2, similar spectral properties as Cy5, a widely used red cyanine dye. A compara- Andrea Markelz1. tive study of quantum yield and lifetime was done between Dy630 and Cy5 to 1 2 Physics, State Univ of New York-Buffalo, Buffalo, NY, USA, Department see the potential of this dye as a protein-induced fluorescent enhancement of Biochemistry and Biophysics, University of Rochester Medical Center, (PIFE) probe. The effect of viscosity on cis-trans isomerization was more pro- Rochester, NY, USA. nounced for Dy630 than Cy5, displaying potential to be a better red PIFE probe. Global structural vibrations at terahertz (THz) frequencies have been associated Concentration-dependent fluorescence enhancement studies were done with protein function and allosteric control. A chief obstacle to utilizing this showing that these dyes interact with nucleobases (dNMPs) more strongly control mechanism has been measurement of specific motions. Recently it than Cy5. To investigate these interactions further, dye covalently attached was shown that while the vibrational density of states, and isotropic absorption to the DNA was observed using single-molecule fluorescence. Two fluores- spectra are broad and featureless, collective vibrations can be isolated based on cence intensity states for Dy630 and Dy632 were observed, whereas one single their directionality using aligned samples (realized with protein crystals) and state was observed for Cy5. This could be explained by a stronger pi- pi inter- anisotropic THz microscopy [1]. However the assignment of resonant bands action possible due to the presence of additional aromatic ring in Dy dyes, giv- to specific structural motions was complicated by the high symmetry of the ing rise to two modes of dye-DNA interaction. The single-molecule analysis tetragonal crystals used, and the slow experimental method. To structurally gives a short-lived (0.87 s), strongly interacting component and a long-lived map the vibrations of the chicken egg white lysozyme (CEWL) we measure (3.6 s), weakly interacting component for Dy630. The comparison of dwell anisotropic absorption of triclinic crystals using our new technique: ideal polar- time between the sulfonated (Dy632) and non-sulfonated (Dy630) dye shows ization varying anisotropic THz microscopy (IPV-ATM). The low symmetry that the non-sulfonated dye is more ‘‘sticky’’ on DNA, which is supported triclinic crystals provide absolute protein orientation, and the near field IPV- by the lifetime measurements. Therefore, a thorough investigation of such in- ATM rapidly measures broadband terahertz linear dichroism of the microcrys- teractions between biomolecule and probe is needed before performing single- tals. All measurements were performed at room temperature under 100% hu- molecule experiments like PIFE and Foster resonance energy transfer (FRET) midity conditions. The unit cell parameters of triclinic lysozyme nitrate to stay away from artifacts. crystals, a = 28.5A, b = 32.7A, c = 35.1A, a = 88.2, b = 108.9, g = 111.9 , belonging to the P1 space group, were determined by X-ray diffraction 2800-Pos before and after THz measurements. The intramolecular vibrational absorbance Novel Fluorescence Tool for Measuring Protein Binding Kinetics and En- of the triclinic crystals has a more complex polarization dependence than the ergy Transfer over Full Fluorescence Spectral Ranges higher symmetry tetragonal crystals, as expected. While the tetragonal crystals Karen E. Steege Gall, Alex Siemiarczuk. 1 1 have two strong bands at 45cm and 55cm , the triclinic crystals have a se- Fluorescence Division, Horiba Scientific, Piscataway, NJ, USA. 1 1 ries of narrow bands between 40 and 60cm and a prominent band at 30cm . For decades, fluorescence spectroscopy has been used to measure protein bind- We compare the measured spectra to normal mode ensemble averaged calcula- ing kinetics with single point intensities over time. The addition of 1,8-anilino- tions to assign the observed resonances, and isolating which collective motions naphthalene sulfonate (ANS) to bovine serum albumin (BSA) protein is a impact the catalytic site. common experiment to demonstrate protein-small molecule binding. Typically, [1] K. A. Niessen et al., Biophysical Journal 112, 933 (2017). the demonstration detects the emission intensity of ANS over a few seconds af- ter adding BSA and ANS together. In other demonstrations, a fluorometer scans 2798-Pos the whole steady state emission spectrum of the solution at different concentra- The Effects of Carbamazepine, an Anti-Epileptic Drug, on Structure and tions of ANS, using one concentration at a time. We use a novel fluorometer Surface Roughness of Healthy Rat Bone Tissues: An FTIR Microspectro- with CCD detection to measure the entire fluorescence emission spectrum of scopy and AFM Microscopy Study both BSA and ANS molecules simultaneously and dynamically as the binding Sebnem Garip, Feride Severcan. occurs over time. The fluorescence spectrum measured in time-based mode Faculty of Medicine, Altinbas University, Istanbul, Turkey. shows that ANS and BSA not only bind, but also undergo energy transfer, Carbamazepine (CBZ) which has been one of the most widely used and not seen by single wavelength fluorescence kinetic methods previously em- effective antiepileptic drugs for epilepsy treatment, is also used for the treat- ployed. Denatured protein does not show the binding of ANS at all over the ment of neuropsychiatric disorders, neuropathy and depression in recent same time frame, which demonstrates that ANS does bind to hydrophobic cav- years. In literature, it was not clarified that the effects on bone metabolism ities in BSA that are not available once the protein has been degraded. This new in epileptic patients are due to anti-epileptic drug treatment or also from technique in fluorescence spectroscopy can be employed to other protein bind- epileptic seizures. In the current study, we aimed to clarify the effects of Car- ing studies as a very powerful tool for energy transfer elucidation and binding bamazepine on healthy bones by investigating the structural and mechanical kinetics studies.

BPJ 9475_9479 Wednesday, March 6, 2019 565a

2801-Pos Understanding the function and metabolism of cellular lipids is crucial for Twin-FRET: A New Molecular Ruler for Biomolecules characterization and treating various diseases. Several methods for studying Sankar Jana1, Marta Diez-Castellnou2, Euan R. Kay2, Carlos Penedo3. the distribution, dynamics, and function of lipids are developed, e.g., fluores- 1School of Biology, University of St Andrews, St Andrews, United Kingdom, cence resonance energy transfer (FRET), super-resolution and electron mi- 2EaStCHEM School of Chemistry, University of St Andrews, St Andrews, croscopy. Nevertheless, a label-free approach for probing lipids in the United Kingdom, 3School of Biology, School of Physics and Astronomy, living, healthy or diseased cells is still lacking. We show here the direct, University of St Andrews, St Andrews, United Kingdom. label-free probing of lipid composition in the Leishmania-infected macro- Conformational changes in biomolecules underpin all biological processes and phage cells in vitro by surface-enhanced Raman scattering (SERS).1 SERS being able to quantify these structural changes in solution is crucial to under- is used as a sensitive tool to study the structure and function of macromole- stand biological function. By carefully positioning two fluorophores within cules inside the infected cells, as it provides comprehensive information on the biomolecule, it is possible to use fluorescence resonance energy transfer the molecules in the nm-scale proximity of gold nanoparticles that are used (FRET) as a molecular ruler to measure the desired distance. In current as nanoprobes. As revealed by electron microscopy, the gold nanoparticles FRET assays, the biomolecule needs to be labelled with two different chemical access parasitophorous vacuoles and parasites inside the infected macrophage fluorophores acting as a donor-acceptor FRET pair. Incorporation of these two cells. Spectra acquired from the parasitophorous vacuoles and the parasite it- chemically different species at specific positions within the biomolecule is self provide information on the distribution of the lipid molecules that are challenging due to limited chemical labelling strategies. Here, we present a important for the virulence of Leishmania, e.g., cholesterol and ergosterol.2 radically different strategy for measuring distances in biomolecules. We have Spectra of proteophosphoglycans, an important hallmark of the infection, developed the concept of TWIN-FRET which removes the need for two are detected inside the different compartments. In conclusions, SERS enables different fluorophores attached to the biomolecule by chemically encoding us to study the interaction and distribution of molecules in Leishmania in- the FRET pair within the structure of the fluorophore itself. We have designed fected cells and similar disease models with disturbed lipid metabolism. 1. and synthesised a fluorescent molecule to prove this concept. The fluorophore Zivanovi c V et al., Anal. Chem., 2018, 90, 8154-8161. 2. Subha S, PLoS Path- (FH) has an acid-base equilibrium with a ground state pKa8.9. We have de- ogens, 2011, 7, 9. rivatized the fluorophore to its succinimide ester, and used this derivative, to label a duplex DNA with two molecules of the same fluorophore at specific po- 2804-Pos sitions. Our results demonstrate the transfer of non-radiative energy from the The Fluorescence Lifetime of Bound NADH: Clues from the Phasor Plots neutral (FH) to the anionic (F) state of the fluorescent molecule. We further Suman Ranjit, Leonel S. Malacrida, Enrico Gratton. demonstrate the use of TWIN-FRET to measure nanometer-size distances Dept Biomed Eng, Univ Calif Irvine, Irvine, CA, USA. þ within the DNA duplex, and we obtained distance values similar to those ob- Changes in metabolism in cells are often studied by the ratio of NAD to tained using a conventional FRET pair (Alexa488-Cy3). By removing the NADH. This ratio is correlated to free and enzyme bound NADH, which can need to introduce two different chemical structures within the biomolecule, be used as a measurement of metabolic states in cells using fluorescence life- we greatly simplified the methodology to measure nanometer-size distances time imaging (FLIM). Binding of NADH to the enzyme causes separation be- in biomolecules. We expect this technique to be widely used in structural tween nicotinamide and adenine moieties of Nicotinamide adenine and biophysical studies of nucleic acids, proteins and interactions between dinucleotide (NADH) from their collapsed structure in solution and results in them. increase of fluorescence quantum yield and lifetime. The extent of increase in fluorescence lifetime is dependent on the apoenzyme and presence of auxil- 2802-Pos iary ions. Reports from past studies show distinctive discrepancies in calcula- Evaluation of Cell Culture Media using Absorption and Transmission tion of the bound NADH lifetime, often related to complications in sample Fluorescence Excitation Emission Matrix (A-Teem)Spectroscopy preparation and limitations in data acquisition. In this work, we show that in Marinella Sandros1, Boqian Yang1, Karoly Csatorday1, Adam Gilmore1, presence of oxalic ion, proper preparation of lactate dehydrogenase (LDH) Alvin Togonon1, John Bobiak2. bound NADH has a lifetime of 3.4 ns and is positioned on the universal 1Horiba Scientific, Piscataway, NJ, USA, 2Bristol-Myers Squibb, Devens, semi-circle of the phasor plot, representing mono-exponential lifetime. MA, USA. Improper preparation results in a mixture of species, with phasor positions in- With regards to protein production using mammalian cell culture, it is impor- side the universal semi-circle. Measurement in cellular environment show tant to utilize the correct cell culture media for use in production processes. similar trend and a linear trajectory between free NADH and cellular NADH Cell culture media is usually prepared as an aqueous solution and should pro- components, which when extrapolated to the universal semi-circle shows a life- vide everything cells need for optimal growth as well as product yield and time of 3.4 ns at the crossing point. These results suggests that 3.4 ns can be quality. This could include amino acids, glucose, vitamins and various other used as a bound NADH lifetime and phasor approach can correlate lifetime nutrients. The exact composition and concentrations of components found contributions to concentration fractions of free and bound species. The effects in a given type of cell culture media vary depending on the unique needs of different types of FLIM acquisitions are also discussed in context. This work of a given cell line and are usually considered proprietary information by their is supported by NIH grant P41-GM103540. manufacturers. In any given bioreactor process, it is important to identify the proper type of cell culture media and its quality because even subtle variations 2805-Pos in composition could have a noticeable impact in the growth rate of the cell Investigation of the Structural Effects of Radiotherapy Dose Rate on Rat culture and its yield. Therefore, the composition and quality of cell culture Lung Tissue: An FTIR Imaging Study media in bioreactors must be tightly screened in order to maintain an optimal Ipek Ozyurt1, Sebnem Garip1, Fatma Kucuk Baloglu2, Faruk Zorlu3, bioreactor process. As a result, methods of identifying and analyzing the qual- Feride Severcan1. 1Faculty of Medicine, Altinbas University, Istanbul, Turkey, 2Biology, ity of cell culture media has become an important focus. Existing solutions 3 include methods such as chromatographic separations and mass spectrometry. Giresun University, Giresun, Turkey, Radiation Oncology, Hacettepe However, these methods are generally considered to be too expensive and University, Ankara, Turkey. time consuming for routine analysis of cell culture media samples. As a result, Radiotherapy is widely used to cure cancer. During treatment, ionizing radia- the industry has begun to turn to spectroscopic methods such as fluorescence tion is applied by a linear accelerator to the area that is being treated. During for cell culture media analysis due to rapid testing, minimal sample handling, this process normal cells can also be damaged by radiotherapy. The lung is and relatively lower cost when compared to other methods. One method of one of the most sensitive organs to ionizing radiation. Radiation-induced particular interest is Absorption and Transmission Fluorescence Excitation lung disease (RILD) is regarded as the result of an abnormal healing response Emission Matrix (A-TEEM) Spectroscopy. Here we evaluated if we can and it can lead to pulmonary fibrosis. Dose amount of radiation plays a key role differentiate different classes of commercially available cell culture media on healing process and prevalence of side effects on normal tissues. The aim of and assess their stability over time. this study is to evaluate the effect of dose rate on rat lung tissue by using FTIR microspectroscopy. A total of 25 animals were randomly devided into three 2803-Pos groups. Group 1: control group, sham irradiated. Group 2: receiving a single Characterization of Lipids in Leishmania Infected Cells by SERS Micro- dose of 12 Gy in DR of 300 monitor unit (MU) / min. Group 3; receiving a sin- scopy gle dose of 12 Gy in DR of 600 MU/min. At the 6th and 16th week of the RT, Vesna Zivanovic1, Geo Semini2, Michael Laue2, Daniela Drescher1, animals from each group were sacrificed for evaluation. For this purpose FTIR Toni Aebischer2, Janina Kneipp1. microspectroscopy was used to detect the dose rate-induced changes in the con- 1Dept Chemistry, Humboldt-Univ zu Berlin, Berlin, Germany, 2Robert Koch- centration of biomolecules such as lipids and proteins and protein/ lipid and Institut, Berlin, Germany. saturated/ unsaturated lipid ratios. These results indicated a remarkable

BPJ 9475_9479 566a Wednesday, March 6, 2019 increase in total lipid amount in both group 2 and 3 depending on dose rate. The rapid and high-resolution volume imaging of biological samples. The Open- current study clearly revealed the power of FTIR microspectroscopy in the pre- SPIM framework [1] was introduced as an open access blueprint to aid cise determination of dose rate induced biomolecular changes in rat lung tissue. wide adoption of light sheet methods. Because of the inherent trade off in field of view (FOV) and resolution when generating the exciting light sheet, the 2806-Pos original OpenSPIM has a limited FOV and modest axial resolution. Building The Disruption of Beta Sheets in Amyloidogenic Sequences by Gly-Gly-Ala on OpenSPIM and existing work on scalable light sheets [2], we introduce an 1 2 1 Sarah A. Petty , Andrew T. Mullin , Sam A. Michelhaugh , open source Extended Field of View Single Plane Illumination Microscope Benjamin R. Fitzgerald1. 1 2 (eFOV-SPIM). eFOV-SPIM has an adjustable field-of-view that enables Dept Chemistry, College of the Holy Cross, Worcester, MA, USA, Dept both high-resolution imaging of in vivo dynamics and large cleared tissue Biology, College of the Holy Cross, Worcester, MA, USA. samples on the same platform. eFOV-SPIM requires minor modification to Alpha-synuclein is an intrinsically disordered neuronal protein whose misfold- the OpenSPIM excitation pathway combined with dedicated in vivo and ing and aggregation is linked to the onset of Parkinson’s disease. Deletion of cleared tissue sample chambers and sample mounts. Here we present the op- residues 67-71 (GGAVV) has been seen to prevent aggregation of alpha- tical characterization of eFOV-SPIM system as well as experimental demon- synuclein, suggesting a potential role of this sequence in nucleating the aggre- stration of single-cell tracking in Danio rerio and near-isotropic imaging of gation of the full protein. We have established that certain short peptides based optical cleared tissue. on this deleted sequence have the ability to aggregate in isolation, whereas 1. P. G. Pitrone, J. Schindelin, L. Stuyvenberg, S. Preibisch, M. Weber, K. W. others remain monomeric and unstructured as in the full-length protein. In Eliceiri, J. Huisken, and P. Tomancak, ‘‘Openspim: an open-access light-sheet this work, we examine the ability of the disordered peptide, GGA to reverse microscopy platform,’’ nature methods 10, 598 (2013). 2. K. M. Dean and R. the aggregation of the beta-sheet forming peptide AVV under various condi- Fiolka, ‘‘Uniform and scalable light-sheets generated by extended focusing,’’ tions. Further, we will discuss the scope of this structural disruption by GGA Opt. express 22, 26141-26152 (2014). based on our experiments with several other beta-sheet peptides derived from alpha-synuclein including the structural similar AVA and other more var- 2809-Pos ied peptide sequences. Rotational and Translational Diffusion in Concentrated Ficoll Solutions Peptides were synthesized using solid phase Fmoc chemistry and were capped Elton Jhamba1, Hacene Boukari2. at both termini to eliminate charge effects. Fourier transform infrared spec- 1Health Sciences, University of New Mexico, Albuquerque, NM, USA, troscopy was used to determine the secondary structure by examining the fre- 2Division of Physical and Computational Sciences, Delaware State quency of the Amide I mode. Combinations of disordered and structured University, Dover, DE, USA. peptides were mixed at different concentrations and in different ratios. The We combined fluorescence correlation spectroscopy (FCS), fluorescence IR spectra of the mixtures were compared to the weighted average of the in- anisotropy (FA), and fluorescence imaging microscopy to measure the rota- dividual spectra to assess the effect of any interaction between the two tional diffusion and the translational diffusion of different nanoprobes peptides. (Alexa488, rhodamine 6G, fluorescein, FITC-Ficoll70) and microbeads in z 2807-Pos non-fluorescent -hence ‘‘invisible’’- aqueous Ficoll (MW 70 kDa) solu- Fluorescence Lifetime Imaging of Tetracycline-Stained Retinal Hydroxy- tions. We first noticed that the emission spectrum and the lifetime of apatite: An Early Biomarker for Age-Related Macular Degeneration? each fluorophore is not significantly altered by the increase of the Ficoll Richard B. Thompson1, Henryk Szmacinski1, Kavita Hegde2, Adam Puche3, concentration. Multiple tracking of the microbeads indicated a linear Trevor McGill4, Martha Neuringer5, Imre Lengyel6. time-dependence of the mean-squared displacements, indicating a diffusion 1Dept Biochem/Molec Biol, Univ Maryland Sch Med, Baltimore, MD, USA, mechanism. Further, changes of the translational diffusion of the microbe- 2Coppin State University, Baltimore, MD, USA, 3Dept Anatomy/Neurobiol, ads with increase of Ficoll concentration could be readily attributed to Univ Maryland Sch Med, Baltimore, MD, USA, 4Dept Ophthalmology, changes of the bulk viscosity of the Ficoll solutions, consistent with the Oregon Health Sciences University, Portland, OR, USA, 5Oregon Health Stokes-Einstein relation. However, FCS and FA measurements of the Sciences University, Portland, OR, USA, 6Queen’s University Belfast, nanoprobes showed that the changes of the translational and rotational Belfast, United Kingdom. diffusion coefficients with increase of Ficoll concentration could not be ac- Age-related macular degeneration (AMD) is the most common cause of loss of counted for by the corresponding changes of the bulk viscosity of the Ficoll vision in the elderly in the developed world, affecting over ten million in the solutions as would be suggested by the Stokes-Einstein relations for both United States alone. It is widely held that the atrophy of the light-sensitive cells diffusions. So, instead, we analyzed the diffusion data -rotation and trans- and their associated neurons in the retina has its origin in protein- and lipid-rich lation- with the entropic model proposed by de-Gennes and fit the concen- a n deposits between the retinal pigment epithelium (RPE) and Bruch’s mem- tration dependence with a stretched exponential [exp(- c )]withnbeing a branes; the best known of these deposits are called drusen. Recently, we found related to the quality of the solvent and being proportional to the size that drusen contained microscopic spherules of hydroxyapatite (HAP; of the nanoprobes. Remarkably, for both sets of rotational and translational Ca (PO ) OH), the hard form of calcium phosphate found in bones and teeth, data, the fits yielded n-value close to one, indicating a theta-like behavior of 5 4 3 a and that these spherules became coated in vivo with proteins characteristic of the host Ficoll-water system. However, the -value for translation was drusen such as amyloid beta, vitronectin, and complement factor H, a variant larger than that of rotation, indicating dissimilar local entropic effects on of which is the strongest genetic risk factor for AMD (Thompson, et al., the rotation and translation. PNAS 2015 PMID: 25605911). These findings and others led us to propose that the growth of drusen is nucleated by the HAP spherules, and thus they 2810-Pos might be an early biomarker to be used for screening for AMD. We found Automation of a Laser Tweezers Raman Spectroscopy Apparatus for Bio- that the HAP stained by certain tetracyclines exhibited substantial increases logical Investigations in fluorescence lifetime upon binding, significantly longer than the lifetime Nathaniel W. Scott, Scott Hancock, Brooke C. Hester, Jennifer L. Burris. of the retina background fluorescence imaged by Schweitzer, Zinkernagel, Physics and Astronomy, Appalachian State University, Boone, NC, USA. and their colleagues. Thus we were able to image tetracycline-stained drusen The Biophysics and Optical Sciences Facility (BiyOSef) maintains a in vitro from aged human and macaque retinas using FLIM. Recent results custom-built and partially automated Laser Tweezers Raman Spectroscopy will be shown. (LTRS) apparatus. Laser tweezers utilizes optical forces produced through refraction and reflection to trap particles in a laser beam, while Raman spec- 2808-Pos troscopy measures a unique spectral fingerprint of materials due to unique In Vivo Cell Tracking and Cleared Tissue Imaging with Extended Field of shifts in vibrational molecules of the materials. Our apparatus combines View Selective Plane Illumination Microscopy both methods to increase our resolution of the Raman fingerprint. This Leonardo A. Saunders1,2, Devin Pace3, Arianna Gentile1, Dominik Stich1, method allows for the capture, manipulation, and study of biological cells, Angeles B. Ribera4, Douglas P. Shepherd1. microbes, proteins, and other micron-sized particles in a fluid. Through 1Pharmacology, University of Colorado Anschutz, Aurora, CO, USA, the use of optical trapping of individual particles and confocal methods, 2Integrated Sciences, University of Colorado Denver, Denver, CO, USA, we can effectively reduce noise that can interfere with our Raman signal 3Physics, University of Colorado Denver, Denver, CO, USA, 4Physiology thereby isolating the Raman signal. The alignment of the apparatus takes and Biophysics, University of Colorado Anschutz, Aurora, CO, USA. considerable time, requires months of training, and has the potential to be Single Plane Illumination Microscopy (SPIM), a type of light sheet fluores- inconsistent when done manually. Through automation, we have been able cence microscopy (LSFM), is continuously evolving to new demands for to maximize our resolution and minimize the time necessary to achieve

BPJ 9475_9479 Wednesday, March 6, 2019 567a results. We present here the analysis of the data collected and processed tion of the spectrometer lead to a temporal resolution of about 6 ps. Without through our automated LabVIEW program. doubt, this allows investigating with high precision fast transfer processes or binding formation by anisotropy measurements in molecule systems or build- 2811-Pos ing blocks. Diamond B23 Beamline for Synchrotron Radiation Circular Dichroism The performance of such a spectrometer in terms of time resolution, ability (SRCD): High Throughput CD (HTCD) and CD Imaging (CDi) Applica- to measure long decays (e.g., phosphorescence) and record time-gated tions spectra using laser drivers with burst capabilities will be demonstrated. Rohanah Hussain, Tamas Javorfi, Charlotte Hughes, Giuliano Siligardi. Moreover, the combination of fluorescence spectrometer with confocal mi- Diamond Light Source, Didcot, United Kingdom. croscope systems allows the detection of time-resolved emission spectra High throughput (HTCD) using 96 and 384 multiplate for the optimisation of with high spatial resolution and all advantages of a microscope (e.g., protein crystallization conditions, protein-ligand binding screening, and CD scanning). Imaging (CDi) of thin films of materials (proteins, nucleic acids, carbohydrates, The achieved sensitivity allows to also quantify the tiniest changes in lumines- polymers with embedded drugs, organic LED, and in general any chiral mate- cence intensities as well as photoluminescence lifetimes. rial with or without achiral substrates) are the results of the unique highly colli- mated microbeam light generated at B23 beamline at Diamond Light Source in 2814-Pos the vacuum UV to visible wavelength range (130-650nm). B23 is the only FRET at the Single Molecule Level using Molecular Brightness and Fluo- bespoke beamline worldwide to characterise the structural and conformational rescence Correlation Spectroscopy 1 1 1 1 properties of biologically important molecules using micro-devices unattain- Robert C. Miller , Rowan Simonet , Christin Libal , Cody Aplin , 1 2 1 1 able with bespoke bench-top CD instruments. Recent applications and latest Anh Cong , Margaret Gurumani , Emma Kauffman , Hong Bok Lee , 3 1 1 beamline upgrades will be discussed. Arnold J. Boersma , Erin D. Sheets , Ahmed A. Heikal . 1Department of Chemistry and Biochemistry, Univeristy of Minnesota Duluth, Duluth, MN, USA, 2Department of Chemistry and Biochemistry, 2812-Pos College of St. Scholastica, Duluth, MN, USA, 3DW1-Leibniz Institute for Widefield Multi-Frequency Fluorescence Lifetimeimaging using a Two- Interactive Materials, Aachen, Germany. Tap CMOS Camera Withlateral Electric Field Charge Modulators Fo¨rster resonance energy transfer (FRET) is a noninvasive, quantitative method Hongtao Chen1, Ning Ma1, Keiichiro Kagawa2, Shoji Kawahito2, to study intermolecular interactions and conformational changes. The energy Michelle Digman1, Enrico Gratton1. transfer efficiency of FRET pairs are routinely quantified using steady-state 1 Dept Biomedical Engineering, Univ Calif Irvine, Irvine, CA, USA, fluorescence spectroscopy or time-resolved fluorescence for which the main 2 Shizuoka University, Hamamatsu, Japan. challenge of these approaches is the averaging over a larger ensemble of mol- Widefield frequency-domain fluorescence lifetime imaging microscopy (FD- ecules. Here, we present a new approach for determining the energy transfer FLIM) measures fluorescence lifetime of entire images in a fast and efficient efficiency of hetero-FRET probes at the single-molecule level, using time- manner. We report a widefield FD-FLIM system based on a CMOS camera resolved fluorescence correlation spectroscopy (FCS) and molecular brightness equipped with two-tap true correlated double sampling (CDS) lock-in pixels (i.e., the number of fluorescence photons per molecule emitted while diffusing and lateral electric field charge modulators. Due to the fast intrinsic response in the open observation volume). In this approach, the molecular brightness of and modulation of the camera, our system allows parallel multi-frequency the donor, in the presence and absence of the acceptor, is related to the energy FLIM in one measurement via Fast Fourier Transform. We demonstrate transfer efficiency. We used the engineered hetero-FRET probe, RD, which that at a fundamental frequency of 20MHz, 31-harmonics (620MHz) can consists of a mCerulean-linker-mCitrine construct, as the model system. Laser be measured with 64 phase images per laser repetition period. As a proof intensity-dependent FCS experiments were carried out on both the intact and of principle, we analyzed cells transfected with Cerulean and with a cleaved RD indicating an energy transfer efficiency of (50 5 10)% for RD construct of Cerulean-Venus that shows Fo¨rster resonance energy transfer in buffer, corresponding to a distance of 7 5 2 nm between the donor and at different modulation frequencies. We also tracked the temperature change acceptor. These results demonstrate that our FCS approach is applicable to of living cells via the fluorescence lifetime of Rhodamine B at different hetero-FRET constructs in well-controlled environments. Further it should be frequencies. Furthermore, we can rapidly distinguish the pre-implantation useful in living cells because it has the added advantage of requiring low embryo development stages at the optimal frequency by the fluorescence expression levels of the FRET sensor, in contrast to more conventional lifetime differences of their intrinsic fluorescence in 2D phasor plots. These FRET methods. results indicate that our widefield multi-frequency FD-FLIM system is a valuable tool in the biomedical field. [Work done in part with funds from 2815-Pos NIH P41-GM103540]. The Fluorescence Study of the Complexation of Nanoemulsio and Proto- porphyrine IX Maurice O. Iwunze. 2813-Pos Dept Chem, Morgan State Univ, Baltimore, MD, USA. Sensitive Time-Correlated Single Photon Counting Systems for Luminces- Nanoemulsion is observed to be fluorescent and has a quantum yield of 0.135. It cence Spectroscopy of Small Molecules and Building Blocks is used as a vehicle for drug delivery. Most water insoluble materials including 1 1 1 1 Christian Oelsner , Eugeny Ermilov , Frank Birke , Felix Koberling , drugs often use nanoemuslion for solubilization. On the other hand, protopor- 2 1 3 1 Matthias Patting , Marcus Sackrow , Nick Bertone , Michael Wahl , phrin IX, a photosensitizer and belongs to a heme group of compounds found in 1 Rainer Erdmann . all living organisms and virtually insoluble in water, but was observed to be 1PicoQuant GmbH, Berlin, Germany, 2PicoQuant, Berlin, Germany, 3 liberally soluble in nanoemulsion. However, when this species is mixed with PicoQuant Photonics North America Inc., Kirkland, QC, Canada. nanoemulsion, the quenching of the fluorescence of nanoemulsion is observed. Detection sensitivity is a key parameter to meet today’s demand for handling The quenching mechanism is assumed to be complexation quenching and the smallest analyte amounts and short measurement times in the optical evaluation observed fluorescence data obeyed the Stern-Volmer equation. The complexa- of pharmaceuticals and biotechnology products. tion constant is determined as 4.96 x 105. The introduction of TCSPC based data acquisition has proven to yield a major sensitivity increase and very high dynamic range - it is the ideal method for 2816-Pos measuring weak luminescence. The achievable signal-to-noise ratio of this Spectral Phasor Analysis on Nanosecond-Gated Autofluorescence Reveals method is significantly higher than of methods based on analog detection. Real Time Information on Cellular NAD(P)H Conformation during This allows for accurate quantification of extremely low concentration levels Chemically-Induced Metabolic Response down to femtomolar range. Paul K. Urayama, Audrey Short, Martin Heidelman, Max Kreider, Here we present different kinds of hardware for a state of the art photolumi- Andrew I. Rodriguez, Chong Kai Wong, Nazar Al Aayedi, Zhifan Cai. nescence spectrometer for steady-state, anisotropy, and time-resolved mea- Dept Physics, Miami Univ, Oxford, OH, USA. surements. We discuss critical components affecting sensitivity and present Cellular reduced pyridine nucleotides NADH and NADPH are metabolic co- a step by step rundown how to find optimal measurement conditions in order factors that exist in multiple conformations, significant because the proportions to reach ultimate temporal and spectral performance limits. The combination of these forms respond differently to metabolic conditions. Real time moni- of a double monochromator in subtractive mode for emission selection, new toring approaches sensitive to conformation support the use of NADH and hybrid PMA detector without after pulsing, PicoHarp 300 TCSPC electronics NADPH as metabolic indicators and endogenous biomarkers. Here, we and deconvolution of the fluorescence decay with instrument response func- demonstrate an approach combining nanosecond-gated spectral detection

BPJ 9475_9479 568a Wednesday, March 6, 2019 with spectral phasor analysis on UV-excited autofluorescence in Saccharo- 2819-Pos myces cerevisiae suspensions. The approach provides real time, dynamical in- Sers as an Effective Probe to Adsorption and Conformation of Biomole- formation on cellular pyridine nucleotide conformation to chemically-induced cules on the Metal Surfaces metabolic change. We demonstrate the ability to sense a broad range of re- Qing Huang. sponses, including concentration-dependent-differences in the acute toxicity Chinese Acad Sci, Hefei, China. response to cyanide, discrimination between NADH- and NADPH-linked re- Adsorption/conformation of biomolecules on metal surfaces is a long-standing sponses, and the ability to sense metabolic response under pressurized (several issue which requires development of new tools and methods to settle the exist- hundred atm) conditions. Emphasis is placed on interpreting the non-two- ing controversies. In this regard, surface enhanced Raman spectroscopy (SERS) component behavior in the spectral response in terms of the affected biochem- emerges as an alternative approach which may provide a solution for it is highly ical pathways. sensitive with even single molecular detection ability and it can provide struc- tural information which also depends on the adsorption and conformation of the 2817-Pos examined molecules on the metal surface. Therefore, our group combined Infrared Spectroscopy Offers Tremendous Potential in Cancer Diagnosis SERS experiments with other methods such as density functional theory Feride Severcan1, Sherif Abbas2, Dilek Yonar3, Salih Emri4. 1 2 computation to analyze conformations and adsorptions of small biomolecules Faculty of Medicine, Altinbas University, Istanbul, Turkey, Biomedical such as tyrosine [1], adenine [2] and cysteine [3], as well as the larger biomol- Sciences, Middle East Technical University, Ankara, Turkey, 3Faculty of 4 ecules such as some sequences of DNA molecules as they are attached to the Medicine, Yuksek Ihtisas University, Ankara, Turkey, Chest Diseases, metal surfaces and interact with other molecules (e.g. coralyne and PCB-77) Yeditepe University, Istanbul, Turkey. [4-6] and ions (e.g. Hg2þ) [7]. With this idea, we are also developing this All over the word, scientists are actively engaged in the characterization, approach in combination with more advanced tools to investigate more com- screening and diagnosis of different pathological conditions. Ideally, the plex biomolecules. aim is to monitor the system of interest, without disturbing it, in a sensitive, References: rapid and operated manner at minimum cost. Infrared spectroscopy is one of [1] G. Yao et al. Spectrochimica Acta Part A: Molecular and Biomolecular the few analytical techniques that fulfill all of these requirements. Recently, Spectroscopy 2015, 151: 111-123. the power of infrared spectroscopy in diagnosis of bladder cancer from the [2] G. Yao, et al. J. Phys. Chem. C 2017, 121, 9869-9878. bladder wash was reported by our group [Gok et al., 2016]. Here, we will [3] G. Yao and Q. Huang. J. Phys. Chem. C 2018, 122, 15241-15251. show the potential of infrared spectroscopy coupled with chemometrics in [4] Y. Lu, Qing Huang. Analytical Methods 2013, 5:3927-3932. the determination of disease specific diagnostic biomarkers and differentia- [5] Y. Lu, et al. Analyst 2014, 139, 3083-3087. tion of malignant pleural mesothelioma (MPM), lung cancer (LC), metastatic [6] K. Sun, et al. ACS Appl. Mater. Interfaces 2016, 8:5723-5728. lung cancers and benign pleural effusions from pleural fluids and serum. [7] Y. Lu, et al. Sensors and Acutators B: Chemical 2018, 258:365-372. Spectral analysis indicated significant differences in lipid, protein, nucleic acid, glycogen content, lipid order and fluidity and protein structure espe- 2820-Pos cially for MPM group. Disease induced specific biomarkers have also Structural and Spectroscopic Study of the Tyrosine Kinase Inhibitor been determined for different groups. Unsupervised (hierarchical cluster PD-153035 analysis, HCA and principal component analysis, PCA) and supervised Muhammad Khattab1, Daryll Knowles2, Feng Wang1, Andrew Clayton1. (Soft Independent Modeling by Class Analogy, SIMCA) chemometric ana- 1Swinburne University of Technology, Melbourne, Australia, 2Australian lyses methods were applied to the infrared spectra. Based on the spectral dif- Custom Pharmaceuticals, Sydney, Australia. ferences successful differentiations with 85-100% sensitivity and 84-100% Our study revolves around investigating the spectroscopic properties of the tyro- specificity for pleural fluid, with 71-95% sensitivity and 80-94% specificity sine kinase inhibitor, PD-153035, both experimentally and theoretically. The for serum were obtained when MPM, LC and control samples (benign tran- UV-Vis absorption spectrum of PD-153035 exhibited four absorption peaks at sudative effusion for pleural fluid and healthy control for serum) were taken ca. 220, 250, 330, and 340 nm. The position and relative optical density of into consideration. The differentiation with 80-85% sensitivity and 84-88% the two lowest energy bands at 330 and 340 nm were significantly altered de- specificity for pleural fluid was achieved with the inclusion of all the groups. pending on solvent used in UV-Vis measurements. We therefore prompted to This study demonstrated that ATR-FTIR spectroscopy coupled with chemo- computationally examine PD-153035 structures. The potential energy surface metrics is a novel, rapid, non-invasive diagnostic method with higher sensi- scan revealed four energetically stable conformers of PD-153035 calculated tivity, specificity and accuracy in differentiation of different malignant and through rotation of the dihedral angle between the anilino and quinazolinyl moi- benign lung diseases. (This study was supported by TUBITAK- SBAG- eties. Two structures have planar conformations while the other two have twisted 113S294 project). conformations. The energy difference between the global minimum structure and the highest energy conformer was estimated at 2.12 kcal/mol. The 2818-Pos HOMO-LUMO energy gap was calculated at (4.3140 5 0.0196) eV for the Early Warning Detection of Carcinogens and Other Contaminants for four conformers. The time-dependant Density Functional Theory (td-DFT) cal- Surface Water Treatment Plants using Simultaneous Absorbance- culations employing B3LYP/6-311þþG** level of theory revealed that the 330 Transmittance and Fluorescence Excitation-Emission Spectroscopy nm peak can be attributed to the planar conformers while the 340 nm peak can be Adam M. Gilmore, Linxi Chen. due to the twisted conformers. The computed oscillator strength of the planar Fluorescence, Horiba Instruments Inc., Piscataway, NJ, USA. isomers in various solvents had values double that of the twisted isomers. Taken Most surface water treatment plants remain prone to contaminations by together, these results showed how the absorption spectrum is sensitive to petroleum-derived carcinogens because they lack detectors upstream or even PD-153035 conformation. Hence, our findings assume relevance in understand- at their intakes. Early spill warnings require that water-soluble fluorescent or ing the structure and environment of PD-153035 in the ATP binding pocket of its chromophoric components, including Benzene, Toluene, Ethylbenzene and target proteins. Our future work is to identify the fluorescence spectra of Xylenes (BTEX), be discriminated from the high fluorescent backgrounds of PD-153035 in different solvents and when it bounds to its target proteins. the natural Dissolved Organic Matter (DOM) components. Benzene is believed Acknowledgment: Authors acknowledge with gratitude the financial support to be the most toxic BTEX component and has a low (around 1%) fluorescence from Excellerate Australia and iMOVE. quantum yield in water; benzene’s EPA regulated maximum contamination limit in the distribution system is 5 mg/L. In contrast, typical surface water 2821-Pos DOM concentrations range from 1 to 20 mg/L and most DOM components Fluorophore-Induced Plasmonic Current have higher fluorescent quantum yields than BTEX compounds. Here we pre- Josh Moskowitz1, Christopher D. Geddes2. sent a sensitive method for rapid (3-5 min) reagent- and extraction-free detec- 1Chemistry, UMBC, Baltimore, MD, USA, 2UMBC, Baltimore, MD, USA. tion of all BTEX compounds in typical raw surface water with respective Fluorophore-induced plasmonic current is generated when a near-field excited Limits of Detection (LOD) and Quantification (LOQ) of 1 and 3 mg/L. The fluorophore in proximity to a metal nanoparticle film induces plasmon reso- method uses a patented simultaneous Absorbance-Transmittance and fluores- nance in the metal, subsequently generating electrical current, only if certain cence Excitation-Emission (A-TEEM) instrument. Importantly the method em- conditions are met. This highly novel technology allows for the detection of ploys an exclusive automated, sequential and targeted variable selection Partial fluorescent molecules without the need for traditional detectors such as photo- Least Squares (PLS) library analysis. The instrument can be equipped for auto- multiplier tubes, avalanche photodiodes, and linear arrays. In this paper, we mated surface water sampling and html based communication at the plant describe our recent photophysical experiments which describe how plasmonic intake or upstream for early warning of carcinogens and other contamination current can be generated. For example, we discuss the excitation polarization, events. fluorophore oscillator strength, nanoparticle spacing, and solution dielectric. In

BPJ 9475_9479 Wednesday, March 6, 2019 569a addition, we show that the background current from common matrices, such as heme extraction (IsdH) has been resolved, the underlying mechanism of urine, blood, and faeces contributes little to background plasmonic current in heme transfer is unknown. Previous NMR and molecular dynamic (MD) plasmonic immunoassays, which is in stark contrast to traditional fluorescence studies have revealed the second NEAT domain (N2) is mobile compared to assays that reply on optical based detection. the remainder of the protein–suggesting a conformational selection mechanism in binding to hemoglobin. Here, we used MD and the goal-oriented enhanced 2822-Pos sampling method FAST (fluctuation amplification of specific traits) algorithm Beating Nyquist Limits for the Measurement of Fluorophore Blinking to model this motion and used the results to construct a Markov state model to Rates using Image Correlation and Camera Detection predict metastable configurations prior to binding. We refine our predictions 1 1 2 1 Simon Sehayek , Yasser Gidi , Viktorija Glembockyte , Hugo Bradao , with SAXS and PRE-NMR experiments using an ensemble-based Bayesian Paul Wiseman1, Gonzalo Cosa1. 1 2 fitting algorithm. In using this approach, we are able to rigorously combine McGill University, Montreal, QC, Canada, Ludwig-Maximilians- the results of computational and experimental studies to identify pathways be- University of Munich, Munich, Germany. tween stable configurations and predict their timescales. This work lays the We present an ensemble autocorrelation technique for rapidly measuring on- foundation for future work on mutations targeting N2 motions that link these and off-blinking rate constants from an image time series of fluorescent probes motions to extraction and provide insights to the Isd heme extraction that is significantly faster than individual single-molecule trajectory analysis mechanism. approaches. We demonstrate that the use of this technique allows for the extrac- tion of characteristic blinking times which are faster than camera detector expo- 2825-Pos sure times, which cannot be accessed by threshold based single molecule Combining Cryo-EM and Simulation to Understand Ligand Binding in approaches due to aliasing. We test our technique on a wide set of computer Pentameric Ligand Gated Ion Channels simulations, as well as on TIRF image series of surface-immobilized labeled E. Joseph Jordan1, Christian Blau2, Erik R. Lindahl1. DNA. We analyzed DNA-Cy5 complex in the presence of varying concentra- 1Dept Biochemistry & Biophysics, Stockholm University, Solna, Sweden, tions of Ni(II) ions, which act as triplet-state quenchers. In general, we have 2Theoretical Computational Biophysics, Stockholm University, Solna, found good agreement between our technique and a single-molecule based Sweden. method. Furthermore, we recover blinking times which are shorter than the de- Pentameric ligand gated ion channels (pLGICs) are important in neuronal tector exposure time from one of the datasets. Finally, we analyze STORM communication between synapses. Endogenous ligands are known to mediate data, and show it is possible to recover the characteristic on-times, using the both the opening and closing of pLGICs and these proteins are also the target autocorrelation analysis. for a number of neurotoxins, animal venoms, as well as pharmacological agents. In the past decade a number of crystal structures have elucidated Posters: Molecular Dynamics III gating mechanisms as well as open, closed, and desensitized states, often in the presence of agonists or antagonists. More recently, cryo-electron micro- 2823-Pos scopy (cryo-EM) has also produced high resolution structures of channels Estimation of Time-Varying Single Particle Tracking Models using Local in the pLGIC family and has shed further light on ligand mediated activation. Likelihood Given the importance of these proteins to pharmaceutical intervention, a Boris I. Godoy, Nicholas A. Vickers, Sean B. Andersson. method to understand how ligands bind to pLGICs could have important clin- Mech Eng, Boston University, Boston, MA, USA. ical impacts. We have recently implemented a tool that allows inclusion of Single particle tracking (SPT) is a class of experimental techniques and math- cryo-EM densities as an input to the popular molecular dynamics package ematical algorithms for following the motion of very small particles moving in- GROMACS. Using a fitting procedure that uses successively higher resolution side living cells, including viruses, proteins, and strands of RNA, to mention a density maps derived from experimental cryo-EM data, we demonstrate the few. ability to drive a simulation to a conformation corresponding to EM density. There are several models in the literature to describe the motion of such small Here we use recently solved cryo-EM structures of members of the pLGIC particles. Amongst the most common ones is the so-called simple diffusion family to demonstrate the use of this procedure to give insight into modes model, which corresponds to a random walk with noisy observations. of ligand binding in this important class of molecules. The atomic structures Maximum Likelihood (ML) is an estimation method to determine the value of derived from this method are then compared to the models built directly from parameters in a model. It has been utilised in previous works in the context of the EM density as well as to available crystal structures. We believe this novel SPT to estimate key parameters in motion models. However, these works pose technique has much promise to help elucidate mechanisms of drug binding in the limitation that the parameters are considered time-invariant. the increasing number of systems that have been characterized in a ligand An early attempt to include time-varying parameters (TVP) is SPT considers a bound state via cryo-EM. probability of change in a jump Markov model, however, it does not allow to continuously track TVP, requiring the inclusion of probabilities that might have 2826-Pos no physical meaning. GPU Accelerated Computation of Isotropic Chemical Shifts Offers New Here, the estimation problem of TVP is posed as a local time-invariant likeli- Dimension of Structure Refinement in Largescale Molecular Dynamics hood function, which is optimised (locally) to obtain parameters within a win- Simulation 1 2 2 2 dow of nominated span. This local likelihood function is centred at time t. To Alexander J. Bryer , Eric F. Wright , Mauricio Ferrato , Thomas Huber , K((u(i) - t)/h) u(i) Edwin Ortiz2, Robert Searles2, Sunita Chandrasekaran2, Juan R. Perilla1. develop the idea, we introduce a weight , where is the data 1 h t Chemistry and Biochemistry, University of Delaware, Newark, DE, USA, inside the window, is the window size. and is a chosen point inside the 2 window. The estimation algorithm continues when the time points are Computer Science and Engineering, University of Delaware, Newark, DE, increased by one unit and terminates when the last data point is included in USA. the window. Semi-empirical chemical shift prediction, where molecular coordinates and Results of the estimation considering time-varying diffusion have been carried empirical data are reduced to backbone and sidechain chemical shifts out with different window lengths. Our estimation algorithm is capable of pro- through input to differentiable functions, offers a unique opportunity to vali- ducing a TVP for this simple motion model, having, as expected, less variance date and refine protein structure during largescale molecular dynamics (MD) when considering longer windows. simulation but poses the challenge of optimizing performance to levels com- parable to modern GPU-enabled MD engines. The software PPM_One pre- 2824-Pos dicts backbone and sidechain chemical shifts with competitive accuracy to Combining Goal-Oriented Enhanced Sampling and Bayesian Ensemble purely data-driven counterparts while obviating the need for deep network Modelling of SAXS and NMR Data to Model the Solution Ensemble of processing and homology assignment. To poise PPM_One for practical Staphylococcus Aureus IsdH application in largescale biomolecular modeling, a newly optimized GPU- Joseph A. Clayton, Jeffery M. Wereszczynski. accelerated version of PPM_One was developed. Code refactoring and par- Dept. Physics, Illinois Inst Tech, Chicago, IL, USA. allel GPU-processing through the directive-based OpenACC API provide a Iron is an important resource for metabolism, but is not always readily available 67-fold speedup between unaccelerated and Nvidia V100 accelerated inside organisms. The pathogen Staphylococcus aureus obtains iron from its PPM_One test-cases, comparatively, in a system of 21 million protein atoms. host by removing heme from hemoglobin through the iron-regulated surface The core functions that comprise the prediction model and compute pertur- determinate (Isd) pathway. While the structure of the protein responsible for bation due to Hydrogen bonding, magnetic anisotropy and ring currents were

BPJ 9475_9479 570a Wednesday, March 6, 2019 profiled at 271x, 322x, and 211x speedups, respectively. Remaining compo- development of vaccines offers great potential, with the lipopolysaccharides nents of the predictive model saw similar individual speedups through (LPS) on the surface of K. pneumonia representing a potential antigen. How- profiling. Implementing the newly optimized functional core into NAMD ever, variability in the composition of the LPS occurs, a factor that may is NMRForces, a global force module that enables users to compute back- impact the scope of potential Kleb vaccines. To understand the potential bone and sidechain chemical shifts at each timestep and force atoms along impact of this variability on antigenicity a detailed molecular picture of the prediction model’s gradient with a magnitude proportional to deviance the conformational differences associated with the addition of a 1– 4 branch from experimental chemical shift data. will be presented. Using molecular dynamics simulations in conjunction with enhanced sampling methods the full range of conformations accessible 2827-Pos to the unbranched (Kp4) and branched (Kp24) LPS of Klebsiella p. will be Self-Assembly of 2D Viral Capsids with Oscillatory Interactions determined. The impact of branching will be presented in the context of the Jacob R. Swartley1, Jessica Niblo1, Zhongmin Zhang1, Kateri H. DuBay2. 1 2 accessibility of the monoscaccharide components of the LPS that may Chemistry, Univ Virginia, Charlottesville, VA, USA, Univ Virginia, impact antigenicity. Charlottesville, VA, USA. Self-assembly is foundational to all life; it is the route by which many impor- tant biostructures are formed. Environmental changes which occur together 2830-Pos with processes of self-assembly have been shown to dramatically impact Influence of Cholesterol on PI(4,5)P2 Clustering in Model Membranes the characteristics of the emergent organized structures. Viral capsids are a Kyungreem Han1, Anne-Marie Byrant2, Richard M. Venable1, particularly compelling example of the formation of a biostructure within Arne Gericke2, Richard W. Pastor1. complex environments. Furthermore, they provide a unique, self-limiting as- 1Laboratory of Computational Biology, National Heart, Lung and Blood sembly process that can be probed in great detail using computational algo- Institute, National Institutes of Health, Bethesda, MD, USA, 2Department of rithms. Here, we have employed a coarse-grained computational model to Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, investigate the relationship between viral capsid formation and oscillations MA, USA. in the attractions among its components. It has been demonstrated on similar Phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2), a phospholipid compo- models in the past that capsid formation is isolated to a narrow range of inter- nent of cell membranes, plays important roles in many cellular processes. component attractions under static conditions. However, with our model we Transitions between non-clustered and clustered states are necessary for observe an oscillation-dependent shift of the range of maximal capsid forma- the lipid to carry out its multiple roles in a context-dependent manner. tion. Our results provide insight into how the complex environmental dy- Among many factors that influence clustering, specific ion effects have namics within a cell may influence the assembly of essential biological recently begun to be explored. Given that the headgroup is highly nega- structures. tively charged at physiological conditions, the cation-headgroup interac- tion is essential for screening the negative charge. The electrostatic 2828-Pos interaction is significantly influenced by interaction with other lipids. In Search of a Structural Pattern in Crazy Sugarsidentification of Confor- The present study examines the roles of cholesterol in the PI(4,5)P2 clus- mation Clusters of the Oligosaccharides Within Glycoproteins with LEUS ter formation via computational and experimental approaches. All-atom Aysegul Turupcu, Chris Oostenbrink. molecular dynamics simulations were conducted on pure PI(4,5)P2 and Molecular Modeling and Simulation, University of Natural Resources and PI(4,5)P2/cholesterol mixed monolayers with different ion compositions Life Sciences, Vienna, Austria. and the trajectories were analyzed using graph theory. The trajectories Oligosaccharides within glycoproteins, also called glycans, have been show that cholesterol forms hydrogen bonds with oxygen atoms on the recognized to have critical biological roles: in regulation of the immune phosphate diester group and the protonated phosphate monoester groups system, cellular growth, cell-cell adhesion, infection, inflammation and attached to the PI(4,5)P2 headgroup. This leads to the redistribution of malignant transformations. Their structural diversity has enabled them to ions at the membrane surface and the reorganization of connectivity have this broad range of functionalities. Glycans can be built from mono- among PI(4,5)P2 headgroups. Graph-theoretic analysis reveals that cluster mers that differ in size, linkage types, linkage positions and charges. size distributions and structural properties (i.e., degree distributions and Furthermore, there is a lack of reliable 3D structural information from clustering coefficients) are different between the pure PI(4,5)P2 and experimental techniques because of their flexible nature. To tackle this PI(4,5)P2/cholesterol mixed monolayers. The clustering trends in the sim- problem, we used enhanced molecular dynamics simulation techniques ulations are supported by area/PI(4,5)P2 measurements obtained from a to offer 3D models in the absence of experimental structural data. With Langmuir trough. this aim, ‘motion libraries’ of all disaccharides constituting the most com- mon glycans were constructed using an enhanced sampling method, local elevation umbrella sampling (LEUS). In the build-up phase, biased poten- 2831-Pos tials were constructed for the glycosidic linkages of disaccharides, which Molecular Simulation Studies of E. Coli O171, O175, and O181 LPS and were subsequently used to sample the conformational space for larger ol- V. Cholerae O1 LPS Symmetric Bilayers Emanuel Luna1, Seonghoon Kim1, Wonpil Im2. igosaccharides. First, the effect of consecutive linkages on the disaccha- 1 2 ride free-energy landscape was assessed at the trisaccharide level. Next, Biological Sciences, Lehigh University, Bethlehem, PA, USA, BioS and some of the most common human glycans, such as complex and high- BioE, Lehigh Univ, Bethlehem, PA, USA. mannose N-glycans, were modelled. In addition to these glycans, the Understanding Gram-negative bacterial outer membrane (OM) components effect of core-fucosylation is investigated on these models. The disaccha- and their behaviors is an essential step in discovering how bacteria sur- ride motion libraries are used to sample the conformation space of these vives and how to prevent it from causing damage to its host. Lipopolysac- oligosaccharide units along the glycosidic dihedral angle. Several clus- charides (LPS), present in all Gram-negative bacterial OMs, serve the role tering schemes are used to identify different structural patterns observed of protecting the bacteria from such external threats. Antibiotic perme- in the different glycan types. Here, we show preliminary results for solu- ability and immune system recognition are dependent on LPS structure tion structure clusters of the most common glycans found in human and composition; therefore, a solid understanding of their behavior is glycoproteins. imperative in discovering methods to treat these pathogens. To that end, utilizing CHARMM-GUI, we have simulated three symmetric bilayers 2829-Pos for E. coli Lipid A, R1 core, and 10 repeating units of O171-, O175-, Impact of Branching on the Conformational Heterogeneity of the Lipo- and O181-antigens, respectively. We used the CHARMM36 force field, polysaccharide from Klebsiella Pneumonia: Implications for Vaccine and each system were simulated for 500 ns utilizing NAMD in an environ- Design ment of constant pressure (1 bar) and temperature (303.15 K). The same Asaminew H. Aytenfisu1, Raphael Simon2, Alexander D. MacKerell3. process has been applied to generate and simulate five different V. cholera 1Department of Pharmaceutical Sciences, School of Pharmacy, University of types (lipid A types 1 through 5, Core A, and 30 repeating units of O1- Maryland, Baltimore, MD, USA, 2University of Maryland School of Medicine antigen). The sugar conformations, hydrophobic and O-antigen thickness, Center for Vaccine Development, University of Maryland, Baltimore, MD, density profiles of individual components along the membrane normal, USA, 3Dept Pharm Sci, Univ Maryland, Baltimore, MD, USA. solvent-accessible O-antigen surface areas, ion behaviors, and overall Klebsiella pneumonia is a bacterial pathogen for which the development of LPS conformational preferences are then determined and presented from resistance to antibiotics is a concern. Towards addressing this concern, the these generated systems.

BPJ 9475_9479 Wednesday, March 6, 2019 571a

2832-Pos yers, Ca2þ bound to the bottom outer leaflet is pulled through the pore by Interaction of Free Docosahexaenoic Acid with Lipid Bilayer: A Molecular the external field and remains bound to the bottom inner leaflet. Free Ca2þ Dynamics Study can enter one pore loosely bind to the pore wall, exit the pore, migrate across Olivia White, Mohammad Alwarawrah. the intracellular compartment, and then pass through the second pore in the Biological Sciences, University of South Florida St Petersburg, St Petersburg, same way. FL, USA. Docosahexaenoic acid (DHA) is an omega-3 fatty acid that has 22 carbons and 2835-Pos 6 double bonds which makes it the longest and most unsaturated fatty acid Discontinuous Wrapping Transition of Nanoparticle by Tensionless Lipid found in membranes. DHA is a primary structural component of the cerebral Membranes Eric J. Spangler1, Mohamed Laradji2. cortex, skin, and retina cells membranes and essential for their function. Previ- 1 2 ous studies suggest that free DHA affect the membrane instability; however, Biomedical Engineering, Univ Memphis, Memphis, TN, USA, Department their interactions with phospholipids are poorly understood. Therefore, we of Physics, Univ Memphis, Memphis, TN, USA. used conventional molecular dynamics simulations to systematically investi- Using molecular dynamics simulations of a coarse-grained implicit-solvent gate the interaction between free DHA and phosphatidylcholine (POPC) in model, we studied the wrapping of spherical nanoparticles, with diameters lipid bilayer. Our results show that free DHA alter the physical properties of ranging between 10 and 40 nm, by tensionless lipid membranes. We found lipid bilayer through decreasing the area per lipid and increasing the bilayer that, for small nanoparticles, the amount of wrapping increases continuously thickness. As the DHA concentration increases, the average area per lipid with increasing adhesion strength, in agreement with earlier studies. However, shows a monotonic decrease. On the other hand, the average bilayer thickness for larger nanoparticles, the degree of wrapping exhibits an abrupt transition shows an increase when the DHA concentration below 20%. Interestingly, from weakly wrapped states to highly wrapped states. The discontinuity in increasing the DHA concentration beyond 20% does not affect the average the degree of wrapping increases with increasing adhesion strength. These re- bilayer thickness. Furthermore, our simulations show that free DHA increases sults therefore asymptotically approach the mean field predictions, based on the the spacing between lipids headgroups through spanning its chain laterally in Helfrich free energy, that large spherical nanoparticles are either unbound or the hydrophobic region of the bilayer. This mechanism facilitates the translo- completely wrapped. cation of free DHA across the lipid bilayer. We hypothesis that free DHA 2836-Pos does not require a complex mechanism to translocate across the cytoplasmic A New Lipid Force Field (FUJI) for Lennard-Jones PME membrane. Hideaki Fujitani. RCAST, Univ Tokyo, Tokyo, Japan. 2833-Pos Standard molecular dynamics simulations estimate van der Waals interactions Molecular Structure of the Long Periodicity Phase in the Stratum Cor- only of the atoms within a short distance around 1 nm. Because this cutoff neum method is insufficient for accurate evaluation of energy and pressure, the Eric Wang, Jeffery B. Klauda. isotropic dispersion correction was always included. To explore the anisotropic Dept Chem/Biomolec Eng, Univ Maryland, College Park, MD, USA. characters of membrane, Lennard-Jones particle mesh Ewald (LJ-PME) was The long periodicity phase (LPP) of the stratum corneum (SC) is studied using applied without the isotropic dispersion correction. As the popular AMBER all-atom molecular dynamics simulations of sandwich models on the micro- and CHARMM lipid force fields were developed under the cutoff scheme, their second timescale. The interior of the sandwich structure is fluid but transitions lipid bilayers unacceptably shrank under LJ-PME. We developed a new lipid into the gel phase due to free fatty acid (FFA). Ceramides (Cer) transition from force field (FUJI) based on the AMBER force field scheme including Lipid14 hairpin to extended conformations, and the mechanism is characterized by van der Waals parameters. Point charges were calculated by restrained electro- initial anchoring of the head-proximal carbons. FFA translocates through the static potential (RESP) from Hartree-Fock calculations on many conformers of interior on the hundred-nanosecond timescale, and cholesterol (Chol) is ori- lipids. Using high level ab initio molecular orbital calculations, we obtained ented perpendicular to the normal at the bilayer-monolayer interface. Strong torsion energy profiles of 12 dihedral angles of phospholipid and derived their agreement with experiment is noted based on electron density and neutron scat- molecular mechanical dihedral parameters from the ab initio torsion profiles tering length density (NSLD), and the high disorder of Cer linoleate supports utilizing fast Fourier transform (FFT). Incorporating these first principle param- infrared spectroscopy. Cer preferentially clusters over Chol and FFA in the eters to a new lipid force field without any fittings to experimental data, we suc- outer leaflet. To further validate the model, ethanol permeability was calculated cessfully derived desirable lipid characters, such as area per lipid and lateral using umbrella sampling, which agrees excellently with experiment (logP of diffusion coefficients, using LJ-PME without the dispersion correction. 6.5 cm/s vs. 6.7 cm/s in experiment) and suggests that the limiting factor AcrABZ-TolC is an efflux protein complex in Escherichia coli, extruding a is the LPP. Furthermore, a leapfrog mechanism of ethanol permeation into vast variety of antimicrobial agents from the cell. We performed molecular dy- the skin is proposed. These are the first experimentally verified sandwich namics simulations for AcrABZ-TolC with inner and outer membranes of models of the LPP and will aid the design and optimization of transdermal POPE, which had more than 7 million atoms including water and ions. The in- drug delivery systems. ner and outer membranes showed undulatory fluctuations, however, the observ- ables such as membrane thickness and NMR order parameters of acyl tails did 2834-Pos not differ from those obtained by a small unit cell of pure POPE bilayer. Microsecond Kinetics of Ion Transport and Membrane Interface Binding in Electrically Stressed Lipid Bilayers 2837-Pos Federica Castellani1, Esin B. Sozer2, P. Thomas Vernier2. Extending the Amber Lipid Framework for Atomistic Modeling of 1Batten College of Engineering and Technology, Old Dominion University, Organic-Lipid Conjugates Norfolk, VA, USA, 2Frank Reidy Research Center for Bioelectrics, Old Rachel J. Dotson, Gary Angles, Sally C. Pias. Dominion University, Norfolk, VA, USA. Dept of Chemistry, New Mexico Tech, Socorro, NM, USA. Strong transmembrane electric fields promote the formation of lipid pores We present a method for extending the Amber Lipid Framework, currently through which drugs and other normally impermeant materials can enter cells. Lipid14/Lipid17, in order to model lipid-conjugated organic molecules using The mechanisms underlying this process, called electroporation, have not been all-atom molecular dynamics simulations. The approach retains the ‘‘plug- clearly established. We investigate this phenomenon at the experimentally and-play’’ modularity of the Amber Lipid Framework, using a fixed-charge inaccessible nanoscale with atomically detailed molecular simulations. Here capping strategy along with the RESP protocol to fit charges for molecular dy- þ þ we describe the previously unexplored microsecond kinetics of K ,Ca2 , namics simulations. It allows the use of atom types derived from the lipid and Cl binding to phospholipid bilayers and transport through lipid pores in force field or from other compatible parameter sets, such as the General þ double bilayer systems. Ca2 is a key component in the operation of numerous Amber Force Field. Here, we highlight application of the strategy to modeling regulatory and signaling pathways. During and after membrane permeabiliza- an electron paramagnetic resonance (EPR) spin-label probe as well as lipid- þ tion, Ca2 is electrically and diffusively driven into the cell by the transmem- linked prodrug compounds. We validate the behavior of the simulation brane electric field and the large extracellular:intracellular concentration models through comparison with experimental data. The simulations enable þ gradient. Little is known about the evolution of the three-dimensional [Ca2 ] physical characterization as well as visualization of the lipid conjugates and profile during the nanoseconds and microseconds after a porating electric pulse. their manner of incorporation in lipid bilayers. Moreover, the simulations þ Does Ca2 diffuse freely into the cytoplasm (available for signaling) or is it are found to be insightful for interpreting experimental data and valuable as bound quickly to the intracellular interface of the membrane? Preliminary an- a tool for smart design of additional lipid-conjugate compounds. The method D swers to these questions will be presented. Summary results of Ca2 binding of extending the Amber Lipid Framework is straightforward, although and transport dynamics. In a double bilayer system with pores in both bila- nontrivial to implement.

BPJ 9475_9479 572a Wednesday, March 6, 2019

2838-Pos ticles, thus fostering their aggregation on the membrane surface and driving CHARMM-GUI Synthetic Polymer Modeler for Modeling and Simulation the membrane composition to be locally asymmetric, causing membrane of Synthetic Polymers instability. Yeol Kyo Choi, Tibo Duran, Wonpil Im. Biological Sicences and Bioengineering, Lehigh University, Bethlehem, PA, USA. Posters: Electron Microscopy Synthetic polymers are made of small structural units (monomers) that are con- nected by covalent bonds and have been used extensively in technology and 2841-Pos everyday life. Thus, characterizing their structural and dynamical properties Leginon’s Extended Image Shift Mode Increases the throughput for Single at the molecular level is essential to design new materials and understand their Particle Data Collection roles in a variety of application. However, systematic generation of synthetic Edward T. Eng, Anchi Cheng, William J. Rice, Mykhailo Kopylov, polymer structures is challenging because there are great variations in arrange- Laura Y. Kim, Ashleigh M. Raczkowski, Daija Bobe, Kelsey Jordan, ment of monomers and linkage methods (i.e., branching and tacticity of mono- Kotaro Kelley, Clinton S. Potter, Bridget Carragher. mers). In this study, to facilitate the generation of all-atom and corresponding Simons Electron Microscopy Ctr, New York Structural Biology Ctr, New coarse-grained structures of various polymer structures, we present the devel- York, NY, USA. opment and case studies of Synthetic Polymer Builder in CHARMM-GUI Recent advances in instrumentation have made determination of near-atomic (http://www.charmm-gui.org), a web-based interface to facilitate a building resolution structures by cryo-electron microscopy (cryoEM) almost routine. of complex simulation systems. In particular, detector improvements now give us the ability to capture informa- tion from vitrified biological macromolecules nearly to the Nyquist limit, and 2839-Pos improved lens stability allow maintenance of good alignments over the data Computational Study of Calcium Phosphate Mineralization in Extracel- collection period. While instrumentation has greatly improved, sample prepa- lular Vesicles ration remains a significant bottleneck to the generation of high resolution Rudramani Pokhrel, Bernard S. Gerstman, Joshua D. Hutcheson, structures. For cases where sufficient optimization is not possible, due to the Prem P. Chapagain. source material being precious or limiting, it is imperative to exhaustively Dept Physics, Florida Intl Univ, Miami, FL, USA. collect on the available samples to push research projects forward. To increase Calcification in bone, cartilage, and cardiovascular tissues involves the release the throughput of data collection we utilize image shift data collection. Leginon of specialized extracellular vesicles (EVs) that promote mineral nucleation. has the ability to minimize the coma and astigmatism introduced during this > m The small size of the EVs, however, makes molecular level studies difficult, mode of data collection with image shifts 15 m from the optical axis. and consequently uncertainty exists on the role and function of these structures From our internal tests we can acquire an image every 20s and reach sub- ˚ in directing mineralization. The lack of mechanistic understanding associated 3A resolutions for single particle reconstructions. For more conservative data with the initiators of ectopic mineral deposition has severely hindered the collection strategies, moderate image shift configurations may be chosen to ˚ development of potential therapeutic options. Here, we used multiscale molec- routinely achieve 2A reconstructions. ular dynamics simulations to investigate the calcification within the EVs. Re- 2þ 2– 2842-Pos sults show that Ca –HPO4 and phosphatidylserine complexes facilitate 3D-Structual Modeling of Differentiation and Developmental Process the early nucleation. Use of coarse-grained simulations allows investigations 2þ 3– using Advanced Electron Microscopy and Light Microscopy of Ca –PO4 nucleation and crystallization in the EVs. Systematic variation Takako Ichinose1, Takeshi Itabashi1,2, Hikari Mori3, Junpei Kuroda4, in the ion-to-water ratio shows that the crystallization and growth strongly Masaki Imayasu5, Sei Saitoh6, Shigeru Kondo4, Atsuko H. Iwane1,2. depend on the enrichment of the ions and dehydration inside the EVs. Our in- 1Cell Field Struc., BDR, RIKEN, Higashi-hiroshima, Japan, 2Hiroshima vestigations provide insights into the role of EVs on calcium phosphate mineral Univ., Higashi-hiroshima, Japan, 3Cell Field Struc., BDR, RIKEN, Suita, nucleation and growth in both physiological and pathological mineralization. Japan, 4Pattern Formation, Grad. Sch. Fronti., Biosci., Osaka Univ., Suita, Japan, 5Central R&D Lab, Menicon, Kasugai, Japan, 6Fujita Health Univ., 2840-Pos Toyoake, Japan. The Key Role of Temperature and Lipid Composition in Modulating the Sequential 2D-EM images and 3D reconstruction will provide us several infor- Intake of Gold Nanoparticles into the Plasma Membrane mation not only to the 3D-structural shape and connection. Last annual meeting 1 2 3 Fabio Lolicato , Loic Joly , Hector Martinez-Seara Monne , we had selected C. merolae, as a model organism for mitosis, presented the 4 5 6 7 Giovanna Fragneto , Jaakko Akola , Marco Maccarini , Ilpo Vattulainen . interaction between individual several organelles during mitosis cycle and us- 1University of Helsinki, Helsinki, Finland, 2Chemistry Department, 3 ing FIB-SEM and 3D-reconstruction technique. Applying these techniques, we Politecnico di Milano, Milan, Italy, Institute of Organic Chemistry and selected C2C12 cell for muscle differentiation and Zebrafish caudal fin for Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech 4 5 developmental process of bone formation, and presented the interaction be- Republic, Institut Laue-Langevin, Grenoble, France, Department of tween individual several cells and in cell during these important processes us- Physics, Norwegian University of Science and Technology, Trondheim, ing FIB-SEM/ SBF-SEM as compensating for the dynamic morphological Norway, 6Laboratoire TIMC-IMAG, Universite Grenoble Alpes, Grenoble, 7 changes by laser microscope in this meeting. For differentiation of myoblasts France, Department of Physics, University of Helsinki, Helsinki, Finland. cells were growth into reach semi-confluence on a specific chamber consisting Monolayer-protected gold nanoparticles are emerging as promising candidates of 10% FBS-DMEM and then stretched (1Hz, 20% elongation) for 48 hrs, for drug delivery due to their ability to permeate through plasma membranes. finally changed into the differentiation culture medium containing 2% horse Understanding the molecular mechanisms of such complex systems is crucial serum. The cells, at 6 days after differentiation induction were observed their to control cell permeation and to develop efficient biomedical delivery applica- dynamic morphological changes containing to Sarcomere structure compared tions based on nanoscale gold nanoparticles. Here, neutron reflectometry (NR) to before differentiation. As the differentiation process progressed, the cell and molecular dynamics (MD) simulations were used to shed light on the inter- has multi-nuclei, the number and the size of mitochondria increased and formed action between cationic gold nanoparticles (AuNPs) and model lipid mem- thicker cell body and cell. We would also like to discuss about mitochondrial branes and the consequences thereof. Atomistic simulations predicted that remodeling and regarding the role of phagosome associated with during the dif- there is a free energy barrier that has to be overcome to enforce AuNPs to parti- ferentiation process of C2C12 cells. On the other hand, for clarifying the mech- tion into a DSPC bilayer. NR experiments confirmed the prediction, showing anism about the cell autonomous generation of collagen crystal in caudal fin we that AuNP encapsulation takes place only at temperatures higher than 330 are making a 3D structural model of the relationship between actinotrichia and K. Meanwhile, in a mixture of DSPC-DSPG (3:1), experiments showed that its surrounding cells. The main components of actinotrichia are several type II the adsorption of AuNPs to the membrane is weak; the nanoparticles were collagens. Based on the results of the LM and EM 3D structural model, we readily released during annealing. Coarse-grained MD simulations used to would also like to discuss the directionality, extension of actinotrichia and for- interpret this behaviour indicated that in this mixture DSPGs migrate around mation of its bundles for spread radially straight bone. AuNPs, thereby weakening their interaction with the surface and leading to detachment of AuNPs at high temperatures. NR experiments also confirmed 2843-Pos this by showing that in the presence of negative lipids (DSPG), desorption of Single Particle Cryo-EM Workflow: Structures of Apoferritin and AuNPs is associated with a reduced coverage of the floating bilayer, suggesting Aldolase that some lipids, which comprise both DSPC and DSPG, are extracted and left Daija Bobe, William J. Rice, Edward T. Eng, Laura Y. Kim, the bilayer. Finally, the results indicated that the crowding of lipids over the Mykhailo Kopylov, Ashleigh M. Raczkowski, Bridget Carragher, nanoparticles shields the electrostatic interaction between cationic nanopar- Clinton S. Potter.

BPJ 9475_9479 Wednesday, March 6, 2019 573a

Simons Electron Microscopy Center, New York Structural Biology Center, transfer of DNA from one cell to another. The T-pilus, expressed by Agrobac- New York, NY, USA. terium tumefaciens, transfers T-DNA from bacterial cells to plant cells, while Single particle cryo-electron microscopy (cryo-EM) on a high voltage trans- the RP4 pilus, used to transfer DNA between bacterial cells, is encoded by the mission electron microscope (TEM) is frequently being used as a method for conjugative plasmid, RP4, and can be expressed by multiple species of gram- obtaining near-atomic resolution 3D macromolecular structures. It is important negative bacteria. These pili are composed of thousands of subunits called pi- to understand the resolution limitations per instrument, which can be done with lins that are arranged into macromolecular assemblies with helical symmetry. the use of a robust, biological specimen that is routinely used for cryo-EM data CryoEM is the gold-standard for determining the complex arrangement of sub- collection and processing. Because of the variability in sample preparation units in macromolecular structures. Recently a variety of bacterial pili have (from specimen purification to image processing), a standardized workflow is been investigated using cryoEM resulting in electron density maps at near- greatly sought out. Here, we present a single particle workflow using two of atomic resolution. One such pilus is the F-pilus which, like the T- and Rp4- our test proteins, apoferritin and aldolase, collected on a 300kV FEI Titan pili, transfers DNA from one cell to another. Previous studies have indicated Krios. We include details on sample preparation, grid purification, data collec- that both the T-pilus and RP4-pilus are composed of cyclic subunits, which tion and image processing in order to improve and standardize an overall serve as initial evidence that the structure of these two macromolecules may pipeline. prove to be different than F-pili, which is composed of linear subunits. Other than F family pili there is little structural information regarding pili with the 2844-Pos ability to transfer DNA. Additionally, the structural similarities among DNA- Easing Exhaustive Rigid-Body and Flexible Fitting in UCSF Chimera transferring pili have yet to be investigated thoroughly. In this study we use cry- Pablo Solar, Pablo Chacon, Jose Ramon Lopez-Blanco. oEM to elucidate the structures of these pili, providing novel insights into how Biological Physical Chemistry, Rocasolano Institute of Physical Chemistry, they transfer their DNA. C.S.I.C., Madrid, Spain. Build macromolecular models from cryo-electron microscopy (EM) density 2847-Pos maps requires fitting strategies to localize available atomic structures. To this Towards a Solution of the A-B-Z Question using Z-Disks Isolated from the end, we developed several approaches that are efficient enough to run as com- Flight Muscle of Lethocerus Indicus mand line tools on commodity hardware. For rigid-body fitting, ADP_EM em- Fatemeh A. Abbasi Yeganeh1, Corinne Summerill1, Kenneth A. Taylor1, ployed spherical harmonics to speed up the rotational part of the exhaustive, Hamidreza Rahmani2, Dianne Taylor1, Zhongjun Hu3. whereas for flexible fitting, iMODFIT exploit Normal Model Analysis to 1Institute of Molecular Biophysics, Florida State Univ, Tallahassee, FL, reduce the explored conformational space. Here we present the update and inte- USA, 2Physics, Florida State Univ, Tallahassee, FL, USA, 3Florida State gration of such tools into UCSF Chimera enabling interactive macromolecular Univ, San Jose, CA, USA. fitting into this powerful visualization tool which is the facto standard of the The recent publication of a 3-D image of isolated Z-disks from Apis mellifera, EM community. flight muscle[Rusu et al., J. Muscle Res Cell Motil (2017)], the first of its kind utilizing isolated Z-disks, confirmed many details of the previously 3-D im- 2845-Pos J. Cell Biol. Structural Analysis of Mouse Platelets using Serial Block-Face Scanning age obtained from plastic sections[Cheng and Deatherage, Electron Microscopy (1989)]. However, they used relatively harsh treatment of high KCl/KI to 1 1 1 1 isolate Z-disks. Here we combine 1.4M NaCl and 10mM pyrophosphate Kenny Ling , Yajnesh Vedanaparti , Michael P. Tobin , Rohan P. Desai , Lethocerus indicus Guofeng Zhang1, Irina D. Pokrovskaya2, Brian Storrie2, Maria A. Aronova1, applied to flight muscle to remove the thick filaments Richard D. Leapman1. and 4.5min exposure to calcium insensitive gelsolin to remove the thin fila- 1NIBIB, NIH, Bethesda, MD, USA, 2Department of Physiology and ments from the remaining of the I-Z-I-brushes. We are improving the resolu- Biophysics, UAMS, Little Rock, AR, USA. tion by adding more tilt-series to our previous analysis. By comparing the Fourier transforms and subvolume averages, we conclude that Z-disks from Platelets are small, anucleate cells in mammalian blood that maintain hemosta- honey-bee Lethocerus indicus sis and aggregate to seal leaks at sites of vascular injury, a vital element of and are similar but not identical. The large sol- wound healing. Upon detecting vascular lesions, blood platelets become acti- vent channel that characterizes the honey-bee Z-disk is less prominent in Lethocerus, which is reflected in a much weaker to nearly absent 1,0 spot in- vated and release components, such as the Von Willebrand factor, from at least Lethocerus three different storage compartments: alpha-granules, dense granules, and lyso- tensity in transforms of Z-disks, whereas the corresponding spots somes. Abnormalities in platelet functions can contribute to thrombosis, athero- in the honey-bee transform are strong. There is ample evidence that the actin sclerosis and other diseases. Obtaining cellular structure is critical for gaining filaments in the overlap zone of both bee and waterbug flight muscle present a helical array of target zones around each thick filament. However, Squire[J. better understanding of platelet physiology. We used Serial Block-Face Scan- Molec. Biol. ning Electron Microscope (SBF-SEM) to elucidate the 3-D ultrastructure of 72, (1972)] has pointed out that the P312 symmetry assumed mouse blood platelets in both the activated and inactivated (resting) states. In for the honey-bee Z-disk reconstruction from plastic sections when extrapo- SBF-SEM an in-situ ultramicrotome cuts slices of heavy-atom stained cells lated into the A-band, implies rings rather than helices of target zones sur- that are embedded in a resin block. The top surface of the block is then imaged rounding the thick filament. This discrepancy might be resolved if there are using a back-scattered electron detector, and the process of slicing and imaging three-fold screw axes instead of three-fold rotation axes at the lattice and is repeated until the desired volume of cells is obtained. A stack of images can trigonal positions of the flight muscle Z-band; corresponding more to space then be aligned, and ultrastructure visualized using segmentation software. group symmetry P3121. Based on the segmentation and measurements on images from 4 different platelet activation states, we observed that rapid swelling of alpha-granules is 2848-Pos accompanied by matrix decondensation that occurs first in peripheral granules, Investigating the Structural Mechanism of the Stalled Bacterial Ribosome whereas condensed granules were situated more centrally. Decondensed gran- Bound to a Drug that Targets Trans-Translation 1 2 3 3 ules appeared almost adjacent or connected to the plasma membrane. Based on Atousa Mehrani , Eric D. Hoffer , Tyler D.P. Goralski , Kenneth C. Keiler , Christine M. Dunham2, Scott Stagg1. these findings, we propose that the granule matrix organization is an important 1 factor for platelet function, i.e., for timed-release properties of alpha-granule Department of Chemistry & Biochemistry, Florida State University, Tallahassee, FL, USA, 2Department of Biochemistry, Emory University cargo, which could be physiologically important to thrombus formation, wound 3 healing and angiogenesis. School of Medicine, Atlanta, GA, USA, Department of Biochemistry and This work was supported by the Intramural Research Program of the National Molecular Biology, Pennsylvania State University, University Park, PA, Institutes of Biomedical Imaging and Bioengineering, NIH (RDL) and NIH USA. grants R01 HL119393 and R56 HL119393 (BS). Ribosomal stalling occurs when ribosome is trapped at the 3’ end of an mRNA in absence of a stop codon in the decoding center. Since the accumulation of 2846-Pos stalled ribosomes is toxic for the cell, many bacteria have evolved an initial Structural Studies of the T- and RP4-Pili using Cryo-EM rescue mechanism known as trans-translation to skip ribosome stalling, and Mark A. Kreutzberger1, Spencer Hughes2, Vincent Conticello2, ensure the controlled protein synthesis. Ribosome rescue has been proposed Edward H. Egelman3. to be of high importance in many pathogenic strains, and therefore it has 1Mol. Physiology and Biophysics, Univ Virginia, Charlottesville, VA, USA, been considered as a target for the development of new antibiotics. Small mol- 2Chemistry, Emory University, Atlanta, GA, USA, 3Mol. Physiology and ecules have been synthesized that possess antibiotic properties and inhibit Biophysics, The University of Virginia, Charlottesville, VA, USA. trans-translation in Escherichia coli (E. coli) ribosomes. The inhibition mech- Bacterial pili are a diverse group of filamentous appendages with a variety of anisms for these compounds are unknown and structural analysis is required in functions including but not limited to the adhesion to host surfaces and the order to characterize them. Here, we have determined the structure of stalled

BPJ 9475_9479 574a Wednesday, March 6, 2019

70S E-coli ribosomes bound to a trans-translation inhibitor. Using cryogenic 2851-Pos electron microscopy, we have resolved the binding site of the drug and have High Resolution Cryo-Electron Microscopy of Clathrin Cage Networks observed novel conformational changes in the ribosomal protein L27. We Sarah M. Smith1, Kyle L. Morris2, Mary Halebian1, Corinne J. Smith1. will discuss how these observations help explain the mechanism by which 1School of Life Sciences, University of Warwick, Coventry, United drugs can target trans-translation without affecting normal translation. These Kingdom, 2Department of Molecular & Cell Biology, University of studies will further help to improve the design of antibiotics that target rescue California, California, CA, USA. pathways in bacteria. Clathrin-mediated endocytosis is a fundamental cellular process by which mol- ecules are selectively imported into cells, and it plays a central role in a wide 2849-Pos range of processes in eukaryotic organisms, such as: nutrient uptake, synaptic Characterization of the DE64 Direct Electron Detector vesicle recycling and signaling determination of cell polarity. It is also linked to Joshua H. Mendez1, Scott M. Stagg2,3. numerous pathological conditions including: neurodegeneration, heart disease 1Physics, Florida State University, Tallahassee, FL, USA, 2Chemistry and and tumour formation. Clathrin is key to the assembly of a protein coat which Biochemistry, Florida State University, Tallahassee, FL, USA, 3Institute of surrounds the invaginated plasma membrane during cargo internalisation. To Molecular Biophysics, Florida State University, Tallahassee, FL, USA. achieve this, clathrin interacts with a diverse set of adaptor proteins. Using The significant advantages new direct electron detectors (DEDs) provide over cryo-electron microscopy, we aim to determine the structure of self- the previous generation of detectors has made them very popular in the field of assembled clathrin cages at atomic resolution to gain further insight into how cryogenic electron microscopy. With this, the resolution of maps has this protein interacts with its cellular partners. Clathrin has an inherent ability improved greatly, and has pushed the resolution to the sub 3 A˚ domain. to adopt multiple, large, self-assembled architectures in the cell and it is this significant heterogeneity DEDs form images in one of two different modes: integrating mode, where that has made the pursuit of high-resolution structures extremely challenging the deposited charge is accumulated as electrons hit the detector and counting of clathrin cages . However, our single-particle cryo-EM mode where each individual electron is counted as a single event the moment analysis of whole assembled clathrin cages bound to adaptor protein, AP180, they hit the detector. It has been shown that counting mode can result in better has confirmed that this protein promotes the formation of small cage structures. ˚ quality images as measured by the detective quantum efficiency (DQE) at the Our current resolution is 6.4 A which has been derived from 97820 indepen- expense of speed. The image quality improvements are most dramatic at low dent, asymmetric units of a minicoat hub region. At this resolution we are spatial frequency, while at high spatial frequencies the integrating and count- beginning to unveil details on the interactions of clathrin terminal domain ing are more comparable. A next generation DED called the DE64 has been within the minicoat hub region. We are also continuing to employ the latest im- introduced that is capable of high-quality imaging in both integrating and age processing methodologies to tackle sample heterogeneity to improve our counting modes. Here we have characterized the potential for acquiring resolution. high quality images with the DE64 by measuring its noise power spectrum 2852-Pos (NPS), modulation transfer function (MTF) and detective quantum efficiency Hunting for the Adhesion Molecule, Retinoschisin, in Retina using Cemovis (DQE). These metrics measure the detectors theoretical best performance, but Bernard Heymann1, Christopher K.E. Bleck2, Robert N. Fariss3, imaging speed and throughput are also important practical considerations for Alexandr Smirnov3, Dennis C. Winkler1, Camasamudram Vijayasarathy4, single particle reconstruction cryo-EM. Thus, we also characterized Rick Huang5, Altaira D. Dearborn1, Paul A. Sieving3, Alasdair C. Steven6. throughput and resolution for single particle reconstructions of apoferritin 1NIAMS, Natl Inst Hlth, Bethesda, MD, USA, 2NHLBI, Natl Inst Hlth, collected in integrating mode, integrating mode with a Volta phase plate, Bethesda, MD, USA, 3NEI, Natl Inst Hlth, Bethesda, MD, USA, 4NIDCD, and counting mode. We will discuss the throughput and resolution for the Natl Inst Hlth, Bethesda, MD, USA, 5Janelia, HHMI, Ashburn, VA, USA, DE64 and how these influence the choice of data collection mode depending 6LSBR, NIAMS NIH, Bethesda, MD, USA. on the sample. In studying biologically relevant molecules, the ultimate aim is to visualize them in their native environment. One technique that has the potential to 2850-Pos resolve individual molecules in cells and tissues is cryo-electron microscopy Hybrid Analysis of Mab Flexibility by Electron Microscopy and Scattering of vitreous sections (CEMOVIS) combined with tomography. We are inter- Thomas E. Cleveland1, Travis Gallagher1, Jianfang Liu2, Gang Ren2, ested in revealing the disposition of the retinal adhesion protein, retinoschisin John Marino1. (RS1), within the retina. Immuno-fluorescence and immuno-electron micro- 1 Institute for Bioscience and Biotechnology Research, NIST, Rockville, MD, scopy showed that RS1 is abundant in the intercellular spaces between the inner 2 USA, The Molecular Foundry, Lawrence Berkeley National Laboratory, segments of photoreceptors, and between bipolar cells. Loss-of-function muta- Berkeley, CA, USA. tions in RS1 cause X-linked retinoschisis (XLRS), a debilitating disease asso- The biopharmaceutical industry is one of the most valuable business sectors of ciated with the formation of retinal cavities or cysts (schisis) and vision loss in the US economy. In 2016, ten of the 15 top-selling drugs were protein bio- young males. We solved the structure to 4.1 A˚ by cryo-electron microscopy logics; and of these, five were monoclonal antibodies (mAbs). Bio- (cryoEM), a 16-mer composed of two octameric rings with a total weight of pharmaceuticals also represent at least 40% of the medicines in development 380 kDa. From this structure it is clear that many mutations in the subunit in- today, and the global market for protein biologics in 2022 is projected to be terfaces would preclude assembly of the rings. However, other mutation sites over $200B. A key measurement gap in the biopharmaceutical industry is are exposed at the periphery and it was not evident how they would cause the inability of current methods to fully characterize the structure of highly flex- disease. In further cryoEM experiments, we observed that RS1 double rings ible, protein-based drugs, such as mAbs. Crystallization is generally not suc- self-assemble into extensive branched networks, suggesting a mechanism cessful due to their high flexibility; and even when successful, captures only for its adhesive function. To ascertain whether this assembly also occurs a single conformational snapshot of the molecule. Similarly, the continuum in situ, we are examining mouse retinal tissue using CEMOVIS and cryo- of conformational states does not allow conventional multi-particle averaging electron tomography. We obtained images of 50 nm cryosections that show in CryoEM. clear distinction of microtubular protofilaments separated by 5 nm. This res- Using the NISTmAb (RM8671), a model drug-like monoclonal antibody olution is clearly sufficient to see the RS1 double ring and whether it assembles reference material, we have developed hybrid methods to combine EM into networks in the intercellular space between photoreceptors and bipolar with small-angle x-ray and neutron scattering in order to build more com- cells. plete models of antibody structure and flexibility in solution. In this method- ology, Individual Particle Electron Tomography (IPET) is initially used to 2853-Pos build a collection of conformational states of the antibody. These states The Structural Basis for Release Factor Activation during Translation can then be used to model scattering data under physiologically or pharma- Termination Revealed by Time-Resolved Cryogenic Electron Microscopy ceutically relevant solution conditions (e.g., different formulations). We Ziao Fu1, Gabriele Indrisiunaite2, Sandip Kaledhonkar3, Binita Shah4, have applied these methods to the NISTmAb reference material itself, as Ming Sun4, Bo Chen3, Robert A. Grassucci5, Mans Ehrenberg6, well as to soluble aggregates naturally present in small amounts in the prod- Joachim Frank5. uct, and to a NISTmAb/antigen complex. Ultimately, the ability to measure 1Columbia Univ, New York, NY, USA, 2Department of Cell and Molecular the detailed conformational distribution of biotherapeutics may allow this Biology, Uppsala University, Uppsala, Sweden, 3Dept Biochemistry and behavior to be correlated with attributes of pharmaceutical significance, Molecular Biophysics, Columbia Univ, New York, NY, USA, 4Department such as stability, efficacy, and immune tolerance. This understanding could of Biological Sciences, Columbia Univ, New York, NY, USA, 5Department allow for the more rational design of stabile and effective biopharmaceutical of Cell and Molecular Biology, Columbia University, New York, NY, USA, molecules and formulations. 6Dept Molec Biol, Uppsala Univ, Uppsala, Sweden.

BPJ 9475_9479 Wednesday, March 6, 2019 575a

Upon encountering a stop codon in the aminoacyl site (A site) of the mRNA for computer aided image segmentation and volume rendering. In this work, translating ribosome, the ribosome recruits a class-1 release factor (RF), we have investigated effective means of improving fine-alignment in three di- which induces hydrolysis of the ester bond between peptide chain and mensions to facilitate 3D image data reconstruction, visualization and peptidyl-site (P-site) tRNA. This process, called termination of translation, quantification. is under strong selection pressure for high speed and accuracy. Class-1 RFs The focal charge compensation module on the Zeiss Sigma VP/Gatan 3View (RF1, RF2 in bacteria, eRF1 in eukarya and aRF1 in archaea), have SBEM system greatly reduces charging artifacts and the need for coarse im- structural motifs that recognize stop codons in the ribosomal decoding age stack alignment. However, some lesser but important misalignments center (DC) and a universal GGQ motif for induction of ester bond hydro- remain, especially for backscattered electron imaging at multiple primary en- lysis in the peptidyl transfer center (PTC) 70 A˚ away from the DC. The ergies to extract sub-slice depth information, which require image stack finding that RF2 is compact with only 20 A˚ between its codon reading alignment with single voxel precision. For this fine alignment, we have tested and GGQ motifs came therefore as a surprise. Cryo-electron microscopy three available programs: scale-invariant feature transform (SIFT), patch- (cryo-EM) then showed that ribosome bound RF1 and RF2 have extended based cross correlation in IMOD, and TeraSticher, which can be applied to structures suggesting that RF1 and RF2 are compact when entering the very large datasets. A workflow of fine alignment of SBEM image stacks is ribosome and switch to the extended form in a stop signal-dependent proposed. manner. FRET, cryo-EM and X-ray crystallography have lent indirect support to this proposal and a rapid kinetics study further suggested that 2856-Pos the extended forms of class-1 RFs are short-lived intermediates with life- Polarization Agents for Sensitivity-Enhanced NMR Spectroscopy in Cells time on the millisecond time-scale. However, direct demonstration of Byung Joon Lim, Galia T. Debelouchina. such a switch in conformation on the native pathway to RF-dependent Dept Chem & Biochem, Univ Calif San Diego, La Jolla, CA, USA. termination is missing due to its transient nature. Here we use time- Nuclear magnetic resonance (NMR) has large potential for in cell structural resolved cryo-EM to visualize compact and extended forms of RF1 biology - it is compatible with physiological conditions and it can ˚ and RF2 at 3.5 and 4 A resolution, respectively, in the authentic codon- provide structural and dynamic information of biological macromolecules recognizing complex on the pathway to termination. The ensembles of mol- at atomic resolution with minimal perturbation. One severe limitation, how- ecules in the compact state are predominant at 20 ms after RF-binding to ever, is the low sensitivity of NMR compared to other cell compatible bio- the ribosome and are within 100 ms transformed into ensembles with physical techniques. This limitation can be overcome by using a extended RFs. methodology called dynamic nuclear polarization (DNP). DNP relies on the large Boltzmann polarization of unpaired electron spins, which is 660 2854-Pos times higher than the polarization of the 1H nuclear spins typically used Cryo-Electron Tomography, Faster: Development of a Fast-Incremental in protein NMR spectroscopy. In a DNP experiment, the electron spin polar- Tilting Scheme for Rapid Tomogram Acquisition ization is transferred to the nuclear spins resulting in large sensitivity en- Georges Chreifi1, Songye Chen1, Lauren Ann Metskas1, hancements and unprecedented savings in data acquisition time. Our David Mastronarde2, Grant J. Jensen1. group is developing polarization agents for DNP-enhanced NMR spectros- 1Biology and Biological Engineering, California Institute of Technology, copy in cells. These polarization agents contain unpaired electron spins Pasadena, CA, USA, 2Molecular Cellular & Developmental Biology, and are stable radicals or biradicals compatible with the cellular environ- University of Colorado Boulder, Boulder, CO, USA. ment. Furthermore, we are developing strategies to efficiently target Cryo-electron microscopy enables high-resolution imaging for both protein them to specific locations in the cellular milieu with the goal to polarize structure determination and cellular imaging studies. When a sample’s and enhance the NMR spectra of selected proteins in the cell. We will ultrastructure is unique, cryo-electron tomography can generate a volume present our progress towards these goals and outline examples of how image from a single sample, allowing the study of cellular architecture these novel reagents can aid NMR structural studies in the cellular or polymorphic viruses. This technique requires tilting the microscope stage environment. between each exposure, such that a collection of exposures is built into a stack that can be reconstructed into a three-dimensional volume. 2857-Pos Proteins within these volumes can be averaged, with sub-tomogram aver- Improved De Novo Main-Chain Tracing Method Mainmast for Multi- aged structures capable of reaching high resolution. However, each tomo- Chain Modeling, Local Refinement, and Graphical User Interface gram can require between 20-60 minutes of data collection time, Genki Terashi1, Yuhong Zha2, Daisuke Kihara1,3. restricting the number of tomograms that a user can collect in time- 1Dept Biol Sci, Purdue University, West Lafayette, IN, USA, 2School of limited microscopy sessions. As the final resolution of sub-tomogram aver- Computer Science, Carnegie Mellon University, Pittsburg, PA, USA, 3Dept ages is dependent upon the number of particles in the dataset, the number of Comp Sci, Purdue University, West Lafayette, IN, USA. tomograms collected is severely limiting in cases where protein copy num- The significant progress of the cryo-EM poses a pressing need for software ber is low. for structural interpretation of EM maps. Particularly, protein structure The Jensen lab has developed a ‘‘fast-incremental’’ tilt scheme, whereby a modeling tools are needed for maps determined around 4 A˚ resolution, single tomogram can be collected in 3-10 minutes. An FEI Titan Krios where finding main-chain structure and assigning the amino acid sequence cryo-electron microscope and Gatan K2 camera are operated in continuous into EM map are still challenging problems. We have developed a de acquisition mode, using SerialEM software to blank the electron beam dur- novo modeling tool named MAINMAST (MAINchain Model trAcing ing stage movement; the resulting acquisition is similar in geometry and from Spanning Tree, http://kiharalab.org/mainmast/) for EM maps for this exposure to a conventional tomogram. We find that the precision of a resolution range (Nature Communications, 2018). MAINMAST builds single-tilt stage and a camera with fast frame capture rates and high sensi- main-chain traces of a protein in an EM map from a tree structure con- tivity compensate for the lack of time-consuming stage tracking and adjust- structed by connecting points with a high density in the map without refer- ments during data collection, to a resolution of roughly 2.5 nm. Further ring to known protein structures or fragments.Here, we report substantial hardware and software development will improve the resolution and utility improvements of MAINMAST in three aspects. The largest improvement of this method. is that the method now can perform automatic map segmentation and structure modeling for symmetrical multi-chain complexes. The tree 2855-Pos structure that connects dense points are traced for multiple chains simulta- Improved Visualization of Structure at the Nano-Scale in Entire Eukary- neously in a symmetric fashion. Moreover, the accuracy of a model is signif- otic Cells by Fine Alignment of Serial Block Face Sem Image Stacks icantly improved by a new implementation of local sequence matching Qianping He, Matthew D. Guay, Guofeng Zhang, Richard D. Leapman. and structure refinement. The local matching protocol is also useful for NIBIB NIH, Bethesda, MD, USA. identifying missing regions in a structure model, i.e. regions with a low Serial block face scanning electron microscopy (SBEM) provides nanoscale density, in an EM map. Finally, we developed a software plugin of 3D ultrastructure of eukaryotic cells and tissue volumes. It allows automated MAINMAST for the UCSF Chimera, so that users can monitor structures acquisition of thousands of sequential images at different depths within a spec- at each step of a modeling procedure. The major functionalities include to imen block without layer-to-layer warping problems and section loss. Howev- generate and to display tree structures from local dense points in the map, er, image drift, jitter and distortion can still occur due to mechanical main-chain traces, and reconstructed all-atom models. Through the inter- instabilities and build-up of surface charge. These artifacts pose challenges face, users can easily control parameters of MAINMAST and save and to 3D image stack alignment, which is crucial for post data analysis, especially restore sessions.

BPJ 9475_9479 576a Wednesday, March 6, 2019

2858-Pos channels, a two-fold symmetry was observed in hBK. Within the tetramer, Structural Insights into Entry and Antibody Neutralization of Eastern two opposing subunits in the Ca2þ sensing gating ring rotate around the center Equine Encephalitis Virus of mass of each subunit, which results in the movement of the assembly inter- Syed Saif Hasan1, Chengqun Sun2, Arthur Kim3, Yasunori Watanabe4, faces, flexible interfaces and Ca2þ binding sites. Despite the significant move- Chun-Liang Chen1, Thomas Klose1, Geeta Buda1, Max Crispin4, ment, the local conformation of the assembly interfaces and Ca2þ binding sites Michael S. Diamond3, William B. Klimstra2, Michael G. Rossmann1. remains the same among the four subunits. 1Purdue Univ, West Lafayette, IN, USA, 2Univ Pittsburgh, Pittsburgh, PA, USA, 3Washington Univ, St. Louis, MO, USA, 4Univ Southampton, 2861-Pos Southampton, United Kingdom. Cryo-EM Imaging of Kv1.2 Channels with Membrane Potential Applied Alphaviruses are enveloped pathogens that cause arthritis and encephalitis. Hideki Shigematsu1, Youshan Yang2, Yangyang Yan2, Fred J. Sigworth2. 1Life Science Research Infrastructure Group, RIKEN SPring-8 Center, Host entry of alphaviruses involves binding to plasma and endosomal mem- 2 branes followed by low pH-triggered membrane fusion. Here we present Hyogo, Japan, CM Physiology, Yale Univ, New Haven, CT, USA. a 3.5-6.5A˚ resolution (average 4.4A˚ ) cryo-electron microscopy (cryoEM) We use cryo-EM methods to image Kv1.2 potassium channels reconstituted structure of Eastern Equine Encephalitis virus (EEEV), an alphavirus that often into small (40-50 nm) liposomes. A zero-potential structure at 7.5 angstrom res- causes fatal encephalitis in humans. Motifs for potential binding of host mem- olution agrees well with the crystal structure of the detergent-solubilized brane receptors were identified. The capsid protein structure showed an RNA ‘‘native’’ channel complex (S. Long, E. Campbell and R. MacKinnon, 2005). genome-binding segment and an inherently unstable nucleocapsid core primed Flux assay results confirm that a negative membrane potential can be estab- for disassembly. CryoEM structures of five different Fab-EEEV complexes lished in this system, and we now report results from our imaging these chan- derived from neutralizing antibodies showed how the icosahedral structure of nels in what should be their resting state. alphaviruses poses steric restrictions on interactions with antibodies. These re- sults also identified molecular mechanisms of host cell entry that likely are 2862-Pos conserved among enveloped icosahedral viruses. Reconstruction of Average Subtracted Tubular Regions (RASTR) Peter S. Randolph, Scott Stagg. 2859-Pos Inst. of Molecular Biophysics, Florida State Univ, Tallahassee, FL, USA. Endophilin B1 and Membrane Remodeling at the Brink of Death Tubular structures are common occurrences in biological systems. Tubules Veer Bhatt, Robert Ashley, Anna C. Sundborger-Lunna. can be formed from lipid membranes that have been deformed by a protein Cryo-EM and Molecular Cell Biology, Hormel Inst, Austin, MN, USA. lattice or only ordered protein oligomerization as is the case of filaments. Mitochondria are highly dynamic organelles constantly undergoing fission In many cases there is substantial heterogeneity in the widths of the tubules, and fusion events. Dysregulation of mitochondria dynamics is associated the proteins that make up the tubules, or proteins that decorate the tubules. with changes in organelle function, transport, location, and quality control. This heterogeneity can create a challenge when attempting to use cryo-EM As such regulators of mitochondria dynamics are critical for cell health. Mem- to determine the structures of the tubular proteins. The common method of brane remodeling Bin/Amphiphysin/Rvs (BAR) protein endophilin B1 has using helical symmetry to resolve the structure will average out areas of het- been implicated in these processes, although the underlying mechanisms are erogeneity, removing areas of interesting. We have developed a method called poorly understood. BAR proteins mediate remodeling of cellular membranes Reconstruction of Average Subtracted Tubular Regions (RASTR) to deal with by inducing varying degrees of membrane curvature, a property that is pro- heterogeneity by treating the tubules not as helical filaments and relying on moted by protein dimerization and assembly of crescent-shaped BAR domains symmetry, but instead focusing on a segment of one surface of the tubule. that enforce their shapes onto membranes. Knockdown of endophilin B1 leads During RASTR, we first align the tubules vertically, then determine the to inhibition of apoptosis and autophagy, suggesting that endophilin B1- azimuthal global average of the tubules. This is then reconstructed in 3D mediated membrane remodeling coordinates mitochondria dynamics with which gives a smoothed-out representation of the surface of the tubules. A critical cell death processes. We have solved the structures of endophilin segment of the smooth 3D tubule is masked out leaving a tubule with a B1 tetramers in solution and assembled on mitochondrial outer membrane- missing segment on one surface. This is then projected and subtracted from like liposomes by cryo-EM. Our 3D reconstructions indicate that endophilin individual cryo-EM images of the tubules, resulting in images of the tubule B1 promotes remodeling of membranes by assembling into tetramers that with everything but one surface strongly down-weighted. The up-weighted further organize into small assemblies or helical scaffolds on mitochondrial surface can then be treated as a single particle and can be aligned and classi- membranes. Our previous work has revealed that endophilin A1 controls fied by single particle methods. A benefit of our method is that it limits the recruitment of dynamin 1 to sites of plasma membrane fission, and our prelim- degrees of freedom of alignment, which can allow us to drop particles that inary 3D reconstruction of endophilin A1-dynamin 1-decorated lipid tubes align outside a narrow range. Here we will present our results comparing indicate that endophilin A1 assembles into a membrane template for dynamin the reconstruction of generated model data and real data of the same tubules 1. Similarly, endophilin B1 promotes recruitment of dynamin 2 to constricted (VIP tubules). Golgi tubules upon autophagosome formation. Based on these findings, we propose that endophilin B1 is critical for mitochondria dynamics and Bax- 2863-Pos a mediated apoptosis due to its unique ability to remodel the mitochondrial Structural Insights into the Disease-Causing Mutant -Actinin 4 K255E outer membrane, which controls recruitment and organization of effector pro- Bound to F-Actin teins, Bax and dynamin family members, and activation of down-stream mito- Weili Zheng1, Joan L. Arolas2, Slobodan Vujin2, Kristina Djinovic-Carugo2, Edward H. Egelman1. chondrial events. Our findings provide unique mechanistic insights into novel 1 2 mechanisms of BAR protein-mediated membrane remodeling controlling Univ Virginia, Charlottesville, VA, USA, University of Vienna, Vienna, cell fate. Austria. a-actinin 4 is a member of the spectrin family of actin-binding proteins, 2860-Pos involved in the regulation of cell motility and adhesion. It forms an anti- An Intermediate State of Human BK Channel Reconstituted in Liposomes parallel rod-shaped heterodimer with the N-terminal actin-binding domain Lige Tonggu, Liguo Wang. (ABD) at two ends, which mediate cross-linking F-actin to form actin bundles. Dept Bio Struct, Univ Washington, Seattle, WA, USA. Several mutations in the a-actinin 4 gene (ACTN4) ABD have been found to Voltage-gated and ligand-modulated ion channels play critical roles in excit- cause a kidney disease called familial focal segmental glomerulosclerosis able cells such as neurons and muscle cells. To understand the interplay among (FSGS). It has been reported that the K255E mutation significantly increase voltage-sensing, ligand-binding and channel opening, the structures of ion the binding affinity of a-acyinin 4 to F-actin leading to abnormal aggregates channels under various transmembrane potentials need to be determined. To in podocytes. However, the relationship between the mutation-induced human achieve that, a method called ‘‘random spherically constrained’’ (RSC) kidney disease and the increased affinity of a-actinin 4 binding to F-actin are single-particle cryo-EM was developed. The RSC method establishes an asym- still unknown, partly due to the lack of an atomic model of the complex of metric lipid environment for membrane proteins and makes it possible to apply F-actin and a-actinin 4. Here, we present a 4.2 A˚ cryo-EM structure of a-actinin the desired transmembrane potential to trigger voltage-sensitive membrane 4 bound to F-actin, which provides structural insight into the mode of interac- proteins including voltage-gated ion channels. Here, the RSC method was em- tion between F-actin and a-actinin 4 ABD. Residues involved in a-actinin 4 ployed to study the human large conductance voltage- and calcium-activated ABD binding to F-actin are highly conserved among other spectrin family pro- potassium (hBK or hSlo1) channels reconstituted into liposomes. The hBK teins. This thus provides a framework for future therapeutics in treating a-ac- structure in an intermediate state has been determined at 3.5 A˚ resolution. tinin 4-associated diseases and a general perspective for how other actin cross- Instead of the common four-fold symmetry observed in ligand-modulated ion linking proteins bind to F-actin through ABDs.

BPJ 9475_9479 Wednesday, March 6, 2019 577a

Posters: Biosurfaces integration of specific GBPs into the E. coli outer membrane protein in- creases binding to both Au(111) surfaces and AuNPs. Second, from molec- 2864-Pos ular dynamics (MD) simulations of select free GBPs in aqueous solution and Investigating the Biophysical Changes in Prey Cells under Attack by Wild on Au(111), we provide insights into the intermolecular interactions and dy- Bdellovibrio namics driving peptide binding to gold that will be utilized to drive the Ciara Dwyer, Catherine B. Volle. rational design of inorganic-binding peptides. To conclude, we will discuss Biology, Cottey College, Nevada, MO, USA. these findings in the greater context of our work in living materials, namely Bdellovibrio bacteriovorus is a Gram-negative predatory bacterium that our efforts in fashioning sense and respond materials such as designer coat- preys on other Gram-negative bacteria. Bdellovibrio is a key component ings and self-healing composites. in ecosystems as diverse as sewage, ocean waters, and even the human intes- tine. It serves as an ecological regulator, ensuring that bacteria don’t over- 2867-Pos grow in a given environment, and represents a novel approach to Probing Biophysicochemical Interactions at Nano-Bio Interface of Perov- combating antibiotic-resistant bacteria. While the molecular mechanism un- skite Tandem Biosolar Cells derlying bdellovibrio recognition of its prey is still unknown, once bdellovi- Subhabrata das1, Teguh Citra Asmara2, Zhaoning Song3, Andrivo Rusydi2, brio has recognized a prey cell, it buries into the periplasmic space and Bernardo Barbiellini4, Ponisseril Somasundaran1, secretes enzymes that cause the prey to round up. The rounded, infected Venkatesan Renugopalakrishnan5. prey cell is now called a bdelloplast, and bdellovibrio scavenges nutrients 1Langmuir Center of Colloids and Interfaces, Columbia University, New from the prey. Previous work using atomic force microscopy has shown York, NY, USA, 2Condensed Matter and Material Physics, National that during predation, E. coli become less stiff and the outer membrane be- University of Singapore, Singapore, Singapore, 3Physics and Astronomy, comes sticky and starts to pull apart. However, most research performed University of Toledo, Toledo, OH, USA, 4Physics, Lappeenranta University with bdellovibrio uses a lab strain, either 109J or HD100, which have of Technology, Lappeenranta, Finland, 5Northeastern University, Boston, been selected for predation ability. Therefore, the biophysical changes MA, USA. observed in the prey cell in these experiments may represent an extreme. A first-principles study of the dielectric spectrum from Spectroscopic El- By isolating wild bdellovibrio from freshwater sources and repeating the ex- lipsometry associated with the photoactive chromophore in bacteriorho- periments, we will provide information on the variation of biophysical dopsin (bR) and its various mutant (Cys-35, D96N, triple glutamic changes in the prey cell. mutationsE9Q/E194Q/E204Q) forms allowed the explanation of the effec- tive coupling existing between the p electrons of the chromophore and the 2865-Pos local environment of the Schiff base when adsorbed to the metal substrate. Characterizing the Persistance and Adhesion of Multi-Species Biofilms The role of the counter-ion on the electronic structure of the chromophore During and After Predation by Bdellovibrio bulk is investigated by means of Gradient Projector Augmented density Celestine Ooko, Catherine B. Volle. functional theory (GPAW-DFT) calculations. Experimental Spectroscopic Biology, Cottey College, Nevada, MO, USA. Ellipsometry data have been interpreted in the light of these theoretical Bdellovibrio bacteriovorus is a Gram-negative predatory bacterium that findings with an interesting accumulation of points along the optical preys on other Gram-negative bacteria, but not other Bdellovibrio. Bdellovi- band-gap. XANES and Photoemission measurements are further performed brio acts as an ecological regulator, making sure that populations of Gram- to study the bio-physicochemical interactions at the nano-biointerface for negative bacteria don’t over grow. Importantly, Bdellovibrio can hunt both bR adsorbed on Au and Mesoporous TiO2 Substrates. The HOMO- free-swimming planktonic bacteria as well as bacteria that have attached LUMO interaction with the bR environment can stabilize the chromophore. to a surface and formed an organized community called a biofilm. Biofilms Based on the spectroscopy results and DFT Calculation, a new FTO/TiO2/ form when planktonic bacteria attach to a surface and begin secreting exo- perovskite/Spiro//bR/Au Solar cell architecture could be proposed. The role polymeric substance (EPS), a combination of DNA, proteins, and polysac- of the traditional Semiconducting quantum dots (SQDs) in terms of charides. The EPS provides a protective layer surrounding the cells, their spintronic functionality and photosensitization will now be performed making biofilms hard to remove by physical and chemical means. Previous by the organohalide Perovskite in the proposed tandem solar cell research has investigated changes in a single species biofilm under attack by architecture. Bdellovibrio, however most biofilms are composed of multiple species. Thus we have created model biofilms containing two different species of bacteria, Bioengineering the Gram-negative E. coli ZK1056 and the Gram-positive Staphylococcus aureus . By allowing Bdellovibrio to hunt in the biofilm over time we will 2868-Pos determine how the biofilm responds to loss of one species, whether the bio- Design and Implementation of 3D-Printable Optomechanical Components film will collapse with the removal of one species or if the other species will Ryan Bullis, Julie Gunderson. simply increase growth to replace the lost species. Furthermore, using Physics, Hendrix College, Conway, AR, USA. atomic force microscopy, we will determine if the EPS adhesion changes 3D printing is an additive manufacturing modality with a range of applica- during Bdellovibrio predation, and explore the use of adhesion mapping as tions that have grown dramatically over the last several decades. As Fused a method of characterizing biofilms. Filament Fabrication (FFF) 3D printers have become more affordable, many scientists and engineers have begun printing customized parts for ex- 2866-Pos periments and prototypes. FFF is a 3D printing technique in which thermo- Computational and Experimental Approaches to Understand a Living plastic is extruded by a robot onto a hard, flat surface in successive layers to Biotic-Abiotic Interface using Gold Binding Peptides create an object from a Computer Aided Design (CAD) file. Because many Meagan C. Small1, Deborah A. Sarkes2, Hong Dong2, scientific applications require parts that are expensive to purchase or manu- Dimitra N. Stratis-Cullum2, Margaret M. Hurley1. facture, 3D printing custom parts for scientific instrumentation can save 1Sensors and Electronic Devices Directorate, U.S. Army Research valuable (shipping and/or manufacturing) time and money and requires Laboratory, Aberdeen Proving Ground, MD, USA, 2Sensors and Electronic only one moderately priced printer. Thus, 3D printing has emerged as a Devices Directorate, U.S. Army Research Laboratory, Adelphi Laboratory viable low-cost method to produce custom scientific instruments, therefore, Center, MD, USA. increasing the progress of research. Here, we present a library of 3D print- Bio-abiotic hybrid materials have many potential applications in medicine, able optomechanical components for use in research-grade optical systems. electronics, and nanotechnology. Peptides with predictable and controllable These components are fully compatible with commercial optomechanical binding to inorganic surfaces are of great interest, in which bioengineered components and include posts, post holders, bases, lens mounts, kinematic proteins can be introduced into bacterial cells to confer nonnative function- mirror mounts, filter mounts, and micrometers. These components were ality and thereby alter whole-cell behavior. With this is mind, part of our tested for their optical stability and durability in home-built optical systems work has been focused on engineering E. coli to display metal binding pep- constructed entirely from 3D printable optomechanical components, and we tides using bacterial display system eCPX. Here, we present our efforts in demonstrate that these systems performed comparably to their more expen- understanding cell binding to Au(111) surfaces as well as gold nanoparticles sive, commercially available counterparts. Thus, we expect our library of 3D (AuNPs) through a series of known and designed gold binding peptides printable optomechanical components to find utility in scientific research (GBPs) that have been engineered into eCPX. First, we demonstrate that laboratories.

BPJ 9475_9479 578a Wednesday, March 6, 2019

2869-Pos surface come from a coat or shield of carbohydrates present on it. These carbo- Macrophage Checkpoint Blockade and Tumor Mechanics in a Cell-Based hydrates are attached to proteins embedded within the virus envelope or mem- Immunotherapy brane. Current research in this field focuses on the chemical recognition of the Lawrence J. Dooling, Jason C. Andrechak, Charlotte R. Pfeifer, sugars or proteins on the virus coat by receptors on host cells. Our goal is to Dennis E. Discher. interrogate the physical interactions of the virus coat in order to research University of Pennsylvania, Philadelphia, PA, USA. broad-spectrum strategies that will disrupt the protection afforded by carbohy- Receptors that help immune cells recognize ‘self’ cells can be functionally drate shield. The specific aim of the presented research is to test the propensity blocked with antibodies, and a fraction of cancer patients show durable cures of sugar residues constituting the virus glycan shield to self-interact. The pseu- through blockade of such T-cell checkpoints. Macrophages possess a similar dovirus HIV pol- env- / VSV-G has a glycan shield that composed of mannose, checkpoint that we block in combination with tumor opsonization in order to galactose, sialic acid, and glucosamine. target injected macrophages for phagocytosis of cancer cells. The approaches We synthesized gold nanoparticles coated with these sugars and tested the require immune cell infiltration, which motivates study of the physical proper- interaction with the pseudovirus. UV-VIS spectrophotometer results show ties of tumors. We are specifically blocking signal regulatory protein alpha that the plasmon resonance peak of the sugar-coated nanoparticles shifted in (SIRPa) on macrophages, which binds the ubiquitous ‘self’-marker CD47, the presence of several of these sugars, but not in the presence of the control and we inject these cells together with a pro-phagocytic antibody against citrate-coated nanoparticles. The shift in colors is consistent with the coating tyrosinase-related protein 1 (Tyrp1) on syngeneic B16 melanoma in an immu- on the nanoparticles being modified, possibly by binding to viruses. The UV nocompetent mouse. This widely used preclinical murine model for evaluating studies were complemented by Dynamic Light Scattering studies which immunotherapies is sometimes described as a ‘solid’ tumor, but macropipette show a larger aggregate virusþnanoparticle species developing when there is aspiration of fresh tumors reveals high compliance and largely irreversible a plasmon shift. AFM imaging was performed to deconstruct the structure of deformation. Such properties will generally modulate macrophage infiltration, the aggregates. Preliminary results indicate that the sialic acid and mannobiose interactions, and phenotype. As we continue to study such effects, we already residues on the surface of a virus have a greater propensity for self-interactions, find that systematic injection of engineered macrophages into mice with subcu- and may play a role in host-virus interaction since host cells are also abundant taneous B16 tumors results in delayed tumor growth and even some durable in these residues. cures. 2872-Pos 2870-Pos The Treatment of MDA-MB-231 Breast Cancer Cells with Biocompatible Probing the Role of HIV Antigen Nanoscale Organization on B-Cell Acti- Manganese Iron Oxide Nanoparticles as Drug Carriers vation with DNA Origami Negin Farzad1, Christina Zito2, Saion K. Sinha3. Remi Veneziano1, Tyson Moyer2, Matthew B. Stone2, Sudha Kumari2, 1Biomedical Engineering, Univ New Haven, West Haven, CT, USA, 2Dept William R. Schief3, Mark Bathe2,4, Darrell Irvine5,6. Biology & Environmental Science, Univ New Haven, West Haven, CT, USA, 1Department of Bioengineering, George Mason University, Fairfax, VA, 3Dept Electrical & Computer Engineering, Univ New Haven, West Haven, USA, 2Department of Biological Engineering, Massachusetts Institute of CT, USA. Technology, Cambridge, MA, USA, 3Department of Immunology and Targeting cancer cells and killing them without affecting normal healthy cells Microbial Science, IAVI Neutralizing Antibody Center, Scripps Research is one of the primary objective in cancer treatment. Thus, developing better tar- Institute, La Jolla, CA, USA, 4Broad Institute of MIT and Harvard, geted drug delivery mechanisms, will enhance the efficacy of the use of already Cambridge, MA, USA, 5Department of Materials Science and Engineering used chemotherapeutic drugs significantly by decreasing its harmful side ef- and Department of Biological Engineering, Massachusetts Institute of fects. Our earlier studies have demonstrated that polymer coated Magnetic Technology, Cambridge, MA, USA, 6Koch Institute for Integrative Cancer Nanoparticles (MNP) together with a chemotherapeutic drug, are 50% more- Research, Cambridge, MA, USA. efficient drug delivery agent compared to the intrinsic drug itself. We have HIV germline-targeting antigens are promising immunogens in development also observed that the interaction of the applied DC magnetic field with the for HIV vaccines, which have the goal of priming progenitor B-cells capable MNP is primarily decided by its Curie temperature. of evolving toward the production of broadly neutralizing antibodies In the current paper we report the results by expanding the scope of this study. (bNAbs). Amongst these antigens, eOD-GT8 engineered from the envelope This was achieved by changing three parameters, a different MNP (MnFe2O4) glycoprotein gp120 has been previously reported to be highly immunogenic having a lower Curie temperature but a stronger magnetic susceptibility, a only when presented in a multimeric form. B-cell activation is initiated upon different cell-line (MBA-MD-231 breast cancer cells) which has been reported engagement of cell membrane expressed B-cell receptors (BCR) by antigens. to show difference in similar studies, and finally including Electro-magnetic Although the intracellular signaling cascade following antigen binding is well field as the coupling agent in place of DC magnetic field. understood, very little is known about the early events of recognition and Manganese iron oxide (MnFe2O4) nanoparticles were coated with PEG (Poly- binding of antigens by the BCR. In particular, the importance of antigen ethylene glycol) and Sodium Oleate and their biocompatibility were tested on nanoscale organization and valency remain not well understood even though cells compared to treated cells with naked nanoparticles. The MNP were sub- these parameters may be critical for the development of a successful vaccine. sequently coupled with doxorubicin and magnetically delivered to MDA-MB- Here, to gain more insight into the molecular mechanisms leading to B-cell 231 breast cancer cells. Furthermore the in vitro viability of the cells were stud- activation, we use scaffolded DNA origami nanoparticles to test various ied both on coupling the MNPs with DC and AC electromagnetic field. The re- models of BCR-mediated activation. DNA nanoparticles offer the unique op- sults of this study show that the Mn-MNPs are more biocompatible than other portunity to organize HIV antigens in a precise nanoscale organization and MNPs on this particular cell line. Also it is observed that AC electromagnetic with control of valency. We used a combination of cytoplasmic calcium field have more efficient drug delivery mechanism than DC magnetic field. influx assay and fluorescence imaging to monitor antigen binding and subse- quent B cell activation for multi-dimensional presentation of the eOD-GT8 2873-Pos immunogen. Thus, we determined that an inter-antigen distance greater Ultrasound-Mediated Targeted Drug Delivery in T-Cells Alina Karki1, Emily Giddings2, Mercedes Rincon2, Junru Wu1. than 28 nm and a copy-number of eOD-GT8 above 5 per nanoparticles is suf- 1 2 ficient to trigger maximal B-cell activation. Taken together, our results Physics, University of Vermont, Burlington, VT, USA, Medicine, demonstrate the usefulness of DNA origami nanoparticles to probe the role University of Vermont, Burlington, VT, USA. of nanoscale viral antigen presentation on immune cell activation. Moreover, Ultrasound in mega-hertz range, interacting with the gas encapsulated micron- this study opens the way to the development of new vaccines for HIV and sized bubbles makes temporary mini pores on the nearby cell membranes by the other infectious diseases. shear stress. Thus, specific drugs could be delivered inside the cells via these mini holes. This process is called the sonoporation and is one promising non- 2871-Pos viral approach for the targeted drug and gene delivery. Small interference Self-Interactions of a Virus Glycan Shield RNA (siRNA) has been used to silence expression of molecules in cells in vitro. Eric E. Ogharandukun1, Hashanthi K. Abeyratne-Perera2, The delivery of siRNA to different tissues have been limiting factors for their Preethi Chandran1. use as therapeutics. Similarly, T-cells are extremely difficult to be transfected. 1Chemical Engineering, Howard University, Washington DC, DC, USA, Considering the fact that small interference RNA (siRNA) delivery, particu- 2Biochemistry, Howard University, Washington DC, DC, USA. larly in T-cells have been challenging in medical field by various viral and Viruses are able to infect cells because they possess a cell-surface component non-viral methods, we have performed in-vitro experiments using this tech- which enables them to bind on host cells, escape the surveillance of the immune nique to deliver siRNA molecules inside the mouse and human T-cells. We system, and survive harsh environments. These physical properties on virus have optimized the ultrasound parameters (acoustic frequency intensity, duty

BPJ 9475_9479 Wednesday, March 6, 2019 579a cycle, time duration of ultrasound excitation), concentration of DEFINITYÒ efficiency, the effect of membrane composition and domain formation on microbubbles and siRNA per sample to provide efficient siRNA drug delivery fusion efficiency has not been as widely explored. Here, we demonstrate that in human and mice T-cells. fusion efficiency between small unilamellar vesicles containing surface bound DNA tethers can be tuned by altering vesicle membrane characteristics, such as 2874-Pos phase segregation and hydrophobic mismatch. Using FRET assays for lipid Stable Hybrid Nanopores for Biomolecule Sensing mixing, we demonstrate that vesicle membranes containing liquid ordered 1 2 3 1 Mehrnaz Mojtabavi , Sandra Greive , Benjamin Cressiot , Xinqi Kang , and disordered phases improve the rate and efficiency of membrane fusion. Alfred Anston2, Meni Wanunu3. 1 We find that hydrophobic mismatch between domains further enhances vesicle Department of Bioengineering, Northeastern University, Boston, MA, USA, fusion kinetics. The enhancement of fusion due to hydrophobic mismatch was 2Department of Chemistry, University of York, York, United Kingdom, 3 reduced when phase segregation was absent and membranes were homoge- Department of Physics, Northeastern University, Boston, MA, USA. nously mixed, emphasizing the importance of domain formation in enhancing Nanometer-sized pores (nanopores) have emerged as a novel single-molecule membrane fusion. Finally, we demonstrate that fusion efficiency correlates detection technique to probe biomolecules (protein, DNA, and RNA) electri- with the efficiency of content mixing. Using phase-segregated vesicles, we cally and/or optically whilst being threaded into the pore. Since then, demonstrate the initiation of model biological reactions driven through naturally-occurring pores known as biological nanopores (membrane proteins DNA-mediated vesicle fusion. This study shows how membrane biophysics and bacteriophage portal proteins) along with synthetically-assembled nano- can be used to enhance DNA-mediated fusion events and advances our capabil- pores known as solid-state nanopores have been employed to study various bio- ities in controlling localized reactions, an important step in developing more molecular characteristics. A third class of nanopores, known as hybrid complex vesicle-based chemical systems. nanopores, exploits the robust framework and mechanical stability of SS nano- pores with atomically reproducible and tunable characteristics of biological 2877-Pos nanopores. Biological nanopores supported by robust synthetic membrane Does Membrane Asymmetry Affect Nanoparticle-Membrane Interactions eliminates fragility of lipid bilayers and arbitrary geometry of SS nanopores. Saeed Nazemidashtarjandi, Amir M. Farnoud. Our group recently demonstrated the lipid insertion and stable formation of hy- Ohio Univ, Athens, OH, USA. drophilic G20C portal protein transmembrane channels, derived from the G20C Plasma membrane damage is one of the primary mechanisms through which thermostable virus. Herein, we demonstrate the improved performance of our engineered nanomaterials cause cytotoxicity. Despite a number of studies, system by substituting the lipid bilayer support of the protein by thin SiN x the role of the structural features of the plasma membrane, in particular mem- free-standing membrane and making further modifications to chemically fix brane phospholipid asymmetry in regulating nanoparticle-membrane interac- the protein on the membrane support. Finally, we report the application of tions has remained obscure. In the current study, the role of membrane our hybrid system in nanopore-based sensing of biomolecules. phospholipid asymmetry, differences in the lipid composition of exofacial 2875-Pos and cytofacial leaflet of the plasma membrane in regulating nanoparticle- A Fatty Acid Induces the Functional Assembly of a Channel Protein into membrane interactions was investigated. Vesicles mimicking the exofacial Phospholipid Vesicles (Vexo) and cytofacial leaflets (Vcyto) of the plasma membrane of erythrocytes Claire Hilburger, Kamryn Lewis, Miranda Jacobs, Neha P. Kamat. were exposed to silica nanoparticles (50 nm), with different surface functional Biomedical Engineering, Northwestern University, Evanston, IL, USA. groups, plain, or carboxyl-, amine-, and polyethylene glycol (PEG)-modified The release of encapsulated cargo is an important step towards the design of and the effect of particles on vesicle integrity was examined. Symmetric and intelligent drug delivery systems or vesicle-based bioreactors. Controlling the asymmetric vesicles with similar outer leaflet, but different inner leaflet lipid integration of pore forming channels into phospholipid vesicle membranes pro- compositions, as well as live erythrocytes, were also used to elucidate the vides a route to initiate content release that could be coupled to gene-expression role of membrane asymmetry and individual leaflets in regulating systems. Here, we present the design of phospholipid vesicles that release cargo nanoparticle-membrane interaction. Vexo and Vcyto vesicles showed significant upon the temporally controlled assembly of pore-forming channels. Our difference in their interactions with nanoparticles. Vexo vesicles showed drastic approach was to design a synthetic vesicle in which membrane composition leakage of intravesicular content after exposure to plain and amine-modified could be remotely changed to enable the assembly of a pore-forming channel particles while none of the particles caused significant leakage in Vcyto vesicles. protein, a-hemolysin (aHL), and trigger content release. We first observed FRET experiments revealed that nanoparticle-induced disruption by plain and that a membrane lacking a sufficient concentration of cholesterol is not compat- amine-modified particles occurs via pore formation. In agreement, FRET assay ible with channel assembly and we hypothesized that increasing membrane and GUV images revealed significant localization of plain and amine-modified fluidity, through the addition of oleic acid (OA) micelles could promote chan- particles at the surface of the Vexo vesicles. However, no localization was nel assembly. Using a dye release assay, we first confirmed that increasing OA observed with the Vcyto vesicles. Interestingly, symmetric and asymmetric ves- content in phospholipid membranes leads to pore assembly. Next, we demon- icles were disrupted similarly by plain and amine-modified particles. Impor- strated that remote addition of OA micelles to inactive membranes results in tantly, hemolysis was observed after the incubation of erythrocytes with fatty acid membrane uptake and channel assembly. We then showed that our plain and amine-modified particles, consistent with leakage assays using Vexo system provides a route to control the integration of aHL into vesicle mem- vesicles. In conclusion, these results suggest that the exofacial leaflet of the branes when the channel protein is produced through a cell-free expression sys- plasma membrane is the primary regulator of nanoparticle-induced membrane tem. We remotely added OA micelles to vesicles present in a cell-free damage in erythrocytes. expression system where the amount of protein expressed is difficult to control. OA addition led to functional pore assembly and dye release. We ultimately 2878-Pos demonstrate a new way to utilize remote changes in membrane composition Engineering a Coiled Coil Protein as pH Sensor to control aHL assembly and content release. Our study contributes to the Ameed Hashmi, Mourad Sadqi, Victor Mun˜oz. design of vesicle-based technologies and also reveals new insight into the rela- Univ CA Merced, Merced, CA, USA. tionship between membrane composition and cholesterol-dependent cytolysin To design our pH sensor, we decided on using an anti-parallel coiled-coil pro- a channel assembly. tein. The protein consists of two -helices connected by a loop, adopting a super-coiled tertiary structure. Designing and engineering of coiled-coil pro- 2876-Pos teins are already parametrized, so length of the helices can be controlled as Tuning Membrane Composition to Enhance DNA-Mediated Vesicle desired. The approach for our sensor design incorporates the ‘binding coupled Fusion to folding’ design-principle with grafting of histidines (H). The partially pro- 1 2 Justin A. Peruzzi , Neha P. Kamat . tonated side chain of histidine has a pKa close to physiological pH and can 1Chemical and Biological Engineering, Northwestern University, Evanston, switch between protonated (general acid) and unprotonated (general base) IL, USA, 2Biomedical Engineering, Northwestern Univ, Evanston, IL, USA. states. This switching from an aromatic (at higher pH) to positively charged Bilayer vesicles have great potential as bioreactors that can act in a variety of (at lower pH) state, can have a role in (un)stabilizing the folded structure of biological environments. As synthetic vesicles continue to be designed with the protein. We mutated H into the hydrophobic core to manipulate the increasingly complex activities, such as housing cascaded reactions or genetic coiled-coil structure stability, resulting in an intrinsically unstable native state. circuit-driven protein production, an important question has arisen about how The protein populates the unfolded state when the concentration of protons is to control the progress of such reactions. Vesicle fusion driven through lipid- low and as the concentration of protons is increased, the protein ensemble shifts conjugated DNA tethers is a promising route to control content mixing between to the folded state. To monitor this transition, we used quenching of fluorescent specific vesicles. While most studies on DNA-tethered fusion have focused on dye (Atto 655). As the protein folds, photo-induced electron transfer (PET) oc- how the geometry and chemistry of DNA oligomers may impact vesicle fusion curs between the excited state dye and the ground-state of tryptophan (W). Our

BPJ 9475_9479 580a Wednesday, March 6, 2019 results show quenching of about 80% of florescence intensity as the pH is low- significantly than Cis- BSBCA22-peptide. Furthermore, we design the opti- ered from 8-4, also corroborating our circular dichroism studies. mized inhibitor peptide to be modified with bifunctional and monofunctional azobenzene derivatives. 2879-Pos Engineering a Cytochrome with a Tunable Bandgap Potential 2881-Pos Samuel D. Fontaine1, Coleman Swaim2, P. Raj Pokkuluri3, 4 Photo-Control of Small GTPase RAS using Photo Responsive Peptide Oleksandr Kokhan . Inhibitor Which Mimic aH Helix of SOS 1Chemistry, James Madison University, Harrisonburg, VA, USA, 2James Masahiro Kuboyama1, Nobuyuki Nishibe1, Kenichi Taii1, Madison Univ, Harrisonburg, VA, USA, 3Argonne National Lab, Lemont, IL, Toshio Nagashima2, Toshio Yamazaki2, Kazunori Kondo3, USA, 4Dept Chem/Biochem, James Madison Univ, Harrisonburg, VA, USA. Shinsaku Maruta1,3. We are exploring fundamental factors controlling electron flow in proteins and 1Graduate School of Engineering, Soka University, Tokyo, Japan, 2RIKEN trying to apply these principles to create bio-hybrids that mimic properties of 3 Center for Life Science Technology, Yokohama, Japan, Faculity of Science conventional semiconductors. To this end, we developed and extensively char- and Engineering, Soka University, Tokyo, Japan. acterized 12 point mutations of PpcA, a 3-heme member of the cytochrome c7 Small GTPase Ras is known as a central regulator of several cellular signaling family native to Geobacter sulfurreducens. These mutations were engineered to which are related to cell viability, growth, differentiation and apoptosis. Ras is influence the redox potential (Em) of the middle heme (heme III) in PpcA by already studied well at molecular level and they have two state, GTP binding using four different strategies: performing charge reversal mutations, (active) state and GDP-binding (inactive) state. These switching of Ras activity decreasing solvent access to the heme plane with bulky residues, altering the was regulated by two factors, GEFs and GAPs. GEFs serves as Ras activator native bis-histidine axial ligation of the heme, and by attempting to form and they replace GDP from Ras and attach GTP to Ras alternatively. While, hydrogen bonds with the propionates of the heme. The latter strategy is ex- GAPs serves as Ras inactivator and they accelerate Ras GTPase. Previously pected not only to increase Em but also to introduce a redox Bohr effect. it has been reported that SOSaH mimicking peptide which has cyclic structure Out of 12 mutants, 11 were expressed in E.coli in sufficient quantities and at N-terminal region exhibited stablea-helix structure, and inhibit Ras GDP- show thermal stability in temperature-dependent CD experiments comparable > GTP exchange activity competitively with SOS. In this study, we focused on to wild-type protein (Tm 90 C). HPLC-ESI-MS was used to confirm both the interaction between Ras and aH-helix which locates Ras interface of SOS the purity and the mass of the expressed mutants. Small-angle X-ray scattering (Ras-GEF) in order to photo-regulate Ras function artificially. Several kinds confirmed that the mutant proteins were folded correctly and formed the ex- of aH mimicking peptides which have two cysteine to be crosslinked were syn- pected compact globular structures. Peroxidase activity assays were used to thesized. The cysteine residues of the peptides were crosslinked by bifunctional study flexibility and solvent exposure of heme binding pockets. Optical redox azobenzene derivatives, azobenzene di-maleimide (ABDM) intramolecularly. titrations have shown our ability to obtain reliable and reproducible data The ABDM incorporated into the peptides showed cis-trans isomerization thereby allowing us to measure the effect of the mutations on the electrochem- upon ultraviolet (UV) light and visible light irradiations. CD spectral analysis ical properties of all 3 hemes and to understand the underlying principles and revealed that trans-ABDM-aH peptide forms partially ahelix and cis-ABDM- viable approaches in tuning relative heme redox potentials. Successful develop- aH peptide exhibit random secondary structure. SOS dependent GDP-GTP ex- ment of this project may lead to biological semiconductors with much smaller change reaction of Ras was inhibited by trans- ABDM-aH peptide. Further- footprints and selectively tunable bandgap properties. more, Ex vivo experiment revealed the SOS mimicking peptides exhibit cell 2880-Pos membrane permeation indicating the photochromic peptides are applicable to Photo-Control of Small GTPase RAS GDP-GTP Exchange Reaction using regulation of cell function. Novel Peptide Inhibitor Modified with Azobenzene Derivatives Nobuyuki Nishibe1, Masahiro Kuboyama1, Kenichi Taii1, 2882-Pos Toshio Nagashima2, Toshio Yamazaki2, Shinsaku Maruta1,3. Photo-Control of RAS GDP-GTP Exchange using the SOSaH Mimicking 1Graduate school of Engineering, Soka University, Tokyo, Japan, 2Center for Peptides Modified with Spiropyran Derivative Life Science Technologies, RIKEN, Yokohama, Japan, 3Faculty of Science Kenichi Taii1, Nobuyuki Nishibe1, Kei Sadakane2, Shinsaku Maruta1,2. and Engineering, Soka University, Tokyo, Japan. 1Graduate School of Engineering, Soka University, Tokyo, Japan, 2Faculty of Previously we have demonstrated that guanine nucleotide exchange activity of Science & Engineering, Soka University, Tokyo, Japan. SOS for small G protein Ras is photoreversibly controlled by the peptide Ras is a kind of GTPase protein, and it regulates cellular signal transduction. mimicking SOSaH helix region, which is modified with azobenzene- di- For example, some information such as cell growth and cell differentiation maleimide (ABDM). Recently, a novel peptide inhibitor of Ras (KRpep-2d) from in vitro to in vivo of downstream by cycling between GTP active form was reported to inhibit the guanine nucleotide exchange of Ras with GEF and GDP inactive form. GDP-GTP exchange of Ras is induced by binding of potently. The peptide contains two cysteine residues and the two cysteine res- GEF(Guanine Exchange Factor) to Ras. Recently some kinds of peptide which idues are intramolecularly crosslinked by S-S bond resulting in formation of cy- interfere binding of GEF resulting in inhibition of GDP-GTP exchange, were clic structure. The cyclic conformation is essential to exhibit inhibitory activity. reported. Synthetic peptide mimicking aH helix of SOS inhibited the Ras In this study, we employed water soluble bifunctional azobenzene 2,20-bis(sul- GDP-GTP exchange in the presence of SOS (Son Of Sevenless). Photochromic fonate)-4,40- bis(chloroacetamide)azobenzene (BSBCA22) to introduce photo- molecule, spiropyran isomerize between ring-closed spiro (SP) and ring- switching into SOSaH peptide and KRpep-2d. Peptides modified with opened merocyanine (MC) forms photo-reversibly upon visible and ultraviolet BSBCA22 showed absorption spectral change accompanied by UV and visible lights. It is known that the zwitterion of MC forms exhibit dimerization. Previ- light irradiations, reflecting cis-trans isomerization. CD spectral analysis indi- ously, it has been demonstrated that the peptides modified with spiropyran de- cated that the unmodified peptide showed random secondary structure. NMR rivatives change their secondary structure accompanied by formation of spectra of BSBCA22- SOSaH peptide clearly revealed the two cysteine resi- spiropyran dimerization photoreversibly. In this study, we designed and synthe- dues in the peptide intramolecularly crosslinked. CD and NMR analysis sug- sized the peptides which have two cysteines and analogous to the SOS aH-he- gested BSBCA22- SOSaH peptide formed partially a-helix structure and UV lix. The two cysteine residues of the peptides were modified with SP- and visible light irradiations induced the change of secondary structure of the maleimide(SP-MA) stoichiometrically. The spiropyran incorporated into the modified peptide. Moreover, photoreversible control of GDP-GTP exchange peptide exhibited SP-MC isomerization upon visible light and ultraviolet light of Ras using peptides modified with BSBCA22 was also examined using fluo- irradiations. The secondary structural change of the SOS mimic peptide induce rescent GDP analog. BSBCA22- KRpep2d inhibited GDP-GTP exchange of by photo-isomerization of SP-MA were examined. Furthermore, photoreversi- Ras much more potently than intact peptide which does not form cyclic confor- ble inhibition of Ras GDP-GTP exchange in the presence of SOS with the mation. Trans-BSBCA22-peptide inhibited the GDP-GTP exchange more photochromic peptide was analyzed using fluorescent GDP analogues.

BPJ 9475_9479