Wednesday, February 15, 2017 465a

Symposium: Dynamics and Allostery example, in-silico models of human cardiac electrophysiology are being considered by the FDA for prediction of proarrhythmic cardiotoxicity as a 2285-Symp core component of the preclinical assessment phase of all new drugs. How- Coupled Residue-Residue Dynamics in Protein Allosteric Mechanisms ever, one issue that exists with current models is that they each respond Donald Hamelberg. differently to insults such as drug block of ion channels or mutation of cardiac Department of Chemistry, Georgia State University, Atlanta, GA, USA. . Clearly this poses a problem in relation to the utility of Although the relationship between structure and function in biomolecules is these models in making quantitative predictions that are physiologically or well established, it is not always adequate to provide a complete understanding clinically meaningful. To examine this in detail we tested the ability of three of biomolecular function. The dynamical fluctuations of biomolecules can also models of the human ventricular action potential, the O’hara-Rudy the Grandi- play an essential role in function. Detailed understanding of how conforma- Bers and the Ten Tusscher models, to reproduce the clinical phenotype of tional dynamics orchestrates function in allosteric regulation of recognition different subtypes of the long QT syndrome. All models, in their original and catalysis at atomic resolution remains ambiguous. The overarching goal form, produce markedly different and unrealistic predictions of QT prolonga- is to understand how biomolecular dynamics are coupled to function by using tion. To address this, we used a global optimization approach to constrain ex- atomistic molecular simulations to complement experiments. In this talk, we isting in silico models to clinical datasets. After optimization, all models have will discuss computational studies on members of a ubiquitous family of en- similar current densities during the action potential, despite differences in ki- zymes that catalyze peptidyl-prolyl bonds and regulate many sub-cellular pro- netic properties of the channels in the different models, and closely reproduced cesses. We analyze large amount of time-dependent multi-dimensional data the prolongation of repolarization seen in clinical data. We suggest that models with a coarse-grained approach and map key dynamical features within individ- optimized using this approach can be utilized with more confidence in clinical ual macrostates by defining dynamics in terms of residue-residue contacts. The and preclinical applications such as prediction of proarrhythmic risk as part of effects of substrate binding are observed to be largely sensed at a location over in silico drug screening, examining pathogenic interactions of electrical 15 A˚ from the active site, implying its importance in allostery. Using NMR ex- dysfunction and structural alteration in the myocardium and assessing the periments, we confirm that a dynamic cluster of residues in this distal region is impact of genetic variants in ion channel genes in contributing to rhythm directly coupled to the active site. Furthermore, the dynamical network of inter- disturbances. residue contacts is coupled and temporally dispersed. Mapping these dynamical 2288-Symp features and the coupling of dynamics to function has crucial ramifications in Towards in Silico Drug Trials using Human Multiscale Cardiac Models understanding allosteric regulation in enzymes and in general. Blanca Rodriguez. 2286-Symp University of Oxford, Oxford, United Kingdom. Entropy in Molecular Recognition by Proteins In silico physiological modelling and simulation provide a cheap, useful tool Joshua Wand. for drug safety and efficacy assessment. In this presentation, we will show ev- Biochemistry & Biophysics, University of Pennsylvania, Philadelphia, idence of the potential of human multiscale models of the heart for the evalu- PA, USA. ation of drug safety and efficacy. At a fundamental level, biological processes are most often controlled using mo- We will illustrate how in silico trials can be conducted to assess drug safety and lecular recognition by proteins. Protein-ligand interactions impact critical events efficacy with consideration of population variability and disease conditions. ranging from the catalytic action of enzymes, the assembly of macromolecular Methodological progress for in silico trials is based on the maturity of multi- structures, complex signaling and allostery, transport phenomena, force genera- scale models of human physiology, availability of human recordings to cali- tion and so on. The physical origin of high affinity interactions involving proteins brate the human models from the ionic to the whole organ level, and our continues to be the subject of intense investigation. Conformational entropy rep- ability to simulate the consequences of diseases such as myocardial ischemia, resents perhaps the last piece of the thermodynamic puzzle that governs protein heart failure and inherited disease conditions. We will show results using structure, stability, dynamics and function. The presence and importance of in- experimentally-calibrated populations of models and high performance ternal conformational entropy in proteins has been debated for decades but has computing simulations of diseased human , and their comparison to resisted experimental quantification. Over the past few years we have introduced, experimental datasets. In summary, in silico trials using human multiscale car- developed and validated an NMR-based approach that uses a dynamical proxy to diac models provide a useful, mechanistic tool for the preclinical assessment of determine changes in conformational entropy. This new approach, which we drugs, which enriches and complements current approaches. term the NMR ‘‘entropy meter,’’ requires few assumptions, is empirically cali- brated and is apparently robust and universal. Using this ‘‘entropy meter,’’ it 2289-Symp can now be quantitatively shown that proteins retain considerable conforma- Predictive Computational Pharmacology: From Atom to Rhythm tional entropy in their native functional states and that this conformational en- Colleen E. Clancy. tropy can play a decisive role in the thermodynamics of molecular recognition Department of Pharmacology, University of California, Davis, Davis, by proteins. Recent results show that changes conformational entropy of a pro- CA, USA. tein upon binding a high affinity ligand is highly system specific and can vary Common paroxysmal electrical diseases that affect millions of people world- from strongly inhibiting to even strongly promoting binding and everything in wide are notoriously difficult to manage with drug therapy, and some drugs in- between. Thus one cannot possibly understand comprehensively how proteins tended for therapy can even exacerbate disease. A vital hindrance to safe and work without knowledge of the breadth and underlying principles of the role effective drug treatment of excitable disorders is that there is currently no of conformational entropy in protein function. This approach also allows for way to predict how drugs with complex interactions and multiple subcellular the refinement of empirical coefficients that relate changes in accessible surface targets will alter the emergent electrical activity of cells and tissues. Our area to changes in the entropy of water and the determination of the loss of work involves the development of a novel quantitative systems pharmacology rotational-translational entropy in high affinity protein complexes. Supported approach derived from a combination of experiments, computational biology, by the NIH and the Mathers Foundation. high performance computing and clinical observation that allows for probing the mechanisms of action of drugs in the settings of one of the most common Symposium: Computational Cardiology excitable diseases: cardiac arrhythmias. These new tools can be applied to pre- clinical screening of compounds for therapeutic benefit or harm. A computer- 2287-Symp based approach can be used to determine mechanisms of drugs, with a specific Using Clinical Datasets to Optimize Models of Human Ventricular Elec- focus to conduct failure analysis for once promising drugs that have failed clin- trophysiology: Implications for In Silico Drug Screening ically. Finally, models are applied to demonstrate utility in guiding therapy for Adam P. Hill1, Stefan A. Mann2, Mohammad S. Imtiaz1, Matthew D. Perry3, specific clinical situations and to identify optimal ‘‘polypharmacy’’ to inform Jamie I. Vandenberg3. the common practice of clinical empirical mixing and matching of drugs to 1Computational Cardiology, Victor Chang Cardiac Research Institute, create multidrug therapeutic regimens. The computational processes that we Sydney, Australia, 2Cytocentrics Bioscience GmbH, Cologne, Germany, have developed are paradigms for how the explosion in systems and computa- 3Molecular Cardiology and Biophysics, Victor Chang Cardiac Research tional biology can be utilized to assist drug-screening, determination of mech- Institute, Sydney, Australia. anisms and to guide therapy. The eventual goal is a scalable, automated Recent advances in Computational Cardiology mean we can now examine the platform that will interact with other cutting edge technologies to serve pur- causes, mechanisms and impact of cardiac dysfunction in silico, particularly in poses in industry, academia and in clinical medicine that will be widely regards to risk stratification and treatment of heart rhythm disturbances. As a expanded to pharmacology of other common disorders of excitability such as result, computational cardiology stands at the threshold of clinical utility. For , ataxia and even pain.

BPJ 7875_7883 466a Wednesday, February 15, 2017

Platform: TRP Channels channel and more specifically in proximity to transmembrane segment 4 (S4). We show three residues (W982, D988 and G991) that seem to be 2290-Plat involved in the shaping of the alternative ion permeation pathway. The results obtained in this study are in line with the location of other non-canonical pores Permeation, Gating, and Modulation of the TRPA1 Channel in Long- þ þ 2þ Timescale Molecular Dynamics Simulations described in mutated voltage-gated K -, Na - and Ca -channels and propose Heidi Koldsø1, Morten Ø. Jensen1, Vishwanath Jogini1, David E. Shaw1,2. the potential occurrence of non-canonical pores in other ion channels that are 1D. E. Shaw Research, New York, NY, USA, 2Department of Biochemistry lacking the typical gating charges found in voltage-gated channels. and Molecular Biophysics, Columbia University, New York, NY, USA. 2293-Plat The cation channel TRPA1, a member of the transient receptor potential (TRP) ion Dynamic Solvation of Protein Cavities Underlies TRPV1 Gating channel superfamily, is a promising drug target in pain and inflammatory diseases, Marina Kasimova1, Aysenur Yazici2, Daniele Granata3, Tibor Rohacs2, but its function and dynamics—including ion permeation and channel gating—are Vincenzo Carnevale3. not well understood at the structural level. We have used the recently published 1Department of Theoretical Physics, KTH, Stockholm, Sweden, cryo-EM structure of the closed, non-conducting state of TRPA1 (Paulsen et al., 2Department of Pharmacology, Physiology & Neuroscience, Rutgers - New Nature, 2015) as a starting point to study the functional states and activation of Jersey Medical School, Newark, NJ, USA, 3Department of Chemistry, this channel using long-timescale, all-atom molecular dynamics simulations Temple University, Philadelphia, PA, USA. enabled by special-purpose hardware. A key functional region of TRPA1—the TRPV1 is a member of transient receptor potential (TRP) channels family, channel’s selectivity filter (SF)—was unstable in simulations started from the which promotes nonselective cationic current across the membrane in response cryo-EM model of the closed state, even when using extensive restraints. We con- to multiple activating stimuli such as , temperature, extracellular pH structed an alternative model of the SF region based on a high-resolution crystal and voltage. Since TRPV1 is directly involved in nociception, it is among the structure of a closely related ion channel. This SF model, which is compatible primary targets for pain-relief drugs. Despite its importance, the gating mecha- with experimental data, remained stable on simulation timescales of 100 msor nism of TRPV1 remains unexplored. Here, we applied a combined computa- longer, allowing us to study ion permeation in TRPA1 at the single-ion level. To tional (molecular dynamics and metadynamics simulations) and experimental study ion permeation across the open channel, we introduced a mutation approach to shed light on the open-to-closed transition of this channel. In partic- (V961R) within the TRPA1 internal gate, leading to a rapid pore opening. Upon ular, we found that the gating of TRPV1 is controlled by a strictly conserved subsequent back mutation to the wild-type protein, the pore remained in an open, asparagine (N676) in the middle of the S6 helix: this residue is able to rotate conducting conformation. Additional simulations under an applied membrane po- from a conformation facing the S4-S5 linker to one facing the central pore. tential, moreover, led to direct observation of inward and outward ionic currents This rotation is correlated with the dehydration of small protein cavities between and suggested a novel structural determinant of channel gating within the SF region. the linker and the S6 helices and hydration of the central pore. Free energy cal- We also studied modulation of the open and conducting channel by performing free culations confirm that the two conformational states are stable and are separated binding simulations with the TRPA1 inhibitor, A-967079, which, intriguingly, was by a relatively low free energy barrier. Based on our findings, we propose a observed to bind in a location previously hypothesized to be a potential binding site. model of TRPV1 gating in which dehydration of the protein pockets determines 2291-Plat a rotation of N676; the latter triggers water flow inside of the channel and, ulti- Ion Permeation Mechanism in TRPV6 Ca2D Channel mately, the opening of the gate. Our model is in line with the known experi- Serzhan Sakipov1, Alexander I. Sobolevsky2, Maria Kurnikova1. mental data and has been tested with mutagenesis experiments. 1Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA, 2Biochemistry 2294-Plat and Molecular Biophysics, Columbia University, New York, NY, USA. Exploring Functional Roles of TRPV1 Intracellular Domains with Transient receptor potential (TRP) channels function as sensors for various Unstructured Peptide-Insertion Screening physical and chemical stimuli and are vitally important players in regulation Linlin Ma1,2, Fan Yang1, Simon Vu1, Jie Zheng1. of key biochemical processes in a variety of cells. TRPV5 and TRPV6 subtypes 1Physiology and Membrane Biology, University of California, Davis, Davis, þ of TRP channels are the main Ca2 entry channels in epithelia Their pores are CA, USA, 2Institute for Molecular Bioscience, University of Queensland, þ selective for Ca2 and permeable for a variety of monovalent and divalent cat- St Lucia, Australia. þ ions. An exact mechanism of Ca2 permeation and selectivity through the TRPV1 is a polymodal nociceptor for diverse physical and chemical stimuli TRPV5/6 ion channels is unknown. Recently, a crystal structure of the that interact with different parts of the channel protein. Recent cryo-EM studies TRPV6 channel, the first high-resolution structure of a TRP channel, has been revealed detailed channel structures, opening the door for mapping structural solved in complex with different ions bound in the channel pore [1]. Analysis elements mediating activation by each stimulus. Towards this goal, here we þ of the structure suggested that Ca2 permeates TRPV6 channel by a knock-of have combined unstructured peptide-insertion screening (UPS) with electro- mechanism. We have created a fully atomistic model of the trans-membrane physiological and fluorescence recordings to explore structural and functional (TM) domain of the TRPV6 channel in lipid and water, and performed molecular roles of the intracellular regions. We found that most of the tightly packed pro- þ þ þ þ dynamics simulations of Na ,Ca2 ,Ba2 , and Gd3 ion permeation through the tein regions did not tolerate structural perturbation by UPS when tested, indi- selectivity filter of this channel. As a result, our MD simulations directly demon- cating that structural integrity of the intracellular region is critical. In strate the key features of ion permeation through TRPV6 channel pore. In partic- agreement with previous reports, Ca2þ-dependent desensitization is strongly þ þ ular, at low calcium concentrations, we observed interplay of Na and Ca2 dependent on both intracellular N- and C-terminal domains; insertions of an un- þ permeation that follows the knock-off mechanism. We also compare Ca2 structured peptide between these domains and the transmembrane core domain þ þ permeation with permeation of Ba2 and channel block by Gd3 . nearly eliminated Ca2þ-dependent desensitization. In contrast, channel activa- 1. Saotome, Kei, Singh, A. K., Yelshanskaya, M. V., Sobolevsky, A. I. (2016) tions by capsaicin, low pH, divalent cations, and even heat are mostly intact in Crystal structure of the epithelial TRPV6. Nature 534, 506-511 mutant channels containing the same insertions, suggesting that the transmem- 2292-Plat brane core domain of TRPV1 is responsible for sensing these stimuli. Localization of an Alternative Ion Permeation Pathway in TRPM3 2295-Plat Katharina Held, Annelies Janssens, Thomas Voets, Joris Vriens. Structural Insight into the Molecular Mechanism of TRPA1 Inhibition KU Leuven, Leuven, Belgium. and Activation Transient receptor potential (TRP) channels are cationic channels that are Amrita Samanta1, Janna Kiselar2, Seungil Han3, Vera Moiseenkova Bell4. involved in a broad array of functions, including homeostasis, motility and sen- 1Physiology and Biophysics, Case Western Reserve University, Cleveland, sory functions such as transduction, nociception, and temperature sensing. OH, USA, 2Center for Proteomics and Bioinformatics, Case Western Reserve In general, the activation of these channels leads to the opening of their central University, Cleveland, OH, USA, 3Pfizer, New London/Norwich, CT, USA, channel pore resulting in a cationic influx and a subsequent depolarization of 4Pharmacology, Case Western Reserve University, Cleveland, OH, USA. the cell. TRP melastatin 3 (TRPM3) is a member of the melastatin family of Pain, while serving the beneficial function of provoking our attention to TRP channels and was shown to be expressed in sensory where it dangerous situations, is an unpleasant sensory and emotional experience. Tran- acts as noxious heat sensor and nociceptor. Activation of TRPM3 results typi- sient Receptor Potential Ankyrin 1 (TRPA1) is a member of the transient recep- cally in an outward-rectifying current-voltage (I-V) curve due to the opening of tor potential (TRP) cation channel family and is localized in nerve fibers called the central pore of the channel. Recently, it was shown that certain agonists can ‘‘nociceptors’’ where it plays a key role in the transduction of chemical, inflam- induce the opening of an alternative ion permeation pathway next to the central matory and neuropathic pain signals from the periphery to the . pore, resulting in outward- and inward-rectifying currents. Here we use muta- TRPA1 is a non-selective ion channel, which is activated by a variety of genesis studies to localize the alternative pore of TRPM3. Our data suggest the structurally diverse compounds. TRPA1 agonists are exogenous electrophilic location of the alternative pore in the voltage-sensing domain (VSD) of the compounds and endogenous thiol-reactive inflammatory mediators, all of

BPJ 7875_7883 Wednesday, February 15, 2017 467a which target and activate the channel via a cluster of cysteine residues at the N- membrane area. Previous tether pulling experiments provided insights on mem- terminus. Activation is clearly dependent on a thiol-reactive moiety, thus ex- brane mechanical properties, and the curvature effects on phase behaviour and plaining the structural diversity of this group. Although near-atomic resolution distribution of coexisting phases. However, detailed information on tether structures of TRPA1 were resolved recently by cryo-EM, both in the presence properties and variations in composition is challenging to obtain experimen- of agonist and antagonists, detailed mechanisms of channel activation and in- tally due to the small diameters and dynamic nature of tethers. hibition by these modulators could not be determined. Here we provide a molecular view on curvature-induced lipid sorting in plasma To determine the structural details of TRPA1 conformation upon agonist and membrane tethers. We pulled tethers from an idealized plasma membrane antagonist binding, we used limited proteolysis and mass spectrometry. Our model using molecular dynamics simulations with the coarse-grained Martini study indicates that conformational rearrangements of the N-terminus ankyrin model. The membrane consists of 63 lipid types with an asymmetric distribu- repeats and the linker region between C-terminus and the transmembrane tion of components between the leaflets [JACS, 2014, 136, 14554]. The tethers domain contribute to the channel modulation. are formed by applying an external constant force to a lipid patch in the direc- tion normal to the bilayer plane [Biophys J, 1012, 102, 1866]. Pulling is per- 2296-Plat formed both from the inner and outer leaflets, corresponding to the direction Inhibition of TRPM3 Ion Channels by G-Protein Beta-Gamma Subunits in and out of the cell, respectively. As a result of pulling, we observe re- Yevgen Yudin1, Doreen Badheka1, Istvan Borbiro1, Aysenur Yazici1, distribution of different lipid types along the regions of different curvature Tooraj Mirshahi2, Tibor Rohacs1. without macroscopic phase separation. Depending on the direction of pulling, 1Department of Pharmacology, Physiology and Neuroscience, Rutgers the distribution of lipids and the tether properties differ. Univeristy, Newark, NJ, USA, 2Weis Center for Research, Geisinger Clinic, Danville, PA, USA. 2299-Plat þ Transient Receptor Potential Melastatin 3 (TRPM3) is a non-selective, Ca2 Calculating Membrane Area Compressibility from MD Simulations: permeable cation channel activated by heat, and chemical activators such as a Novel Computational Framework pregnenolone sulphate and CIM0216. Here we show that activation of receptors Milka Doktorova, George Khelashvili, Harel Weinstein. coupled to heterotrimeric Gi proteins inhibits TRPM3 currents in a mammalian Physiology and Biophysics, Weill Cornell Medical College, New York, expression system. This inhibition was alleviated by co-expression of proteins NY, USA. that act as sinks for the bg subunits of heterotrimeric G-proteins (Gbg). Co- Like thin elastic sheets, lipid bilayers can bend and stretch, and are characterized expression of Gb1g2, but not a constitutively active mutant of Gao inhibited by mechanical constants such as bending rigidity (Kc) and area compressibility pregnenolone sulphate-induced TRPM3 currents. TRPM3 was also inhibited (Ka), which define the free energy of bilayer deformation and thus have direct im- by purified Gbg proteins applied to excised inside out patches, indicating a direct plications in quantifying the energetics of protein-membrane interactions. effect. Baclofen and somatostatin, agonists of Gi coupled receptors, inhibited Limited experimental data for Kc and Ka of different lipid mixtures makes þ Ca2 signals induced by pregnenolone sulphate and CIM0216 in dorsal root gan- such calculations challenging. Existing computational methods for calculating glion (DRG) neurons. The GABAB receptor agonist baclofen also inhibited Ka from MD simulations exhibit strong dependence on bilayer size, or require CIM0216-induced currents in DRG neurons, and nocifensive responses elicited multiple simulations with constraints on the area, or tension. We present a novel by this TRPM3 agonist in vivo. Our data show that Gi-coupled receptors regulate computational framework that is independent of system size and requires analysis TRPM3 channels via direct inhibition by Gbg. only of a single sufficiently converged simulation trajectory. The method is based 2297-Plat on Helfrich’s theory of elasticity by relating relative changes in area to relative Photopharmacological Modulation of Lipid-Gated TRPC Channels as a changes in thickness. The distribution of local fluctuations in the thickness of Strategy to Govern Neuronal Excitability each bilayer leaflet is used to construct a potential of mean force and recover Oleksandra Tiapko1, Gema Guedes de la Cruz2, Toma Glasnov2, the bilayer Ka from a quadratic fit. The rapid calculation utilizes freely available Helmut Kubista3, Michaela Lichtenegger1, Klaus Groschner1. software packages. We present results for a variety of single and multicomponent 1Medical University of Graz, Graz, Austria, 2University of Graz, Graz, bilayers of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin, Austria, 3Medical University of Vienna, Vienna, Austria. utilizing simulation results at different temperatures and bilayer sizes. The calcu- Lipid-gated, TRPC channel isoforms 3, 6 and 7 are widely expressed in the brain. lated compressibility moduli range from 170 to 320 mN/m and agree for the Although, cellular regulation and signalling functions of these proteins are well majority of tested cases with values reported from experiments and other compu- characterized in overexpressing systems, their role in native cells is incompletely tational methods. We further validate our method in a comparison with an exist- understood. Recent evidence suggests a complex, inverse coupling between ing polymer brush model and confirm the linear relationship between thickness ^ TRPC3 constitutive activity and hippocampal excitability, potentially involving and (Kc/Ka) (1/2), all calculated from the simulation trajectories. 2þ þ promotion of a Ca -activated K conductance and afterhyperpolarization. 2300-Plat Constitutive Ca2þ entry through TRPC3/6 might be enforced in a positive feed- þ Calcium-Induced Sculpturing of the Plasma Membrane: Lipid Microclus- back loop, which involves linkage to K efflux and maintenance of electrochem- ters Cast by Anionic Lipids ical driving force. Nevertheless, acute suppression of hippocampal function in Michael J. Hallock1, Alex Greenwood2, Yan Wang3, James H. Morrissey3, response to activation of TRPC3 channels via receptor-mediated pathways has Emad Tajkhorshid4, Chad Rienstra5, Taras V. Pogorelov6. not unambiguously been demonstrated. To identify the functional consequences 1School of Chemical Sciences, Beckman Institute, University of Illinois at of TRPC3 activation in without interference of parallel signalling Urbana-Champaign, Urbana, IL, USA, 2Chemistry, University of Illinois at events from receptor/phospholipase C activation, we used derivatives of the Urbana-Champaign, Urbana, IL, USA, 3Biochemistry, University of Illinois novel, selective TRPC3/6 agonist GSK1702934A. GSK1702934A (10 mM) at Urbana-Champaign, Urbana, IL, USA, 4Biochemistry, Biophysics, reversibly suppressed firing of primary cultures of murine hippocampal neurons, Beckman Institute, University of Illinois at Urbana-Champaign, Urbana, IL, while, this effect was absent in hippocampal neurons lacking TRPC3/6 ex- USA, 5Chemistry, Biophysics, Biochemistry, University of Illinois at pression. Our results encouraged the design and generation of photochromic Urbana-Champaign, Urbana, IL, USA, 6Chemistry, Biophysics, Beckman ligands for TRPC activation based on the GSK1702934A structure. Inclusion Institute, NCSA, School of Chemical Sciences, University of Illinois at of an azobenzene moiety into ligands has successfully been used in photophar- Urbana-Champaign, Urbana, IL, USA. macology of a variety of channels and receptors. An azobenzene-modified The plasma membrane of the cell is a complex, tightly-controlled heteroge- GSK1702934A derivative (optoGSK1) was found suitable for fast and reversible neous environment. While anionic lipids constitute a small part of all phospho- control of heterologously expressed TRPC3 and TRPC6 channels in HEK293 lipids in the plasma membrane, they play key functional roles in regulation of cells, and enable highly efficient, optical control of neuronal firing. vital cellular processes such as blood clotting, signaling, vesicle fusion, and apoptosis. Lipid composition and distribution in the plasma membrane are Platform: Membrane Structure II: Simulations highly dynamic, with anionic phosphatidylserine (PS) lipids typically found on the interior leaflet. In turn, the concentration of divalent Ca2þ ions, an 2298-Plat important cellular messenger, is three orders of magnitude greater outside of Curvature-Induced Lipid Sorting in Plasma Membrane Tethers the cell, spatially separating the ions from anionic lipids. PS lipids can become Svetlana Baoukina1, Helgi I. Ingolfsson2, Siewert J. Marrink2, externalized to the outer leaflet of the plasma membrane in the case of cell D. Peter Tieleman1. injury, disease, or apoptotic changes allowing them to interact with Ca2þ, re- 1University of Calgary, Calgary, AB, Canada, 2University of Groningen, sulting in changes in the plasma membrane structure and initiation of signaling Groningen, Netherlands. cascades. Nonetheless, the molecular details of lipid-Ca2þ interactions are not Membrane tethers are nanotubes formed by lipid bilayers. They are efficient completely understood. We performed molecular dynamics simulations of bi- structures for cellular transport and communication, and for storage of excess nary mixtures of PS and phosphatidylcholine (PC) lipids using highly mobile

BPJ 7875_7883 468a Wednesday, February 15, 2017 membrane mimetic model (HMMM) that enabled extended sampling. Simula- automatically insert proteins into the membrane, allowing even more realistic tions revealed that lipid microclusters are formed by higher order (three or models to be generated. Overall, xMAS Builder enables visualization and analysis more) lipid oligomers coordinated by Ca2þ, with two distinct conformations of immense membrane structures at an unprecedented atomistic level of detail, and of the PS headgroup serving as building blocks. In parallel, 13C-dephased it provides the first fundamental steps necessary to simulate these systems. 31P-observed solid-state nuclear magnetic resonance (SSNMR) rotational- echo double-resonance (REDOR) experiments were performed with spin- 2303-Plat diluted Nanodisc samples to report on 31P-13C distances. Experimental data Nanoscale Structure of Lipid Bilayers Revealed by In-Silico and Experi- are consistent with both intra- and inter-molecular lipid headgroup character- mental Small Angle Neutron Scattering þ Mitchell Dorrell1, Frederick Heberle2, John Katsaras2,3, Edward Lyman1,4. istic distances of approximately 4.8 /- 0.5 A that match well with all-atom 1 MD results. To our knowledge, this is one of the first joint MD and high- Department of Physics and Astronomy, University of Delaware, Newark, DE, USA, 2Biology and Soft Matter Division, Oak Ridge National resolution SSNMR studies to characterize how lipid microclusters are formed 3 at the atomic level and how they reshape the plasma membrane. Laboratory, Oak Ridge, TN, USA, Department of Physics and Astronomy, The University of Tennessee, Knoxville, TN, USA, 4Department of 2301-Plat Chemistry and Biochemistry, University of Delaware, Newark, DE, USA. Microscopic Characterization of Membrane Insertion and Dynamics of an Through the combination of simulations and numerical analysis with small Isoprenoid Lipid angle neutron scattering, we probe the lateral organization of lipid bilayer mix- Po-Chao Wen, Emad Tajkhorshid. tures. Small Angle Neutron Scattering (SANS) is an excellent complement to Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA. optical techniques, as it reveals molecular scale structural details without fluo- Bacterial cell wall synthesis requires the transport of peptidoglycan components rescent probes. On its own, however, SANS is not sufficient to determine the or- across via the carrier lipid, undecaprenyl pyrophosphate (UPP). ganization of the membrane. We therefore take a two-pronged approach, The essential role of UPP in bacterial cell growth renders itself a valuable drug comparing molecular dynamics simulations of ternary mixtures to experimental target for antimicrobial treatments (e.g. direct binding by bacitracin, nisin, and data obtained for the same mixtures. In-silico neutron scattering experiments on clausin) in addition to its metabolic enzymes. After synthesized by a soluble simulated membranes are compared to the experimental data, simultaneously enzyme, UPP needs to be incorporated into the membrane before entering the validating the simulation data and providing a molecular scale model of the cell-wall biosynthesis pathway. We have conducted a large set of molecular dy- experimental system. Our all-atom molecular dynamics simulations use pre- namics simulations of UPP in solution and in membrane with several different existing software packages (e.g., Gromacs) to produce atomic trajectories of initial configurations, and have captured its spontaneous (unbiased) membrane ten microseconds in length. These trajectories are processed and analyzed using insertion from the solution phase. Once establishing hydrophobic contacts with custom software to generate scattering intensities for comparison to experiment, acyl chains of phospholipids, UPP rapidly partitions into membrane at a converged assuming laterally averaged scattering within nanoscopic domains. Simulated depth, regardless of initial UPP placement in solution or anywhere within the scattering intensities are fit to experimental data to extract information about membrane. Both phosphate groups of UPP partition to the same depth as the the lateral structure. In this way, the transverse variation in scattering length den- phospholipid phosphates, with the average P-P vector only <20 deviated from sity across the bilayer — which, under fully deuterated solvent, is much more the membrane plane. Most importantly, the terminal prenyl group exhibits a broad significant than lateral contributions — is used to infer lateral structure. distribution spanning the entire hydrophobic layer of the membrane, with a pref- erence at the central region between two leaflets. Due to the longer chain length, 2304-Plat UPP exhibits slightly larger area footprint than a typical phospholipid molecule, Energy Landscape of Pore Formation in Bilayer Lipid Membrane and the terminal prenyl group exhibits greater sub-ms fluctuations (larger apparent Sergey A. Akimov1, Pavel E. Volynsky2, Timur R. Galimzyanov1, lateral diffusion coefficient at 10-ns scale) than terminal methyl groups of phos- Peter I. Kuzmin1, Konstantin V. Pavlov1, Oleg V. Batishchev1. 1 pholipid tails, in contrast to the consistently lower lateral diffusion coefficient of Bioelectrochemistry, A.N. Frumkin Institute of Physical Chemistry and 2 pyrophosphate than that of phospholipid phosphates. Since the undecaprenyl tail Electrochemistry of RAS, Moscow, Russian Federation, Shemyakin- is highly diffusive while the pyrophosphate head group appears more stationary, Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of it is possible that the molecular recognition of UPP by its metabolic enzymes, Sciences, Moscow, Russian Federation. transporters and inhibitors would be primarily based on head group interactions. Lipid bilayer constitutes a major structural component of plasma membranes. Amphiphilic nature of lipid molecules, which contain both polar and hydropho- 2302-Plat bic parts, determines low permeability of lipid bilayers for broad range of sub- Atomistic Modeling of Organelle-Scale Membrane Structures of Arbitrary stances and thus allows the membranes to perform barrier function effectively in Size, Lipid Composition, and Geometric Complexity the cells. Membrane permeation, both natural and artificial, may take place via Noah Trebesch1,2, Emad Tajkhorshid1,3. appearance of through pores resulting from application of an external stress. The 1 Center for Biophysics and Quantitative Biology, University of Illinois at subtle rearrangement of lipid molecules leading to hydrophilic (inverted) pore 2 Urbana-Champaign, Urbana, IL, USA, Beckman Institute for Advanced formation in the intact membrane is still an open question. We developed a Science and Technology, University of Illinois at Urbana-Champaign, model of membrane poration under applied lateral tension or transmembrane 3 Urbana, IL, USA, Department of Biochemistry and Beckman Institute for voltage basing on continuum elasticity theory. The formation of large hydro- Advanced Science and Technology, University of Illinois at Urbana- philic pore was assumed to pass through an intermediate stage, hydrophobic Champaign, Urbana, IL, USA. defect. This intermediate stage is characterized by presence of hydrophobic The structure of the membranes that surround most organelles and cells are highly belt aligned by lipid tails in the equatorial plane of the pore. There were found complex geometrically and are immensely large from the perspective of atomistic two energy barriers for pore formation: i) transition of the hydrophobic defect modeling and simulation. Terasaki ramps, a structural motif from endoplasmic re- to the hydrophilic pore; ii) expansion of the pore to supercritical radii. The bar- ticula with a helicoidal structure similar to that of a parking ramp, exemplify these riers monotonously decrease upon application of transmembrane voltage. The properties. Starting from a surface mesh reconstruction of a Terasaki ramp derived application of lateral tension results in increase of the first barrier and decrease from electron microscopy, we have developed the first atomistic model of an of the second one. In this case the waiting time to pore formation proved a non- organelle-scale membrane structure, enabling us to computationally probe the monotonous function of the lateral tension. We analyzed molecular dynamics interplay between atomic-scale biochemistry and organelle-scale biophysics for trajectories of pre-formed pore closure in membranes composed of lipids with the first time. Measuring approximately 1.97mmby1.59mmby0.61mm, our model different spontaneous curvatures. Analysis of pore formation trajectories for contains ~31.7 million lipids (~3.9 billion atoms) and is composed of seven several lipid species with different spontaneous curvatures and elastic moduli different lipids (POPC, cholesterol, etc.) in experimentally-determined propor- under various external conditions provided instrumental insights into the mech- tions. Because existing atomistic modeling techniques can only produce mem- anisms underlying experimentally observed phenomena. The work was sup- branes with extremely simple shapes (i.e., planes or spheres) and cannot utilize ported by the Russian Foundation for Basic Research (project # 15-04-07816). experimental low-resolution reconstruction data, building this model required sig- nificant methodological development, which we have consolidated into a general 2305-Plat tool called xMAS (Experimentally-Derived Membranes of Arbitrary Shape) Atomistic Simulations Provide Insight into Electron Paramagnetic Reso- Builder. Starting from an experimental (or synthetic) mesh, xMAS Builder nance Probe Behavior analyzes the mesh to determine the number of lipids needed and their Gary Angles, Sally C. Pias. orientations, and it uses molecular dynamics simulations of Lennard-Jones particles Chemistry, New Mexico Institute of Mining and Technology, Socorro, restrained to the mesh to optimize the lipid placement. It then chooses lipids of the NM, USA. appropriate type from a set of conformational libraries, and it eliminates clashes Oxygen permeation through cell membranes is critical to normal tissue func- between lipids using an energy minimization technique. Soon, it will be able to tion and to tumor oxygenation. Probe-based experiments using electron

BPJ 7875_7883 Wednesday, February 15, 2017 469a paramagnetic resonance (EPR) oximetry have provided valuable estimates 2308-Plat of membrane oxygen permeability. Yet, the estimates incorporate a good Hydrophobic Interfaces, Key Regions for Assembly of Transmembrane deal of uncertainty due to ambiguity in probe positioning. We use atomistic Proteins : A Study of E. Coli Z molecular dynamics simulations to examine the behavior of a probe Victoria Schmidt, James N. Sturgis. targeted to the headgroup region of zwitterionic phospholipid bilayers. Our CNRS and Aix-Marseille univ., Marseille, France. simulations are validated by detailed comparison with experimental data, Stable folding of membrane proteins depends on the formation of stable inter- including overall agreement with the EPR oximetry measurements. The faces in the hydrophobic core of the membrane, and the exclusion of lipid mol- simulations predict that the tempocholine ‘‘headgroup’’ probe oversamples ecules. There is very little information on this aspect of membrane protein the hydrophobic tail region of the bilayer, leading to an underestimate of folding for polytopic membrane proteins. We use Aquaporin Z as a model to resistance to permeation in the headgroup region and an overestimate of study the structure and importance of these interfaces. are mem- bilayer permeability. This work highlights the value of using atomistic simu- brane proteins with 6 transmembrane helices, that act as channels in biological lations to test assumptions made in interpreting probe-based experimental membranes for small uncharged molecules. Members of this family of proteins measurements. assemble in the membrane to form tetrameric complexes. In order to better un- derstand the assembly of these proteins we have constructed a series of desta- bilized mutants by modifying the interface between monomers in Aquaporin Z Platform: Protein Assemblies from E. coli. We have characterized these mutants to understand how these modifications modulate in vivo and in vitro characteristics of the protein. 2306-Plat In vivo we have evaluated membrane insertion and the formation of tetramers In Situ Mechanical Interrogation of Single Nuclear Lamins Suggests the and toxicity. In vitro we have evaluated the tertiary and quaternary structure, Lamina is a Robust Framework stability and activity of the protein in detergents or lipid bilayers. Tanuj Sapra1, Zhao Qin2, Markus Buehler2, Ohad Medalia1. 1University of Zurich, Zurich, Switzerland, 2MIT, Cambridge, MA, USA. 2309-Plat The nuclear lamina is a major structural element of the nucleus, predominately Hydrogen-Deuterium Exchange Mass Spectroscopy to Determine Struc- composed of the intermediate filament lamin proteins, enveloped by the nuclear ture and Structural Dynamics of Protein Complexes membrane. Underlining its importance to the mechanical and structural integ- Emanuele Paci. rity of the nucleus, a persistent goal has been to examine the lamin network University of Leeds, Leeds, United Kingdom. in situ. Here we apply an integrative approach combining techniques from Hydrogen deuterium mass spectrometry (HDX-MS) is a growing technique to mechanobiology (atomic force microscopy), visual proteomics (cryo-electron probe structure and dynamics of proteins and protein-ligand complexes. It is tomography) and network analysis (molecular dynamic simulations) to under- particularly powerful to probe disordered or partly disordered states, when stand the design principle of the lamin network. A detailed analysis of the no high-resolution structural techniques are applicable. It is now increasingly mechanical failure under nano-Newton forces of the endogenous lamins of used as a fast and inexpensive approach to map how protein and ligands bind Xenopus laevis oocytes revealed the lamin filaments to be strong, stiff and when structures are not available or when dynamical changes and allostery is tough, equivalent to natural silk and the synthetic polymer KevlarÒ. The com- involved. HDX-MS provides, as a function of time, the change in mass of bined approach is unique, and provides understanding of the structure-function random segments of the polypeptide chain where deuterated amide protons ex- of proteins involved in diseases from a materials science perspective, the under- change with solvent’s hydrogens or vice-versa. The main drawback of the tech- lying goal of ‘materiomics’. nique is that the information provided is intrinsically low-resolution and it is not directly interpretable in structural terms, the main reason being that ex- 2307-Plat change depends simultaneously on local structure and dynamics. Nano-Space Video Imaging Reveals Structural Dynamics of Fibrous We have developed a technique that allows accurate estimation of the ex- Protein Assembly and Relevant Enzymes change kinetics for any segment of the polypeptide chain based from ensem- Takahiro Watanabe-Nakayama1, Noriyuki Kodera1, Hiroki Konno1, bles of structures. The technique we developed builds up on the observation Kenjiro Ono2, David B. Teplow3, Masahito Yamada1, Toshio Ando1. that the rate of exchange of a single amide proton (or equivalently, its protec- 1Kanazawa University, Kanazawa, , 2Showa University, , Japan, 3 tion factor) can be estimated from the protein structure by estimating the acces- UCLA, Los Angels, CA, USA. sibility to the solvent and the probability of the amide being involved in a Protein assembly into fibrous structure is ubiquitous in biological systems. hydrogen bond. However, structural dynamics in assembling processes of these structures I will present published and unpublished results that demonstrate the potential and mechanisms of relevant enzymatic reactions still have remained unclear of the technique to study the functional dynamics of a hexameric helicase [1] due to insolubility of the high order structures. In this presentation, we report and to assess the quality of a model for a protein complex. two application results of high-speed atomic force microscopy (HS-AFM) 1. Radou, G., et al., Functional dynamics of hexameric helicase probed by observation of such systems. HS-AFM visualize structural dynamics of all ob- hydrogen exchange and simulation. Biophys J, 2014. 107(4): p. 983-90. jects in an observation field regardless of their solubility. Bacterial collagenases are widely applied in many fields due to their high 2310-Plat activity and specificity; however, little is known about the mechanisms by Molecular Coevolution of Fli Proteins Provides a Guide to Accurate which bacterial collagenases degrade insoluble collagen. HS-AFM visualized Models of Flagellar Protein Complexes and Dynamics ColG moved ~14.5 nm towards the collagen N terminus over ~3.8 s in a manner Faruck Morcos. depending on a catalytic zinc ion, suggesting that ColG couples its movement Biological Sciences, University of Texas at Dallas, Richardson, TX, USA. and function with high efficiency. In addition, HS-AFM demonstrated ColG en- Elucidating the molecular mechanics of bacterial flagellar motor architecture gagements were hindered by the hierarchical structure of collagen. has been a challenging topic for many years. Efforts in structural biochemistry, Amyloid b-protein (Ab) aggregation into amyloid fibrils has been implicated in crystallography, electron microscopy and computational biology brought an the pathogenesis of Alzheimer’s disease. Fibril formation is a complex process, improved picture of motor ring assemblies and dynamics. There are, however, but HS-AFM enables such observation. HS-AFM visualized two different many challenges to be resolved to fully understand the formation of this multi- growth modes of the 42-residue form of the amyloid b-protein (Ab1-42), one protein macro complex. There are only a few experimentally determined 3D producing straight fibrils and the other producing spiral fibrils. Each mode de- coordinates of the flagellar motor switch proteins. Proteins FliF, FliG, FliM pended on initial nucleus structure, but conformational switching was observed and FliN constitute the rotor of the flagellar motor as well as scaffold for the sometimes. The frequency of this switching phenomenon was varied in Type III ATPase secretion complex responsible for assembly of external response to changes in micro environment around fibril ends. Our data provide flagellar structures. Ring formation has been studied at lower resolutions the new insights into the template-dependent mechanism of Ab1-42 fibril compared to the individual monomers leaving open questions about the com- formation. plex connectivity and the dynamics of switching and rotation. Some remaining References questions are related to the nature of oligomerization and dimerization. T. Watanabe-Nakayama et al. High-speed atomic force microscopy reveals We have studied the amino acid coevolution of Fli proteins, particularly strongly polarized movement of clostridial collagenase along collagen fibrils. FliM (middle and C-terminal domains) as well as protein FliN to elucidate Sci. Rep. 6:28975 (2016). important interactions conserved through evolution. We combine such signals T. Watanabe-Nakayama et al. High-speed atomic force microscopy reveals with molecular dynamics to accurately recapitulate previous known complexes structural dynamics of amyloid b1-42 aggregates. Proc. Natl. Acad. Sci. involving FliN homodimers as well as to provide support for some putative U. S. A. 113(21):5835-40 (2016). models of FliM-FliN dimers as well as distinct signatures for tetrametic

BPJ 7875_7883 470a Wednesday, February 15, 2017 complex formation that could lead to an updated model of the complete distal interactions between several proteins, located in three subcellular compart- C-ring. There is evidence that FliM middle domain is involved in dimeric in- ments, is then used to send this external signal towards the inside of the bacte- teractions but no structure of this complex has been clearly determined. Using ria. This signaling pathway is involved in the regulation of expression and coevolving interactions we propose a model that is consistent with known in- is crucial for the adaptation of bacteria to their environment. We have been direct experimental data and points towards conformational heterogeneity. studying a heme acquisition system (Has) developed by several commensal Our results that originate from coevolutionary analysis support a larger picture and pathogenic bacteria to acquire heme as iron source. Although various pro- of C-ring formation and aim to close the gap between high-resolution mono- teins involved in this process have been identified, signal transduction through mers and the coarse grained understanding of macro-molecular assembly ob- this family of transporters is not well understood [1, 2, 3, 4]. Here, using an tained with current methods. integrative approach (NMR, Xray, SAXS and electron microscopy) we have studied the structure of the transporter HasR, captured in two stages of the 2311-Plat signaling process, i.e., before and after the arrival of signaling activators Characterization of the Conformational Ensemble of Polyglutamine (heme and its carrier protein) [5]. Our results led us to propose a mechanism Peptides via Metadynamics MD Simulations and UV Resonance Raman for the signal transfer form HasR to its interacellular partners. Spectroscopy References Riley J. Workman1, David Punihaole2, Ryan S. Jakubek2, Jeffry D. Madura1. 1 2 [1] Krieg S, Huche F, Diederichs K, Izadi-Pruneyre N, Lecroisey A, Wandersman Chemistry and Biochemistry, Duquesne, Pittsburgh, PA, USA, Chemistry, C, Delepelaire P & Welte W, Proc Natl Acad Sci U S A 106, 1045-1050 (2009). University of Pittsburgh, Pittsburgh, PA, USA. [2] Caillet-Saguy C, Piccioli M, Turano P, Izadi-Pruneyre N, Delepierre M, Understanding the solution-state structures of polyglutamine peptides is crucial Bertini I & Lecroisey A, J Am Chem Soc, (5),1736-44 (2009). to developing fundamental insight into the etiology of at least 10 neurodegen- [3] Amorim GC, Prochnicka-Chalufour A, Delepelaire P, Lefevre J, Simenel C, erative disorders, including Huntington’s disease. Here, we utilize enhanced- Wandersman C, Delepierre M & Izadi-Pruneyre N, PloSOne, 8(3), e58964 sampling and classical molecular dynamics to investigate in detail the model (2013). polyQ peptide, (Q10). Results from this computational work was compared [4] Malki I, Simenel C, Wojtowicz H, de Amorim GC, Prochnicka-Chalufour to UV resonance Raman (UVRR) spectroscopy work performed by Punihaole A, Hoos S, Raynal B, England P, Chaffotte A, Delepierre M, Delepelaire P & et al., part of an ongoing collaboration. Our combined simulation and experi- Izadi-Pruneyre N, PLoS One, 9(4), e89502 (2014). mental results enable us to develop new, molecular-level insights into the [5] Wojtowicz H, Prochnicka-Chalufour A, Cardoso de Amorim G, Roudenko solution-state structures of polyQ peptides. Using metadynamics MD simula- O, Simenel C, Malki I, Pehaud-Arnaudet G, Gubellini F, Koutsioubas A, Perez tions of Q10, we were able to characterize the peptide’s conformational free J, Delepelaire P, Delepierre M, Fronzes R, Izadi-Pruneyre N, Biochem J energy landscape. When compared with UVRR data, this landscape indicates 5,473(14):2239-48 (2016). that Q10 adopts two monomeric conformational states, a collapsed b-strand and a predominately PPII-like structure. Experimentally, these two structures Platform: Single-Molecule Spectroscopy do not readily interconvert, which suggests that a high activation barrier sepa- rates the two states. Using data from the metadynamics simulation, we identi- 2314-Plat fied the height and local metastable minima of this activation barrier. Classical Single-Molecule Dissection of the Role of Directionality in Protein Degra- MD simulations of Q10 have also provided new details regarding the confor- dation by Clp Proteolytic Machines mations and hydrogen bonding environments of glutamine side chains in the Hema Chandra Kotamarthi, Adrian Olivares, Benjamin Stein, PPII and b-strand-like structures in solution-state. Results from our MD simu- Robert Sauer, Tania Baker. lations, as well as the comparison with experimental data collected by Puni- Biology, Massachusetts Institute of Technology, Cambridge, MA, USA. haole et al. will be presented. Protein degradation by AAAþ proteases is an important biological process that maintains protein homeostasis by removing damaged and unneeded proteins.1 2312-Plat In principle, the rate of the proteolysis reaction can be influenced by the stabil- A New Pairwise Shape-Based Scoring Function to Consider Long-Range ity of the protein substrate, the sequence and location of its degradation tag (de- Interactions for Protein-Protein Docking gron), and the chemo-mechanical properties of the AAAþ enzyme. Previous Yumeng Yan, Shengyou Huang. optical-trapping experiments probed single-molecule degradation initiated at School of Physics, Huazhong University of Science and Technology, Wuhan, a C-terminus of substrates with multiple copies of the titinI27 domain by two China. well-characterized AAAþ proteases, E. coli ClpXP and ClpAP. These studies Protein-protein docking is a valuable computational tool to study interactions demonstrated that destabilization of the titin domain by the V15P mutation re- between proteins, of which Fast-Fourier Transform (FFT)-based algorithms sulted in faster enzymatic unfolding, with ClpAP being a faster unfoldase than have been widely used as an initial step of post-docking methods or an ClpXP by a factor of ~3.2,3 In the current work, we use optical trapping to study independent docking approach due to its high computational efficiency and ClpXP and ClpAP degradation initiated at the N-terminus of multi-domain global sampling capability. As the foundation of scoring functions, shape titinI27 substrates. We find that ClpXP unfolds the wild-type and V15P titinI27 complementarity plays a critical role in the success of FFT-based docking domains ~60 times faster in the N-to-C direction than in the C-to-N direction. algorithms. Here, we have presented a new pairwise shape-based scoring func- Indeed, ClpXP unfolding of these domains is so fast that translocation becomes tion to consider long-range interactions for protein-protein docking. The long- rate limiting in degradation. Ensemble assays confirm that ClpXP degradation range shape-based scoring (LSC) function is characterized by a protein core, a of these substrates is much faster when the degron is at the N-terminus. Optical- surface layer where the repulsion component is the sum of the contributions of trapping experiments show that ClpAP unfolds the wild-type and V15P titinI27 neighboring core atoms, and an interacting layer where the favorable compo- substrates ~5 times faster in the N-to-C than C-to-N direction. Thus, the titinI27 nent comes from all the core and surface atoms in the protein. When tested domain has greater mechanical stability when pulled from the C-terminus than on the 176 targets in the protein-protein docking benchmark 4.0 by the from the N-terminus, the location of the degron can have a very large impact on Weng group. Our LSC significantly improved the docking performance in unfolding and degradation rates, and different AAAþ machines can unfold spe- both the success rate and the number of hits in binding mode prediction, cific proteins more efficiently in one direction than the other. compared to the shape-based scoring approaches in other protein-protein dock- 1. Olivares, A.O., T.A. Baker, and R.T. Sauer, Nat Rev Microbiol, 14, 33-44 ing programs. When the top 1000 predictions were considered, LSC obtained a (2016). success rate of 56.3% and an average of 3.62 hits, compared to 48.9% and 2.96 2. Olivares, A.O., et al., Nat Struct Mol Biol, 21, 871-5 (2014). of the hallmark shaped-based docking program ZDOCK 2.1. The improvement 3. Cordova, J.C., et al., Cell, 158, 647-58 (2014). of LSC over other similar approaches provides a new initial-stage protein dock- ing algorithm and also suggests a method to consider the long-range interaction 2315-Plat effect in protein-protein docking. Regulated Snare Folding and Membrane Fusion Yongli Zhang. 2313-Plat Cell Biology, Yale University, New Haven, CT, USA. Structural Basis of the Signaling through a Bacterial Membrane Receptor All actions and thinking depend on delicate machinery at synapses that release HasR Deciphered by an Integrative Approach neurotransmitters with extremely high speed and precision. Core components Nadia Izadi Pruneyre. of the release machinery include SNARE proteins, Munc18-1, and synaptotag- Structural Biology and Chemistry, Institut Pasteur, CNRS, Paris, France. min (Syt). SNAREs couple their stage-wise folding and assembly to synaptic Bacteria use diverse signaling pathways to control gene expression in response vesicle fusion like a zipper, and Munc18-1 and Syt regulate the SNARE assem- to external stimuli. In Gram-negative bacteria, the binding of some nutrients is bly. How these components work together to achieve the high speed and pre- sensed by their specific outer membrane transporters. A cascade of molecular cision required for neurotransmission remain unclear. Using high-resolution

BPJ 7875_7883 Wednesday, February 15, 2017 471a optical tweezers, we demonstrate that SNAREs zipper stage-wise with distinct 2318-Plat kinetics. We directly observed four stages of assembly in SNARE N-terminal, Simulation of FRET Dyes Allows Direct Comparison against Experi- middle, C-terminal, and linker domains (or NTD, MD, CTD and LD, respec- mental Data tively). Results of layer mutations suggest that NTD and CTD are responsible Ines Reinartz, Claude Sinner, Alexander Schug. for vesicle docking and fusion, respectively, whereas MD regulates SNARE as- SCC, KIT, Eggenstein-Leopoldshafen, Germany. sembly and fusion. Munc18-1 intimately regulates SNARE assembly by initi- Single molecule Fo¨rster Resonance Energy Transfer (smFRET) experiments ating SNARE assembly and stabilizing the half-zippered SNARE complex. Our provide valuable insight into protein dynamics. Akin to a molecular ruler, observations clarify the distinct functions of SNARE domains and the essential different protein conformations can be observed by measuring the energy trans- role of Munc 18-1 in synaptic exocytosis. We also developed novel assays to fer depending on the distance between a donor and an acceptor fluorophore. study membrane protein folding and stability based on optical tweezers. We Besides this distance, the energy transfer is also dependent on the mutual orien- applied the assays to folding dynamics of trans-SNAREs bridging two mem- tation of the dyes. Both can be gained from atomistic simulations [1]. branes and interactions of Syt and alpha-SNAP with membranes. Structure based models (SBMs) are based on energy landscape theory and the prin- ciple of minimal frustration [2,3]. With the help of eSBMTools [4] we integrate 2316-Plat FRET dyes into an all-atom SBM. The computational efficiency of these simula- Direct Measurement of Sequence-Dependent Transition Path Times and tion protocols allows simulating such processes on regular desktop computers. We Conformational Diffusion in DNA Duplex Formation developed a method to obtain FRET efficiency histograms from SBM simulations Krishna Neupane1, Feng Wang2, Michael Woodside1. which are directly comparable to experimental measurements [unpublished]. With 1Physics, University of Alberta, Edmonton, AB, Canada, 2National Institute the distance and orientation distributions from our simulations,wewant toimprove for Nanotechnology, Edmonton, AB, Canada. the planning and interpretation of smFRET measurements. As an example, we The conformational diffusion coefficient, D, sets the timescale for microscopic compare distributions from 2-color and 3-color FRET experiments [5] and simu- structural changes during folding transitions in biomolecules like nucleic acids lations [unpublished data] for ClyA in monomer and protomer conformation. and proteins. D encodes significant information about the folding dynamics [1] Hoefling, M., Lima, N., Haenni, D., Seidel, C. A. M., Schuler, B. and such as the roughness of the energy landscape governing the folding and the Grubmuller,€ H., PLoS ONE 6, 2011. level of internal friction in the molecule, but it is challenging to measure. [2] Onuchic, J.N. and P.G. Wolynes, Curr. Opin. Struct. Bio. 14, 2004. The most sensitive measure of D is the time required to cross the energy [3] Schug, A. and J.N. Onuchic, Current opinion in pharmacology 10, 2010. barrier that dominates folding kinetics, known as the transition path time. To [4] Lutz, B., C. Sinner, G. Heuermann, A. Verma, and A. Schug, Bioinformat- investigate the sequence-dependence of D in DNA duplex formation, we ics 29, 2013. measured individual transition paths from equilibrium folding trajectories of [5] Benke, S., Roderer D., Wunderlich B., Nettels D., Glockshuber R. and single DNA hairpins held under tension in high-resolution optical tweezers. Schuler B., Nat. Comm. 6, 2015. Studying hairpins with the same helix length but with G:C base-pair content 2319-Plat varying from 0-100%, we determined both the average time to cross the tran- Single-Molecule Peptide Fingerprinting sition paths, t , and the distribution of individual transit times, P (t). We then tp TP Jetty van Ginkel, Mike Filius, Malwina Szczepaniak, Pawel Tulinski, estimated D from both t and P (t) from theories assuming one-dimensional tp TP Anne S. Meyer, Chirlmin Joo. diffusive motion over a harmonic barrier. t decreased roughly linearly with tp BioNanoScience, Delft University of Technology, Delft, Netherlands. the G:C content of the hairpin helix, being 50% longer for hairpins with only Proteomic analyses provide essential information on molecular pathways of A:T base-pairs than for those with only G:C base-pairs. Conversely, D cellular systems and the state of a living organism. Mass spectrometry is increased linearly with helix G:C content, roughly doubling as the G:C content currently the first choice for proteomic analysis. However, the requirement increased from 0-100%. These results reveal that G:C base-pairs form faster for a large amount of sample renders a small-scale proteomics study such as than A:T base-pairs because of faster conformational diffusion, possibly re- single cell analysis unfeasible. Current detection limits also preclude proteomic flecting lower torsional barriers, and demonstrate the power of transition path analysis of low abundance proteins. We demonstrate the proof-of-concept of a measurements for elucidating the microscopic determinants of folding. single-molecule fluorescence peptide sequencing. We harness AAAþ protease ClpXP to linearize and scan proteins. By sequentially reading out fluorescence 2317-Plat signals from labeled amino acids via FRET, we fingerprint the identity of a pep- Direct Single-Molecule Measurements of Phycocyanobilin Photophysics in tide. The repurposed ClpXP exhibits high processivity, uni-directional process- Monomeric C-Phycocyanin ing with a constant speed, and two orders of magnitude of dynamic range in Allison H. Squires1, Quan Wang2, W.E. Moerner1. sensitivity. It makes a promising approach to sequence full-length protein sub- 1Department of Chemistry, Stanford University, Stanford, CA, USA, strates using a small amount of sample. 2Lewis-Siegler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA. 2320-Plat Phycobilisomes are highly organized pigment-protein antenna complexes An in Vitro Sample Generation Pipeline for High-Throughput Single- found in cyanobacteria, rhodophyta, and cryptophytes that harvest solar energy Molecule FRET Based Screening of Proteins and transport it to the reaction center. A detailed bottom-up model of pigment Kambiz M. Hamadani1, Madeleine Jensen2, Wu Peng3, Jamie H.D. Cate4, organization and energy transfer in phycobilisomes is essential to understand- Susan Marqusee2. ing photosynthesis in these organisms, and may even inform rational design of 1Chemistry and Biochemistry, California State University San Marcos, San artificial light-harvesting systems. However, only allophycocyanin, a two- Marcos, CA, USA, 2Molecular & Cell Biology, University of California, pigment protein which forms the core of the phycobilisome, has been previ- Berkeley, Berkeley, CA, USA, 3Department of Chemical Physiology, The ously characterized at the single-molecule level (Wang and Moerner, PNAS Scripps Research Institute, La Jolla, CA, USA, 4Chemistry, University of 2015). Here, we present the first single-molecule characterization of C-phyco- California, Berkeley, Berkeley, CA, USA. cyanin (C-PC), a three-pigment biliprotein that self-assembles to form the Single-molecule methods access biomolecular distributions, transient states, and mid-antenna rods of cyanobacterial phycobilisomes. Using the Anti-Brownian asynchronous dynamics inaccessible to standard ensemble techniques. Although Electrokinetic (ABEL) trap to counteract Brownian motion of single proteins in extremely powerful, the ability to screen large biomolecular libraries using real-time, we directly monitor, in aqueous solution, the changing photophysical fluorescence-based single-molecule detection platforms is still a challenge due states of individual C-PC monomers from Spirulina platensis by simultaneous to the lack of high-throughput methods for the generation and screening of large readout of their brightness, fluorescence anisotropy, fluorescence lifetime, and libraries of dye-labeled proteins. Here, we demonstrate proof-of-principle that by emission spectra. We are able to resolve single-chromophore emission states combining purified and reconstituted in-vitro translation, quantitative unnatural for each of the three covalently bound phycocyanobilins (a-84, b-84, and amino acid incorporation via sense codon reassignment, and either strain- b-155), providing the first direct photophysical characterization of these chem- promoted or copper-catalyzed azide-alkyne cycloaddition we can overcome these ically identical molecules in intact proteins. Although some observed photo- bottlenecks. We present a purification-free and parallelizable in-vitro approach to physical states of C-PC closely match the predictions of our theoretical generating dual-labeled proteins and ribosome-nascent-chain (RNC) libraries FRET network model, other states exhibit a surprising quenching behavior suitable for single-molecule FRET-based structural phenotyping. Importantly, which can be traced to the b-155 chromophore. These data suggest possible dual-labeled RNC libraries enable single molecule co-localization of genotypes nondegenerate functional roles among the protein’s constituent pigments. with phenotypes, and thus multiplexed single molecule screening of protein (This research is supported by a grant from the Division of Chemical Sciences, libraries (e.g. using zero-mode-waveguides). Such an approach to high- Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. throughput single molecule screening may be useful for the in-vitro directed evo- Department of Energy.) lution of proteins with designer single molecule phenotypes.

BPJ 7875_7883 472a Wednesday, February 15, 2017

2321-Plat of exocytosis and protein discharge. Supported by NIH, RO1-GM110289 to Investigation of DNA Binding, Nucleolysis and Product Release Specificity RWH and DA and 5T32HL7853-17 to KPB. of RNA Guided Endonuclease CRISPR-Cpf1 Family Reveals Important Differences from Cas9-RNA 2323-Plat Digvijay Singh1, John Mallon2, Ramreddy Tipanna3, Anustup Poddar1, Drunken Membranes. Low Doses of Short-Chain Alcohols Inhibit Exocy- Olivia Yang1, Scott Bailey2, Taekjip Ha1. tosis in a Protein-Free Model System 1Department of Biophysics and Biophysical Chemistry, Johns Hopkins Dixon J. Woodbury, Jason Paxman, Brady Hunt, David Hallan, University School of Medicine, Baltimore, MD, USA, 2Bloomberg School of Samuel R. Zarbock. Public Health, Johns Hopkins University School of Medicine, Baltimore, Physiology and Developmental Biology, Brigham Young University, Provo, MD, USA, 3Department of Biophysics, Johns Hopkins University, Baltimore, UT, USA. MD, USA. Although the effects of ethanol on protein receptors and lipid membranes have CRISPR-Cas systems impart adaptive immunity against foreign genetic ele- been studied extensively, ethanol’s effects on vesicles fusing to lipid bilayers ments in bacteria and archaea. In CRISPR-Cas system, DNA interference in- is not known. To determine the effect of alcohols on fusion rates, we utilized volves a nuclease which is guided by RNA to complementary DNA the nystatin/ergosterol fusion assay (Woodbury, 1999. Methods Enzymol. sequences (protospacer) with a requirement that the protospacer be followed 294:319-339) to measure fusion of liposomes to a planar lipid bilayer. Addition by a special protospacer adjacent motif (PAM). This system has been repur- of ethanol excited fusion when applied on the cis (vesicle) side, and inhibited on posed for various genome-engineering applications with Cas9-RNA (Class 2, the trans side. Other short-chain alcohols followed a similar pattern. In general, Type II CRISPR-Cas system) providing an overwhelming bulk of these efforts, the inhibitory effect of alcohols (trans) occurs at lower doses than the excitatory but specificity in DNA targeting continues to be a challenge. Recently a new (cis) effect, with a significant decrease in fusion rates at the legal driving limit of nuclease called Cpf1 of class 2 type V CRISPR-Cas system was identified 0.08% (w/v) ethanol (IC50= 34 mM). Similar inhibitory effects were observed (PAM: 5’-YTN-3’, Protospacer: 24 base-pairs) with robust genome-editing ac- with methanol, propanol, and butanol with ethanol being the most potent. Sig- tivity. AT-rich PAM, longer protospacer, staggered-cleavage at PAM-distal nificant variability was observed with different alcohols when applied to the site are some key differences of Cpf1 from Cas9 (PAM: 5’-NGG-3’, Proto- cis side. The inhibition by trans addition of alcohols implies that they alter the spacer: 20 base-pairs, blunt-cleavage at PAM-proximal site). But little is known planar membrane structure and thereby increase the activation energy required about kinetics and mechanism of Cpf1-RNA DNA interaction and how mis- for fusion, likely through an increase in membrane fluidity. The inhibition by matches influence this interaction and nucleolytic activity despite the impor- ethanol of liposome-membrane fusion is large enough to provide a sufficient bio- tance of competition between dissociation, nucleolysis and product release in physical explanation of compromised neuronal behavior. rapid and accurate targeting. We have used single-molecule fluorescence imag- 2324-Plat ing and biochemical assays to characterize the DNA interrogation by three ma- Lipid Dynamics and the Anthrax Toxin Intracellular Journey jor Cpf1 orthologs. The fluorescent and radio labeling geometries were Nnanya Kalu1, Laura Lucas2, Clare Kenney2, Ekaterina M. Nestorovich1. designed such that they reported on three major Cpf1 activities i.e. binding, nu- 1Biology, The Catholic University of America, Washington, DC, USA, cleolysis and release of nucleolysed products. Single molecule techniques are 2Chemistry, The Catholic University of America, Washington, DC, USA. ideal for developing a kinetic basis of Cpf1 specificity as they can detect Lipid microenvironment has been shown to play a critical role in the intoxica- wide-ranging interactions (transient to long-lived) and identify multiple tion process of many bacterial exotoxins. In this study, we use planar lipid DNA-targeting steps in real-time, which complemented with biochemical as- bilayer technique to gain insight into the role of the lipid dynamics in the says provide a comprehensive picture of Cpf1 activity and specificity. Our anthrax toxin uptake. The tripartite anthrax toxin is secreted as three separate studies reveal important differences between Cpf1 family and Cas9 which proteins, lethal factor (LF), edema factor (EF), and protective antigen (PA), can hold important implications for rapidly expanding genome-engineering that self-assemble at the cell surface to form toxic complexes. The anthrax field. Moreover, the understanding of underlying mechanism of how Cpf1- toxin intracellular journey begins from PA binding to its cellular receptors, pro- RNA recognizes, cuts or rejects DNA targets will aid in further development teolytic cleavage to PA , oligomerization to form heptameric/octameric pre- of improved versions of this enzyme. 63 pores, and LF and EF binding followed by endocytosis. The mild acidic environment of the early endosome causes conformational changes in PA63 Platform: Exocytosis and Endocytosis leading to its insertion into endosomal limiting and intraluminal vesicle (ILV) membranes and ion channel formation. The PA63-mediated delivery of 2322-Plat LF to cytoplasm was suggested to take place later in the endocytic pathway Measurement of the Mobility of Lumenal and Membrane Proteins of via the back-fusion of ILVs. It is well appreciated that the lipid composition Individual Secretory Granules changes dramatically along the endocytic pathway with ILV membranes con- 1 1 1 Prabhodh S. Abbineni , Kevin P. Bohannon , Mary A. Bittner , taining up to 70% of anionic bis(monoacylglycero)phosphate (BMP) lipid. 2 1 Daniel Axelrod , Ronald W. Holz . Here we investigate if the unique stereoconfiguration of BMP contributes to 1Pharmacology, University of Michigan, Ann Arbor, MI, USA, 2 the anthrax toxin properties, such PA63 channel formation and structural dy- Pharmacology/Physics, University of Michigan, Ann Arbor, MI, USA. namics as well as LF binding and translocation. Surprisingly, the channel for- Although many of the proteins of secretory granules have been identified, little mation was suppressed in the BMP membranes compared to PC, a phenomenon is known about their organization and macromolecular structure. Granule- that could not be explained by the negative charge of this lipid because the plasma membrane fusion can only occur when proteins that enable fusion are channel formation was significantly enhanced when tested in PS. At the present at the granule-plasma membrane contact. Thus, the mobility of granule same time, LF was shown to bind about 10 and 100 times less effectively to membrane proteins may be an important determinant of the fusion-readiness of PA63, when the channels were reconstituted into BMP and PS membranes secretory granules. Additionally, granules contents are discharged following respectively, compared with PC. The lipid dependence of the PA /LF binding fusion at highly variable rates (milliseconds to seconds); the mobility of soluble 63 reaction was pH-dependent with PC/PS difference in IC50 nearly disappearing granule proteins, in part, could determine the variable discharge rate. To at pH > 6. These findings suggest that the anthrax toxin complex can be inves- address these issues, we are measuring the mobility of specific (fluorophore- tigated and developed as a robust universal model system to study protein/lipid tagged) membrane and soluble proteins, and aim to examine the functional im- and protein/protein interactions as well as bilayer properties using lipid mem- plications of diffusion rates. We are using a method that is not limited by stan- branes of different composition. dard optical resolution. A bright flash of strongly decaying evanescent field (~100 nm exponential decay constant) produced by total internal reflection 2325-Plat (TIR) selectively bleaches mCherry or GFP-labeled protein proximal to the Actions of Rab27B GTPase on Central Excitatory Synaptic Transmission glass coverslip within individual granules. Fluorescence recovery occurs as un- Meredith M. Njus1, Geoff Murphy2, Stephen I. Lentz3, Stephen A. Ernst4, bleached protein from distal regions within the 300 nm granule diffuses into the Edward L. Stuenkel1. bleached proximal regions. The studies reveal, with 100 ms resolution, granule- 1Molecular and Integrative Physiology, University of Michigan, Ann Arbor, to-granule heterogeneity in the mobility of the lumenal proteins neuropeptide Y MI, USA, 2Molecular Behavioral Neuroscience Institute, University of (NPY), tissue plasminogen activator (tPA) and brain derived neurotrophic fac- Michigan, Ann Arbor, MI, USA, 3Michigan Diabetes Research Center, tor (BDNF), and the transmembrane domain of peptidyl alpha-amidating University of Michigan, Ann Arbor, MI, USA, 4Cell and Developmental monooxygenase (PAM). The mobility of NPY tends to be greater than that Biology, University of Michigan, Ann Arbor, MI, USA. of tPA or BDNF. Initial results suggest limited mobility of the granule mem- Information transfer at central synapses to sustained high frequency neural ac- brane protein. Our successful implementation of these techniques may lead tivity requires coordinated synaptic vesicle (SV) recycling, recruitment and to the identification of heretofore unknown factors that regulate the kinetics conversion of vesicles to release competency. Yet, the mechanism(s) by which

BPJ 7875_7883 Wednesday, February 15, 2017 473a synapses dynamically modify SV recycling to match network demands remains using a combination of chemical stimulation and imaging of individual poorly defined. While specificity and directionality of SV targeting and fusion events. When cells were strongly and repeatedly depolarized, Syt-1 activity-dependent fusion by Rab3 GTPases has been extensively studied, the granules exhibited a greater likelihood of fusion during each stimulus cycle functional significance of the SV localized Rab27B GTPase is lacking. Here than Syt-7 - consistent with a difference in fusion readiness between granule we test the hypothesis that actions of Rab27B on synaptic efficacy are spatially populations. Granules bearing Syt isoforms were differentially dependent on and mechanistically distinct from Rab3 GTPases. Investigations use hippocam- Ca2þ channel activation, as revealed by blockers of channel subtypes. That pal neurons and brain slices from Rab27B wild-type (WT) and knockout (KO) spatial heterogeneity might exist with respect to where granules fuse is apparent mouse models. The KO was verified to lack Rab27B expression through immu- from analyzing exocytosis in the cell footprint. Quantitative image analysis re- nohistochemistry on brain slices and immunocytochemistry on cultured neu- vealed that Syt-7 granules tend to fuse in closer proximity to one another than rons. Immunoblots of WT and KO hippocampal lysate further demonstrated Syt-1 granules. We also analyzed the distribution of Syt-1 and Syt-7 granules no significant effect of the KO on synaptic protein levels. Functionally, we within cells. Granules with Syt-1 were placed further from the cell surface found a significant increase in the frequency, but not amplitude, of spontaneous and potential sites of exocytosis than those with Syt-7. Moreover, Syt-1 gran- mEPSCs in Rab27B KO hippocampal neurons relative to WT when recorded ules were not only more mobile than those with Syt-7, but ‘‘newcomer’’ gran- under voltage-clamp. Moreover, Rab27B KO, led to an increase in presynaptic ules coming from within cells accounted for a greater fraction of all Syt-1 SV release to field stimulation driven AP firing relative to WT controls, as events than for Syt-7. Overall, this study provides evidence that granule pools monitored using vGlut1-pHluorin transfected neurons. Combination of in chromaffin cells may be defined by selective sorting of Syt-1 or Syt-7. Rab27B KO with expression of recombinant RAB3GAP resulted in a further increase in activity driven SV fusion, suggesting that Rab27B as well as 2328-Plat Rab3 may act as a temporary clamp in the SV regulated exocytosis pathway. Distinct Effects of Endosomal Escape and Endosomal Trafficking on Gene To assess effects on synaptic plasticity, extracellular field recording was Delivery via Electrotransfection applied in hippocampal brain slices to determine the influence at the CA3 to Lisa D. Cervia, Chun-Chi Chang, Liangli Wang, Fan Yuan. CA1 synapse of Rab27B on short- and long-term plasticity. Our results demon- Biomedical Engineering, Duke University, Durham, NC, USA. strate that relative to WT the Rab27B KO resulted in a significant decrease in A recent theory suggests that endocytosis is involved in uptake and intracellular LTP, but did not significantly impact short-term potentiation. transport of electrotransfected plasmid DNA (pDNA). Therefore, the current study investigated if approaches used previously to improve non-viral gene de- 2326-Plat livery could be applied to enhancing electrotransfection efficiency (eTE). Re- Molecular Mechanism of Fusion Pore Formation sults from the study showed that photochemically-induced endosomal Satyan Sharma, Manfred Lindau. escape, which could increase poly-L-lysine (PLL)-mediated gene delivery, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany. decreased eTE. The decrease could not be prevented by endonuclease inhibitors The SNARE proteins, synaptobrevin 2 (syb2), SNAP-25, and syntaxin-1A (stx- (aurintricarboxylic acid and zinc ion). Chemical treatment of cells with an en- 1A) form a coiled-coil complex through their SNARE motifs. SNARE proteins dosomal trafficking inhibitor that blocks early endosome progression, are considered to be the minimal membrane fusion machinery required for the Bafilomycin A1, also resulted in a significant decrease in eTE. However, treat- synaptic vesicle exocytosis. Despite of our improved understanding of various ments with lysosomotropic agents (chloroquine and ammonium chloride) had proteins in fusion machinery, a clear picture of the fusion at the molecular level little effects on eTE. These data suggested that endosomes played key roles is still unresolved. To study the role of SNARE’s in membrane fusion, reconsti- in protecting and intracellular trafficking of electrotransfected pDNA. tution experiments with SNARE containing liposome/liposome or, more 2329-Plat recently, nanodisc/liposome systems are employed. Utilizing coarse-grained Dynamics of Clathrin-Mediated Endocytosis within a Developing molecular dynamics, simulations of SNARE-mediated fusion of a ~13nm Organism nanodisc with a multicomponent asymmetric planar bilayer is studied. The lipid Joshua P. Ferguson1, Nathan M. Willy1, Spencer P. Heidotting1, composition of the nanodisc, represented by 5 lipid types, was chosen based on Scott D. Huber1, Matthew J. Webber2, Comert Kural1,2. that of a synaptic vesicle. The composition of the bilayer, consisting of 21 lipid 1Department of Physics, The Ohio State University, Columbus, OH, USA, types, was based on composition of a synaptic plasma membrane. Four partially 2Biophysics Graduate Program, The Ohio State University, Columbus, unzipped SNARE-complexes bridged the nanodisc and the planar bilayer. The OH, USA. transmembrane domain (TMD) of syb2 traversed the nanodisc membrane. The Endocytic clathrin-coated structures (CCSs) mediate the internalization of t-SNARE complex (stx-1A and SNAP-25) was anchored in the planar bilayer receptor-bound macromolecules from the plasma membrane of eukaryotic by the stx-1A TMD and SNAP-25 lipid anchors. In all of twelve 2.5 us long in- cells. Current knowledge of clathrin-mediated endocytosis (CME) dynamics dependent simulations brief pore formation and lipid mixing of the distal leaflets is based on imaging assays designed for tracking of fluorescently labeled was observed but only four showed formation of a stable water-filled pore. The coat components at cell-substrate interfaces. Due to technical limitations, simulations revealed that zipping of SNARE complexes pulls the syb2 C termi- similar experimental and analytical approaches cannot be applied to cells nus deeper into the membrane inducing local curvature. The TMD polar residues within physiological contexts, such as tissues. Here, we utilize the growth Y113, S115 and T116 of syb2 and S281, T 282 and G288 of stx-1A form a hy- rate of fluorescence signals from short CCS intensity trace fragments to avoid drophilic core between the two distal leaflets facilitating lipid mixing and leading the need for determining the entire lifetime of individual endocytic assemblies. to fusion pore formation. Two SNARE protein mutations inhibiting fusion exper- This technique allows us to monitor spatiotemporal changes in CME dynamics imentally produced only proximal leaflet lipid mixing and no fusion pore forma- in real-time with less susceptibility to errors associated with particle detection tion in the simulations. Supported by ERC ADG 322699. and tracking. By compiling growth rates into slope histograms, we find that physical factors impeding CME dynamics result in deceleration of CCS growth 2327-Plat and dissolution rates. Finally, we confirm the applicability of this approach to Chromaffin Cell Isoforms form Functionally and Spatially in vivo systems by demonstrating a reduction in CME dynamics in the amnio- Separable Granule Pools serosa tissue of Drosophila melanogaster embryos during dorsal closure. This Tejeshwar Rao1, Alexandra Ranski1, Peter Dahl1, Julia Bourg2, approach paves the way for elucidating how mechanical cues affect the forma- Edwin Chapman3, Sarah Veatch2, David Giovannucci4, Arun Anantharam1. tion and dissolution of CCSs during the development of multicellular 1 2 Pharmacology, University of Michigan, Ann Arbor, MI, USA, Biophysics, organisms. University of Michigan, Ann Arbor, MI, USA, 3Neuroscience, University of Wisconsin, Madison, WI, USA, 4Neuroscience, University of Toledo, Platform: Nucleic Acid Structure & Dynamics Toledo, OH, USA. Chromaffin granules have long been divided into ‘‘pools’’ whose membership 2330-Plat is based on functional criteria. However, what distinguishes these pools, at the Conformations of Single-Stranded Nucleic Acids in Solution molecular level, has been more difficult to define. A key difference between Alex Plumridge1, Steve Meisburger2, Lois Pollack3. granules may be whether they harbor synaptotagmin-1 (Syt-1) or 1Cornell University, Ithaca, NY, USA, 2Princeton University, Princeton, NJ, synaptotagmin-7 (Syt-7). We previously showed that Syt-1 and Syt-7 are rarely USA, 3Cornell Univeristy, Ithaca, NY, USA. found together on the same granule and confer different properties to the fusion Single-stranded nucleic acids are prevalent throughout biology: DNA replica- event. The major goal of this study was to assess whether Syt isoforms are tion requires unzipping the DNA double helix, while many RNAs require flex- sorted into functionally or spatially separable pools. A useful tool in this ible, non-base paired linkers to form compact states. Elucidating the structure context is provided by monitoring the kinetics of capacitance increases in and properties of these disordered-regions is critical to understanding their bio- response to electrophysiological stimulation. We approximated this approach logical roles; however, as a result of the high flexibility of these single stranded

BPJ 7875_7883 474a Wednesday, February 15, 2017 molecules, structural investigations are particularly challenging. Recently, we 2334-Plat have paired SAXS with novel ensemble methods to visualize the numerous Pore Translocation of DNA Chains with Physical Knots states adopted by these molecules in solution. This approach reveals distribu- Antonio Suma, Cristian Micheletti. tions of structural parameters, conformations and intricate details pertaining Physics and Chemistry of Biological Systems, Scuola Internazionale to the phosphate backbone and base-stacking interactions for free strands. The Superiore di Studi Avanzati (SISSA), Trieste, Italy. new information provided can forward our understanding of a number of impor- Long biopolymers have a large probability of being knotted. These form of self- tant problems, including RNA folding, protein binding of single-strand nucleic entanglement can arise spontaneously both in the context of nucleic acids and acids, the polymer nature of nucleic acids and molecular force-field validation. proteins [1]. Here we focus on the impact of such knots during the translocation through narrow pores, as they can affect both biological processes and experi- 2331-Plat mental setups in general. With the aid of coarse-grained molecular dynamic sim- The Conformations of Confined Polymers in an External Potential ulations, we study systematically the pore translocation of flexible chains tied in Greg Morrison1, Dave Thirumalai2. 1 2 different knot types, including composite ones. We find a rich phenomenology as Physics, University of Houston, Houston, TX, USA, Chemistry, University a function of knot complexity and driving force [2, 3] and show that it can be ratio- of Texas at Austin, Austin, TX, USA. nalized within a simple theoretical framework, which can also be used as a guide The confinement of biomolecules is ubiquitous in nature, such as the spatial for experimental designs. We will also report on preliminary results for more constraints of viral encapsulation, histone binding, and chromosomal packing. detailed models of systems that have been recently characterized experimentally. Advances in microfluidics and nanopore fabrication have permitted powerful [1] S. Jackson, A. Suma and C. Micheletti. Curr. Opin. Struct. Biol., 2016, In new tools in single molecule manipulation and gene sequencing through molec- press. ular confinement as well. In order to fully understand and exploit these systems, [2] A. Rosa, M. Di Ventra and C. Micheletti. Phys. Rev. Lett., 2012, 109, the ability to predict the structure of spatially confined molecules is essential. In 118301. this talk, I describe a mean field approach to determine the properties of stiff [3] A. Suma, A. Rosa and C. Micheletti. ACS Macro Lett., 2015, 4, 1420. polymers confined to cylinders and slits, which is relevant for a variety of bio- logical and experimental conditions. I show that this approach is able to not 2335-Plat only reproduce known scaling laws for confined wormlike chains, but also pro- Single Molecule Fluorescence Studies on Nucleosome Dynamics vides significant improvement over existing weakly bending rod approxima- Kathrin Lehmann, Alenxander Gansen, Katalin Toth, Jo¨rg Langowski. tions in determining the detailed chain properties (such as correlation DKFZ, Heidelberg, Germany. functions). Using this approach, we also show that it is possible to study the ef- Nucleosomes are the basic packaging unit of chromatin compaction. They have fect of an externally applied tension or static electric field in a natural and significant influence on gene expression and regulate DNA accessibility. Two analytically tractable way. These external perturbations can alter the scaling copies of each core histone protein (H2A, H2B, H3, H4) build up the protein laws and introduce important new length scales into the system, which may octamer, around which 147 bp of DNA are wrapped. be relevant for histone unbinding and single-molecule analysis of DNA. We study the disassembly process of reconstituted Xenopus laevis nucleosomes 2332-Plat containing mutated histones or modifications in comparison to the wild type. Single Molecule Visualization of Topology-Mediated Interactions in In vitro nucleosome disassembly may be forced by increasing salt concentration Supercoiled DNA and may be followed by Fo¨rster resonance energy transfer (FRET) between flu- Shane Scott1, Zhi Ming Xu1, Fedor Kouzine2, Daniel Berard1, orophores. Therefore, nucleosomes were fluorescently labeled at various posi- Cynthia Shaheen1, Laura Saunders1, Barbara Gravel1, Catherine Leroux1, tions of the DNA and the histone proteins. This tool set was used to analyse Christopher Cayen-Cyr1, David Levens2, Craig Benham3, Sabrina Leslie1. the functional relevance of the a3-domain of H2A, which might take part in a 1Physics, McGill University, Montreal, QC, Canada, 2National Cancer long range interaction with the N-tail of H3. This was first observed in molecular Institute, Bethesda, MD, USA, 3Genome Center, UC Davis, Davis, CA, USA. dynamic simulations on ‘tailless’ variants of H3 and H2A suggesting conforma- We directly visualize the weak and slow interactions between specific unwinding tional changes affecting two arginines of H2A (R81 and R88). The altered sites on supercoiled DNA, and site-specific probes designed to bind to these sites, conformation of those amino acids lead to conformational changes of the pro- as a function of DNA supercoiling and temperature. We use Convex truding part of the DNA and to an altered electrostatic environment in vicinity Lens-induced Confinement microscopy to confine the DNA molecules within of the a3-domain of H2A. To investigate the influence of the electrostatic envi- a sealed, glass array of nanoscale pits, embedded in a coverslip. Throughout ronment of this region on DNA breathing, unwrapping, and stability of entire nu- our wide-field observations of single-molecule binding and diffusive trajectories, cleosomes we generated recombinant H2A proteins incorporating site-specific the DNA molecules are free to explore all possible configurations, which we mutations (R81A, R88A, R81AR88A, R81E, R88E, R81ER88E). Our results > > > show is influential on the observed dynamics. The increase in binding rate that show a decreasing stability associated with position (Wt R88 R81 > we observe, with both temperature and supercoiling, is consistent with Z-DNA R81R88) and charge (RA RE) of the amino acids. Although the sequence formation playing a key role in governing DNA structural mechanics. This alter- of the disassembly steps remains unaltered, the distribution of the intermediate nate structure suppresses supercoil-induced DNA unwinding in DNA at low tem- state is affected in the same manner. peratures, for a wide range of negative superhelicities. The new single-molecule Furthermore we developed a labelling strategy to analyse the dynamics and in- methodology that we present may be used to visualize a wide range of molecular tranucleosomal interactions of the N-terminal tail of H3. Combining this with interactions that are challenging or impossible to access with other methods such our H2A-mutants or other modified histones will allow the analysis of long as optical or magnetic tweezers. These include interactions that are highly depen- range effects on the H3-tail dynamics and intranucleosomal interactions. dent on molecular topology, proceed over many seconds to minutes, or are rare. 2336-Plat 2333-Plat Shape-Programmed Hierarchical Self-Assembly of Designed DNA Build- Exploring DNA and RNA Structures in Solution with Infrared ing Blocks into Massive Three-Dimensional Finite-Size Objects Spectroscopy Klaus Franz Wagenbauer, Christian Sigl, Hendrik Dietz. Allison L. Stelling1, Hashim Al-Hashimi2. Physik, TU Munchen,€ Garching bei Munchen,€ Germany. 1Biochemistry, Duke University, Durham, NC, USA, 2Dept. of Biochemistry, Large and discrete biological assemblies such as the flagellar motor or viral Duke University, Durham, NC, USA. capsids form by hierarchical self-assembly of many different protein subunits Infrared (IR) spectroscopy was used to locate the Hoogsteen (HG) signature for where some of these subunits often occur in multiple copies. For assemblies GC pairs in both a chemically trapped HG bp that has been shown by NMR to such as capsids the shape of the subunits and their interaction patterns effec- form a single HG pair, and in an all-GC duplex previously shown to have HG tively encode the geometry of the final assembly. Creating artificial objects bps under low salt and ion buffer conditions. The IR signals arise from the with similar complexity through accurate design of the shape of molecular chemical bonds themselves, and are thus quite sensitive to even subtle changes building blocks represents an important design challenge that tests our in geometry. The signals are compared to those from the GC alternating repeat understanding of molecular structure, molecular flexibility, and molecular DNA sequence, which enabled the identification of HG-specific IR bands in GC interactions. bps chemically trapped within a WC duplex. These IR signals will be used to We have recently described that discrete three-dimensional (3D) DNA compo- identify HG pairs in large protein DNA complexes whose size renders them nents can specifically self-assemble in solution on the basis of shape- intractable for study with other spectroscopic methods. complementarity and without base pairing (1). Using this principle, we IRmethods are also being developed toexamine interactions that occur when RNA previously produced homo- and heteromultimeric objects, including interacts with metals or small molecules. These signals can in turn be used to dif- micrometer-scale one- and two-stranded filaments and lattices, as well as re- ferentials between specific and non-specific binding for a range of ligands. configurable devices, including an actuator, a switchable gear, an unfoldable

BPJ 7875_7883 Wednesday, February 15, 2017 475a nanobook, and a nanorobot. These multidomain assemblies were stabilized via divided into two homologous copies of an S1-S6 -like 6-TM domain. short-ranged nucleobase stacking bonds that compete against electrostatic A functional TPC channel assembles as a dimer - the equivalent of a repulsion between the components’ interfaces. voltage-gated tetrameric cation channel. Plant TPC channel (TPC1) is local- Building on these techniques, in the present study we report on progress with ized to the vacuole membrane and is responsible for generating the slow vacu- the hierarchical self-assembly of discrete three-dimensional finite size objects olar (SV) current observed long before its molecular identification; therefore, whose shapes are encoded in the geometry of its building blocks. We created plant TPC1 is also called SV channel. Plant TPC1 is a non-selective cation gear-like objects that form from self-limiting oligomerization of V-shaped channel, permeable to various monovalent cations as well as Ca2þ and likely building blocks. The diameter, the number of subunits per ring, and the shape plays an important role in regulating cytosolic ion concentrations. The chan- of the distribution of subunits per ring are tightly controlled by the angle and nel is voltage-gated and its voltage-dependent activation can be modulated by rigidity of the V building blocks. The rings may be hierarchically assembled both cytosolic and vacuolar Ca2þ. Recently, we determined the first crystal into giant, giga-dalton scale tubes. We also describe progress on the self- structure of a plant vacuolar two-pore channel from Arabidopsis thaliana, assembly of 3D polyhedral cages such as cubes and dodecaheder from appro- AtTPC1, and were also able to functionally characterize the channel activity priately shaped building blocks. using patch clamp recording. Our structural and functional studies demon- (1) T. Gerling, K. Wagenbauer, A. Neuner, and H. Dietz strate that Ca2þ activates the channel by triggering conformational changes ‘Dynamic DNA devices and assemblies formed by shape-complementary, non- at the EF-hand domain that are coupled to the pair of pore-lining inner helices basepairing 3D components’. Science, vol 347 (2015), p1446-1452. from the first 6-TM domains, whereas only activates the second voltage-sensing domain (VSD2), whose conformational changes may 2337-Plat only be coupled to the other pair of inner helices from the second 6-TM do- The Energy Landscape of Human Chromosomes mains. More importantly, the current AtTPC1 structure is in a closed state Michele Di Pierro. and, for the first time, provides a long-awaited structural view of a channel Center for Theoretical Biological Physics, Rice University, Houston, voltage sensor in the resting state. This major breakthrough on AtTPC1 study TX, USA. provides us with an excellent model system to further investigate the struc- In vivo, the human genome folds into a characteristic ensemble of three- tural mechanisms of channel gating and ion selectivity in the TPC channel dimensional structures. The mechanism driving the folding process remains family. unknown. We report a theoretical model for chromatin (Minimal Chromatin Model) that explains the folding of interphase chromosomes and generates 2340-Symp chromosome conformations consistent with experimental data. The energy Gating Pathways for a Pentameric Ligand-Gated Ion Channel Solved by landscape of the model was derived by using the maximum entropy principle Atomistic String Method Simulations and relies on two inputs: a classification of loci into chromatin types and a Bogdan Lev1, Samuel Murail2, Fre´de´ric Poitevin3, Brett A. Cromer1, catalog of the positions of CTCF-mediated chromatin loops. Chromatin types, Marc Baaden2, Marc Delarue4, Toby W. Allen1,5. which are distinct from DNA sequence, are partially epigenetically controlled 1Science, RMIT University, Melbourne, Australia, 2Laboratoire de and change during cell differentiation, thus constituting a link between epige- Biochimie The´orique, CNRS, UPR9080, Univ Paris Diderot, Sorbonne, netics, chromosomal organization, and cell development. We trained our en- Paris, France, 3Structural Biology, Stanford University, Stanford, CA, USA, ergy function using the Hi-C contact map of chromosome 10 from human 4Structural Biology and Chemistry, Institut Pasteur and UMR 3528 du CNRS, GM12878 lymphoblastoid cells. Then, we used the model to perform molec- Paris, France, 5Chemistry, University of California, Davis, CA, USA. ular dynamics simulations producing an ensemble of 3D structures for all Pentameric ligand-gated ion channels control synaptic neurotransmission via GM12878 autosomes. We used these 3D structures to generate contact an allosteric mechanism, where agonist binding induces global protein confor- maps. We found that simulated contact maps closely agree with experimental mational changes that open an ion-conducting pore. We use an atomistic mo- results for all GM12878 autosomes. The ensemble of structures resulting from lecular dynamics string method to solve for the minimum free energy gating these simulations exhibited unknotted chromosomes, phase separation of pathways for the proton-activated GLIC channel. We describe stable open chromatin types, and a tendency for open chromatin to lie at the periphery and closed states, and uncover conformational changes associated with commu- of chromosome territories. Finally, we analyzed the dynamics generated by nication between agonist-binding extracellular and ion-conducting trans-mem- MiChroM. We found our model to be in excellent agreement with experi- brane domains. Transition analysis is used to compute free energy surfaces that mental observations reporting anomalous diffusion and spatial coherence in suggest pathways, allosteric stabilization with pH, and intermediate states that chromatin in vivo. facilitate channel closing in the presence of agonist. We describe a switching Symposium: Channel Gating Mechanisms mechanism that senses proton binding to induce asynchronous pore-lining M2 helix movements. These results provide new insight into the allosteric 2338-Symp mechanisms for the super-family of pentameric ligand-gated channels, with po- Dynamic Coupling between the Gates in the NaK Channel tential applications in improved anesthetics, neuromodulatory drugs, anti- Katherine Henzler-Wildman. parasitics and pesticides. Biochemistry, University of Wisconsin, Madison, WI, USA. Flux-dependent inactivation that arises from functional coupling between the 2341-Symp inner gate and the selectivity filter is widespread in ion channels. The struc- Fine Tuning HCN Channel Activity tural basis of this coupling has only been well characterized in KcsA. Here Anna Moroni. we present NMR data demonstrating structural and dynamic coupling be- Biosciences, University of Milan, Milan, Italy. tween the selectivity filter and intracellular constriction point in the bacterial HCN (hyperopolarization-activated cyclic-nucleotide gated) channels are acti- non-selective cation channel, NaK. High-resolution crystal structures are vated by membrane hyperopolarization and further modulated by binding of available for a number of NaK mutants with different numbers of ion bind- cAMP to the CNBD (cyclic nucleotide binding domain). Understanding the ing sites in the selectivity filter and different ion selectivity properties. NMR molecular mechanisms of regulation of this family of ion channels is critical spectra of non-selective and potassium-selective NaK mutants indicate that as it pertains to the physiological processes as well as to diseases associated the equilibrium distribution of structural states for the entire channel varies dysfunctions in the cardiac and neuronal If/Ih currents. In the brain, cAMP with the ion-selectivity of the channel. Our results highlight the tight struc- modulation of HCN isoforms 1 and 2 is controlled and fine tuned by the tural and dynamic coupling between the selectivity filter, the surrounding auxillary protein TRIP8b while, in the heart, cAMP regulation of the cardiac scaffold, and the inner gate. NaK offers a distinct and valuable model to isoform HCN4 is prevented by cyclic dinucleotides, an emerging class of sec- study this physiologically essential connection between ion conduction and ond messengers in mammals. Starting from a structural and functional channel gating. description of the molecular determinants of the aforementioned regulatory systems, we are developing new tools (peptides, chimeric channels and opto- 2339-Symp genetic approaches) to control and analyse cAMP action on HCN channels. Structure of the TPC1 Channel from Arabidopsis Thaliana Our goals are: i) to fully describe the cAMP-induced conformational changes Jiangtao Guo1, Weizhong Zeng1, Youxing Jiang1,2. which affectpore gating; ii) to understand the physiological role of TRIP8b 1University of Texas Southwestern Medical Center, Dallas, TX, USA, modulation of HCN channel gating in vivo; iii) to identify terapeutical ap- 2Howard Hughes Medical Institute, Dallas, TX, USA. proaches based on the control of cAMP modulation. Our results on the regu- Two-pore channels (TPCs) belong to the family of voltage-gated tetrameric latory network of HCN activity highlight an efficient mechanism for cation channels and are ubiquitously expressed in organelles of animals and preventing b-adrenergic stimulation on If and identify potential drug targets plants. Each TPC subunit contains 12 transmembrane segments that can be in HCN channels.

BPJ 7875_7883 476a Wednesday, February 15, 2017

Symposium: Anomalous Dynamics in Biological 2345-Symp The Genome in the Nucleus: Snaky, Soft and Well-Organized Systems Yuval Garini. Physics, Bar Ilan University, Ramat Gan, Israel. 2342-Symp The DNA in a human cell is ~2 meters long. Although there are no definite Anomalous Diffusion and Compartmentalization on the Surface of structures that maintains the order in the nucleus, the genome is well organized Mammalian Cells and chromosomes stays isolated in ‘chromosome territories’. What are the Diego Krapf1,2. mechanisms that organizes the DNA in the nucleus? 1 Electrical and Computer Engineering, Colorado State University, Fort We used a whole spectrum of dynamic methods in live cells for exploring the 2 Collins, CO, USA, School of Biomedical Engineering, Colorado State organization mechanisms. This includes single particle tracking (SPT) and University, Fort Collins, CO, USA. continuous photobleacing (CP) and the data is analyzed according to diffusion Tracking individual proteins on the surface of live mammalian cells reveals analysis methods that we developed. In normal cells, all the sites in the complex dynamics involving anomalous diffusion and clustering into nano- genome exhibit anomalous diffusion (viscoelastic) with a power law of scale domains. Theoretical models show that anomalous diffusion can be ~0.3-0.5 and the diffusion was found to belong to the family of fractional caused by various processes. By performing time series and ensemble analysis Brownian motion anomalous diffusion. We identified a protein, lamin A, of extensive single-molecule tracks, we show that different subdiffusive pro- which provides the elasticity to the genome. Removal of lamin A drastically cesses simultaneously coexist in the plasma membrane. Interestingly, ergo- changes the diffusion type from slow anomalous diffusion to fast and normal dicity breaking is found to be maintained by immobilization events that diffusion. We suggest a mechanism that explains the organization mainte- take place when proteins are transiently captured within endocytic pits. nance of the chromosomal territories and is strongly supported by the whole Furthermore, using a combination of super-resolution imaging and single-par- set of our dynamic data. ticle tracking, we observe that the actin cytoskeleton introduces barriers lead- Single molecule methods confirm the binding of lamin A to DNA as demon- ing to the compartmentalization of the plasma membrane and that proteins are strated through atomic force microscopy and tethered particle motion. transiently confined within actin domains. In addition to subdiffusive pro- cesses we find superdiffusive motion performed by membrane-targeting pro- teins. We observe that peripheral proteins often dissociate from the Platform: Ligand-gated Channels II membrane to perform three-dimensional Brownian motion in the bulk until re-association. These bulk excursions induce long jumps with step sizes 2346-Plat distributed according to power laws, which can be modeled by superdiffusive Analysis of Gating of Acid-Sensing Ion Channels (ASICs) under Rapid and Levy flights. Slow pH Changes Omar Alijevic1, Jan Kucera2, Stephan Kellenberger1. 1Pharmacology and Toxicology, University of Lausanne (UNIL), Lausanne, 2343-Symp 2 Zooming in on Anomalous Diffusion with Variable-Lengthscale Fluores- Switzerland, Physiology, University of Bern (UB), Bern, Switzerland. To sense extracellular pH changes, neurons express the acid-sensing ion cence Correlation Spectroscopy þ Cecile Fradin. channels (ASICs) that are Na -selective ion channels transiently activated Physics and Astronomy, McMaster University, Hamilton, ON, Canada. by rapid extracellular acidifications (pH50 of activation: ~pH 6.7-4). Ob- The diffusion of macromolecules in cells, and in general in crowded or servations of ASIC currents induced by rapid acidifications suggest that complex fluids, is often found to be anomalous. However, given that each during a slowly developing acidification, ASICs would open not at all or different experimental technique usually probes diffusion at a single length- only briefly, due to channel desensitization. Slow acidifications may occur scale, it remains difficult to determine which anomalous diffusion model is during high neuronal activity, cerebral ischemia and inflammation. The the most appropriate for each particular system. Here we show that vari- response of ASICs to such a slow acidification has however not been reported able-lengthscale fluorescence correlation spectroscopy, where the volume of yet. We used patch-clamp electrophysiology on ASIC1a or ASIC3 expressed observation is varied over several orders of magnitude, combined with a in CHO cells to build their kinetic models. We used these kinetic models to numerical inversion procedure of the correlation data, allows retrieving the predict the response of ASIC1a and ASIC3 to slow pH changes. We then mean-squared displacement of molecular diffusion processes for up to five experimentally validated the modeling results at different speed of acidifica- decades in time. This procedure thus bridges the gap between diffusion exper- tion and found that in response to an intermediate slow acidification (5-10s) iments performed at different lengthscales. We discuss how it may help from pH7.4 to pH6, the kinetics of both ASIC isoforms were dramatically discriminate between different anomalous diffusion models and present appli- decelerated, resulting in current responses that in terms of the amount of charges conducted by the channels were similar to those elicited by a rapid cations for the study of diffusion in cross-linked gels, entangled polymer so- > lutions and cells. acidification. Slower acidifications ( 10s) induced responses that were iso- form specific. We investigated the relevance of these small and slow ASIC1a currents by testing the neuronal excitability of mouse cortical neurons in 2344-Symp response to slow acidifications. We observed that the intermediate slow Anomalous Diffusion in Membranes and the Cytoplasm of Biological acidifications (5-10s) increased by more than 15 times the neuronal excit- Cells ability in terms of neuronal firing duration compared to a rapid acidification. Ralf Metzler. Slower acidifications (>10s) resulted in the complete absence of neuronal Inst Physics & Astronomy, U Potsdam, Potsdam-Golm, Germany. excitations. These neuronal responses were furthermore successfully modeled A surging amount of experimental and simulations studies reveals persistent by integrating our ASIC1a kinetic model into the classical Hodgkin- anomalous diffusion in both cellular membranes and the cytoplasm. The anom- Huxley neuronal model. These findings suggest that intermediate slow acidi- alous diffusion is observed for micron-sized objects down to labelled single fications induce stronger ASIC1a-mediated neuronal excitation than a rapid molecules such as green fluorescent proteins. acidification. This talk will first present results from large scale computer simulations and stochastic analysis of the motion of lipids and embedded proteins in lipid 2347-Plat bilayer model membranes, indicating that increased disorder leads to longer A Selectivity Filter at the Lower End of ASIC1a and longer lasting anomalous diffusion. In particular, the motion of Timothy Lynagh1, Emelie Flood2, Celine Boiteux2, Matthias Wulf1, lipids and proteins can become non-Gaussian. In the membranes of Janne M. Colding1, Vitaly V. Komnatnyy1, Toby W. Allen2, living cells anomalous diffusion of embedded protein channels can last over Stephan A. Pless1. several hundreds of seconds. In particular, this anomalous diffusion can 1University of Copenhagen, Copenhagen, Denmark, 2RMIT University, become non-ergodic and exhibit ageing, two topics explained and discussed Melbourne, Australia. in this talk. Acid-sensing ion channels (ASICs) mediate sodium currents in response to The findings of anomalous diffusion in membranes will be complemented by a increased extracellular proton concentrations. It has long been assumed brief summary of anomalous diffusion in the cellular cytoplasm, referring to that sodium selectivity in ASICs is mediated by a constriction in the both subdiffusion of passive tracers and superdiffusion due to active motion middle of the channel pore (the GAS belt), based on homology with other in cells. ENaC/DEG family members and on ASIC1 X-ray structures. We performed

BPJ 7884_7890 Wednesday, February 15, 2017 477a a novel functional assessment of the GAS belt in ASIC1a by incorporating that also ENaCs are trimers (Jasti et al. Nature 449, 316-323, 2007). The abg unnatural amino acids (UAAs) into the channel to modify the main ENaC was initially determined to be organized as a tetramer (Firsov et al. chain atoms implicated in ion conduction. We also engineered channels EMBO J 17, 344-352, 1998), as recently corroborated by chemical cross- containing either one, two or three mutant subunits (concatemers) to assess linking (van Bemmelen et al PLoS ONE 10, e0135191, 2015). Here we the contribution of individual subunits. Together with molecular dynamics used affinity chromatography combined with blue native polyacrylamide gel simulations of ion permeation through ASIC1, this revealed that the electrophoresis (BN-PAGE) to assess the oligomeric state of the human GAS belt does not determine sodium selectivity. We therefore screened the ENaC expressed in Xenopus laevis oocytes. BN-PAGE has reliably predicted channel via conventional mutagenesis for other determinants of selectivity, the oligomeric state of a variety of proteins including the trimeric architecture identifying a ring of glutamate residues at the lower end of the pore. of P2X receptors (Nicke et al EMBO J 17, 3016-3028, 1998; Aschrafi et al. J Results from UAA incorporation, concatemeric channels, and energetics of Mol Biol 342, 333-343, 2004). Two-electrode voltage-clamp electrophysi- ion binding to this part of the pore suggest that these carboxylate side ology confirmed that N-terminal hexahistidine-tagging did not strongly affect chains directly determine selective sodium conduction. These results suggest the amiloride-sensitive Naþ channel function of either subunit within the het- that the sodium selectivity filter of ASIC1a resides at the lower end of the eromeric abg or dbg complex. Dodecyl maltoside-purified abg and dbg channel pore. ENaC complexes each migrated in the BN-PAGE gel as a defined band with a mass close to that of the a1 glycine-gated receptor (GLYRA1). As a 2348-Plat homopentamer, the GLYRA1 has a calculated mass of 260 kDa (52 kDa Single Molecule Kinetic Measurements and Hidden Markov Models for per protomer including one N-glycan). The abg and dbg ENaC protomers P2X1 Receptors produced by partial denaturing migrated at ~85 kDa in the BN-PAGE gel, James A. Brozik1, Adam O. Barden1, Ashish Bhattari1, Brian N. Webb1, as assessed by comparison with the migration of GLYRA1 monomers and di- Andrew J. Thompson2. mers as molecular mass markers. Evidently, a total mass of ~260 kDa of both 1Chemistry, Washington State University, Pullman, WA, USA, abg and dbg ENaC can be best explained by a trimeric complex of homolo- 2Pharmacology Department, Cambridge University, Cambridge, United gous 85 kDa protomers. A homogenous trimeric assembly state could also be Kingdom. shown by BN-PAGE for the plasma membrane-bound forms of singly ex- Purinergic receptors are ubiquitous throughout the human body and partici- pressed full-length human ASIC1 and ASIC2. Altogether, our data are consis- pate in the regulation of vast numbers of physiological processes. In partic- tent with a conserved trimeric architecture of channels of the Deg/ENaC ular, ATP binds to P2X1 receptors causing a two stage allosteric superfamily. modulation for each bound ATP. This modulation opens an integral pore causing the entry of calcium into cells and initiates numerous downstream 2351-Plat processes. We will present results from stochastic single molecule fluores- Mapping the Flexibility of AMPA Type Ionotropic Glutamate Receptors cence studies that show multiple discrete ATP binding states associated Jelena Baranovic1,2, Andrew J.R. Plested1,2. with individual P2X1 receptors. These observations have been incorporated 1Leibniz-Institut fur€ Molekulare Pharmakologie (FMP), Berlin, Germany, into a Hidden Markov Model(s) that: (1) takes into account discrete 2Cluster of Excellence NeuroCure, Charite´-Universit€atsmedizin, Berlin, ATP binding states, (2) accounts for the photophysical properties of the Germany. probe molecules, (3) extracts the most likely elementary rate constants, and Ionotropic glutamate receptors (iGluRs) are postsynaptic ion channels (4) predicts the most likely state occupancies. These results have been used commonly found in the excitatory synapses of the vertebrate central nervous to create a minimal potential energy surface that describes the operation system. Through their ion channel activity, three members of the iGluR fam- P2X1 as a molecular machine. Some recent super-resolution data that maps ily, AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), out the spatial arrangement of ATP on individual receptors will also be kainate and NMDA (N-methyl-D-aspartate) receptors, shape the communica- presented. tion between neurons. The three members share common domain architecture with amino-terminal and ligand-binding domains (ATDs and LBDs) 2349-Plat forming the extracellular part of the receptor, C-terminal domain (CTD) the Structural Basis and Molecular Mechanism Underlying P2X7 Receptor intracellular part and the transmembrane region (TM) harboring the ion Antagonism channel. Among them, AMPA receptors are the first and fastest to react to Akira Karasawa, Toshimitsu Kawate. presynaptically released glutamate. Recent advances in cryo-electron micro- Molecular Medicine, Cornell University, Ithaca, NY, USA. scopy generated structures of these tetrameric receptors in various states of P2X7 receptors are ATP-gated ion channels expressed in immune cells such as activation. One of the prevailing features of the structures was an open macrophage and microglia. Pharmacological inhibition of these receptors atten- arrangement of the extracellular domains in agonist-bound states. In uate neuropathic and inflammatory pain in animal models, however, the mech- contrast, crystal structures of full-length receptors and isolated domains anism of inhibition is poorly understood largely due to lack of an atomic identified more compact arrangements of the LBD layer. Here, we assess to resolution structure. Here we present the crystal structures of a mammalian what extent the LBD layer moves during activation by glutamate using P2X7 receptor in the presence of five structurally distinct antagonists. Despite bifunctional methanethiosulfonate (bis-MTS) cross-linkers as molecular structural diversity, all five compounds bind to a novel antagonist binding site rulers. The cross-linkers come in different lengths spanning distances from juxtaposed to the ATP-binding pocket. Consistent with these structures, func- 8 to over 20 A˚ ngstroms, thus enabling cross-linking of domains that move tional studies demonstrate that the P2X7 specific drugs allosterically and non- apart from each other during activation. The cross-linkers were attached to competitively prevent the narrowingof inter-subunit spaceupon ATP binding. the receptors through introduced cysteine residues and the activity of cross- These insights may contribute the development of P2X7-specific drug for linked receptors was measured in electrophysiological recordings. The chronic pain therapy. cross-linkers predominantly captured the receptors in non-active, agonist- bound (desensitized) states. Bis-MTS compounds did not modify the activity 2350-Plat of fully activated or unliganded receptors. Thus, the extreme flexibility of Blue Native Page Evidence that Plasma Membrane-Bound Human ENaC AMPAR extracellular domains appears to be primarily a characteristic of their Channels Share a Trimeric Architecture with Full-Length Human ASIC1 desensitized states. and ASIC2 Anke Dopychai1, Ralf Hausmann1, Linda Kruger€ 1, Stefan Grunder€ 2, 2352-Plat Gunther€ Schmalzing1. Probing a Molecular Lock in a Primitive NMDA Receptor 1Molecular Pharmacology, RWTH Aachen University, Aachen, Germany, Alvin Yu1, Robert Alberstein2, Alecia Thomas2, Richard Grey2, 2Department of Physiology, RWTH Aachen University, Aachen, Germany. Mark L. Mayer3, Albert Lau1. The epithelial Naþ channel (ENaC) and the acid-sensing ion channels 1Department of Biophysics and Biophysical Chemistry, The Johns Hopkins (ASICs) belong to the Degenerin/ENaC superfamily of voltage-insensitive University School of Medicine, Baltimore, MD, USA, 2Laboratory of Naþ channels that are gated by diverse exogenous stimuli. ENaCs assemble Cellular and Molecular Neurophysiology, National Institutes of Health, from three homologous subunits (abg or dbg) to form a heteromultimer, Bethesda, MD, USA, 3Laboratory of Cellular and Molecular but high-resolution structures are lacking. ASICs consist of several isoforms Neurophysiology, National Institutes of Health, Baltimore, MD, USA. (ASIC1-5) that assemble as homo- and heterooligomers. X-ray crystallog- Glycine-activated ionotropic glutamate receptors (iGluRs) found in cteno- raphy at 1.9 A˚ resolution revealed chicken ASIC1 as a homotrimer, implying phores are evolutionary precursors to NMDA receptors, which are important

BPJ 7884_7890 478a Wednesday, February 15, 2017 in regulating synaptic plasticity. Recent crystal structures of ctenophore sensitive, site-directed fluors corroborate the dual modes of bilayer binding iGluRs, which bind glycine with unusually high affinity, reveal a ctenophore- by both enzymes, and also disclose cell membrane binding and trafficking specific inter-lobe salt bridge in the ligand-binding domain (LBD) that is into live cells within minutes. Bicelles allosterically activate wild-type notably absent from vertebrate iGluRs. Using molecular dynamics umbrella proMMP-7. NMR of inactivated proMMP-7 complexes reveals bicelle- sampling simulations, we show how perturbations to this strategically posi- induced allosteric rearrangement of the auto-inhibitory peptide and suggests tioned salt bridge disrupt the conformational propensities of the LBD, signifi- a path of allosteric transmission to it from the bilayer interface. (Supported cantly altering receptor kinetics and thermodynamics. We compare our by NIH grant GM057289.) computational results with electrophysiological, biochemical, and crystallo- graphic analyses. 2355-Plat Oxidized Lipids: Their Role in Regulation of Mitochondrial Apoptosis 2353-Plat Artur Dingeldein1,Jo¨rgen Aden1, Tobias Sparrman1, Radek Sachl2, Investigating the Scrambling Mechanism of TMEM16 Scramblases Sarka Pokorna2, Martin Hof2, Gerhard Gro¨bner1. Mattia Malvezzi, Rabia Iqbal, Ashley Brown, Anant Menon, 1Chemistry, Biophysical Chemistry, Umea˚, Sweden, 2J. Heyrovsky´ Institute Alessio Accardi. of Physical Chemistry, Prague, Czech Republic. Weill Cornell Medical College, New York, NY, USA. Virtually every biological process involves membranes. The mitochondrial Ca2þ-dependent phospholipid scramblases collapse the lipid asymmetry on outer membrane (MOM) plays a key role in the intrinsic (mitochondrial) the membrane, causing phosphatidylserine to be externalized and act as a pathway of apoptosis - programmed cell death. Opposing members of the trigger for blood and an ‘‘eat-me’’ signal for macrophage engulf- Bcl-2 protein family are the main regulators of this intrinsic pathway. They ment during apoptosis. Members of the TMEM16 family can be either Ca2þ- meet at the MOM and arbitrate a life or death decision. Elevated oxidative gated ion channels or dual function channel/scramblases. Recently, the crystal stress level trigger a cell death decision which is then executed by permeabi- structure of a fungal TMEM16 ion channel/scramblase, nhTMEM16, was lization of this outer membrane and the release of apoptotic factors such as solved revealing a hydrophilic cavity, ~11A˚ wide, at the protein/lipid inter- cytochrome c. In recent years we have developed a realistic, MOM-like mem- face. This led to the proposal that phospholipid scrambling occurs via a brane system, an ideal system for elucidating the role of oxidized lipids on the ‘‘credit-card’’ mechanism, in which the polar headgroups traverse the mem- regulation of mitochondria-mediated apoptosis. By using solid state NMR ap- brane via the cavity while the hydrophobic tails remain embedded within proaches in combination with calorimetry and fluorescence based methods we the hydrocarbon core. We tested this scrambling mechanism by investigating provide a molecular description of the structural and dynamic organization of how the size of the phospholipid headgroups affects scrambling. We gener- the mitochondrial outer membrane system prior and upon oxidative stress. In ated NBD-labelled phosphatidylethanolamine (PE) phospholipids conjugated particular we provide new knowledge not only about the overall architecture with PEG molecules of increasing size and test whether afTMEM16 was behind the membrane’s basic function, but also about changes upon the gen- able to scramble them. Surprisingly, NBD-PE-PEG2000 lipids (~40A˚ diam- eration of specific oxidized lipid species under oxidative stress, and the con- eter), NBD-PE-PEG3400 lipids (~50A˚ diameter) and NBD-PE-PEG5000 sequences for the membrane perforating activity of the apoptotic Bax protein. lipids (~60A˚ diameter) are all scrambled with rates similar to normal phos- We discovered that the MOM environment is severely altered in the presence pholipids. As all these lipids exceed the width of the cavity by 4-6 times, it of oxidized phospholipids (OxPls). Here, we use NMR spectroscopy to study is highly unlikely that their translocation occurs via the hypothesized mecha- the impact of specific OxPl species and the relative abundance in the mem- nism. Rather, these results suggest that lipid scrambling does not require that brane. In addition, we observed that the presence of OxPls enhances the asso- phospholipids permeate through the lipid cavity. Furthermore, titrating unla- ciation of Bax protein with ‘‘damaged’’ MOMs and their perforation. Our beled PE-PEG2000 up to 15 mole% does not affect scrambling of NBD- preliminary results show this process to be dependent on the OxPl species. PE, suggesting that there is no competition between PE and large PE-PEG Currently we will investigate the impact of Bax on the membrane organiza- lipids. We propose that TMEM16 scramblases locally deform the membrane, tion in more detail; in particular the penetration depth and pore formation pro- for example by thinning the bilayer, thus lowering the energy barrier for lipid pensity of Bax as a function of OxPl species These experiments will be translocation and allowing scrambling to occur outside -but in the vicinity- of complemented by fluorescence leakage assays to determine leakage rates the lipid cavity. Alternatively, it is possible that the cavity expands to accom- and pore sizes. By this research we provide a basic understanding of the mo- modate large headgroups and that the PEG polymers partially uncoil during lecular mechanism by which OxPls are involved in regulation of apoptosis at permeation. the mitochondrial outer membrane by modulating the Bax ability to perforate the membrane to release apoptotic factors; execute via membrane leakage a death sentence at the organelle level; an understanding also suitable to Platforms: Protein-Lipid Interactions: develop future therapy strategies against Bcl-2 overexpressing, currently Structures untreatable tumors.

2354-Plat 2356-Plat Transient Interactions of Metalloproteinases with Bilayers, Captured by Spectroscopic Investigation of a-Synuclein 71-82, a Peptide Derived from a Paramagnetic NMR, Include Dual Modes of Binding, Electrostatic Protein Involved in Parkinson’s Disease Recruiting, and Allostery Benjamin Martial,E´ milie Bruneau, Laurie Be´dard, Thierry Lefe`vre, Steven R. Van Doren, Stephen H. Prior, Yan G. Fulcher, Jia Xu, Miche`le Auger. Tara C. Marcink, Rama K. Koppisetti. Chemistry, Universite´ Laval, Que´bec, QC, Canada. Biochemistry, University of Missouri, Columbia, MO, USA. Our research project focuses on a specific peptide of a-synuclein which is an Mammalian matrix metalloproteinases (MMPs) 7 and 12 are water-soluble amyloid protein known to be involved in the neurodegenerative Parkinson’s and diffuse in the extracellular matrix, but have also been observed at cell sur- disease. Aggregates of this protein are found in Parkinson’s disease patients’ faces. NMR and fluorescence reveal these mammalian proteases as having brain, and more specifically, in the nerve tissues. This protein can be found in dual modes of binding bilayers. Paramagnetic NMR relaxation enhancements several forms, comprising monomers, oligomers and protofibrils. The second- (PREs) from spin-labeled phosphatidylcholine (PC) suggest two equal popu- ary structure of these compounds has been described as a parallel b-sheet lations of MMP-12 bound to small disk-like bicelles, one near the b-sheet structure. All along this 140 amino acids protein, a specific portion plays an and the other near the a-helices (Nat Commun. 5:5552). Both surfaces are important role in the aggregation process, namely the non-amyloid-b compo- electropositive and appear to form salt bridges and hydrogen bonds with phos- nent (NAC, sequence 61-95). In the core of this NAC, the amino acid phoester portions of head groups in molecular dynamics simulations used for sequence 71-82 appears to be crucial in the fibril formation process. In the structural refinement. PREs from spin-labeled PC implicate two loops at edges present study, we have investigated the secondary structure and thermal sta- of the b-sheet of proMMP-7 in superficial contact with the head groups bility of the peptide a-syn 7182, as a function of concentration, ionic of zwitterionic bicelles. Incorporation of cholesterol sulfate (CS) appears to strength and temperature, as well as its interactions with phospholipid model (i) rotate proMMP-7 by 80, (ii) draw it about 11 A˚ into the anionic membranes using various spectroscopic techniques. The data show that a-syn bicelles through attraction to a swath of multiple positive charges, and (iii) 7182 is mainly disordered in solution with the presence of a few b-sheet enable Trp and Ile side chains to insert among the acyl chains of the phospho- structure elements. The peptide reversibly forms intermolecular b-sheets lipids (Structure. 23:2099). In the complexes with anionic bicelles, the lipid with increasing concentration and ionic strength, and decreasing temperature, head groups are ordered in the interface with proMMP-7. Thus, electrostatics suggesting that it is subjected to a thermodynamic equilibrium between a promote the binding of both MMP-7 and 12 to bilayers. Environment- monomeric and an oligomeric form. This equilibrium seems to be slightly

BPJ 7884_7890 Wednesday, February 15, 2017 479a affected by the presence of zwitterionic membranes. Conversely, the influence 2359-Plat of the peptide on zwitterionic lipid bilayers is small and concentration- Role of PIP2-Dependent Membrane Interactions in Vinculin Activation, dependent. By contrast, a-syn 7182 is strongly affected by anionic vesicles, Motility and Force Transmission exhibiting a dramatic conformational change and reflecting an extensive and Sharon L. Campbell1, Peter M. Thompson1, Srinivas Ramachandran1, irreversible self-aggregation in a parallel b-sheet conformation. The aggre- Lindsay Case2, Nikolay Dokholyan1, Caitlin Tolbert1, Clare M. Waterman2. gates appear to be located near the membrane surface but do not perturb 1Department of Biochemistry and Biophysics, University of North Carolina, significantly the membrane order. Comparing these results with the literature, Chapel Hill, NC, USA, 2Cell Biology and Physiology Center, National it appears that a-syn 7182 shares several general properties and structural Institutes of Health, Bethesda, MD, USA. similarities with its parent protein. These common points suggest that the Vinculin is a highly conserved scaffolding protein that localizes to focal ad- sequence 7182 may overall contribute to the behavior and properties of hesions (FA) and adherens junctions, where it binds actin and links the actin a-syn. cytoskeleton to the adhesive structure. Vinculin also binds to phosphatidylino- sitol 4,5-bisphosphate (PIP2). However, despite recent structural studies, it is 2357-Plat unclear how vinculin associates with PIP2 in the context of a lipid bilayer and Determinants of Lipid Specificity for the FVII Membrane Binding how binding to PIP2 regulates vinculin localization, activation, turnover at Domain FAs and cell motility. Herein, we incorporate lipid-association data, nuclear Melanie Muller, Emad Tajkhorshid. magnetic resonance (NMR), and computational modeling to generate an University of Illinois, Urbana, IL, USA. experimentally-driven structural model for PIP2-dependent interaction of vin- Coagulation Factor VII (FVII) is a clotting factor which contains a special- culin with the lipid bilayer. According to our model, two basic patches on the ized binding domain known as a GLA domain. Membrane-binding of the vinculin tail drive membrane association: the basic collar specifically recog- FVII GLA domain is critical to regulation of FVII binding and ultimately nizes the PIP2 head group, while the basic ladder drives association with the clotting cascade, given that FVII’s enzymatic activity increases by the lipid bilayer. As mutations within the basic collar impair vinculin- several orders of magnitude upon its lipid-specific binding to the membrane. mediated actin crosslinking, basic ladder mutations that disrupt PIP2- While membrane binding of other GLA domains has been found to be spe- dependent liposome association were made to evaluate the cellular cific to phosphatidylserine (PS), our experimental collaborators have demon- consequences of vinculin binding to PIP2. Results obtained from cellular strated that FVII is instead most specific to phosphatidic acid (PA). expression of the PIP2 deficient vinculin mutants suggest that PIP2-binding Understanding the molecular determinants of this lipid binding specificity is not required for localization of vinculin to FAs or FA strengthening. would substantially aid in design of novel treatments for diseases such as Rather. PIP2-binding is specifically required for vinculin activation and turn- thrombosis. We have developed the first model of the FVII GLA domain over at FAs to promote its association with the force-transduction FA bound to PA lipids in a 1:1 PA: phosphatidylcholine (PC) membrane. The nanodomain. GLA domain was placed in one of nine distinct orientations above one of 2360-Plat three independently generated bilayers, producing 18 independent models Flexibility Dictates Function in an Exceptional Curvature Sensing Helix which were each simulated for 100 ns. The highly mobile membrane mimetic Erin R. Tyndall1, Edward Kim2, Kumaran S. Ramamurthi2, Fang Tian1. (HMMM) model was used to accelerate the binding process and enhance 1Department of Biochemistry and Molecular Biology, The Pennsylvania sampling of membrane interactions. We developed an additional model of State University, Hershey, PA, USA, 2National Cancer Institute, NIH, the FVII GLA domain bound to a 1:1 PS:PC membrane using the same Bethesda, MD, USA. protocol, and compared the binding modes of PS and PA lipids to FVII, Cellular function requires many processes that organize, deform and curve the allowing us to identify potential PA specific binding sites. In addition, membrane including autophagy and endocytosis. Recently it has been shown relative strength of binding by FVII to each membrane model was assessed that membrane curvature is responsible as a regulator of activity and localiza- using repeated non-equilibrium simulations which gradually removed the tion of proteins such as SpoVM. The small peptide SpoVM is responsible for protein from its membrane patch, reversing the binding process, and then beginning the forespore coat in Bacillus subtilis, which it does by localizing comparing the non-equilibum work. We then identified lipid interactions in exclusively to that slightly curved convex membrane structure (K. Ramamurthi each model which contribute the most to total work required. We have iden- et al. Science 2009). It is the first peptide found to recognize shallow curvature. tified key interactions which likely contribute to the generation of the differ- Recently we have determined 3D structures of this protein and that of its non- ential specificity of FVII for PA lipids, and thus to the regulation of FVII in functional mutant SpoVMP9A (Gill et al. PNAS 2015). The SpoVM structure coagulation. consists of 3-turn a-helix coupled to a flexible N-terminal loop, which is unique when compared to the standard straight amphipathic helix of other curvature 2358-Plat sensing helices. Additionally SpoVM’s helix has an unbalanced non-polar Structure of the Sar1 Lattice and its Role in COPII Vesicle Formation and face, with a very small polar side featuring only one charged residue. We Scission have found that the charge, length of helix and flexibility N-terminal tail all Peter Randolph1, Hanaa Hariri2, Scott Stagg1. contribute to correct localization of SpoVM. However it is not clear how 1Institute of Molecular Biophysics, Florida State University, Tallahassee, 2 much flexibility of the N-terminal and helical length influence the curvature FL, USA, UT Southwestern, Dallas, TX, USA. sensitivity of SpoVM. Sar1 GTPase initiates formation of the COPII coat which generates trans- By replacing residues in the non-function SpoVMP9A with prolines; we have port vesicles, shuttling nascent proteins from the endoplasmic reticulum found that while the SpoVMP9A/K7P mutant remains non-functional, the (ER) to the Golgi apparatus. Sar1, bound to GTP, associates with the ER SpoVMP9A/K10P mutant regains curvature sensitivity. Using NMR, we investi- membrane forming a lattice, and recruiting the first structural component gated the structural and dynamic differences in both functional and non- of COPII, Sec23 (as part of the Sec23/Sec24 dimer). Sec23 stimulates functional mutants, as well as the functional SpoVMP9G. We found that the GTP hydrolysis activity of Sar1, dissociating Sar1 from the mem- flexibility and freedom of the N-terminal are critical factors for the function- brane. While the mechanism of COPII vesicle formation is understood, ality and localization of the protein. We will present both in vivo and in vitro few details have been determined for COPII scission. Multiple studies data demonstrating the specific impact of these mutations on the structure have suggested Sar1 plays a role in COPII vesicle scission, though this func- and molecular dynamics, as well as protein function. tion needs clarification. Disruption of COPII formation through errant function of Sar1 can lead to disease states including craniolenticulo- 2361-Plat sutural dysplasia, congenital dyserythropoietic anemia, or chylomicron Rhodopsin Dimerization in Membrane Bilayers Revealed by Small Angle retention disease, though the direct mechanisms of Sar1’s role in these dis- Neutron Scattering eases is unknown. Here we present our examination of the role of Sar1 in Olivier Soubias1, Jonathan D. Nickels2, Walter E. Teague1, Kirk G. Hines1, both vesicle formation and scission, supplying basal understanding of Kevin L. Weiss3, John Katsaras3, Klaus Gawrisch1. Sar1 function, which will be necessary for determining the disease mecha- 1NIH, Bethesda, MD, USA, 2Joint Institute For Neutron Science, Oak Ridge nisms. We will be presenting our analysis of the formation of the Sar1 lat- National Laboratory, Oak Ridge, TN, USA, 3Oak Ridge National Laboratory, tice through a combination of structural and biophysical techniques, Oak Ridge, TN, USA. including cryo-electron microscopy, and comparison crosslinking with G Protein Coupled Receptor (GPCR) dimerization has emerged as an essen- mass spectrometry. tial mechanism regulating GPCR biosynthesis, maturation, ligand binding,

BPJ 7884_7890 480a Wednesday, February 15, 2017 coupling with G protein and downstream signaling in many cell-signaling gests that tau and a-Syn interact to form pathological co-amyloids that are pathways. However, determining the oligomeric state of a GPCR in a associated with several neurological ailments like Parkinson’s disease, Alz- membrane is in itself a challenge, due to the complex nature of the plasma heimer’s disease, Down syndrome, multiple system atrophy etc. However, membrane and spontaneous receptor collisions. The protonated G protein- the molecular determinants of the tau-a-Syn interactions remain elusive. coupled membrane receptor bovine rhodopsin was reconstituted into proteoli- We are investigating the intermolecular association of tau k18 fragment and posomes composed of perdeuterated lipids extracted from E. coli bacteria a-Syn using a range of biophysical tools involving atomic force microscopy grown on fully deuterated medium. It was observed that intensity of small- and multi-parametric fluorescence spectroscopy and imaging. Our results angle neutron scattering (SANS) from entire proteoliposomes in 98% D2O demonstrate that the early intermolecular contact formation between tau buffer, measured at angles q<0.008 A˚ 1 reached a minimum at a protein/ k18 and a-Syn plays a key role in directing the course of de novo co- deuterated lipid molar ratio near 1/500, while yielding maximal scattering aggregation in which the tau k18:a-Syn concentration ratio critically governs contrast for incorporated opsin (bleached rhodopsin) over the wider angular the rate of amyloid formation. Our two-color confocal imaging confirmed range of 0.008 A˚ 1

BPJ 7884_7890 Wednesday, February 15, 2017 481a increase in solution turbidity. Phase separation is typically triggered in and contrasting multiple interactions between IDPs and their structured part- response to a stimulus such as change in protein concentration, ionic ners, utilising the Bcl-2 family as a model system. strength, or temperature, and may be reversible or precede gelation, fibre formation, or aggregation. Phase separation is a well-known property of 2368-Plat synthetic polymers, and is exploited for material design, for example, The Measles Virus Phosphoprotein: An Intrinsically Disordered Chap- capsules for drug delivery and fast-setting hydrogels, but is not well erone that Regulates Nucleocapsid Assembly understood for proteins. Some examples of phase-separating protein-based Sigrid Milles, Malene Ringkjøbing Jensen, Guillaume Communie, systems include lens gamma crystallin (implicated in cataract formation), Damien Maurin, Guy Schoehn, Rob WH Ruigrok, Martin Blackledge. ‘membraneless organelles’ (transiently formed compartments that regulate Institut de Biologie Structurale, Grenoble, France. molecular interactions), and self-assembling elastomeric proteins, including Measles virus, a negative strand RNA virus, packages its genome into some spider silks, insect resilin and vertebrate elastin, for which phase large, helical superstructures formed by the nucleoprotein (N) that assembles separation is on-pathway for the formation of elastic materials. Here we on the RNA genome. Although a vital step in viral replication, the assembly studied the phase separation of model polypeptides based on the protein process of N into nucleocapsids (NC) has largely remained obscure since elastin. These polypeptides contain both regions of well-defined secondary overexpression of N in eukaryotic and prokaryotic expression systems leads structure and intrinsic disorder. Droplet growth and biophysical properties to NC formation within the expression host. We now show how the presence were monitored using a variety of microscopy and optical spectroscopy of a short 50 residue, intrinsically disordered, peptide from the measles techniques. We report the effect of amino acid sequence mutations on the phosphoprotein (P) can prevent premature assembly during expression 0 size, stability, reversibility and interactions of phase-separated droplets, and by forming a monomeric N P construct that self-assembles into NC-like par- address implications for functional tunability, including loading of small ticles upon addition of RNA in vitro. This approach allowed us, for the first molecules. time, to study NC assembly and its kinetics by NMR and fluorescence spec- troscopies, and revealed a remarkable dependence on RNA sequence despite 2366-Plat the fact that N has to encapsidate the entire viral genome. An integrated Modeling the Early Stages of Aggregation in Disordered Elastin-Like structural analysis of N0P comprising the full intrinsically disordered tails Proteins of P and N of more than 400 residues in length allowed us to delineate 1 2 1 2,3 Yue Zhang , Valeria Zai-Rose , Cody J. Price , Gene L. Bidwell III, , the importance of PTAIL within the assembly process beyond its first 50 John J. Correia2, Nicholas C. Fitzkee1. residues combining information from NMR and fluorescence spectroscopies, 1Department of Chemistry, Mississippi State University, Mississippi State, as well as electron microscopy and small angle X-ray scattering. Since MS, USA, 2Department of Biochemistry, University of Mississippi Medical other negative strand RNA viruses share similar protein architectures, these Center, Jackson, MS, USA, 3Department of Neurology, University of results promise to have implications on a large number of important human Mississippi Medical Center, Jackson, MS, USA. pathogens. Elastin-like proteins (ELPs) are known to aggregate reversibly above a defined transition temperature. Because aggregation occurs reversibly, ELPs 2369-Plat 1-40 1-42 can be a useful system to study the early stages of this process in proteins. Ramachandran Map Analysis of the Monomeric Ab and Ab Previous studies suggested that, as temperature increases, ELPs experience Peptides by Solution NMR an increased propensity for type II b-turns. However, how the ELPs behave Julien Roche. during the aggregation itself is still elusive. Here, we investigate a hypothesis: BBMB, Iowa State University, Ames, IA, USA. 1-40 aggregation is initiated at the b-turn positions. To test this hypothesis, we To explore in detail the structural propensity of the monomeric Ab and 1-42 designed an ELP construct (ELP40, 16 kDa) suitable for NMR measure- Ab peptides in solution we recorded a large set of NMR parameters, 3 ments. We observe that this construct does indeed exhibit increase b-turn including chemical shifts, NOEs, and J couplings. JHNHa couplings were propensity at higher temperature. Based on this observation, a series of struc- measured with high precision using a recently developed TROSY-based 1-40 tural ensembles were generated incorporating differing amounts of b-turn experiment. Systematic comparisons show that at neutral pH the Ab and 1-42 bias, from 1% to 50% throughout the chain. To mimic the early stages of Ab peptides populate almost indistinguishable coil-like conformations. aggregation, two monomers were paired, assuming preferential interaction NOESY spectra collected at very high resolution remove assignment ambigu- at b-turn regions. This approach was justified by the observation that ities and show no long-range NOE contacts. Six sets of backbone J couplings 3 3 3 1 2 1 1-40 buried hydrophobic turns are frequently observed to interact in the PDB. ( JHNHa, JC´C´, JC’Ha, JHaCa, JCaN and JCaN) recorded for Ab were Following dimerization, the ensemble-averaged hydrodynamic properties used as input for the recently developed MERA Ramachandran-map analysis, were calculated for each degree of b-turn bias, and results were compared yielding residue-specific backbone f/c torsion angle distributions that closely with analytical ultracentrifugation (AUC) experiments on ELPs in at various resemble random coil distributions, the absence of significantly elevated pro- temperatures. We find that the temperature dependence of the sedimentation pensity for b-conformations in the C-terminal region of the peptide, and a a coefficient (s20,W) can be reproduced by increasing the b-turn content in the small but distinct propensity for L at K28. Our results suggest that the structural ensemble. This analysis allows us to estimate the secondary struc- self-association of Ab peptides into toxic oligomers is not driven by elevated ture and weak associations under experimental conditions. Moreover, our propensities of the monomeric species to adopt b-strand-like conformations. models can be used to generate structural hypotheses for the early stages of Instead, accelerated disappearance of Ab NMR signals in D2O over H2O, 1-42 IDP self-association. In addition, because disordered proteins frequently particularly pronounced for Ab , suggests that intermolecular interactions exhibit weak biases in secondary structure propensity, these structurally- between the hydrophobic regions of the peptide dominate the aggregation biased ensemble calculations may be a promising method for modeling process. IDPs generally. 2367-Plat Platform: Cardiac Muscle Regulation What Encodes Coupled Folding and Binding Reactions: IDPS or Partner Proteins? 2370-Plat Michael D. Crabtree1, Carolina A.T.F. Mendonca1, Quenton Bubb1, Cardiac Excitability at the Intercalated Disc is Maintained via a Small Sarah L. Shammas2, Jane Clarke1. Isoform of Obscurin 1Department of Chemistry, University of Cambridge, Cambridge, United Heather R. Manring, Maegen A. Ackermann. Kingdom, 2Department of Biochemistry, University of Oxford, Oxford, Physiology and Cell Biology, The Ohio State University Wexner Medical United Kingdom. Center, Columbus, OH, USA. Anfinsen (1961) demonstrated that the information for a protein to fold is The cardiac intercalated disc (ID) is a highly ordered, multi-functional mem- entirely contained within its sequence. More recently, the importance of brane domain essential for both myocyte structure and electrical activity. The unfolded, but functional, proteins has been recognised. These so-called intrin- primary cardiac voltage-gated , Nav1.5, is concentrated at the sically disordered proteins (IDPs) lack a three-dimensional structure, but may ID and responsible for cardiac excitability and conduction. Aberrant Nav1.5 fold upon interacting with a partner protein. Where is the folding information activity is directly responsible for human conduction abnormalities and poten- for these coupled folding and binding reactions contained? Is the amino acid tially fatal arrhythmias. Herein we describe a novel mechanistic system gov- sequence of the IDP the sole determinant of the folding reaction? Or are they erning Nav1.5 localization, stability, and function. Obscurin polypeptides are a dictated by the partner protein? We examine these questions by comparing family of multifunctional adapter proteins originating from a single gene that

BPJ 7884_7890 482a Wednesday, February 15, 2017 are highly expressed in the heart. Through their interaction with ankyrins, ob- modeling is of major clinical interest. We aimed to characterize MI-induced scurins directly link a host of critical membrane and membrane associated ion changes in titin-based cardiomyocyte stiffness and to elucidate the role of titin channels, transporters, signaling, and cytoskeletal molecules. Here we report in ventricular remodeling of remote myocardium directly after MI. Titin that obscurin-80, a small obscurin isoform that contains the canonical properties were analyzed in Langendorff-perfused mouse hearts after 20’ ankyrin-binding domain, preferentially localizes to the ID along with ischemia/60’ reperfusion (I/R) and mouse hearts that underwent ligature of ankyrin-G, which has been previously shown to target Nav1.5 to the ID. the left anterior descending coronary artery (LAD) for 3 or 10 days. Cardio- Our novel findings indicate that obscurin-80 directly interacts with ankyrin- myocyte passive tension (PT) was significantly increased after I/R as well as 3 G and complexes with endogenous Nav1.5. Notably, obscurin-80, along and 10 days after LAD-ligature. Three days after MI collagen I and III are with ankyrin-G and Nav1.5 localize normally to the ID in a mouse model lack- strongly reduced within the infarct zone. The increased titin-based PT was ing giant obscurins, supporting a role of small but not giant obscurin proteins caused by hypophosphorylation of the N2-Bus and hyperphosphorylation of in Nav1.5 targeting. Moreover, during murine development, obscurin-80 and the PEVK-region and was mediated by elevated levels of interleukin-6 via ankyrin-G protein expression is elevated at early embryonic stages likely activation of PKCa. Three days after MI titin stiffening was accompanied facilitating the proper localization of Nav1.5 to the ID. Targeted knockdown by a significantly increased abundance of the titin degradation band T2, of obscurin-80 will determine its role in mediating Nav1.5 function and thus indicating elevated titin turnover. K48-specific titin ubiquitination levels knockdown-rescue experiments are aimed to assess the role of the interaction and trypsin-like proteasomal activity were increased while proteasomal con- between obscurin-80 and ankyrin-G. Taken together our recent findings have tent seems to be unchanged 3 days after MI. K63-specific titin ubiquitination uncovered a novel mechanism for maintaining Nav1.5 activity within the car- was unchanged 3 and 10 days after MI. These data indicate that the acceler- diomyocyte and therefore supporting the maintenance of normal cardiac ated titin turnover after MI is at least partially performed by the ubiquitin pro- conductance. teasome system. We conclude that titin-based cardiomyocyte stiffening acutely after MI is an important mechanism of the surviving myocardium 2371-Plat to adapt to the increased mechanical demands after myocardial injury. This Contractile Force and Kinetics in Failing versus Non-Failing Human adaptation also includes higher turnover of titin and increased proteasomal Myocardium activity. Jae-Hoon Chung, Brit Martin, Benjamin Canan, Mohammad Elnakish, Nancy Saad, Ahmet Kilic, Mei-Pian Chen, Nima Milani-Nejad, Peter Mohler, 2373-Plat Vadim Fedorov, Paul Janssen. Mechanistic Role of Type 1 Inositol 1,4,5-Trisphosphate Receptor in the The Ohio State University, Columbus, OH, USA. Regulation of Vascular Tone in Heart Failure In sharp contrast to (often-inbred) laboratory animals, including animal Gaetano Santulli1, Jessica Gambardella1, Steven Reiken1, Qi Yuan1, models of disease, the human patient population has a large variation due Ryutaro Nakashima1, Frances M. Forrester1, Alain Lacampagne2, to genetic factors and various disease pathologies. Previous studies on con- Andrew Marks1. tractile function of human myocardium have resulted in inconsistent results. 1Columbia University, New York, NY, USA, 2Montpellier University - In a survey of 11 studies performed on isolated multicellular human myocar- Inserm, Montpellier, France. dium, 2 studies found failing myocardium to have a decreased active devel- Patients with heart failure (HF) have augmented vascular tone which in- oped force compared to non-failing controls, while 2 other studies found creases cardiac workload, impairing cardiac output and adversely affecting failing myocardium, at rest, to have increased contractile force, while the re- outcomes. However, the molecular mechanisms underlying the maladaptive maining 7 studies did not observe a statistical difference. Regarding relaxa- vascular responses observed in HF are not fully understood. Vascular tion kinetics, only 1 study found a slower 50% relaxation time, while 6 smooth muscle cells (VSMC) are the main cell type involved in vasoconstric- studies reported no difference, and the remaining 5 studies did not report tion and Ca2þ is an essential player in such a process. To dissect the this parameter. Low n-numbers, coupled with the large variation in the as- functional role of intracellular Ca2þ in the increased vascular tone observed sessed parameters, unfortunately resulted in a lack of statistical power that in HF, we generated VSMC-specific type 1 inositol 1,4,5-trisphosphate re- allows for unambiguous interpretation of these past studies. In order to ceptor (IP3R1) KO mice, which displayed blunted responses to angiotensin address whether or not isolated human myocardium exhibits functional def- II (ATII), both in vivo and ex vivo. We evaluated vascular responses in icits under baseline conditions, between 2010 and 2016 we examined con- different models of HF and we observed that VSMC-KO mice exhibited a tractile function in isolated human multicellular preparations that were significantly attenuated progression towards decompensated HF, with non-failing (50þ muscles from 35þ different hearts), and that were classified decreased afterload and attenuation of interstitial fibrosis. Notably, there as end-stage failing (40þ muscles from 30þ different hearts). We found that were no differences in the activation of the adrenergic and the renin- active developed force at optimal preload was not different (P=0.33) in angiotensin-aldosterone system. In vitro, we determined that VSMC isolated failing versus non-failing myocardium (average 16.5 5 2.2 vs. 19.6 5 from KO mice displayed significantly blunted Ca2þ responses to PE and 2.2 mN/mm2). In contrast, 50% relaxation time (from peak tension) was ATII compared with flox littermates, whereas no difference was detected significantly slower (P= 0.0022) in failing versus non-failing myocardium when incubating with IP3R inhibitors. These results were confirmed in (average 144 5 4.2 vs. 127 5 3.2 ms). We concluded that the myocardium denuded mesenteric arteries preparations. Mechanistically, we established that is contracting and still viable in human end-stage heart failure, at least that in HF there is an activation of myosin light chain kinase (MLCK) in under resting conditions (1 Hz, 60 bpm), suffers from an impairment of con- VSMC, which is markedly blunted in KO mice. Taken together, our data tractile relaxation kinetics, but not from a deficit of active contractile force indicate that antagonism of the IP3R1 system may be of benefit in reversing production. increased vascular tone in patients with HF. Vascular IP3R1 may thereby represent an important, novel target for therapeutic blockade in the pharma- 2372-Plat cological treatment of HF. Adaptive Ventricular Remodelling after Myocardial Infarction Involves Titin-Based Cardiomyocyte Stiffening and Elevated Titin Turnover 2374-Plat Sebastian Ko¨tter1, Malgorzata Kazmierowska1, Christian Andresen1, Troponin I Tyrosine Phosphorylation Modulates Cardiac Contraction Katharina Bottermann1, Maria Grandoch2, Simone Gorressen2, Elizabeth A. Brundage, Vikram Shettigar, Hussam E. Salhi, Andre Heinen1, Jens M. Moll3,Jurgen€ Scheller3, Axel Go¨decke1, Jonathan P. Davis, Mark T. Ziolo, Brandon Biesiadecki. Jens W. Fischer2, Joachim P. Schmitt2, Martina Kruger€ 1. Physiology and Cell Biology, The Ohio State University, Columbus, 1Cardiovascular Physiology, University of Dusseldorf,€ Dusseldorf,€ Germany, OH, USA. 2Pharmacology and Clinical Pharmacology, University of Dusseldorf,€ Phosphorylation of the troponin inhibitory subunit (TnI) represents a funda- Dusseldorf,€ Germany, 3Biochemistry and Molecular Biology II, University of mental mechanism of the heart to modulate cardiac contractile function in Dusseldorf,€ Dusseldorf,€ Germany. response to altered physiological or pathological demand. The phosphorylation Myocardial infarction (MI) increases the wall stress in the viable myocardium of TnI at tyrosine 26 (Tyr-26) represents the first myofilament tyrosine phos- and initiates early adaptive remodeling in the left ventricle to maintain cardiac phorylation identified in the human heart. We demonstrate the phosphorylation output. Later remodeling processes include fibrotic reorganization potentially of Tyr-26 decreases calcium sensitive force development and accelerates cal- leading to cardiac dysfunction. Understanding the titin and extracellular cium dissociation from the thin filament supporting the role for Tyr-26 phos- matrix based mechanisms that support cardiac function in the early phase phorylation in contractile regulation. Additionally, TnI Tyr-26 and the post-MI and to identify the processes that initiate transition to maladaptive re- basally phosphorylated Ser-23/24 exhibit functional and signaling integration.

BPJ 7884_7890 Wednesday, February 15, 2017 483a

That is, the combination of TnI Tyr-26 with Ser-23/24 phosphorylation further lecular level studies with phosphorylated C0C3 will reveal cMyBP-C’s full accelerates calcium dissociation beyond that of Ser-23/24 or Tyr-26 alone, range of contractile fine tuning. without additionally decreasing calcium sensitivity. Furthermore, the presence of Ser-23/24 phosphorylation promotes Src family tyrosine kinase mediated 2377-Plat phosphorylation of TnI Tyr-26. Taken as a whole, these data support tyrosine The Role of on the Cardiac Myofilament Maria Papadaki1, Ronald Holewinski2, Marisa Stachowski1, Jonathan Kirk1. kinase mediated myofilament phosphorylation of TnI as a mechanism of car- 1 diac function modulation. Cellular and Molecular Physiology, Loyola University Chicago, Maywood, IL, USA, 2Cedars Sinai Medical Center, Los Angeles, CA, USA. 2375-Plat Methylglyoxal (MGO), a byproduct of glycolysis and a reactive carbonyl Acetylation of K326 and K328 on Actin Boosts Contractile Properties of species (RCS), is significantly elevated in diabetes. MGO adds post- Muscle In Vitro and In Vivo translational modifications by reacting with arginine and lysine residues to William Schmidt, Meera C. Viswanathan, D. Brian Foster, form irreversible carbonyl adducts. At basal concentrations, MGO is Anthony Cammarato. removed by glyoxal-1 (GLO-1), but at concentrations observed in diabetes, Cardiology, Johns Hopkins University, Baltimore, MD, USA. GLO-1 cannot meet the demands and MGO modifications can accumulate. In K326 and K328 of actin establish critical electrostatic interactions with nega- the heart, MGO has been previously found to act on the tively charged amino acids of tropomyosin. Recently, these residues were and SERCA to depress calcium transients, and other proteins to induce car- shown to be acetylated in guinea pig hearts, a post-translational modification diomyocyte apoptosis. However, the effect of MGO on the cardiac myofila- (PTM) that could weaken actin-tropomyosin contacts, and possibly modulate ment has never been studied. In single skinned mouse myocytes, 20 minute contraction. Expression of K326Q, K328Q, and K326Q/K328Q acetyl- exposure to 100 mM MGO reduced maximal calcium activated force (Fmax) mimetic cardiac actin, in indirect flight muscles of Drosophila, resulted in by 12 5 0.04 % (n = 8, p = 0.048) and Ca2þ sensitivity by 45 5 0.11 % severe hypercontraction when modest amounts (~20%) of K328Q actin (n = 8, p = 0.02). With 10 mM MGO, Ca2þ sensitivity was decreased by were present. Furthermore, beating fly hearts expressing similar levels of 67 5 0.2 % (n = 9, p<0.01) but there was no effect on Fmax. Agreeing K328Q or K326Q/K328Q actin displayed significantly prolonged systolic in- with data indicating that MGO modifications are irreversible, the functional tervals (20450.01 and 21150.01 msec respectively) relative to controls effect of MGO on skinned myocytes did not recover during washout. Using (16150.01 msec, p<0.001). Despite nearly equivalent baseline shortening mass spectrometry and immunoprecipitation assay, we identified MGO mod- velocities for WT- and K328Q-expressing hearts (450524.2 vs. 449523.9 ifications on several myofilament proteins in mouse myocytes treated with mm/s respectively), exposure to a viscous afterload resulted in an approxi- MGO including actin, myosin and myosin essential light chain. In a pilot mate 45% reduction in velocity for control myocardium compared to only study, we were able to detect similar MGO modifications on myofilament 30% for K328Q-containing hearts (p<0.05). Thin filaments were reconsti- proteins from human diabetic cardiomyopathy tissue that were not present tuted using vertebrate cardiac troponin-tropomyosin and K328Q, K326Q or in control hearts (n = 2/group). Our data show that these modifications would WT actin. The pCa50, calculated from in vitro sliding velocities, was signif- have significant implications on myofilament function in patients suffering icantly leftward-shifted for K328Q relative to WT thin filaments (6.2550.03 from diabetic cardiomyopathy and other diseases affecting vs. 6.0650.02 mm/sec, p<0.001), while that for K326Q filaments was indis- metabolism. tinguishable. These results are consistent with acetylation-induced increases in crossbridge cycling, power production and sensitivity of force develop- Platform: /Intercellular ment. Lastly, to determine if the PTM may be a regulatory mechanism ex- ploited in vivo, we measured the relative amounts of acetylated actin in Calcium Channels and Sparks and Waves guinea pig and human ventricles during cardiac failure. We discovered ~75% more acetylated actin in guinea pig hearts subjected to aortic constric- 2378-Plat tion (p<0.001), and ~21% more in human dilated cardiomyopathy samples High-Throughput Screens to Discover Inhibitors of Leaky Ryanodine (p<0.05), relative to controls. In sum, acetylation of actin K326 and/or Receptor Calcium Channels K328 could be a novel means to enhance performance in failing hearts. Robyn T. Rebbeck1, Megan V. Ryan1, Gregory D. Gillispie2, This work was supported by 5T32HL007227-38 and 1R01HL124091. David D. Thomas1, Donald M. Bers3, Razvan L. Cornea1. 1Bioc/Mol Bio/Biophys, University of Minnesota, Minneapolis, MN, USA, 2376-Plat 2Fluorescence Innovations, Inc., Minneapolis, MN, USA, 3Pharmacology, Direct Single Molecule Visualization of Cardiac MyBP-C N-Terminal University of California - Davis, Davis, CA, USA. Fragment Interactions with Suspended Thin Filaments Using fluorescence lifetime detection of FRET, we have developed and vali- 1 2 2 Alessio V. Inchingolo , Michael J. Previs , Samantha E. Beck Previs , dated high-throughput screening (HTS) methods to discover compounds that Neil M. Kad1, David M. Warshaw2. 2þ 1 modulate an intracellular Ca channel, the ryanodine receptor (RyR), for School of Biosciences, University of Kent, Canterbury, United Kingdom, 2þ 2 therapeutic applications. Intracellular Ca regulation is critical for striated Department of Molecular Physiology and Biophysics, University of muscle function, and RyR is a central player. Under resting Ca2þ, increased Vermont, Burlington, VT, USA. propensity of channel openings due to RyR dysregulation is associated with Cardiac Myosin-Binding (cMyBP-C) is a sarcomeric protein bound severe cardiac and skeletal myopathies, diabetes and neurological disorders. by its C terminus to the thick filament backbone, while its N terminus can This leaky state of the RyR is an attractive target for pharmacological agents bind to either the myosin head region or the thin filament. These N-terminal to treat such pathologies. Our FRET-based HTS detects RyR binding of acces- cMyBP-C interactions modulate cardiac contractility by activating the thin 2þ sory proteins or FKBP12.6. Under conditions that mimic a patho- filament (actin-troponin-tropomyosin) at low Ca , and by modulating logical state, we carried out a screen of the 1280-compound small-molecule contraction velocity at high calcium. To understand the dynamic nature of library of pharmaceutically active compounds, to identify modulators of these interactions, we directly visualized the impact of Cy3-labelled N-termi- 2þ RyR in either skeletal or cardiac SR membrane preparations. This screen nal cMyBP-C fragments (C0C3-Cy3) on Ca - dependent activation of regu- yielded several compounds that reproducibly altered FRET with RyR1 (skel- lated thin filaments (RTFs) in vitro. As a measure of activation we observed etal isoform) and/or RyR2 (cardiac isoform). Ongoing studies focus on the the binding of fluorescent myosin-S1 to RTFs suspended between 5 mm silica mechanisms of compound effect on FRET and on the correlation of this struc- beads adhered to the coverslip surface of a microfluidic flow cell. The pres- tural readout with effects on RyR function. This work was supported by ence of 1 mM C0C3 resulted in a 2-fold increase in bound myosin-S1 at American Heart Association Postdoctoral Fellowship 16POST31010019 (to low Ca2þ (pCa 7). Interestingly, the dynamics of C0C3-Cy3 binding to 2þ RTR) and Grant-in-Aid 15GRNT25610022 (to RLC), and NIH grants RTFs show Ca -dependence. Specifically, at pCa 7 C0C3-Cy3 binding is R01HL092097 (to RLC/DMB), R37AG26160 (to DDT), and R42DA037622 5 3 reduced, however 24 6% of the fragments diffuse randomly (~1x10 (to GDG/DDT). mm2/s) along the RTF. Whereas at high Ca2þ (pCa 4), although C0C3-Cy3 binding is greatly increased, only 7 5 2% of fragments remain diffusive. 2379-Plat These data suggest that cMyBP-C may compete with tropomyosin for its Improvements of the Kinetics of Red Fluorescent Calcium Indicators þ binding site on RTFs, evidenced by its diffusive behavior at low Ca2 . How- Silke Kerruth, Catherine Coates, Katalin Torok. þ ever, once strongly-bound, cMyBP-C sensitizes RTFs to Ca2 , possibly by Molecular and Clinical Sciences Research Institute, St. George’s University displacing tropomyosin from the ‘blocked’ position; enabling myosin to of London, London, United Kingdom. bind. cMyBP-C phosphorylation results in reduced actin-binding affinity by Genetically encoded calcium indicators (GECIs) are widely used for moni- affecting cMyBP-C’s N-terminal structure. Therefore, we expect similar mo- toring calcium signalling in various cell types. They are based on calmodulin

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(CaM), circular-permuted fluorescent protein (cpEGFP or others) and the light- molecule (STIM), the ER Ca2þ sensor that stimulates store-operated Ca2þ entry chain myosin kinase peptide RS20. Although genetically engineering has led to (SOCE), accumulated after depletion of ER Ca2þ stores. Our results show that 2þ a broad variety of different GECIs and also red probes suitable for optogenetics, IP3-evoked Ca signals are initiated by immobile IP3R clusters tethered near there are only few variants with fast kinetics to monitor action potentials [1]. the ER-PM junctions at which SOCE occurs. We suggest that this organization We have performed mutations on two red fluorescent GECIs, jRCaMP1a and may both optimize delivery of IP3 to IP3Rs and allow effective regulation of jRGECO1a [2], to weaken the interaction between CaM and the RS20 target SOCE by local depletion of Ca2þ stores. peptide, to fasten the kinetics of these slow probes. 2382-Plat In total we made and biophysically characterized 17 variants. At physiological 2D ionic strength and 20C, jRCaMP1a shows a biexponential fluorescence rise Characterization of Different Localized Ca Signals in Skeletal Muscle with rates 5251 (47%) and 2.350.1 s1 (53%) and a dynamic range of Fibers 1 1 2 6.850.3. Fluorescence decay is biexponential with a fast (2.250.1 s1 Mikhail Svirin , Tihomir Georgiev , Enrique Pe´rez Jaimovich , 1 (17%)) and slow phase (2.250.1 (17%) and 0.3250.01 s1 (83%)). The Rainer H A Fink . 1Medical Biophysics Unit, Institute for Physiology und Pathophysiology, variant termed jRCaMP1afast has similar rise kinetics to jRCaMP1a with rates 2 1551 (47%) and 2.150.1 s1 (53%) but decays 21-fold faster (6.9 s1) with Heidelberg University, Heidelberg, Germany, Facultad de Medicina, 5 Universidad de Chile, Santiago de Chile, Chile. dynamic range of 4.5 0.2. 2þ jRGECO1a (dynamic range 12.351.2) has single exponential kinetics with rise Discrete localized Ca signals (LCS) occur in many cell types. In skeletal and decay rates 15053s1 and 4.350.1 s1, respectively. The variant termed muscle fibers they have been shown e.g. to be elicited as a result of an os- 1 motic shock or mechanical skinning of isolated muscle fibers. Indicating jRGECO1afast has a similar rise rate (15053s ) with a 5-fold faster bi-phasic 1 their possible pathophysiological importance, spontaneous LCS have been decay (koff:2552 (85%) and 1.550.1 s (15%)) and dynamic range observed in the mdx mouse model of human Duchenne muscular dystrophy (12.950.8). The variant termed jRGECO1aultrafast has slower rise kinetics 5 1 5 5 1 and in wild type mouse muscle fibers after severe mechanical stress. In addi- (53 2s ) but a 100-fold faster decay (koff: 515 80 (80%) and 7 1s 2þ (20%)) and a dynamic range of 6.950.1. tion, there is a variety of different types of intricate subcellular Ca The faster kinetics and preserved dynamic ranges of the novel red GECIs make signaling in micro-domains of mammalian skeletal muscle fibers. However, global Ca2þ-transients from the sarcoplasmic reticulum often mask these them useful imaging tools. 2þ This work is funded by BBSRC grant BB/M02556X/1 to K.T. localized Ca signals (LCS). In our study we have focused on the localiza- tion of nuclear Ca2þ signals which are thought to be involved in the regula- 2380-Plat tion of gene expression and that, among other functions, have been shown to Particle-Based Approaches to Clearing Calcium: a Protein Landscape play a role in tumor growth in non-muscle cells. We have identified nucleus- localized and perinuclear-localized Ca2þ signals observed under hypertonic Model of the Sarco/Endoplasmic Reticulum Calcium-ATPase (SERCA) 2þ Pump for Sub-Cellular Stochastic Models stimulation conditions. The Ca signals are in the micrometer range, with 1 2 2,3 full width at half maximum at 2.7550.27 mm and 2.5550.17 mm for nuclear Sophia P. Hirakis , Thomas M. Bartol , Terrence J. Sejnowski , 2þ Rommie E. Amaro1. and perinuclear LCS respectively. Types of Ca channels involved in for- 1Chemistry and Biochemistry, UC San Diego, La Jolla, CA, USA, 2Salk mation of nuclear and perinuclear LCS were investigated utilizing channel Institute, La Jolla, CA, USA, 3Neuroscience, UC San Diego, La Jolla, blockers. Dantrolene (RyR blocker), nifedipine (DHPR blocker) and Xesto- CA, USA. spongin C (IP3R blocker) have been used. We have observed the incidence of LCS associated with nuclei in the presence of each blocker. STIM channel The Sarco/Endoplasmic Reticulum Calcium-ATPase (SERCA) is a membrane- 2þ protein that completes the cycle of Excitation-Contraction Coupling; achieving blockers may provide further insight on the nature of the intranuclear Ca cardiomyocyte ‘‘relaxation’’ by importing Ca2þ to the Sarcoplasmic Reticulum signals which may shed light to new signals influencing a variety of nuclear (SR). SERCA associates with many small molecules and ions during the pump- processes. ing mechanism. Existing models of the SERCA pump mechanism reduce 2383-Plat complexity of intermolecular reactions by combining discrete binding and un- Role of ORAI Proteins in Activation of Endogenous TRPC1-Composed binding steps, effectively reducing the total number of states. To be able to Channels more-closely understand how small perturbations in variables like Ca2þ, þ Alexey Shalygin, Anton Skopin, Dmitrii Kolesnikov, Lyubov Glushankova, ATP, and H concentrations affect the mechanism of SERCA, we developed Elena Kaznacheyeva. an explicit-particle based approach to modeling the SERCA-2a pump. Our Institute of Cytology, Russian Academy of Sciences, Saint-Petersburg, protein-landscape model features twelve discrete states and explores the effects Russian Federation. of pH on the rate of calcium clearing. Depletion of intracellular calcium stores activates store-operated channels. This process induces numerous intracellular signaling events. The most studied 2381-Plat 2D store-operated channels are CRAC channels. Other channels are believed to Ca Signals Originate from Immobile IP3 Receptors at ER-PM Junctions consist of TRPC and Orai proteins. However, their role in molecular composi- Nagendra Babu Thillaiappan, Alap P. Chavda, Stephen C. Tovey, tion of endogenous store-operated non-CRAC channels remains obscure. One David L. Prole, Colin W. Taylor. of the main questions to ask is how TRPC channels are activated after store Department of Pharmacology, University of Cambridge, Cambridge, United depletion. Most studies have used whole-cell patch clamp or calcium imaging Kingdom. 2þ techniques. To discriminate different types of store-operated channels, we used Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ubiquitous intracellular Ca 2þ 2þ single-channel patch-clamp recordings in HEK293 cells. We showed that in channels, which upon activation by IP3 and Ca , release Ca from the endo- 2þ experiments with dominant-negative mutant ORAI E106Q endogenous plasmic reticulum (ER). Regulation of IP3Rs by Ca allows regenerative prop- TRPC1-composed channels were not sensitive to store depletion, but they agation of Ca2þ signals, generating a hierarchy of Ca2þ release events. The 2þ were activated by other pathways. In cells overexpressing STIM2 proteins spatial and temporal organization of these Ca signals contributes to their ca- endogenous TRPC1 channels were activated with a delay (similarly to ORAI pacity to selectively regulate diverse biological functions. The spatial arrange- channels activated by STIM2). In summary, we propose that (i) ORAI does ment and dynamics of IP3Rs are important for producing these regenerative 2þ not serve as a pore forming subunit of endogenous TRPC1 channels, but it is Ca signals. To study the distribution and dynamics of native IP3Rs, we necessary to maintain sensitivity to calcium stores and (ii) TRPC1 channels used transcription activator-like effector nucleases (TALENs) to tag endoge- are activated downstream to ORAI channels after store depletion. This study nous IP3R1 of HeLa cells with EGFP. EGFP-IP3Rs were functional and formed was supported by the Russian Scientific Foundation, Project 14-14-00720 (to small clusters within ER membranes. Most IP3R clusters were mobile, but E. K., D.K. and A.S.); and the Russian Foundation for Basic Research Project some were immobile over protracted periods; with minimal mixing of the mo- 16-04-01792 (A.S. and L.G.). bile and immobile IP3Rs. Single-particle tracking revealed that IP3Rs move by diffusion, and along microtubules by both kinesin-1 and dynein motors. Within 2384-Plat D both mobile and immobile IP3R puncta, some IP3Rs were tightly packed but The Two-Pore Domain K Channel TALK-1 Provides a Countercurrent others were too far apart for their association to be mediated by direct interac- that Facilitates Endoplasmic Reticulum Ca2D Leak 2þ tions between IP3Rs. Simultaneous visualization of EGFP-IP3Rs and Ca sig- Nicholas Vierra, Prasanna Dadi, Sarah Milian, David Jacobson. 2þ nals showed that Ca signals, whether evoked by photolysis of caged IP3 or Molecular Physiology & Biophysics, Vanderbilt University, Nashville, activation of endogenous receptors that stimulate IP3 formation, originate TN, USA. 2þ þ from immobile IP3Rs at ER-plasma membrane (PM) junctions. These Ca The two-pore domain K (K2P) channel TALK-1 modulates insulin secretion 2þ 2þ release sites closely apposed the ER-PM junctions where stromal-interaction by limiting b-cell electrical excitability and cytosolic Ca (Ca c) influx.

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2þ Mouse islets lacking TALK-1 (KO) exhibit accelerated Ca c oscillations and along the pathways hampers their experimental detection, making the 2þ enhanced insulin secretion relative to controls (WT). The Ca c oscillation fre- underlying molecular mechanisms highly elusive. Coarse-grained methods 2þ þ quency is affected by Kslow, a repolarizing Ca -dependent K current. Kslow is have been developed to provide guesses of the routes that lead from 2þ reduced in KO b-cells, possibly contributing to the accelerated Ca c oscilla- one conformer to another, but are severely limited by the difficulty to 2þ tions of KO islets. Since TALK-1 activity is not regulated by Ca c, we as- evaluate the intermediate structures obtained. In our recent work (Orellana sessed whether TALK-1 impacts Ca2þ release from endoplasmic reticulum et al. Nat. Commun. 2016:7, 12575), we apply a novel combination of 2þ 2þ (ER) stores (Ca ER), which modulates Kslow. We find lower basal Ca c in Principal Component Analysis of structurally-rich X-ray ensembles and KO b-cells, and application of the SERCA inhibitor CPA causes a greater in- coarse-grained simulations, in order to dissect the essential motions orches- 2þ crease in Ca c in KO versus WT b-cells. These observations suggest that trating biological functions for a set of highly studied proteins. We show 2þ TALK-1 regulates Ca ER. Therefore, we tested how heterologous expression how the Principal Components of such ensembles decode their core 2þ of TALK-1 in HEK293 cells affects Ca ER. TALK-1 overexpression signifi- motions, providing mathematical reaction coordinates to cluster structures 2þ 2þ cantly increases basal Ca c and reduces the CPA-induced Ca c response. and reveal clearly the interconnecting pathways between different states. However, expression of the K2P channels TREK-1 or TREK-2 does not affect Modeling proteins as elastic networks in a Langevin simulation, we generate 2þ þ Ca ER. Furthermore, K -impermeable mutant TALK-1 channels do not reca- smooth trajectories between end-points that visit spontaneously multiple in- 2þ þ pitulate Ca ER defects, thus, TALK-1 K channel function presumably mod- termediate states, providing a visual description of the sampled routes and 2þ ulates Ca ER. Consistent with an intracellular role for TALK-1, fluorescence their overlap with state-of-the-art atomistic simulations. This combined microscopy reveals expression of TALK-1 in the b-cell ER. Additionally, nu- approach allows to detect previously unnoticed temperature-dependent clear patch-clamp experiments demonstrate the presence of functional TALK-1 gating transitions for the ion channel GLIC, or to identify as on-pathway in- channels on the outer nuclear membrane, which is continuous with the ER. termediates a series of inhibitor-bound structures of the SERCA pump help- Moreover, specific inhibition of endogenous TALK-1 channels with expression ing to understand their sequence along the conformational cycle. The present þ 2þ of a K -impermeable mutant increased human b-cell Ca ER. Together, our work thus provides a powerful theoretical framework to study conforma- 2þ data suggest that TALK-1 channel activity augments islet-cell Ca ER tional changes and exploit the rich structural information stored in the Pro- 2þ ‘‘leak.’’ Altered TALK-1 channel function may perturb islet-cell Ca ER, tein Data Bank. 2þ which can affect Ca c handling, hormone release, and contribute to disease pathogenesis. 2387-Plat Studying Biomolecular Interactions: A Hybrid Approach 2385-Plat Sean A. McKenna1, Manuel Koch2, Janusz M. Bujnicki3, Joerg Stetefeld1, Safety Upgrades of the Mitochondrial Calcium Uniporter Trushar R. Patel4. Ming-Feng Tsai1, Christopher Miller2. 1Chemistry, University of Manitoba, Winnipeg, MB, Canada, 2Institute for 1Brandeis University, Waltham, MA, USA, 2Brandeis University, Waltham, Dental Research and Oral Musculoskeletal Biology, University of Cologne, MA, USA. Cologne, Germany, 3Laboratory of Bioinformatics and Protein Engineering, The mitochondrial Ca2þ uniporter is a Ca2þ-activated Ca2þ channel complex International Institute of Molecular and Cell Biology, Warsaw, Poland, composed of the ion-conducting MCU protein, the MICU1 subunit that medi- 4Alberta RNA Research and Training Institute, University of Lethbridge, ates Ca2þ activation, and an animal-specific ‘‘EMRE’’ protein necessary for Lethbridge, AB, Canada. uniporter function. Interestingly, lower eukaryotes, such as plants and protists, RNA-protein and protein-protein interactions play crucial roles in a number do not have EMRE genes, but their uniporters are fully capable of ion trans- of biological processes. To understand mechanisms underpinning such pro- port. This observation raises a question: What are the physiological benefits cess, structural information on biomolecular complexes are essential. How- for animal uniporters to be absolutely dependent on a new subunit (EMRE) ever, the experimental determination of biomolecular complexes that for function? To approach this problem, I determined the transmembrane provides information on position, orientation and interactions of individual orientation of EMRE in mitochondria, and identified molecular interactions domains is often time-consuming and in many cases not possible. As a result, that allow EMRE to bind MCU and MICU1. The results allow me to put complete high-resolution structures are not yet available for many complexes together a molecular picture describing how uniporter subunits assemble that are crucial for numerous fundamental cellular processes. An emerging into a full protein complex. I then replaced native EMRE in HEK cells alternative to high-resolution structural techniques is to employ a hybrid with mutants unable to bind MCU or MICU1. Experiments show that approach that combines low-resolution shape information about macromole- breaking MCU-EMRE contacts abolishes uniporter activation by EMRE. cules and their complexes from hydrodynamic and SAXS methods, with Moreover, disruption of EMRE-MICU1 interactions greatly reduces MICU1 high-resolution structures (where available), and with computational affinity to the uniporter, thus yielding a group of MICU1-free channel sub- modeling to obtain atomic-level models. Our laboratories have established complexes. These channels constitutively load Ca2þ into the mitochondrial such approach and have used for a variety of complexes involving RNA- matrix, and perturb mitochondrial physiology by uncoupling respiration protein and protein-protein complexes. For example, such hybrid approach from ATP synthesis and by inducing mitochondrial permeability transition. was utilized to study human netrin-4/laminin 𝛾;1 complex, These results therefore establish a concept that EMRE represents a ‘‘safety revealing that the N-terminal globular domains of proteins mediate interac- upgrade’’ in animal uniporters, acting to prevent the dangerous event of tions. Structural guided mutations provided detailed insights on the biolog- MICU1 dissociation. The absolute EMRE-dependence for function ensures ical relevance of this complex.1 Additionally, a high-resolutions structural that only those uniporters properly equipped with the safety module EMRE model between adenovirus VAI RNA and host enzyme, Protein Kinase R can operate. In summary, this work shed light on the molecular design that (PKR) was also investigated using a hybrid approach2,3, demonstrating that allows animal uniporters to handle the sophisticated intracellular Ca2þ only the apical stem of VAI interacts with the double-stranded RNA binding signaling systems in animal cells. motifs of PKR. 1. Reuten R.* and Patel, T. R.* et al. Nature Communications (accepted). Platform: Protein Structure and 2. Dzananovic, E.* and Patel, T. R.* et al. Journal of Structural Biology (2014) 185, 48-57. Conformation III 3. PyRy3D (http://genesilico.pl/pyry3d/).

2386-Plat 2388-Plat Trapping On-Pathway Intermediates for Large Scale Conformational The Molten Globule State of Maltose Binding Protein: Structural Charac- Changes with Coarse-Grained Simulations terization by Epr Spectroscopy Laura Orellana1,O¨ zge Yoluk1, Oliver Carrillo2, Modesto Orozco3, Benjamin Selmke1, Chen Nickolaus1, Peter Borbat2, Jack H. Freed2, Erik Lindahl1. Wolfgang E. Trommer1. 1Science for Life Laboratory, Solna, Stockholm, Sweden, 2Scuola Normale 1Chemistry, Kaiserslautern Technical University, Kaiserslautern, Germany, Superiore, Pisa, Italy, 3Institute for Research in Biomedicine Barcelona, 2Chemistry and Chemical Bology, ACERT, Cornell University, Ithaca, Barcelona, Spain. NY, USA. Conformational changes in proteins are essential to their biological func- Maltose-binding protein (MBP) is a single chain protein composed of two tions, from allosteric regulation to signal propagation, which often involve domains that is in a molten globule state at pH 3 as characterized by ANS interconversions between open/close and bound/unbound pairs as observed binding. DEER measurements of seven spin-labeled double mutants in the by X-ray crystallography. However, the transient nature of the intermediates native state at pH 7 had shown excellent agreement with X-ray data. At

BPJ 7884_7890 486a Wednesday, February 15, 2017 pH 3 corresponding DEER measurements of all the mutants yielded a broad between any DHHC enzyme and protein substrates. Among the DHHC family, distribution of distances. This can be expected if there is no defined tertiary DHHC17 and DHHC13 have special features: they both have six transmem- structure and the individual helices point into all possible directions. Depend- brane helices and a large cytoplasmic domain with seven ankyrin repeats ing on maltose binding in a cleft between the domains, MBP exhibits both, an (ANK). The ANK domain is known to harbor the binding site for several sub- open and a closed conformation with respect to these domains. We have fol- strates. DHHC17, also known as Huntingtin Interacting Protein 14, is highly lowed this substrate-depending conformational change by means of additional expressed in neurons and acts on substrates such as Snap25b and Huntingtin. spin-labeled mutants at or near the active site. In these experiments DQC The importance of DHHC17 and DHHC13 is underscored by the fact that spectroscopy has been particularly helpful as it allows for distance measure- loss of both lead to embryonic lethality in mice. In this study we investigate ments of labels in close proximity. Data show, e.g., that there is a defined the structural and thermodynamic bases of the interaction between the ANK structure of the active site of MBP at both pH values even in the absence domain of DHHC17 (ANK17) with Snap25b. We solved a high-resolution of substrate. crystal structure of the complex between ANK17 and a peptide fragment of Snap25b containing the recognition motif and use a combination of binding 2389-Plat studies in vitro (with purified proteins) and in vivo to validate the structural Model Comparison to Identify Structural Heterogeneity in Protein X-Ray model. Our structure reveals that the N-terminal part of the peptide substrates Crystallography makes key contacts with DHHC17 and through mutagenesis analysis we Nathan S. Babcock1, David A. Sivak1, Daniel Keedy2, James S. Fraser2. discovered single point mutations in DHHC17 that completely prevent binding 1Department of Physics, Simon Fraser University, Burnaby, BC, Canada, to Snap25. Finally, we extend our study to the interaction between ANK17 and 2Department of Bioengineering and Therapeutic Sciences, University of Huntingtin, one of the most prominent substrates of DHHC17. Overall, our California, San Francisco, San Francisco, CA, USA. research represent a first step for the elucidation of the mechanism of action Researchers conventionally infer a single best-fit atomic structure from pro- of DHHC17 and it will be valuable toward the development of regulators of tein X-ray crystallographic data. Higher-resolution and room-temperature da- DHHC17 function. tasets, greater computational power, and a growing appreciation for the dynamic lives of proteins all motivate increased interest in fitting experi- 2392-Plat mental datasets to multiple-structure and generally more complex models. Integrative Structural Biology of a Type II Secretion Pseudopilus A more complex model will fit the data better, but also provides greater ca- Benjamin Bardiaux1,2, Aracelys Lopez-Castilla1,2, Xiong Yu3, pacity to overfit experimental noise. Significant research effort has focused Edward H. Egelman3, Nadia Izadi-Pruneyre1,2, Olivera Francetic1,4, on parameter inference in a given model type, yet comparatively little atten- Michael Nilges1,2. tion has been paid to the selection among distinct model types of varying 1Institut Pasteur, Paris, France, 2CNRS UMR 3528, Paris, France, 3University complexity. Using metrics derived from fundamental statistical principles, of Virginia, Charlottsville, VA, USA, 4CNRS ERL 3526, Paris, France. we develop a model comparison framework for statistically-rigorous detection Type II secretion systems (T2SS), type IV pili and archaeal flagella use a of protein structural heterogeneity. We compare these information criteria to conserved plasma membrane machinery to assemble helical filaments promot- more conventional crystallographic criteria, and we assess their utility for ing macromolecule transport or motility. We had built an atomic model of the judging different model types’ tradeoff of parsimony against quality-of-fit. T2SS pseudopilus based on comparative modelling, low resolution electron We benchmark these criteria against synthetic data and explore their predic- microscopy, and an automated modelling procedure. Our new detailed tions in experimental data sets. NMR structure shows that the model of the globular head had been correctly modelled. The new higher resolution cryo-EM map indicates that the overall 2390-Plat architecture of our model and many of the detailed contacts we described are Single Molecule Identification Against Proteomes using Sub-Nanometer correct. However, the cryo-EM map clearly shows partial melting of the Pores N-terminal helix that is perfectly regular in the X-ray crystal structure of a Eamonn Kennedy1, Mikhail Kolmogorov2, Zhuxin Dong1, Pavel Pevzner2, homologous protein, and predicted to be a regular helix with very high con- Greg Timp1. fidence. The partial melting is likely caused by the environment in the pseu- 1University of Notre Dame, South Bend, IN, USA, 2University of California dopilus that differs from the crystal. While the study indicates potential San Diego, San Diego, CA, USA. problems with current secondary structure prediction algorithms, it clearly The information gleaned from precise, comprehensive whole-protein analysis shows the power of combining even lower resolution electron microscopy will elucidate disease phenotypes. However, conventional tools like top- and modelling. down mass spectrometry (TD-MS) lack the sensitivity to interrogate proteome and discriminate proteoforms without a priori knowledge of the target protein 2393-Plat required for enrichment (including all the different molecular forms for a pro- First Protein Scattering Measurements at LiX Beamline tein product of a single gene, genetic variations, post-translational modifica- Shirish N. Chodankar. tions and alternative splicing). Recently, it was discovered that denatured, National Synchrotron Light Source II, Brookhaven National Lab, Upton, charge linearized single proteins translocating a sub-nanopore exhibit current NY, USA. blockade fluctuations that correlate with the amino acid sequence of the protein. High Brightness X-ray Scattering for Life Sciences (LiX) is a newly commis- Small volume differences (<0.1 nm3), associated with post-translation modifi- sioned beamline at the NSLS-II facility of Brookhaven National Laboratory cations or single residue substitutions can be detected even in a single mole- dedicated to three specific scientific areas: 1) high-throughput solution scat- cule. Here, we describe how proteins can be discriminated with high tering, 2) diffraction from single and multi-layered lipid membranes and 3) accuracy (p < 105) with supervised machine learning using random forest scattering based scanning probe imaging. SAXS of macromolecules in solu- regression, based on improvements that include a revised model of the volume tions has an ever-increasing demand and to better serve user needs, and to allow that accounts for hydrophilicity. This accuracy can be achieved for small automated and high-throughput operation, an automated solution scattering blockade ensembles (n < 10), indicating that the current technology is already setup has been developed at LiX. It consists of two flow-cells, each of this capable of identification at E-value = 0.01 for small (<104 constituents) flow-cell picks sample from 6 PCR tube placed in two rows. In future a robot proteomes. will transfer the holder containing two rows of PCR tube to perform unattended measurements. While the sample is flowed through one of the cell and 2391-Plat measured, the second cell is cleaned and dried and made ready for the next Structural Basis for Substrate Recognition by the Ankyrin Repeat Domain set so that an unhindered and continuous set of measurements could be per- of DHHC17 Palmitoyltransferase formed. Test measurements were performed on standard set of protein Raffaello Verardi, Jin-Sik Kim, Anirban Banerjee. Ribonuclease-A, Hemoglobin, Bovine Serum Albumin (BSA) and b-amylase National Institutes of Health, Bethesda, MD, USA. with molecular weight of 13.7, 64.5, 66.5, 210 KDa respectively. To circum- DHHC enzymes catalyze palmitoylation, a major post-translational modifica- vent X-ray radiation damage, sample is continuously flowed during measure- tion that regulates a number of key cellular processes. There are 24 DHHCs ments. Thanks to brilliance of the new source and the optics at the beamline in mammals and hundreds of substrate proteins that get palmitoylated. How- even for relatively small protein molecule Ribonuclease-A at a low concentra- ever how DHHC enzymes engage with their substrates is poorly understood. tion of 1mg/ml a 5 sec exposure measurement was good enough to obtain sta- There is currently no high-resolution structural information for the interaction tistically relevant data.

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Posters sporadic ALS, further linking ubiquilin-2 to the pathogenesis of ALS. Interest- ingly, most ALS-linked mutations are localized to the proline-rich repeat (Pxx) region that is unique to ubiquilin-2 and not present in the other members of the Protein Structure and Conformation IV ubiquilin family. The consequences of these mutations (specifically to residues P497, P506, P509, P525 and P533) on ubiquilin-2’s structure and function 2394-Pos Board B1 remain unknown. Towards that end, we are using biomolecular NMR spectros- 13 Development and Comparison of Enhanced Sampling Methods for Bio- copy to characterize the Pxx region of ubiquilin-2. C-detect NMR experi- molecular Simulation ments have allowed for the direct and necessary observation of the prolines James Lincoff1, Sukanya Sasmal1, Teresa Head-Gordon2,3. as well as aided in resonance assignment for a significant portion of this 1Chemical and Biomolecular Engineering, UC Berkeley, Berkeley, CA, ubiquilin-2 region. Significantly, even though the Pxx region is intrinisically USA, 2Chemical and Biomolecular Engineering, Chemistry, Bioengineering, disordered, we observed sequence-distant perturbations in several ALS- UC Berkeley, Berkeley, CA, USA, 3Chemical Sciences Division, Lawrence linked mutants. Serendipitously, similar to some other ALS-linked proteins, Berkeley National Laboratory, Berkeley, CA, USA. our ubiquilin-2 constructs exhibit salt and temperature dependent liquid-like Molecular dynamics (MD) simulations generate structural ensembles of proteins phase-separation, a behavior also affected by ALS mutations. We hypothesize at atomic-level resolution, and are particularly valuable for studying intrinsi- that ALS mutations alter the structure and dynamics of the Pxx region. We are cally disordered proteins, whose ensembles cannot be fully characterized exper- working towards modeling the structure of ubiquilin-2 containing the Pxx re- imentally. Standard MD is impractical as trajectories have to be run for gion, as well as identifying protein-binding surfaces involving the Pxx region. intractably long times in order to converge to the correct structural distribution. 2397-Pos Board B4 Enhanced sampling methods, of which temperature replica exchange (TREx) is Energy Landscapes of a Mechanical Prion and their Implications for the the most common, are used to overcome this sampling problem. However, TREx Molecular Mechanisms of Long-Term Memory is also limited due to its very high computational expense for large systems Mingchen Chen. and sub-optimal rate of convergence. We have developed a new enhanced sam- Rice University, Houston, TX, USA. pling method, temperature cool walking (TCW), that demonstrates marked Aplysia cytoplasmic polyadenylation element binding (CPEB) pro- tein, a trans- improvements in both of these areas. Originally developed and tested on a lational regulator that recruits mRNAs and facilitates translation, has been one-dimensional potential, TCW converges to the equilibrium probability distri- shown to be a key component in the formation of long-term memory. Experi- bution of that system more rapidly than optimally scheduled TREx simulations. mental data show that CPEB exists in at least a low-molecular weight coiled- We have extended TCW to peptides, and codes are now available in OpenMM, coil oligomeric form and an amyloid fiber form involving the Q-rich domain an open-source platform for running MD on GPUs. We validate on two test pep- (CPEB-Q). Using a coarse-grained energy landscape model, we predict the tides, alanine dipeptide and met-enkephalin, that TCW more rapidly converges structures of the low-molecular weight oligomeric form and the dynamics of to the equilibrium distributions generated by long standard MD trajectories than their transitions to the b-form. Up to the decamer, the oligomeric struc- tures TREx simulations, at one third the computational cost of TREx. We then apply are predicted to be coiled coils. Free energy profiles confirm that the coiled b TCW to the amyloid beta (A ) family of peptides, known key players in Alz- coil is the most stable form for dimers and trimers. The structural transition heimer’s disease. On these systems, TCW has 13 % the cost used to generate tra- from a to b is shown to be concentration dependent, with the transition barrier jectories of the same length using TREx, and has improved agreement with decreasing with increased concentration. We observe that a mechanical pulling NMR observables, demonstrating that we have overcome the limitation in sam- force can facil- itate the a-helix to b-sheet (a-to-b) transition by lowering the free pling of TREx. The computational savings and improved convergence will energy barrier between the two forms. Interactome analysis of the CPEB protein allow for more ambitious calculations in the future. suggests that its interactions with the cytoskeleton could provide the necessary mechanical force. We propose that, by exerting mechanical forces on CPEB 2395-Pos Board B2 oligomers, an active cytoskele- ton can facilitate fiber formation. This mechan- Structural Characteristics of the RNAse H Domain in HIV-1 Reverse ical catalysis makes possible a positive feedback loop that would help localize Transcriptase the formation of CPEB fibers to active synapse areas and mark those synapses Ryan L. Slack, Naima G. Sharaf, Angela M. Gronenborn, Rieko Ishima. for forming a long-term memory after the prion form is established. The func- Structural Biology, University of Pittsburgh, Pittsburgh, PA, USA. tional role of the CPEB helical oligomers in this mechanism carries with it im- HIV-1 Reverse Transcriptase (RT) is a multi-functional enzyme responsible for plications for targeting such spe- cies in neurodegenerative diseases. the viral DNA polymerase and ribonuclease activities required for viral repli- cation. RT is encoded as a 66 kDa polypeptide in the Gag-Pol polyprotein 2398-Pos Board B5 and matured to form a heterodimer composed of 66 kDa (p66) and 51 kDa Methyl-Labeling Assisted NMR Structure Determination of a 66 KDA (p51) subunits. The maturation process of RT is believed to entail dimerization Growth Factor-Receptor Complex of p66 subunits, followed by cleavage of the C-terminal ribonuclease H domain Andrew Hinck1, Morkos A. Henen1, Christian Zwieb2, Ravindra Kodali1, (RNH) in one of the p66 subunits by viral protease (PR), yielding the p51 sub- Cynthia S. Hinck1. unit within the mature heterodimer. In structures of mature RT, the p51-RNH 1Dept. of Structural Biology, University of Pittsburgh, Pittsburgh, PA, USA, cleavage site in the remaining p66 subunit is buried within the RNH core. 2Dept. of Biochemistry, UT Health Sci. Car. at San Antonio, San Antonio, Therefore the structural determinants which allow for PR recognition and TX, USA. cleavage of a single p66 subunit in the immature p66 homodimer remain un- TGF-b1, TGF- b2, and TGF-b3 are 26 kDa disulfide-linked homodimeric clear. Thus, we performed solution NMR experiments of the immature p66 ho- signaling proteins. They all signal through the TGF-b type I and type II recep- modimer to elucidate the conformation of the RNH domain within this tors, yet TGF-b2, which is well known to bind TbRII several-hundred fold complex. Additional NMR experiments were conducted to investigate the ef- more weakly than TGF-b1 and TGF-b3, has an additional requirement for fect of buffer ionic strength on subunit dimerization. Finally, we introduced the TGF-b type III receptor (TbRIII), a membrane-anchored non-signaling re- mutations within the isolated RNH domain to clarify structural characteristics ceptor that potentiates the binding of TbRII. Though it is known that TbRIII of the cleavage site using NMR and other biophysical methods. (Supported by: has two component domains that bind TGF-b2 non-cooperatively at indepen- the National Institutes of Health P50GM082251 and R01GM105401). dent sites, the structure of these domains bound to TGF-b2 and residues respon- sible for specific binding are not yet known. The objective of this study was to 2396-Pos Board B3 determine the three-dimensional structure of the 66 kDa 2:1 complex formed Effects of ALS Mutations on Structure, Dynamics, and Function of between the TbRIII C-terminal domain (RIIIC2) and TGF-b2, the structures Ubiquilin-2 of which are both known. To obtain the necessary experimental restraints to Carlos A. Castaneda, Thuy P. Dao. determine an accurate three-dimensional structure of the complex, the back- Chemistry and Biology, Syracuse University, Syracuse, NY, USA. bone resonances of unbound TGF-b2 were assigned, extended to the sidechain Amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases are methyls of Ile, Leu, and Val, and confirmed by Ile -> Leu and Leu/Val -> Ile caused by malfunctions in ubiquitin-mediated protein degradation pathways. dropout variants. To identify the interface residues, fully deuterated Ile d1 and Ubiquilin-2 is a multi-domain adaptor protein critical for maintaining pro- Leu and Val proS methyl-protonated proteins were prepared and used to iden- tein homeostasis through the ubiquitin-proteasome system, the endoplasmic tify methyl chemical shift perturbations (CSPs). The results show that the bind- reticulum-associated protein degradation (ERAD) pathway and autophagy. ing site for RIIIc on TGF-b2 lies on the concave surface of the fingers and Mutations in ubiquilin-2 have recently been shown to cause dominant x-linked includes I33, I92, L101. This is consistent with accompanying mutagenesis inheritance of ALS and ALS/dementia. Moreover, wild-type ubiquilin-2 is pre- data, which shows that substitutions that had the greatest effect on binding sent in protein inclusions found in patients diagnosed with either familial or correspond to residues that were perturbed to the greatest extent in the NMR

BPJ 7891_7895 488a Wednesday, February 15, 2017 titration (Ile 33 and Ile 92), or were positioned adjacent to these residues in the tendon, cartilage, arteries and cornea, where it plays a crucial role in providing three-dimensional structure (Glu 99). structural support. Collagen molecules self-assemble to form hierarchical struc- tures, from single molecules to fibrils to fibers and tissues. Structural and me- 2399-Pos Board B6 chanical changes at the molecular level may affect self-assembly of the Tetrahymena Solution NMR Structures of the C-Terminal Domain of molecules and mechanics of the resulting tissue. Despite its significance, the Cytoskeletal Protein TCB2 Reveal Distinct Calcium-Induced Structural mechanics of collagen at the molecular level remain contentious. To rationalize Rearrangements the wide range of values for collagen’s molecular flexibility (where literature 1 2 2 Adina M. Kilpatrick , Jerry E. Honts , Heidi M. Sleister , reports span flexible to rigid rod-like descriptions), here, AFM imaging is 3 C. Andrew Fowler . used to study the effect of salt and pH on molecular conformations of single 1Physics and Astronomy, Drake University, Des Moines, IA, USA, 2Biology, 3 collagen molecules. Single molecules of collagen type I are imaged in solutions Drake University, Des Moines, IA, USA, NMR Facility, University of Iowa at a range of ionic strengths and different pH. Image analysis and statistical Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA. analysis of the chains are performed with our newly developed algorithm. Re- Tcb2 is a calcium-binding protein from the membrane-associated cytoskeleton sults show that collagen’s flexibility depends strongly on ionic strength and pH. of the ciliated protozoan Tetrahymena thermophila, with hypothesized roles in Surprisingly, collagen type and source do not influence molecular flexibility to ciliary movement, cell cortex signaling, and pronuclear exchange. Interest- a significant extent. These findings are interpreted in terms of polymer and ingly, Tcb2 is a major component of contractile fibers isolated from the biochemical models in order to shed light on the factors responsible for the sta- Tetrahymena cytoskeleton; in these fibers, addition of calcium triggers an bility of this fundamental triple helical protein. ATP-independent type of contraction. To gain insight into Tcb2’s structure- function relationship and contractile properties, we determined solution 2402-Pos Board B9 NMR structures of its C-terminal domain (Tcb2-C) in the calcium-free and Structural Characterization of CwsA in a Lipid Bilayer with ssNMR calcium-bound states. The overall architecture is similar to that of other Rongfu Zhang. calcium-binding proteins, with paired EF-hand calcium-binding motifs. The National High Magnetic Field Lab, Tallahassee, FL, USA. calcium-induced changes observed in Tcb2-C are distinct from those seen in Tuberculosis (TB) is a leading infectious disease killer worldwide caused by the prototypical calcium sensor calmodulin (CaM) and are more similar to Mycobacterium tuberculosis (Mtb). According to World Health Organization what is observed in members of the S100 subfamily of EF-hand proteins. (WHO), 9.6 million people became ill with TB and 1.5 million died from the dis- Despite very similar structures in the absence of calcium, Tcb2-C and CaM ease in the year of 2014. The standard drugscurrently used for the treatment of TB respond quite differently to calcium binding, suggesting that the two proteins can be dated back to more than three decades ago, and the widespread of drug- play distinct functional roles in Tetrahymena and likely have different mecha- resistance has been a major concern. In 2014, an estimated 480,000 people devel- nisms of target recognition. A calcium titration of Tcb2-C monitored by 1H-15N oped multidrug-resistant TB (MDR-TB), and among which 9.7% had extensively HSQC spectra is consistent with the domain binding two calcium ions. Many drug-resistant TB (XDR-TB). New anti-TB drugs are thus desperately needed. residues respond monotonically over the range of 0-2 equivalents of calcium, CwsA is a small integral membrane protein that belongs to Mtb cell divisome. while others (mainly from the first calcium-binding loop) respond more to The depletion or overproduction of CwsA has been shown to affect cell wall syn- the addition of the second equivalent. Interestingly, residues from both EF- thesis. The structural study of CwsA in a lipid bilayer environment is crucial to hands are more sensitive to the addition of the first equivalent of calcium, sug- the understanding of its function. Recently, Nabanita Das obtained some prelim- gesting a complex response of Tcb2-C to calcium binding. We are currently inary PISEMA ssNMR data on oriented CwsA samples which showed the using site-directed mutagenesis and fluorescence spectroscopy to investigate approximate TM helix core region range as well as a tilt angle of 10 with respect and quantify Tcb2-C’s calcium-binding properties. Future structural studies to the lipid normal. More detailed topological information of CwsA in a lipid of the full-length protein and the identification of Tcb2 cellular targets will bilayer environment requires the generation of better aligned OS samples. The help establish the molecular basis of Tcb2 function and contractile properties. structural characterization of CwsA will help to unravel the mechanism of Mtb cell division machinery as well as the discovery of new drug targets. 2400-Pos Board B7 Understanding Effect of Geranylation of tRNALys on Ribosome Binding: 2403-Pos Board B10 A Computational Study Determination of the Solution Structure of Isolated Histone H2A-H2B Sweta Vangaveti1, Phensinee Haruehanroengra2, Rui Wang2, Heterodimer by using CS-Rosetta Srivathsan V. Ranganathan1, Jia Sheng2. Tsutomu Yamane, Yoshihito Moriwaki, Hideaki Ohtomo, 1The RNA Institute, Albany, NY, USA, 2Chemistry, State University of New Mitsunori Ikeguchi, Mitsunori Ikeguchi, Jun-ichi Kurita, Masahiko Sato, York, Albany, NY, USA. Aritaka Nagadoi, Hideaki Shimojo, Yoshifumi Nishimura. The anticodon stem loop of the tRNA contains several post transcriptional Graduate School of Medical Life Science, Yokohama City University, modifications which are known to be essential for its structural stability and de- Yokohama, Japan. coding functions. We used a computational approach, to understand the effects CS-Rosetta is a program for protein structure determination from NMR experi- of a naturally occurring modification 2-thiogeranylated-uridine in Escherichia mental parameters and is able to determine solution structures of proteins only coli. In E.coli the presence or absence of the geranyl group at position 34 of from the chemical shift values. However, to determine structures only from the tRNALys(UUU) affects codon bias (AAG, AAA) and frame-shifting during chemical shift values, the target protein have to be a monomer, less than about translation. First we performed MD simulations to investigate the effects of 120 residues, and smaller flexible region. Histone H2A-H2B heterodimer, which the geranyl modification on stability of DNA and RNA duplexes. Our simula- is contained in histone octamer, is out of this limitation. In the present study, we tion results are in agreement with and provide an explanation for the greater sta- applied CS-Rosetta to determine the solution structure of isolated histone H2A- bility of the duplexes containing the geS2(U/T)-G base pair compared to the H2B heterodimer only from chemical shift values. As the results, our solution geS2(U/T)-A base pair, as is observed experimentally by measuring their structures are in good agreement with the chemical shifts values and the chemical melting temperatures. We further performed simulations of the ribosome bound shift indices observed from the NMR experiments. In the solution structures, H2A tRNA and found that the codon-anticodon interaction is strengthened by the and H2B each contain a histone fold, comprising four a-helices and two b-strands, geranylation in case of codon AAG and broken in case of codon AAA which together with the long disordered N- and C-terminal H2A tails and the long N-ter- like the duplexes is the result of an altered base pairing pattern in case of minal H2B tail. The N-terminal aN helix, C-terminal b3 strand, and 310 helix geS2U-G. We also investigated the significance of the length of this conserved of H2A observed in the H2A-H2B nucleosome structure are disordered in modification both in case of duplexes as well as in the context of ribosome isolated H2A-H2B. In addition, the H2A a1 and H2B aC helices are not well binding. Our simulation results are consistent with the experiments and provide fixed in the heterodimer, and the H2A and H2B tails are not completely a platform for gaining insights into the molecular explanation for how the ger- random coils, which are consistent with the results of NMR experiments. anyl modification modulates tRNALys. 2404-Pos Board B11 2401-Pos Board B8 Quantitative NMR Analysis of Concurrent Phosphorylation Events and Nano-Mechanical Studies of Collagen: The Influence of Ionic Strength, pH Phosphorylation-Related Structural Changes in Unstructured Cyto- and Collagen Sources on Molecular Flexibility plasmic Domain of T-Cell Receptor CD3g Subunit Naghmeh Rezaei, Aaron Lyons, Nancy R. Forde. Innokentiy Maslennikov1, Christian Klammt2, Johan Kufs2, Amy Blount2, Physics, Simon Fraser University, Burnaby, BC, Canada. Barbara Varsonofieva2, Bjoern Lillemeier2. Collagen, a key component of the extracellular matrix, is the most abundant 1School of Pharmacy, Chapman University, Irvine, CA, USA, 2Nomis Center protein in vertebrates, and is widely used in tissue engineering. Collagen is for Immunobiology and Microbial Pathogenesis, The Salk Institute for found in almost every connective tissue of the body, including skin, bone, Biological Studies, La Jolla, CA, USA.

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NMR spectroscopy has a unique ability to describe concurrent molecular need for new diagnostic and therapeutic approaches to FLHCC. The results of events, such as phosphorylation, which cannot be characterized individually whole genome sequencing and transcriptome sequencing of FLHCC tumors by other methods. By combining heteronuclear NMR methods and selective show a single, consistent genetic alteration in the FLHCC tumor cells: a dele- protein labelling we aim to build a quantitative approach for the analysis of tion of ~400 kB between the first exon of the heat shock protein, DNAJB1, and multi-site phosphorylation and related structural changes in proteins. Particu- the first exon of the catalytic subunit of PKA, PRKACA, on one copy of chro- larly, we are studying kinetics of phosphorylation, order of the phosphorylation mosome 19. This deletion produces a chimeric gene, which creates a chimera events, and related structural changes within immunoreceptor tyrosine-based protein. This chimeric DNAJB1-PRKACA protein is now recognized as the activation motifs (ITAM) at the mostly unstructured cytosolic domains of driver of FLHCC so inhibition of the DNAJB1-PRKACA chimera offers signal-propagating CD3 subunits of T-cell receptor (TCR) complex. T-cell tremendous potential to cure FLHCC. We are carrying out a structural and activation is triggered by an antigen binding to TCR complex. Subsequent biochemical characterization of DNAJB1-PRKACA and comparing it to native phosphorylation of paired tyrosines within ITAM of six CD3 subunits PRKACA in hopes of providing valuable insights toward developing a drug (CD3g, CD3d, two CD3ε, and two CD3z) modulates their binding to ZAP70 that selectively blocks the FLHCC chimera. The DnaJB1-PKA has similar ki- tyrosine kinase, which is the crucial step in the T-cell activation. nase activity as wild type PRKACA and also forms R2 DnaJB1-PRKACA2 ho- In our NMR-based approach we combined (1) cell-free protein expression sys- loenzymes with PKA regulatory subunits similar to wild type R2-PRKACA2 tem for the selective 15N/13C- and/or 19F-labelling of the cytoplasmic domain holoenzymes even though they elute at different positions though gel filtration, of CD3g subunit, (2) the combinatorial selective 15N/13C labelling for the indicating they have an extended conformation. Both cryo-EM and X-ray crys- accelerated NMR assignment of mostly unstructured CD3g cytoplasmic tallography are being used to elucidate structural differences between wild type domain, (3) 19F-NMR analysis of the kinetics of phosphorylation of two tyro- and mutant holoenzymes. sine residues within ITAM domain of CD3g, and (4) 15N/13C-NMR analysis of phosphorylation-related structural changes. 2407-Pos Board B14 Our data indicate that (1) two ITAM tyrosines have different phosphorylation Effect of Phosphorylation on Supertertiary Structure and Ligand Binding kinetics: N-terminal Tyr160 is phosphorylated much faster and induces the Affinity on Scaffold Proteins phosphorylation of the ‘‘slow’’ Tyr171; (2) the phosphorylation is accompanied Brie´ N. Levesque, Fang Wu, Mark E. Bowen. with a structural rearrangement: the phosphorylation of ‘‘fast’’ Tyr160 triggers Physiology and Biophysics, Stony Brook University, Stony Brook, NY, USA. structural changes within ITAM domain, which, in turn, induces the kinetics of PSD-95 and PSD-93 are multidomain scaffold proteins with a similar tertiary the ‘‘slow’’ Tyr171. The described mechanism of co-regulation of structural structure and a common set of interaction partners. Despite the high sequence changes and phosphorylation events in the ITAM domain of CD3g modulates homology in their protein binding domains these isoforms play opposing roles TCR-ZAP70 complex formation and TCR signalling. in learning and memory. The origin of their functional differences remains a mystery. The most apparent physical difference between them is the length 2405-Pos Board B12 and composition of their interdomain linkers as well as the number and position Different Applications for CAS in Functional Classification of Protein of the phosphorylation sites. In PSD-95, the binding domains partition into two Interfaces structurally-independently supramodules separated by an intrinsically disor- Cameron J. Jones1, Sanjana Sudarshan2, Isha D. Mehta1, Brian W. Beck3. dered linker. PSD-93 has a similar intrinsically disordered region but the super- 1Biology, Texas Woman’s University, Denton, TX, USA, 2Bioinformatics tertiary structure has yet to be determined. We used single molecule and Genomics, University of North Carolina at Charlotte, Charlotte, NC, fluorescence resonance energy transfer (FRET) to compare the supertertiary USA, 3Texas Advanced Computing Center, University of Texas at Austin, structures and found PSD-93 has a greater separation between supramodules. Denton, TX, USA. This could arise from the increased linker length but also a more even distribu- Protein-protein interactions (PPI) play essential roles in virtually all biological tion of positive and negative charges in PSD-93. The number and spatial orga- processes. While modern structural determination methods such as x-ray crys- nization of charges determines the extension of intrinsically-disordered tallography and NMR provide valuable information that can aid in our under- peptides. Interestingly, in vitro phosphorylation by Src kinase decreased the standing of these complexes, they often produce many putative conformations separation in PSD-93 but increased the separation in PSD-95 resulting a similar that require further refinement to determine the biological structure. Here, we separation between supramodules upon phosphorylation. As with structural outline two methods that may aid in the refinement of these potential conforma- studies, binding between PSD-95 and its ligands has been extensively studied tions. Using a manually curated database of both biologically functional and while the affinity of PSD-93 for their shared ligands is has not been measured. biologically non-functional PPI (FLIPdb), we explore the relationship between Using smFRET we found that the two scaffolds have similar binding affinity, binding free energy as determined by computational alanine scanning (CAS) before and after phosphorylation, for their common ligands. Furthermore, we and small translational perturbations of the interacting protein subunits. In found that phosphorylation selectively increased the affinity for some ligands short, the interacting protein subunits are translated along a grid that is coplanar while leaving others unchanged. Our results show that phosphorylation can with their dividing plane and the interface is subjected to CAS at each point remodel the supertertiary structure of multidomain scaffold proteins. Addition- along the grid. The coordinates of the translation are then used in combination ally, phosphorylation can change the specificity by selectively altering binding with the binding free energy to create an energy landscape that is further affinity for specific ligands. analyzed for various features. Analysis of these features by SVM has allowed us to achieve discrimination of biologically functional/biologically non- 2408-Pos Board B15 functional interfaces with an accuracy of seventy-six percent, and suggests Toxic Metal Ions Promote Self-Association and Replace Structural Zinc that biologically functional interfaces are more sensitive to perturbation than Ions in the Regulatory Region of Protein Kinase C 1 1 1 biologically non-functional interfaces. In addition to translational perturbation Taylor R. Cole , Samuel G. Erickson , Min Woo Sung , 2 1 sensitivity, we analyze the size and distribution of energetic hotspots present in Andreas Holzenburg , Tatyana I. Igumenova . 1Biochemistry and Biophysics, Texas A&M University, College Station, TX, each PPI. Using solvent accessible surface area (SASA) as measured by 2 NACCESS, and binding free energy as determined by CAS, residues are clus- USA, Div.of Research, Innovation, and Economic Development, The tered into hotspots using density based clustering. The size and energy density University of Texas Rio Grande Valley, Harlingen, TX, USA. of these hotspots is then analyzed. Analysis of these features by SVM allows us Protein Kinase C (PKC) isoenzymes are a family of kinases that control cell proliferation, migration, and apoptosis. The hallmark of PKC activation is their to discriminate between biologically functional and biologically non-functional 2þ PPI with an accuracy of approximately eighty percent. translocation to lipid membranes that occurs in response to Ca and diacylgly- cerol. Previous studies have shown that PKC activity is modulated by toxic 2406-Pos Board B13 metal ions such as Pb2þ and Cd2þ. Our objective was to understand the effect Fusion Protein, DNAJB1-PRKACA, as a Cancer Driver of these metal ions on the structure and function of the membrane-binding reg- Tsan-Wen Lu1, Michael Cianfrocco2, Ping Zhang3, Susan Taylor1,4. ulatory region of PKC. Our efforts focused on the PKCa region that consists of 1Chemistry and Biochemistry, UCSD, San Diego, CA, USA, 2Cellular and two peripheral membrane-binding domains, C1B and C2. Using a combination Molecular Medicine, UCSD, San Diego, CA, USA, 3National Cancer of biophysical techniques, we demonstrate that interactions of Pb2þ and Cd2þ Institute, Frederick, MD, USA, 4Pharmacology, UCSD, San Diego, with C1B-C2 have profound and distinct consequences for the biophysical and CA, USA. functional properties of this region. While both Cd2þ and Pb2þ can spontane- Fibrolamellar Hepatocellular Carcinoma (FL-HCC), a rare liver cancer disease, ously replace structural Zn2þ ions in C1B, NMR spectroscopy revealed that usually occurs in young adults without any liver disease history. FLHCC does only Cd2þ was able to support the proper fold and ligand-binding function of not respond well to chemotherapy so surgery is still the main treatment. Given the protein. In addition, Cd2þ promotes the formation of well-defined C1B- the low survival rate and lack of available treatment options, there is a pressing C2 oligomers that we were able to detect and characterize using small-angle

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X-ray scattering experiments and negative-stain electron microscopy. In this problem to the solvent quality and degree of polymerisation, and find contrast to Cd2þ,Pb2þ cannot support the proper fold of C1B but acts success- analytical expressions for the energy barriers present in the problem. From fully as a functional surrogate of Ca2þ in driving protein-membrane associa- these, we analyse the kinetic problem of a force-ramp experiment, showing tion. Our work demonstrates the potential diversity of responses of signaling that the rupture force depends on the rate of loading. We then change the sys- proteins to toxic metal ions and suggests that molecular mechanisms of Pb2þ tem by inserting a super-hydrophobic ‘core’ at a given point in the polymer and Cd2þ toxicity are distinct. This work was supported by Welch Foundation chain. This acts as a crude model for a large class of folded biomolecules grant A-1784, NSF CAREER award CHE-1151435, and NIH grant R01 with hydrophobic or hydrogen-bonded cores. Again, we consider applying a GM108998. constant tensile force. Introducing a ‘core’ leads to an intrinsic (quenched) sto- chastic variation in the unfolding rate, even when the positions of the added Aggregates, Chaperones, and Mechanical Forces monomers are fixed along the sequence. This gives rise to non-exponential pop- ulation dynamics, which is consistent with a variety of experimental data. It 2409-Pos Board B16 does not need any structural disorder of the type thought to be at the origin Using Single Molecule Chemo-Mechanical Unfolding to Probe the Effect of of non-exponential relaxation laws. Environmental Conditions on Protein Folding Emily J. Guinn, Bharat Jagannathan, Susan Marqusee. 2412-Pos Board B19 California Institute for Quantitative Biosciences, UC-Berkeley, Berkeley, Mechano-Induced Unfolding of Von Willebrand Factor: A Clinical CA, USA. Example of Protein Destabilization 1 2 2 In vivo, proteins function in a complex environment where they are subject to Camilo A. Aponte Santamaria , Svenja Lippok , Judith J. Mittag , 3 3 4 € 1 stresses like solutes, temperature and strain which can modulate the protein’s Tobias Obser , Reinhard Schneppenheim , Carsten Baldauf , Frauke Grater , Ulrich Budde5, Joachim R€adler2. energy landscape. Perturbing these conditions allows one to explore how pro- 1 teins respond to changes in environment. This also helps to characterize protein Molecular Biomechanics, Heidelberg Institute for Theoretical Studies, Heidelberg, Germany, 2Faculty of Physics and Center for NanoScience, energy landscapes because perturbant effects are related to the structure and en- 3 ergetics of the different protein states along the energy landscape. The effect of Ludwig Maximilian University, Munich, Germany, Department of Pediatric perturbants on protein stability is related to the structure of the native and de- Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 4Theory, Fritz-Haber-Institut der Max-Planck- natured state, while the effect of perturbants on protein kinetics is related to the 5 folding pathway. We have developed a technique called chemo-mechanical un- Gesellschaft, Berlin, Germany, MEDILYS Laborgesellschaft mbH, folding where we combine force and chemical denaturant using optical twee- Hemostaseology, Asklepios Klinik Altona, Hamburg, Germany. zers. We use chemo-mechanical unfolding as well as temperature and point Protein stability can be quantified, in experiments or simulations, by moni- mutations to explore the denatured state and the parallel pathways proteins toring changes in free energy induced by single amino-acid mutations relative fold through. We also compare experiments on- and off- the ribosome to deter- to changes imposed by the same mutation in a reference unfolded peptide. We mine how the ribosome affects the folding pathway. here assessed the destabilization by disease mutations of a protein, for which both the folded and unfolded states play key functional roles in hemostasis. 2410-Pos Board B17 Force-induced unfolding of the giant von Willebrand Factor (VWF) Protein Aging: Loss of Folding Contraction due to Oxidation of Cryptic multimer-protein exposes a cleavage site for enzymatic proteolysis, a critical Side Chains down-regulatory mechanism to prevent the formation of large thrombus aggre- Jessica Valle Orero1, Jaime Andres Rivas-Pardo1, Rafael Tapia-Rojo1, gates. Several naturally occurring mutations modify this process, inducing Ionel Popa2, Daniel J. Echelman1, Julio M. Fernandez1. distinct types of bleeding disorders, by unknown mechanisms. We present 1Biophysical, Columbia University, New York, NY, USA, 2Physics, the first quantitative description of the dramatic destabilization of VWF caused University of Wisconsin Milwaukee, Milwaukee, WI, USA. by a one of such mutations, which strongly accelerates VWF cleavage. Molec- Tensegrity is the property of tissues that allows them to regain their shape after ular dynamics simulations and free energy calculations revealed this mutation a mechanical deformation. Constituent proteins support tensegrity by being to induce structural, dynamic, and mechanical perturbations in the VWF-A2 able to generate a restoring force at any length. A salient feature of tissue aging domain, thereby destabilizing this domain by ~10 kJ/mol promoting its unfold- is the oxidative modification of its proteins, thus compromising the tensegrity ing. In close agreement, fluorescence correlation spectroscopy (FCS) revealed of the system (e.g. sagging skin). Here, we use magnetic tweezers to monitor a 20-fold increase in the cleavage rate for this mutant, compared to the wild- the folding dynamics of single protein L molecules under force over times type VWF. Cleavage was found cooperative with a cooperativity coefficient scales from hours to days. Mechanically unfolded proteins that are maintained n = 2.3, suggesting that the mutant VWF gives access to multiple cleavage sites extended for 22 hours entirely lose their ability to fold. This loss of folding is at the same time. Taken together, the enhanced cleavage activity can be readily triggered by the exposure of the cryptic side chains to the oxidative environ- explained by an increased availability of the cleavage site through A2-domain- ment, as it can be greatly slowed by adding an antioxidant to the solution. fold thermodynamic destabilization. Our study therefore puts forward the com- This phenomenon compromises the tensegrity of the protein by reducing its bination of free energy calculations and FCS, as a powerful way of examining extensibility by 40%. We provide an analytical expression that describes the protein stability in a clinically relevant context. Reference: C. Aponte-Santa- extensibility of a protein under force, combining the entropic elasticity of the marı´a, et al. Biophysical Journal. In revision. polypeptide and the folding collapse. By incorporating an aging factor measured from the loss of protein folding over time, we can predict the loss 2413-Pos Board B20 of tensegrity. Our ability to accurately keep a single protein unfolded for hours Mechanical Architecture and Genesis of Bacterial Pilus Domains to days presents a novel assay for accelerating aging. We anticipate that this Revealed by Single-Molecule Force Spectroscopy will become a useful tool to discern the role of environmental contaminants Alvaro Alonso-Caballero, Raul Perez-Jimenez. to understand the loss of tensegrity in exposed tissues. CIC nanoGUNE, San Sebastian, Spain. Gram-negative bacteria attach to tissues using long filaments called pili. In ur- 2411-Pos Board B18 opathogenic Escherichia coli (UPEC), the pilus type-1 is composed by thou- Thermodynamics and Kinetics of Globular Polymers under an Applied sands of FimA subunits that form the pilus rod followed by the subunits Force FimF-FimG-FimH at the tip fibrillum. The mechanical resistance of the pilus Samuel Bell, Eugene M. Terentjev. is essential for successful attachment to target cells and tissues. With the excep- Cavendish Laboratory, University of Cambridge, Cambridge, United tion of FimH, the contribution of each subunit to the mechanical architecture of Kingdom. the pilus has not been investigated. Here, we use atomic force spectroscopy to We examine the problem of a polymer chain, folded into a globule in poor sol- report a complete nanomechanical map of the pilus. We have used protein engi- vent, subjected to a constant tensile force. This system represents a Gibbs ther- neering to redesign the pilus domains in order to replicate the force vector that modynamic ensemble, and is useful for analysing force-clamp AFM they sense in vivo. All domains show a very high mechanical stability reaching measurements - now common in molecular biophysics. Using a basic Flory forces above 400 pN. We have discovered that the domains follow a mechanical mean-field theory, we account for surface interactions of monomers with sol- hierarchy in which the stability decreases from the pilus rod to the tip. We have vent. Under an increasing tensile force a first-order phase transition occurs. also used force-clamp spectroscopy to investigate with single-molecule resolu- The compact globule ruptures and fully extends, in an ‘all-or-nothing’ unfold- tion the mechanism of domain folding from the stretched state to the folded state ing event. This contrasts with the regime of imposed extension, first studied by assisted by periplasmic oxidoreductases and chaperones, DsbA and FimC. We Halperin and Zhulina, where there is a regime of coexistence of a partial have observed a synergistic effect that allows us to depict a precise kinetic model globule with an extended chain segment. We relate the transition forces in for the mechanogenesis of the pilus subunits.

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2414-Pos Board B21 resolution approach, using both microsecond-scale explicit-solvent all-atom Multiscale Modeling of Protein Unfolding and Translocation by AAAD (AA) and coarse-grained (CG) molecular dynamics (MD) simulations. Our Chaperones in the Degradation Pathway data show that the H4 histone is significantly more rigid compared with the Andrea N. Kravats1, Sam Tonddast-Navaei2, Abdolreza Javidialesaadi3, H3 histone and its variant CENP-A, hence, serving as a reinforcing structural George Stan3. element within the histone core. We report that the CENP-A/H4 dimer is signif- 1Laboratory of Molecular Biology, National Cancer Institute, National icantly more dynamic than its canonical counterpart H3/H4, and our results pro- Institute of Health, Bethesda, MD, USA, 2Department of Biology, Georgia vide a physical explanation for this flexibility. Further, we observe that the Institute of Technology, Atlanta, GA, USA, 3Department of Chemistry, centromere-specific chaperone HJURP stabilizes the CENP-A/H4 dimer University of Cincinnati, Cincinnati, OH, USA. by forming a specific electrostatic interaction network. Finally, replacing AAAþ nanomachines, such as the hexameric ring-shaped Clp (Caseinolytic CENP-A S68 with E68 disrupts the binding interface between CENP-A and protease) ATPases, perform essential actions in the degradation pathway by un- HJURP in all-atom MD simulation, and consistently, in vivo experiments folding and translocating substrate proteins (SPs) into the peptidase compart- demonstrate that replacing CENP-A S68 with E68 disrupts CENP-A’s localiza- ment. Repetitive mechanical forces mediating these actions arise from tion to the centromere. Based on all our results, we propose that, during the sequential ATP-driven axial motions of flexible loops protruding into the nar- CENP-A/H4 deposition process, the chaperone HJURP protects various sub- row central channel. We developed coarse-grained and implicit solvent models structures of the dimer, serving both as a folding and binding chaperone. of ClpY-SPs to study the effect of SP topology, ClpY allostery and direction- ality of the mechanical force on protein remodeling mechanisms. 2417-Pos Board B24 Comparative studies of all-alpha and alpha-beta model SPs reveal conserved Protein Kinase Recognition and Sorting by the HSP90 Kinome-Specific aspects, including unfolding initiated at the tagged C-terminus and discrete Cochaperone CDC37 translocation steps via a power stroke mechanism. Topology-dependent aspects Ioannis Gelis, Dimitra Keramisanou, Adam Aboalroub. include the time scales, the rate limiting steps in the degradation pathway, the Chemistry Department, University of South Florida, Tampa, FL, USA. effect of force directionality, and the translocase efficacy. The Hsp90 chaperone machinery is a ubiquitous mediator of protein homeosta- We probe the role of sequential intra-ring ClpY allostery by considering clock- sis and activity. Despite its essential roles, little is known about the molecular wise (CW), counterclockwise (CCW) and random (R) order of ATP-driven mechanism that controls substrate entry into its chaperone cycle. Here we show conformational changes of subunits. In all three allosteric mechanisms, the that the functional role of the kinome-specific cochaperone, Cdc37, reaches SP undergoes conformational transitions along a common set of pathways, beyond that of an adapter protein and find that it actively participates in the se- consistent with active remodeling work provided by single ClpY loop-SP inter- lective recruitment of only client kinases. The cochaperone recognizes kinase actions. Nevertheless, faster timescales of SP processing in CW compared with specificity determinants in both clients and non-clients and thus it acts as a gen- CCW and R allostery reflect asymmetric collaboration between loops of adja- eral kinase scanning-factor. Kinase sorting within the client to non-client con- cent subunits, which involves CW-compatible torque applied onto the SP. tinuum relies on the ability of Cdc37 to challenge the conformational stability To elucidate the effect of force directionality we contrast ClpY-SP models that of client kinases by locally unfolding them. This metastable conformational mimic laser optical tweezer (LOT) experiments, in which the SP N-terminus is state has high affinity for Cdc37 and forms stable complexes through an restrained, with cellular-like ClpY action, without N-terminus restraints. LOT- extended, multi-domain cochaperone interface. On the other hand, the interac- like one-directional pulling results in probing a strong mechanical direction of tion with non-clients is not accompanied by conformational changes of the sub- I27 and unfolding involves a shearing mechanism, while unrestrained pulling strate and results in substrate dissociation. Collectively, Cdc37 performs a enables ClpY to unfold I27 along a soft mechanical direction via unzipping. quality control of protein kinases, where induced conformational instability acts as a ‘‘flag’’ for Hsp90 dependence and thus stable cochaperone association. 2415-Pos Board B22 2418-Pos Board B25 Evidence for Chaperone Function in Mechanosensation Tracking Structural Changes in the UCS Domain of the Myosin Chap- Miranda Collier, Georg Hochberg, Cameron Turtle, Carin De Villiers, erone UNC-45 by Fluorescence Spectroscopy Katja Gehmlich, Justin L.P. Benesch. Andres Oberhauser1, Paul Nicholls2. Chemistry, University of Oxford, Oxford, United Kingdom. 1Neuroscience and Cell Biology, UTMB, Galveston, TX, USA, 2Internal The small heat-shock proteins (sHSPs; HSPBs) are a family of molecular chap- Medicine, UTMB, Galveston, TX, USA. erones involved in stress response. Their canonical function is to prevent irre- Molecular chaperones are commonly identified by their ability to suppress versible aggregation of denatured or partially denatured proteins. However, heat-induced protein aggregation. The muscle-specific molecular chaperone sHSP binding partners have been identified that interact without prior denatur- UNC-45 is known to be involved in myosin folding and is trafficked to the sar- ation, suggesting a separate function. Several of these partners are involved in comeres A-band during thermal stress. Using a combination of fluorescence, the mechanical translation of chemical signals and are key components in sys- circular dichroism, limited proteolysis and mass spectroscopy to quantitatively tems that underlie cell or tissue viscoelastic properties, such as the muscle analyze the effect of temperature on UNC-45 we previously found that the UCS sarcomere and the actin cytoskeleton. Though sHSP association with the domain undergoes significant structural changes in response to temperatures cellular support network is long established, little is understood about the within a heat-shock range (Bujalowski, et al., FEBS let 2015). In order to purpose or mechanism of these interactions. Here we discuss results that further analyze the regions within the UCS domain that become exposed to demonstrate a propensity for sHSP-mediated stabilization of mechanosensing the solvent we used fluorescent assays based on the environment-sensitive hy- protein domains at the atomic level which leads to stiffening at the macro scale. drophobic fluorescent dye, Bis-ANS. We found that UCS interacts with Bis- We focus on the interactions of HSPB5 (aB-crystallin) with cardiac titin within ANS in 2:1 stoichiometry at 25oC with an apparent Kd of 46nM. We found the sarcomere, and of HSPB1 (HSP27) with the actin-binding protein filamin C, that the number of bound ANS molecules increases ~10-fold when exposed and primarily draw upon native mass spectrometry and gas phase unfolding ex- to temperatures above 35oC. We found that Bis-ANS stably binds to the periments in conjunction with ex vivo tissue assays. We postulate that sHSPs UCS domain after heating (indicated by the asymmetry in the heating and cool- are able to recognize certain proteins prone to mechanical stress and bind ing curves). We further investigated temperature-induced structural changes them proactively such that physiological tensile forces do not lead to full un- tryptophan fluorescence. The UCS domain has three Trp residues, W783, folding, thus both preventing downstream aggregation and aiding cells and tis- W823 and W863. We found that the relative Trp fluorescence signal decreases sues in the maintenance of elastic properties essential to their function. of as the temperature was raised indicating a change from an hydrophobic to an aqueous environment. In order to quantify the energetics of the ANS-UCS 2416-Pos Board B23 interaction we used isothermal titration calorimetry (ITC). Analysis of the calo- Promiscuous Histone MIS-Assembly is Actively Prevented by Chaperones rimetric titration profile showed a monotonic decrease in the exothermic heat of Haiqing Zhao. binding. From these data we estimated an N= 4 (number of binding sites) and University Of Maryland, College Park, MD, USA. DS= þ 70 cal/mol/K, suggesting that hydrophobic forces are dominant in the Histone proteins are essential for the organization, expression, and inheritance of binding of ANS to UCS. In order to approximately map the regions at which genetic material for eukaryotic cells. A centromere-specific H3 histone variant, ANS binds in the UCS domain we used resonance energy transfer (FRET) be- centromere protein A (CENP-A), shares about 50% amino acid sequence iden- tween Trp residues and Bis-ANS. We found that as Bis-ANS is added, Trp fluo- tity with H3. CENP-A is required for packaging the centromere and for the rescence is quenched as energy is transferred to the extrinsic fluorophore in a proper separation of chromosomes during mitosis. Despite their distinct biolog- concentration dependent fashion. We are using these techniques to map the ical functions, previously reported crystal structures of the CENP-A/H4 and H3/ key amino acid regions in the UCS domain that mediate its myosin-binding H4 dimers reveal a high degree of similarity. In this work, we characterize the and chaperoning functions. This work was supported by the American Heart structural dynamics of CENP-A/H4 and H3/H4 dimers based on a dual- Foundation (AHA 13GRNT17290006) and the NIH (1R01GM118534 01).

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2419-Pos Board B26 dimensional lattices with periodic boundary conditions (two lanes model). Unfolding Efficacy of the Immunoglobulin Domain I27 Controlled by One lane represents the transport through the filament and the other lane Force Directionality in Protein Remodeling by CLP ATPase Chaperones mimics a diffusive environment. The filament lane also takes into account Abdolreza Javidialesaadi, George Stan. the filament dynamics. The results are discussed in terms of the efficiency of Department of Chemistry, University of Cincinnati, Cincinnati, OH, USA. the bidirectional transport. Cellular protein quality control comprises a network of chaperones that main- tain the proteome viability by performing key cellular tasks such as degrading 2422-Pos Board B29 or remodeling misfolded proteins. Bacterial Caseinolytic proteases (Clp) which Characterization of Human IgG1 Fc Region Stability and Aggregation are responsible for protein degradation include powerful ring-shaped AAAþ Propensity (ATPases Associated with diverse cellular Activities) motors with a central nar- Evan A. Wells, Anne S. Robinson. row pore that unfold and translocate tagged abnormal proteins. Clp ATPase Chemical and Biomolecular Engineering, Tulane University, New Orleans, machines thread substrate proteins (SPs) through their central channel by using LA, USA. repetitive ATP-driven subunit motions coupled with axial mechanical forces Monoclonal therapeutic antibodies (MAb) are highly valued for their efficacy exerted onto the SP. Here we perform multiscale molecular simulations of in treating ailments ranging from arthritis to cancer. Unrivaled specificity ClpYDI and Titin I27 to mimic and contrast laser optical tweezer (LOT) exper- and infrequent adverse effects benefit patients in ways traditional small mole- iments, in which the SP N-terminus is restrained, with in vivo ClpY-mediated cule therapeutics cannot. However, formation of aggregates during expression, unfolding and translocation, in which the SP is not restrained at the N-terminus. purification, or storage as a drug substance can lead to product loss and This allows us to shed light on the effects of restraining forces and SP mechan- increased immunogenicity. Glycans present on the antibodies also contribute ical direction probed on Clp-mediated unfolding mechanism. The external LOT to both aggregation propensity and antibody effector functions. Understanding restraint limits ClpY-mediated pulling along the N-C direction of the SP, which the importance of glycans in aggregation and protein function is important in yields unfolding of I27 via a shearing mechanism. By contrast, in vivo-like limiting costs and maximizing therapeutic efficacy. Antibodies are comprised ClpY-action results in pulling along softer mechanical directions and I27 is of two heavy chains and two light chains, and regions are alternatively classi- unfolded via an unzipping mechanism. We find that factors that affect these fied into Fab (fragment antigen binding) and Fc (fragment crystallizable) re- distinct mechanisms are SP-ClpY surface interactions, the size of the SP rela- gions. Only heavy chain subunits exist in the Fc region and remain constant tive to the ClpY pore size, the SP mechanical resistance, and the presence of between MAbs of the same isotype (commonly human IgG1). Uniformity of other substrate domains. Fc regions between antibodies allows for creation of fusion proteins containing an Fc region that facilitates affinity purification using a standardized protocol. 2420-Pos Board B27 The Fc region is N-glycosylated at Asn-297, and glycoform identities drive g g Metastabilities in the Human Prion Protein N-Terminal Beta-Sheet are effector functions via interactions with specific Fc receptors (Fc Rs). We Dictated by the 129 Polymorphism have utilized mammalian cells to express the human IgG1 Fc region and will S. Alexis Paz1, Eric Vanden-Eijnden2, Cameron Abrams3. present initial studies on efforts to purify and characterize the aggregation pro- 1Mathematics and Physics, National University, Cordoba, Cordoba, pensity and stability of this antibody fragment under controlled stress condi- Argentina, 2Courant Institute of Mathematical Sciences, New York tions (e.g. thermal, mechanical, etc.). Data collected from these conditions University, New York, NY, USA, 3Chemical and Biological Engineering, has potential applications in building and modifying protein stability models Drexel University, Philadelphia, PA, USA. and could improve the rational design of future recombinant therapeutic We study the thermodynamic stability of the native state of the human prion antibodies. protein using a new free-energy method, replica-exchange on-the-fly paramate- 2423-Pos Board B30 rization. This method is designed to overcome hidden-variable sampling limi- Utilising Fluorescence Microscopy to Visualise the Dynamics and Interac- tations to yield nearly error-free free-energy profiles along a conformational tions of Molecular Chaperones and a-Synuclein coordinate. We confirm that all four (M129V,D178N) polymorphs have a Quill Bowden1,2, Alex MacMillan3, Tobias Rosenkranz2, Till Bo¨cking2. ground-state conformation with three intact beta-sheet hydrogen bonds. Addi- 1Single-molecule Biophysics, University of Wollongong, Wollongong, tionally, they are observed to have distinct metastabilities determined by the Australia, 2Single Molecule Science, University of New South Wales, side-chain at position 129. We rationalize these findings with reference to Sydney, Australia, 3Biomedical Imaging Facility, University of Sydney, the prion ‘‘strain’’ hypothesis, which links the variety of transmissible spongi- Sydney, Australia. form encephalopathy phenotypes to conformationally distinct infectious prion In Parkinson’s Disease (PD), the protein a-synuclein and its early stage oligo- forms and classifies distinct phenotypes of sporadic Creutzfeldt-Jakob disease mers have been implicated as the primary cause of neuronal toxicity. Molecular based solely on the 129 polymorphism. Because such metastable structures are chaperones play an important role in maintaining protein homeostasis in such not easily observed in structural experiments, our approach could potentially diseases. The dynamic nature of the chaperone cycle and its interaction with the provide new insights into the conformational origins of prion diseases and other a-synuclein substrate makes it difficult to characterise by traditional biochem- pathologies arising from protein misfolding and aggregation. ical techniques. Using a range of fluorescence microscopy approaches we show that a-synuclein exists in a concentration dependant dynamic equilibrium 2421-Pos Board B28 between monomeric and oligomeric states, with rapid exchange of subunits Defective Axonal Transport and Alzheimer’s Disease Correlations: A Mo- between species. Dilution leads to rapid monomerisation and loss of structure lecular Motor Point of View followed by a slow recovery of higher order species. Chaperones from the Marcelo Nakaema. Hsp40/Hsc70 machinery interact only with the dissociated monomeric a-syn- School of Sciences and Technology (ECT - UFRN), University of Rio Grande uclein and delay the reassociation of subunits to higher order species impli- do Norte at Natal, Natal, Brazil. cating the multimer form as the potential unknown native state of Alzheimer’s disease (AD) is the principal factor for dementia during old age; it a-synuclein. Using single-molecule techniques we studied the membrane bind- is characterized by a progressive impairment of cognitive skills. Several genetic ing properties of the larger a-synuclein oligomers associated with toxicity. This and environmental factors contribute for the AD onset. Recently, several evi- method allowed us to visualise in real-time the perforation of lipid vesicles in dences were unraveled that correlated defective axonal transport (AT) caused the presence of a-synuclein oligomers and characterise the ability of chaper- by abnormal axonal swellings to the pathogenesis of AD. AT is an active pro- ones to prevent this process. A greater understanding of the mechanism of cess that utilizes the cellular cytoskeleton as a road network for the cytoskeletal chaperone mediated modulation of disease proteins and their preferential inter- motor proteins (dyneins and kinesins superfamilies). There are two types of actions will provide insights into the early stages of the progression of Parkin- AT: the anterograde and the retrograde axonal transport. Dynein molecular mo- son’s disease. tors support the retrograde transport to cell bodies by walking to the minus end of microtubules while the kinesin motors drive the anterograde axonal transport Protein-Small Molecule Interactions II towards the synapses by walking the microtubules to the plus end. The three major components of the neuronal cytoskeleton are microtubules, actin and in- 2424-Pos Board B31 termediate filaments. One major feature about AT that has been challenging Biomolecular Interaction Determination and Quantification by Microscale scientists is its bidirectional transport character and how it is efficiently orga- Thermophoresis nized. It is critical for axonal function the vesicular transport for long distances Wyatt Strutz, Govind Shah. and this transport occurs mainly through the microtubule (MT) tracks. In this NanoTemper Technologies, South San Francisco, CA, USA. work, we model this bidirectional transport using a variant of TASEP (Totally MicroScale Thermophoresis (MST), an immobilization-free technology, is Asymmetric Simple Exclusion Process) which consists of two parallel one used to rapidly quantify biomolecular interactions (pM-mM). MST, the

BPJ 7891_7895 Wednesday, February 15, 2017 493a directed movement of molecules in optically generated microscopic tempera- 2427-Pos Board B34 ture gradients, is monitored by fluorescence. Thermophoretic movement is Porphyrin Induced Multimerization of Solution-State Proteins highly sensitive to binding reactions, which affect the size, charge, conforma- Daniel R. Marzolf, Coleman M. Swaim, Aidan M. McKenzie, tion, and/or hydration shell of target molecules. Here, we demonstrate that Christopher A. Hudson, Nathan T. Wright, Oleksandr Kokhan. MST can be used to quantify interactions between biomolecules of interest, Department of Chemistry and Biochemistry, James Madison University, including proteins, nucleic acids, ions, and more. In one example, the binding Harrisonburg, VA, USA. site of a protein-protein interaction is investigated via protein engineering. As Interactions between charged porphyrins and complimentary or similarly MST is suitable for probe interactions with membrane proteins in detergent or charged proteins provide important models systems for studies of electron liposomes, we also demonstrate the quantification of interactions with GPCRs transfer processes, artificial photosynthesis, and control of protein-protein in- in a label free format using tryptophan fluorescence. Thus, MST is a critical teractions. Typically, the experimental results are analyzed and discussed addition to the biophysical toolbox allowing greater user-flexibility for assuming that the proteins exist in a monodisperse state. Here, we explored analyzing molecular interactions. interaction of four solution-state proteins (horse heart cytochrome c, hen egg-white lysozyme, 3-heme c-type cytochrome PpcA from Geobacter sulfur- 2425-Pos Board B32 reducens, 2-heme cyt c4 from Pseudomonas stutzeri) with several cationic and Does the Presence of a Co-Solvent Alter the Affinity of a Hydrophobic anionic water-soluble derivatives of tetraphenylporphyrin. Combined small- Drug to its Target? and wide-angle X-ray scattering experiments revealed formation of multimers Caroline Senac1, Patrick Fuchs2, Wladimir Urbach1,3, Nicolas Taulier1. with a wide range of complex sizes. Thermodynamic interaction parameters 1Laboratoire d’Imagerie Biome´dicale, Sorbonnes Universite´, UPMC and complex binding stoichiometries were established with isothermal Univ Paris 6, CNRS, INSERM, Paris, France, 2Institut Jacques Monod, calorimetry. Locations of porphyrin binding sites were determined with heter- CNRS UMR 7592, Universite´ Paris Diderot, Paris, France, 3Laboratoire de onuclear single quantum coherence (HSQC) and total correlation spectroscopy Physique Statistique de l’Ecole Normale Supe´rieure, UMR CNRS 8550, (TOCSY) NMRs for PpcA and cytochrome c, while covalent labeling shielding Paris, France. experiments followed by LC-MS analysis of tryptic digests were used to map Among all newly discovered chemical entities about 40% drugs are lipophil- ligand binding sites on cyt c4 and lysozyme surfaces. The obtained results lic. The solubility behavior of these drugs remains one of the most challenging demonstrate that multimerization of solution-state proteins by large water- aspects in the first steps of formulation, especially when drug affinity to its soluble ligands appears to be a wide-spread phenomenon controlled by a deli- target is evaluated. The addition of a water miscible solvent, (co-solvent) in cate interplay of electrostatic and hydrophobic forces. Molecular level mapping which the drug has good solubility is one of traditional techniques for of the binding sites allows us to build a theory explaining the size of the formed enhancing drug solubility and reduce the percentage of poorly soluble drug complexes and provides opportunities for targeted design and assembly of candidates. multi-subunit protein complexes. One of the most frequently used co-solvents is dimethylsulfoxide (DMSO) 2428-Pos Board B35 because of its large solubilisation capacity for poorly soluble drugs and its In Silico Discovery of a Protein-Protein Interaction Inhibitor for Influenza relatively low toxicity. This routinely employed approach is considered to Viruses have a negligible effect on the binding affinity of drug to its therapeutic target. Gregory Mohl, David Busath. To our knowledge this assumption was never checked. To show how DMSO Physiology and Developmental Biology, Brigham Young University, Provo, impacts the binding affinity we will present an in silico study of a model sys- UT, USA. tem mimicking ligand interaction with a hydrophobic pocket that can be The Influenza virus poses a significant public health threat. Frequent mutations found in proteins of therapeutic interest. In particular free energies of binding make the virus a difficult target for antiviral development. Amantadine and ri- in pure water and in a solution of DMSO will be evaluated. If the use of mantadine are now ineffective against most circulating strains due to point mu- DMSO leads to a wrong estimation of drug efficiency the use of co-solvent tations in the M2 channel, and neuraminidase inhibitors are also vulnerable to is thus far from negligible. viral resistance. This highlights the need for the discovery of antivirals that inhibit infection by a new mechanism. We set out to design antivirals that 2426-Pos Board B33 would block influenza replication by inhibiting the interaction of Polymerase Convenient Biological Interaction Analysis with a Regenerative Streptavi- Basic 1 (PB1), a subunit of the viral polymerase complex, and Ran-Binding din Chip Protein 5 (Ran BP5), a host importin, with a small molecule that would bind Lukas Traxler, Claudia Knoglinger, Kristyna Posledni, Andreas Zich, to PB1. It has been shown that PB1 interacts with Ran BP5 for nuclear import, Bianca Ruttmann, Gloria Friedl, Peter Hinterdorfer, Hermann J. Gruber. a necessary step in viral replication. Compounds that might block nuclear Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria. import of the polymerase complex were identified using high-throughput vir- Biological Interaction Analysis (BIA) is an essential tool to characterize the tual screening coupled with in vitro validation assays. The Asinex protein- mechanism of cellular processes. BIA is performed in vivo to identify compo- protein interactions library was docked with Glide and Autodock Vina to the nents which interact, as well as in vitro to measure biophysical parameters like PB1 subunit of 4WSB, the trimeric polymerase complex of bat influenza. association, dissociation, and equilibrium constants. The latter is often per- The proposed binding site was chosen due to its proximity to the bipartite nu- formed in surface plasmon resonance (SPR) biosensors where one of the clear localization signal (NLS), and the top hits were rescreened using a higher two interacting components is immobilized on the sensor surface, while the exhaustiveness to confirm the results. Four compounds were chosen and tested second component is repeatedly injected at different concentrations. Conven- in cell culture for inhibition of viral replication with an immunofluorescence tionally, the biomolecules are immobilized by covalent attachment to the chip assay and for cytotoxicity with a vital dye uptake assay. A lead compound or by biotinylation and subsequent binding to a streptavidin chip. Both was identified that has an EC50 of 13.89 mM against A/California/07/2009 methods are irreversible which means that a new chip must be used for every and an EC50 of 16.19 mM against A/WS/33. This compound also showed new interaction study, as well as for every unsuccessful attempt. Due to the some activity against B/USSR/69 (Brigit) with an EC50 of 37.52 mM. This high price per chip, this hampers the wide-spread use of SPR biosensors in lead compound is amenable to medicinal optimization. the academic community. Here, we present a new streptavidin chip on which any kind of biotinylated molecules (or lipid vesicles) can be immobilized with 2429-Pos Board B36 very high stability, allowing for a wide range of measurement conditions in Binding of Antibiotics to the Multidrug Efflux Pump AcrB of E. coli the interaction studies. When desired, however, the biotinylated molecules Investigated by Molecular Docking can be removed and replaced by other biotinylated probes on a time scale Giuliano Malloci, Giovanni Serra, Andrea Bosin, Attilio Vittorio Vargiu, of minutes. The new chip allows for an unlimited number of regeneration Paolo Ruggerone. cycles inside the biosensor within 3 weeks. Because of the high lateral density Physics, University of Cagliari, Monserrato (Cagliari), Italy. of streptavidin on the flat chip surface, the capture molecules can be immobi- The AcrB multidrug efflux transporter of E. coli is a giant homotrimeric mem- lized at close proximity, allowing to study the cooperativity of multivalent in- brane protein able to recognize and expel a wide variety of compounds out of teractions. The new streptavidin surface can be prepared on all gold-coated the cell [1]. Obtaining the co-crystal structures of AcrB bound to its substrates sensor chips, as used in SPR, quartz crystal microbalance (QCM), and has proven to be difficult, and to date only a few antibiotics have been success- surface-acoustic wave (SAW) biosensors. Due to its low cost and broad fully co-crystallized with the protein. From these studies, two main recognition applicability, the new chip surface allows for unrestricted use and for quick sites were identified, but the small number of available co-crystal structures still switching between different research topics with the identical instrument hinders a full understanding of the molecular recognition mechanism. As a part configuration. This work was supported by the state of Upper Austria (project of an extensive computational research activity on efflux pumps, we performed DK NanoCell). a systematic docking campaign to characterize the putative binding sites of

BPJ 7891_7895 494a Wednesday, February 15, 2017

antimicrobial compounds from different classes, namely carbapenems, cepha- These findings suggest reducing the toxicity associated with Ab42 aggregation losporins, fluoroquinolones, penicillines, tetracyclines, and beta-lactamase in- could be explored to find viable therapeutic strategies for the treatment of AD. hibitors. In particular, for each compound we screened for possible binding sites on the whole protein, including thus possible binding pockets other than 2432-Pos Board B39 those already known. Flexibility has been taken into account by considering en- Determination of Ligand Migration Pathways in Human Cytoglobin 1 2 3 3 sembles of rigid structures for both the ligand and the receptor. Ligand confor- Antonija Tangar , Michael Goncalves , Sophie Bernad , Valerie Derrien , 3 1 mations have been extracted from microsecond-long molecular dynamics (MD) Pierre Sebban , Jaroslava Miksovska . 1Florida International University, Miami, FL, USA, 2TERRA Environmental simulations in explicit water [2]. For the protein we considered a large set 3 of conformations, including structures from both high-resolution X-ray and Research Institute, Miami, FL, USA, Universite Paris-Sud, Orsay, France. MD simulations. We present the results of this systematic investigation by Cytoglobin (Cygb) is one of the latest additions to the vertebrate globin family comparing the patterns of interactions observed for the different compounds. found in virtually all human tissues. Due to its low intracellular concentration, The binding properties of the transporter are discussed in terms of statistics Cygb is not believed to act as a oxygen transport/storage protein and its func- of the most contacted residues. tion remains to be determined. However, in vivo studies have indicated that 1. P. Ruggerone, S. Murakami, K. M. Pos and A.V. Vargiu, Curr. Top. Med. Cygb has a strong cytoprotective role by acting as a peroxidase, nitric oxide di- Chem.13, 3079 (2013). oxygenase and/or reactive oxygen species scavenger. Additionally, affinity for 2. G. Malloci, A. V. Vargiu, G. Serra, A. Bosin, P. Ruggerone, M. Ceccarelli, ligands is modulated by internal disulfide bond, which is believed to act as a Molecules20, 13997 (2015). redox switch and impact Cygb function. In order to probe the mechanism of Cygb interactions with small diatomic ligands, we took a closer look into res- 2430-Pos Board B37 idues found on two main computationally-suggested ligand migration path- Exploring Folate-Small Molecule Interactions in Bacterial Cells ways: Arg84, which is proposed to act as a gate between heme cavity and Deepika K. Nambiar, Robert Shew, Bryan Schwarz, Michael Duff, solvent; and Arg33, found at the end of the tunnel enclosed by helices A, G Timkhite Kulu-Berhane, Elizabeth Howell. and H. Biochemistry, Cellular & Molecular Biology, University of Tennessee Photoacoustic calorimetry, transient absorption and stopped-flow were em- Knoxville, Knoxville, TN, USA. ployed to determine kinetic and thermodynamic profiles for diatomic ligand in- Folate (Vitamin B9) is involved in one carbon transfer reactions required for teractions with human Cygb wild-type, hCygb Arg84Leu, hCygb Arg33Trp, the synthesis of DNA and amino acids. Our current understanding of the folate and hCygb Arg33Trp Arg84Leu. Additionally, impact of mutations on Cygb pathway is mostly based on in vitro studies, which are very different from the stability was determined. Our results indicate that both Arg84 and Arg33 crowded environment in the cell. E. coli produces osmoprotectants during have an important role in both ligand escape and rebinding by modulating dy- times of osmotic stress. This leads to perturbation of water activity inside the namics and energetics of structural changes associated with ligand binding to cell, and an increase in macromolecular crowding. We have shown earlier Cygb. Moreover, Arg84 was found to be a crucial residue in stabilizing that, in vitro, osmolytes weaken the binding of dihydrofolate to dihydrofolate heme pocket. Furthermore, computational approaches (locally enhanced sam- reductase in the folate pathway. We hypothesize that an increased osmolyte pling, MD pocket) were employed to determine most-sampled ligand migration concentration in the cell will also prevent the functioning of other folate pathways and occupancy of internal cavities. pathway enzymes by interaction of osmolytes with the various folate redox 2433-Pos Board B40 states. In this study, we studied the effect of osmotic stress on the folate synthe- Molecular Recognition Mechanism of Hematopoietic Prostaglandin D sizing and metabolizing enzymes such as dihydropteroate synthase (folP), dihy- Synthase with its Cofactor and Substrate drofolate reductase (folA), methylenetetrahydrofolate reductase (metF) and Keisuke Asada1, Shigeru Shimamoto1, Tomohiro Oonoki1, serine hydroxymethyltransferase (glyA) in vivo. Studies were done with Takahiro Maruno2, Yuji Kobayashi2, Kosuke Aritake3, Yoshihiro Urade3, knockout and rescued strains. Protein expression in rescued strains was limited Yuji Hidaka1. to very low levels using a Ptet promoter and a protein degradation tag. We can 1Graduate School of Science and Engineering Research, University of titrate the enzyme activity in the rescued strains by osmotic stress. Osmotic Kindai, Higashi-, Japan, 2Graduate School of Engineering, University stress studies have indicated that the rescued strain was unable to grow in of Osaka, Suita, Japan, 3International Institute For Integrative Sleep higher osmolality conditions when compared to knockout strains. We predict Medicine, University of Tsukuba, Tsukuba, Japan. this is due to an increase in osmolyte concentration in vivo which leads to inter- Hematopoietic prostaglandin (PG) D synthase (H-PGDS) is a Sigma class action of osmolytes with folate intermediates in the pathway. This is turn de- member of the glutathione S-transferase (GST) superfamily, and requires gluta- creases the efficiency of the folate pathway enzyme. Finally, we have varied thione (GSH) as a cofactor. H-PGDS catalyzes the isomerization of prosta- the predominant osmolyte in the cell from trehalose to glycine betaine to study glandin H (PGH ) to prostaglandin D (PGD ), which acts as an allergic if our model holds for these different conditions. 2 2 2 2 and inflammatory mediator. The overproduction of PGD2 by H-PGDS causes allergic and inflammatory reactions in the necrotic muscle fibers of Duchenne 2431-Pos Board B38 muscular dystrophy (DMD) patients. Therefore, H-PGDS inhibitors are thought Attenuating the Toxicity of Amyloid-Beta Aggregation with Specific to have potential for use in drug therapy for DMD. Species The catalytic mechanism of H-PGDS remains unclear, since essential informa- Ryan Limbocker1, Benedetta Mannini1, Michele Perni1, Sean Chia1, tion, such as the binding affinity and stoichiometry of GSH and PGH2 to 1 1 1 1 Gabriella Heller , Francesco S. Ruggeri , Johnny Habchi , Georg Meisl , H-PGDS, have not been precisely determined yet. Therefore, to clarify the issues 1 2 1 Pavan K. Challa , Michael Zasloff , Tuomas P.J. Knowles , related to the cofactor and substrate recognition of H-PGDS, we investigated the Michele Vendruscolo1, Christopher M. Dobson1. 1 interaction between H-PGDS and PGH2 in the presence and absence of GSH. Department of Chemistry, University of Cambridge, Cambridge, United For this purpose, isothermal titration calorimetry (ITC) was performed using a Kingdom, 2Department of Surgery, MedStar Georgetown Transplant substrate mimetics U-44069 in the place of PGH2 since PGH2 is quite unstable Institute, Washington, DC, USA. in most solutions. Recombinant H-PGDS was prepared using an E. coli expres- The deposition into amyloid plaques of the amyloid-b peptide (Ab) is a hall- sion system and used for the ITC measurements. The results showed that mark of Alzheimer’s disease (AD). Increasing evidence suggests that soluble U-44069 binds to H-PGDS in the presence of the cofactor, GSH, but the bind- Ab oligomers are the pathological species primarily responsible for AD onset ing was not significant in the absence of GSH. Therefore, these results suggest and progression. Shifting the aggregation pathway towards the formation of that the GSH binding promotes the interaction between H-PGDS and PGH2, non-toxic aggregated species by specific compounds could thus prove an effec- and that GSH plays an important role, not only for the catalytic reaction but tive therapeutic strategy for AD. Herein, we employ an array of biophysical also for substrate binding. techniques, including fluorescence-based chemical kinetics, Atomic Force Microscopy, Nuclear Magnetic Resonance, and absorbance spectroscopy, and 2434-Pos Board B41 in vivo techniques, including cells and C. elegans, to characterise compounds Lipocalin-Type Prostaglandin D Synthase Possesses Two Binding Sites for for their ability to mitigate the aggregation of Ab. We find structurally similar its Product compounds that 1) regulate protein assembly by modulating the microscopic Shigeru Shimamoto1, Yusuke Nakagawa1, Takahiro Maruno2, processes governing aggregation, 2) interact with primary species resulting Yuji Kobayashi2, Kosuke Aritake3, Urade Yoshihiro3, Yuji Hidaka1. in increased fibrilization in vitro and in vivo, 3) drive the formation of shorter, 1Kindai University, Higashi-osaka, Japan, 2Osaka University, Suita, Japan, more mature fibrils, 4) generate larger, less hydrophobic, and less toxic aggre- 3University of Tsukuba, Tsukuba, Japan. gates from oligomers after incubation with pre-formed oligomers, and 5) in the Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) belongs to the lipoca- case of one species, eliminates Ab42-induced toxicity in a worm model of AD. lin superfamily which consists of transporter proteins for lipophilic ligands in

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the extracellular space, and is referred to as the PGD2-synthesizing enzyme, beta tubulin heterodimer. We then used the model to run a molecular dynamics which is responsible for sleep regulation. Recently, we found that L-PGDS simulation with 20% v/v isopropanol in the simulated environment. Drug bind- binds, not only to its substrate, PGH2, but also to its product, PGD2. However, ing pockets are commonly hydrophobic, and this type of simulation has been the mechanism responsible for the binding of L-PGDS to its product, PGD2, re- shown to prevent hydrophobic collapse in such pockets because it facilitates in- mains unclear. In the present study, to address this issue, we investigated the teractions between the protein residues and the cosolvent. We then selected 10 interactions between L-PGDS and its ligand, such as PGD2, PGF2a and random conformations from this simulation and ran a high-throughput virtual PGE2. The PGD2 molecule contains 9-hydroxy and 11-keto groups which are screen using 100,000 compounds from the ‘‘Leads Now’’ subset of the ZINC located at the relatively opposite positions of PGE2, and PGF2a contains a hy- small molecule library. The top 1000 compounds were docked and re-scored droxyl group in the place of the 11-keto group of PGD2. The binding affinity using the Extra Precision (XP) Glide method. From these results, we used a and stoichiometry to each ligand were analyzed by isothermal titration calorim- chemical similarity clustering algorithm to choose a final set of several dozen etry (ITC), and the binding region for each ligand on L-PGDS was estimated compounds with diverse chemical topologies. The selected compounds were using data obtained from NMR titration experiments. The recombinant L- used to conduct cell-based cytotoxicity assays with cultures of Leishmania tar- PGDS for each experiment was prepared using an E. coli expression system. entolae and Ca46 cells to assess whether the molecules exhibited selectivity for The ITC results showed that L-PGDS binds PGD2 and PGF2a with a stoichiom- the former over the latter. etry of 1 to 2 but binds PGE2 with a stoichiometry of 1 to 1. In addition, NMR 2437-Pos Board B44 spectral data indicated that PGD2 and PGF2a bind to both the catalytic site con- Effects of Azole Drugs on Conformational Dynamics and Energetics Asso- taining the Cys65 and non-catalytic site of L-PGDS, while PGE2 binds only to ciated with Diatomic Ligand Photorelease and Migration within Bacterial the non-catalytic site. These results suggest that the 9-hydroxy of PGD2 plays an important role in binding to the catalytic site of L-PGDS, and that the 9 and Flavohemoglobins David Butcher1, Myriam Moussaoui2, Laura Baciou2, Jaroslava Miksovska1. 11 positions of the cyclopentane ring have little effect on the binding to the non- 1 2 catalytic site. Chemistry, Florida International University, Miami, FL, USA, Chemistry, Universite´ Paris-Sud 11, Orsay, France. 2435-Pos Board B42 Bacterial flavohemoglobins (fHbs) are a widely distributed class of proteins Structural, Thermodynamic and Phosphatidylinositol 3-Phosphate Bind- which are though to protect microbes from nitrosative stress due to the immune ing Properties of Phafin2 response of a host organism via nitric oxide dioxygenase (NOD) activity. There Tuo-Xian Tang. has been interest lately in developing fHbs as targets for antibiotics due to their Biological Sciences, Virginia Tech, Blacksburg, VA, USA. ubiquity in bacteria and the discovery of inhibition of NOD activity of fHbs by < Phafin2 is a phosphatidylinositol 3-phosphate (PtdIns(3)P)-binding protein azole-based drugs which bind with high affinity ( 12 mM). To determine how involved in the regulation of endosomal cargo trafficking and in the lysosomal azole drugs affect fHbs we have used photoacoustic calorimetry (PAC) and induction of autophagy. Binding of Phafin2 to PtdIns(3)P is mediated by both transient absorption spectroscopy (TA) to calculate enthalpy and volume its PH and FYVE domains. However, the structural basis for understanding changes (DH and DV) and kinetic parameters for CO photorelease and rebind- how Phafin2 promotes signaling at endomembrane compartments is unknown. ing to fHbs from Cupriavidus necator (FHP) and Staphylococcus aureus Here, we show that the human recombinant Phafin2 is a moderately elongated (HMPSa) in the presence and absence of the drugs ketoconazole and micona- monomer of ~28 kDa. Circular dichroism (CD) analysis indicates that Phafin2 zole. We observe that binding of drugs to fHbs results in a more exothermic exhibits an a/b structure and predicts ~40% of random coil content in the protein. DH for CO photorelease and faster bimolecular rebinding of CO. We also Heteronuclear NMR data indicates that a unique conformation of Phafin2 is pre- use classical molecular dynamics (cMD) to characterize ligand migration path- sent in solution and that dispersion of resonances suggests that the protein exhibit ways present in FHP. We have conducted cMD simulations of CO-FHP random coil regions, in agreement with CD data. Dynamic light scattering WT and FHP WT containing free CO molecules using a locally enhanced sam- studies show that Phafin2 is relatively stable, displaying a melting temperature pling (LES) algorithm. We have carried out a 10 ns simulation of CO-FHP WT of 35C. The folding-unfolding curves, obtained by the use of urea- and guani- and a 30 ns simulation of the LES-enabled CO:FHP WT. We observe dissoci- dine hydrochloride-mediated denaturation, indicate that Phafin2 undergoes a ation of 18 of 20 LES-enhanced CO molecules from the distal heme pocket. two-state native to denatured transition. Analysis of these transitions shows The primary escape pathways are between the CD-loop and heme-propionate that the free energy change for urea- and guanidine hydrochloride-induced groups and through a pathway exiting the protein matrix near the N-terminal Phafin2 denaturation in water is ~4 kcal mol1. Using SPR analysis, we show end of the A-helix. These pathways are close to the locations of ketoconazole that PtdIns(3)P binding to Phafin2 occurs with moderate affinity. Preliminary and miconazole binding predicted by AutoDock Vina, suggesting that data shows that Phafin2 promotes membrane tethering and tubulation in vitro drug binding inhibits NOD activity by closing migration pathways for O2 in a PtdIns(3)P-dependent manner, suggesting that the protein plays a role in and NO. inducing membrane curvature changes, a pre-requisite to trigger autophagy. 2438-Pos Board B45 Taken together, these studies represent a platform for establishing the structural An Efficient Protein System for Screening Specific MdmX and Mdm2 basis of Phafin2 membrane interactions and the role of its two potentially redun- Inhibitors dant PtdIns(3)P-binding domains in endomembrane compartments. Rong Chen, Jingjing Zhou, Lingyun Qin, Zhengding Su. Hubei University of TEchnology, Wuhan, China. 2436-Pos Board B43 In approximately half of human cancers, the overexpressed Mdm2 and MdmX Targeting Microtubules in Leishmania using a Combined Computational impair the cancer suppression of p53 protein. So far many Mdm2 inhibitors & Experimental Approach have been considered for clinical trials, but the progress in the discovery of Andrew Kalenkiewicz1, Phil Cruz2, Michael Dolan2, Dan L. Sackett1. 1 effective MdmX inhibitors has been left very behind. In this work, we made Unit on Cytoskeletal Dynamics, Eunice Kennedy Shriver National Institute use of the sole tryptophan residue of Mdm2/MdmX-binding peptide (p53p) of Child Health and Human Development, NIH, Bethesda, MD, USA, 2 as fluorescence assay for screening natural compounds. Therefore, a fusion pro- Bioinformatics and Computational Biosciences Branch, National Institute of tein was constructed by fusing p53p to the N-terminal domain of MdmX Allergy and Infectious Diseases, NIH, Bethesda, MD, USA. (N-MdmX) with a flexible amino acid sequence linker and the resultant fusion Previous work has shown that targeting microtubules in protozoan parasites has protein was defined as p53p-N-MdmX. We examined the p53p-N-MdmX the potential to be an effective therapeutic strategy against diseases caused by fusion protein with a potent Mdm2 inhibitor, nutlin-3a. Furthermore, the fusion these microorganisms, such as Leishmaniasis, African sleeping sickness, and protein was evaluated with a small library that contained the currently-available Chagas Disease. Preliminary studies demonstrate that tubulin from these para- Mdm2 inhibitors. Our results indicated that the fusion protein of p53p-N- sites exhibits distinct binding properties compared to tubulin from mammalian MdmX provided an efficient system for screening novel scaffolds of MdmX- species. In particular, the well-known tubulin binding drug colchicine is 10,000 inhibitors and Mdm2-inhibitors. times more potent against human cell lines than Leishmania cultures in assays measuring GI50. Homology modeling suggests that this is due to the fact that 2439-Pos Board B46 the colchicine binding-site in mammalian tubulin is chemically distinct from Ligand Binding Studies of a Plasmid Encoded Dihydrofolate Reductase the analogous site in protozoans. We examined the possibility of reversing by 19F NMR this selectivity and finding a small molecule compound that selectively binds Gabriel J. Fuente Gomez, Michael Duff, Elizabeth Howell. to tubulin from Leishmania. Our approach includes a combination of computa- Biochemistry and Cellular and Molecular Biology, University of Tennessee, tional and experimental analysis to streamline the process of identifying poten- Knoxville, TN, USA. tial novel leads. We first constructed a homology model of Leishmania tubulin Plasmid encoded-R67 dihydrofolate reductase (DHFR) catalyzes the NADPH- based on known co-complex crystal structures of colchicine bound to the alpha/ dependent reduction of dihydrofolate (DHF) to tetrahydrofolate. R67 DHFR is

BPJ 7891_7895 496a Wednesday, February 15, 2017 a homotetramer with a single active site pore and two tryptophans per monomer (CP) is a tube built from four stacked heptameric rings with 3 pairs of catalytic (W38 and W45). W38 and its symmetry-related partners occur at the dimer- subunits equipped with specific proteolytic activities. The catalytic sites face dimer interfaces while the W45 residues occur at the monomer-monomer inter- inside of the tube and are additionally secured by the gates on the tubes’ alpha faces. We have labeled these tryptophans using 19F-labeled indole with the 19F faces and blocking the access to the CP interior. To get to the active centers, atoms at different positions (4-, 5-, 6- or 7-) of the indole ring. In vitro ligand substrates have to bypass the gate, which closed state is affected by binding li- binding studies of NADPþ to the apoprotein or a ternary complex with NADPþ gands to the alpha face or catalytic sites. To gain capability to digest polyubi- and DHF showed characteristic spectra for each complex. The apoprotein gave quitinylated substrates, the CP binds a regulatory particle (RP) or a 19S cap to rise to a sharp and a broad peak. Upon addition of NADPþ, the sharp peak for least one site of the tube. The interface between CP and RP allosterically con- W38 shows line broadening while the broad peak for W45 remains unchanged. trols dynamics of substrates uptake, digest readiness, and efficiency and spec- In the ternary complex, the sharp peak in the apo and binary complex splits into ificity of the active centers. The CP also wears other types of caps that allow it three peaks while the broad peak remains. The appearance of three new reso- to digest efficiently particular populations of substrates and generate certain nances can be explained by how NADPH and DHF bind in the active site types of products. Also in these cases the interface between the protein com- pore. Two symmetry related Lysine 32s at the edge of one side of the pore plexes plays a crucial regulatory role. Here we designed a range of peptides, constrain the position of NADPH by forming ionic interactions with the phos- peptidomimetics and small molecule compounds that affect these protein – pro- phate group. However, on the other side of the pore, the glutamate tail of DHF tein interactions to study selected mechanical aspects of proteasomal regula- is disordered and, switches between ion pairs with the K32s on that side of the tion. Using spectroscopic and microscopic tools we found that binding of pore. This results in two different environments for the nearby W38residue. certain ligands may affect status of the proteasomal gate, alpha face conforma- Our results are consistent with NMR and computational simulations of the tion, stability of the complexes or/and the CP dynamics. These structural glutamate-tail interacting with symmetry related lysine 32 residues at the changes alter the enzyme activity and specificity, which in turn influence intra- edge of the pore. cellular metabolism. We attempted to correlate particular biological effects of the ligands with their mode of molecular action and their chemical properties. Protein Dynamics and Allostery IV 2442-Pos Board B49 2440-Pos Board B47 Incorporation of Multi-State Information Improves Molecular Modelling Mechanism of Allosteric Regulation of Substrate Specificity in Protein of Dynamic Allostery: A Case Study of PDZ Domains Computational Kinase C Quantitative Characterization of Entropic Signaling Pathways in Proteins: Nagarajan Vaidehi1, Hyun Deok Song1, Titu Devamani2, Ruth Sommese2, A Case Study on Human PDZ2 Domain of PTP1E Sivaraj Sivaramakrishnan2. Mohsen Botlani, Sameer Varma. 1Immunology, Beckman Research Institute of City of Hope, Duarte, CA, Cell Biology, Microbiology and Molecular Biology, University of South USA, 2Genetics, Cell Biology and Development, University of Minnesota, Florida, Tampa, FL, USA. Minneapolis, MN, USA. Understanding protein regulatory mechanisms requires assessment of various Protein kinase C (PKC) is a member of the AGC kinase family and it phosphor- states, including inactive, transition and active states, as well as relationships ylates multiple peptide substrates. Kinase inhibitors indiscriminately block the between them. For many proteins, their mechanisms can be understood in terms phosphorylation of all its substrates and by inference all the subsequent of how their 3D structures, or conformations, change during transition and acti- signaling pathways mediated by the kinase. A molecular level understanding vation. Such structural models of regulation, however, cannot be constructed of the mechanism by which PKC regulates its catalytic efficiency towards when structural differences between protein states are small, and differences various substrates is a critical component to designing substrate specific kinase in thermal fluctuations also matter. This challenge faces several major protein inhibitors. Using FRET sensor measurements on PKCa we have shown that families like GPCRs and nuclear transcription factors, whose activities are above a threshold binding affinity, the peptide substrate binding is inversely regulated allosterically by induced changes in both structure and dynamics. correlated to the catalytic turn over by PKCa, for 14 different peptides (1). Methods have been developed to relate inter-state differences to allosteric sig- In this work we have used a combination of internal coordinate molecular dy- nals, but none account for inter-state differences in thermal fluctuations. Formal namics (MD) (GNEIMO) to map large scale conformational changes, followed relationships also exists between signal communication propensity and thermal by all-atom MD simulations to provide insight into how the dynamics of the fluctuation correlations, but they provide information only about how different kinase domain of PKCa modulates the binding affinity and the turnover of regions of a protein communicate with each other in a given state, and do not the phosphorylation of the substrates. The calculated substrate binding affinities provide direct insight into causalities (or regulatory signals). It is, therefore, not correlate with the measured change in FRET in the ATP bound PKCa. The surprising that molecular mechanisms underlying dynamic allostery remain conformational ensemble from the coarse grain internal coordinate dynamics poorly understood. Here we propose an ‘‘inverse’’ machine learning inspired shows enhanced sampling of conformations in which the catalytic Asp in the method to dissect multi-state conformational ensemble information, and a activation loop is close to the Ser that gets phosphorylated in the substrates. parameter-free graph theory approach to systematically probe the effects of At the same time the Ser of the substrate is also close to the gamma phosphate incorporating such information on the molecular description of entropic of ATP. We call this conformation as the ‘‘catalytic conformation’’. We show signaling pathways. Our studies on PDZ domains show that incorporation of that substrates with high activity show a substantial population of this ‘‘cata- multi-state information improves significantly the correspondence with bio- lytic conformation’’ while other catalytically inefficient substrates do not sam- physical experimental data, and provides key molecular insight into the role ple the catalytic conformation. The correlated movement in the torsional angle of dynamics in allosteric regulation. space among the residues involved in the catalysis is high when the kinase is in the ‘‘catalytic conformation’’. We have also mapped the residues located along 2443-Pos Board B50 the allosteric communication pathway from the ATP site to the substrate bind- Dynamical Mechanisms of Allosteric Modulations by Small Molecules on ing site and studied the effect of various inhibitors bound on the allosteric Leukocyte Function-Associated Antigen-1 1,2 communication. Jung-Hsin Lin . 1Research Center for Applied Sciences & Institute of Biomedical Sciences, (1) Sommese R., and Sivaramakrishnan S., J. Biol. Chem. 2016, pii: 2 jbc.M116.737601. Academia Sinica, Taipei, Taiwan, School of Pharmacy, National Taiwan University, Taipei, Taiwan. 2441-Pos Board B48 Integrins are highly important targets for drug design in a wide range of dis- Regulation of Proteasomal Catalytic Activity by Altering its Protein- eases, such as cardiovascular and immunological disorders. Among different Protein Interactions integrins, lymphocyte function-associated antigen-1 (LFA-1; aLb2 or CD11a/ Pawel A. Osmulski1, Przemyslaw Karpowicz2, Matthew B. Giletto3, CD18), an a/b heterodimeric glycoprotein present on all leukocytes, is a cell 3 4 4 1 Corey L. Jones , Kristin E. Cano , Dmitri Ivanov , Tim H. Huang , surface receptor that belongs to the b2 integrin family. aLb2 contains a domain Elzbieta Jankowska2, Jetze Tepe3, Maria Gaczynska1. of approximately 200 amino acids, which is also known as the von Willebrand 1Molecular Medicine, UTHSCSA, San Antonio, TX, USA, 2Department of factor A domain or the inserted (I) domain. The I domain plays an important Biomedical Chemistry, University of Gdansk, Gdansk, Poland, 3Chemistry role in ligand-binding and contains a single divalent cation coordination site, Department, Michigan State University, East Lansing, MI, USA, the metal ion-dependent adhesion site (MIDAS). Binding of aLb2 to ICAM-1 4Biochemistry, UTHSCSA, San Antonio, TX, USA. at MIDAS is associated with effective T-cell activation by antigen-presenting The proteasome is a central protease of the main intracellular pathway respon- cells. It mediates lymphocyte recirculation, firm adhesion of leukocytes to sible for regulated proteolysis that uses polyubiquitinylation for substrate endothelial cells, and their subsequent extravasation to the surrounding tissue. tagging. The basic functional unit of proteasome called 20S or catalytic particle The aI allosteric antagonists are a class of small-molecule inhibitors which

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bind to the C-terminal a7-helix of aL I domain at the lovastatin (L) site. Lova- unique feature of this approach comes from the good agreement of the statin, a drug primarily used for the prevention of cardiovascular disease and peptide-water cross-correlation modes, which involve distortions of the electron the treatment of hypercholesterolemia, was found to inhibit the interaction of density as a result of polarization and hence cannot be captured by fixed charge aLb2 with ICAM-1. In this study, we analyze the effect of lovastatin and several force fields. This is a promising first step towards future studies for simulating related small molecules on the aI domain of aLb2 via several m-second long all- and decomposing the THz observable for larger solutes such as polymers or pro- atom explicit solvent molecular dynamics simulations. We focus on the teins where AIMD studies are currently intractable. MIDAS and the a7 helix of the aI domain, which have been reported to play key roles in binding aLb2 to ICAM-1, and illustrate the allosteric effects 2446-Pos Board B53 induced by these small molecules acting at aLb2. The largest movement was Molecular Allostery in Dengue NS3 Helicase along the ATP Hydrolysis observed at the a7 helix upon the removal of lovastatin. Besides, we also Cycle observe the change of Mg2þ position and the remodeling of MIDAS, as well Russell B. Davidson1, Brian J. Geiss2, Martin McCullagh1. 1 as changes of dynamical modes of the aI domain, elucidated by reference- Chemistry, Colorado State University, Fort Collins, CO, USA, 2 free time-dependent principal component analysis. Microbiology, Immunology, Pathology, Colorado State University, Fort Collins, CO, USA. 2444-Pos Board B51 The flavivirus non-structural 3 (NS3) protein is a viral helicase that plays a The Structural and Dynamic Effects of Inhibitor Binding to Protein pivotal role during the replication of the viral genome. Specifically, this Kinase C bII enzyme uses energy released from ATP hydrolysis to translocate and cause Shashank Jariwala1, Sivaraj Sivaramakrishnan2, Barry J. Grant1. the unwinding of a double-stranded RNA substrate, thereby preparing the 1 Computational Medicine and Bioinformatics, University of Michigan, Ann RNA for the replication machinery. Microsecond molecular dynamic simula- 2 Arbor, Ann Arbor, MI, USA, Department of Genetics, Cell Biology, and tions of the dengue NS3 helicase are performed on seven structures, five of Development, University of Minnesota, Minneapolis, MN, USA. which represent important RNA-bound structures that model the ATP hydroly- Protein kinase C bII (PKCbII) regulates diverse cellular signaling pathways sis cycle (apo, pre-hydrolysis, post-hydrolysis, and product release states). involved in B-cell activation, insulin signaling, and oxidative stress-induced These simulations demonstrate that the RNA binding cleft is affected by the apoptosis. Aberrant PKCbII activity has been implicated in a variety of human different ATP substrates and suggest a hold-release mechanism for the translo- diseases including diabetic retinopathy and B-cell immunodeficiency. Accord- cation of the enzyme along the RNA substrate. Also, comparisons made be- ingly, PKCbII has become a popular target for small molecule based inhibition. tween the five RNA-bound and remaining two RNA-unbound simulations However, the effects of available inhibitors on the conformational dynamics and indicate that the RNA substrate influences the water dynamics of the ATP bind- allosteric couplings essential for PKCbII function remains largely unknown. ing pocket. These results provide molecular insights into the experimentally This lack of knowledge hampers the development of improved inhibitors and observed RNA-enhanced ATPase activity of dengue NS3 helicase. The allo- limits our understanding of how disease-associated mutations in distal sites steries observed between both the ATP and RNA binding sites will be discussed can interfere with inhibitor efficacy. Here we characterize distinct flexibilities in relation to the ATP hydrolysis cycle. and internal dynamical couplings upon inhibitor binding using molecular dy- namics (MD) simulations and bioinformatics analysis. MD simulations revealed 2447-Pos Board B54 increased flexibility of the nucleotide-binding P-loop and the regulatory C-ter- How the Barrierless Folding Helps DNA Recognition: Theoretical Investi- minal tail when bound to the ATP-competitive inhibitor Bisindolylmaleimide gation on Ultrafast Folding Protein Engrailed Homeodomain I (BIM1). Community partitioning of the dynamical cross-correlations, based Xiakun Chu1, Victor Mun˜oz2,3. on network approaches, reveal further variations between ATP and inhibitor 1IMDEA-Nanociencia, Madrid, Spain, 2Centro Nacional de Biotecnologı´a states. Specifically, the inhibitor state exhibits overall dynamical tightening, (CNB), Consejo Superior de Investigaciones Cientı´ficas (CSIC), Madrid, with stronger couplings between communities encompassing the C-terminal Spain, 3School of Engineering, University of California Merced, Merced, tail in the N-lobe and helices aE, aG, aH and activation loop in the C-lobe. In CA, USA. contrast, the ATP state displays distinct couplings between active-site residues Engrailed homeodomain (EngHD), a 3-helix bundle fast-folding protein, has that are lost upon inhibitor binding. Furthermore, mutational simulations of res- been found to play a critical role in transcription regulation during its binding idues exhibiting state specific couplings demonstrate a shift towards inhibitor- to DNA. Evidence shows that protein efficiently recognizes the short target state dynamics, indicating their role in allosteric modulation. Collectively, DNA sequence from the enormous pool of binding sites via the process of this study elucidates the effect of inhibitor binding on conformational dynamics ‘‘facilitated diffusion’’, including sliding and hopping. During the facilitated and furthers our understanding of allosteric mechanisms in PKCbII. diffusion, protein is supposed to go through ‘‘speed’’ mode, where protein nonspecifically interacts with DNA in fast diffusion, and ‘‘stability’’ mode, 2445-Pos Board B52 where protein strongly and specifically scans the base pairs in DNA with slow Studying Solvation of Small Biomolecules via Molecular Dynamics using a displacement. How the conformational dynamics in protein participating into Polarizable Force Field the balance of the two modes to optimize the non-specific searching and specific 1 2 2 Saurabh Belsare , Alexander Esser , Dominik Marx , binding to DNA remains unclear. Here, we developed a variable-barrier struc- Teresa Head-Gordon3. 1 ture-based model to investigate EngHD’ folding and DNA-binding process. UCB-UCSF Graduate Program in Bioengineering, University of California, The folding barriers of EngHD can be achieved from 2-state (>3 kT) to global 2 € Berkeley, Berkeley, CA, USA, Lehrstuhl fur Theoretische Chemie, Ruhr- downhill (~0.5 kT) folding regimes by modulating the interplay between the € 3 Universitat Bochum, Bochum, Germany, Departments of Chemistry, local dihedral and non-local native contact energy. During the DNA binding pro- Bioengineering, Chemical & Biomolecular Engineering, University of cess, the folding energy landscape of EngHD undergoes dynamical changes at California, Berkeley, Berkeley, CA, USA. different diffusion processes. EngHD is found to preferentially fold in sliding, Interactions with solvent have been shown to affect the dynamics of small bio- where EngHD binds to DNA in the specific manner, reminiscent of the ‘‘stabil- molecules. While bulk properties of solvation can be studied via experiments ity’’ mode, while hopping favors unfolded states, in which the fluctuating like spectral measurements, understanding the precise impact of solvent interac- protein-DNA interfaces are formed, corresponding to the ‘‘speed’’ mode. We tions on specific motions of biomolecules, and the spatial extent of impact of the also addressed the important roles of conformational disorder and salt concen- biomolecules on the solvent are accessible through simulations. We have used trations in modulating the two modes during EngHD-DNA recognition process. the polarizable AMOEBA molecular mechanics force field to simulate terahertz Finally, we found folding with marginal barrier possessing significantly confor- (THz) spectra of two zwitterionic peptides, glycine and valine in aqueous solu- mational flexibility, is able to fulfill the two modes at the same time. In summary, tion. Ab initio molecular dynamics (AIMD) simulations have been previously the conformational rheostat in downhill folding protein optimizes the DNA used to study these spectra. An analysis method has been previously developed recognition process by solving the speed-stability paradox. by Marx and co-workers (Sun et. al. JACS 2014) to decompose the THz spec- trum into the component motion modes for specific molecules, as well as modes 2448-Pos Board B55 arising from intermolecular interactions, for AIMD simulations. We present The Folding Mechanism and Kinetics of the Domains of a-Spectrin: here an approach whereby classical MD simulations performed via the Results from a Variational Model AMOEBA forcefield can be analyzed with that mode decomposition method Daniel Gavazzi, John J. Portman. to obtain dynamic modes for the zwitterions. Based on this decomposition, over- Department of Physics, Kent State University, Kent, OH, USA. all we find very good agreement of the AMOEBA classical MD simulations with Although the three domains of a-spectrin (R15, R16, and R17) are highly ho- AIMD. THz spectral assignments and the spectral decomposition of the total mologous, experiments reveal striking differences in their folding mechanism spectrum into intramolecular peptide motions and peptide-water cross-correla- and kinetics. In particular, the R15 domain’s folding rate is measured to be tion modes shows substantial agreement between AMOEBA and AIMD. The three orders of magnitude greater than the other two domains, with R17 being

BPJ 7891_7895 498a Wednesday, February 15, 2017 the slowest. Higher folding barriers as well as increased internal friction due to erted on the substrate. Vinculin undergoes conformational changes in high landscape ruggedness are proposed to be responsible for the diversity of folding stress regions and has been used as a model system for studying molecular me- rates. In this work, we show that a simple analytic model is able to capture sub- chanosensitivity. It has been suggested that a-actinin binding to the vinculin tleties in the folding mechanism of a-spectrin domains despite their structural head induces conformational changes that leads to vinculin activation. In our similarities. Using a simple model for internal friction based on variance in study, we employed an integrated computational approach (all-atomic molecu- native contact energies, the model also gives folding kinetics that qualitatively lar dynamics simulation and normal mode analysis) to study allosteric regulation agree with experimental rates. The model predicts that R16 and R17 have of vinculin activation upon a-actinin binding. Our results demonstrated the mo- higher folding barriers and increased internal friction within the transition state lecular mechanisms of force transmission within the vinculin structure towards ensemble compared to R15. While the model captures the correct range of its tail domain after a-actinin association to the vinculin head. Furthermore, the folding rates for the domains of a-spectrin, the degree that internal friction in- final bound structure of aVBS (a-actinin’s vinculin binding site) to the vinculin fluences folding kinetics of each domain may be sensitive to molecular detail head after 50ns of simulation matched the crystal structure of the complex (ID: beyond the level of approximation in this simple model. 1YDI), which validated our binding trajectory. Also, we confirmed helical bundle conversion of vinculin head upon a-actinin binding. To our knowledge, 2449-Pos Board B56 this is the first study on the molecular basis of mechanosensitivity and force Free Energy Landscapes of Metabotropic Glutamate Receptor Ligand- transmission within the structure of vinculin, which can ultimately be implicated Binding Domains to develop novel therapeutics for controlling various diseases involving pathol- Tyler J. Wied, Albert Y. Lau. ogies in mechanotransduction including cancer. Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, MD, USA. 2452-Pos Board B59 Metabotropic glutamate receptors (mGluRs) are dimeric class C GPCRs that Correlated Motions in the DHFR-NADPH Complex mediate cellular responses to the neurotransmitter glutamate in the central ner- Craig J. Earley1, Paul F. Maxson1, Arish Murda Rakshasa1, vous system. Crystal structures and single-molecule FRET results suggest that Heather A. Carlson2, Michael G. Lerner1. closure of the ligand-binding domains (LBDs) as well as reorientation of the 1Physics and Astronomy, Earlham College, Richmond, IN, USA, dimer interface are necessary conformational transitions for receptor activation. 2Department of Medicinal Chemistry, College of Pharmacy, University of The energetic factors that drive these coupled processes, however, are poorly Michigan, Ann Arbor, MI, USA. understood. Here, we report free energy landscapes, or potentials of mean force Correlated motions are thought to be related to the catalytic step for dihydrofo- (PMFs), for monomer and dimer mGluR3 LBDs in apo and glutamate-bound late reductase (DHFR). We performed several molecular dynamics studies of states. PMFs of LBD monomers suggest the LBDs are bistable: the closed the DHFR-NADPH complex, the analog of anapo structure existing prior tothe and open-cleft conformations are accessible for both glutamate-bound and binding of dihydrofolate. We compare correlated and anti-correlated motions apo LBDs, but with the apo closed-cleft conformation destabilized relative to andtheir timescaleto motions previously observed in the catalytically active the glutamate-bound LBD. state. We also compare correlated motions across several DHFR mutants. 2450-Pos Board B57 2453-Pos Board B60 Quantifying Allosteric Communication via Correlations in Structure and Static and Dynamic Roles of Proteins in Proline Isomerization Reactions Disorder Toshifumi Mori, Shinji Saito. Sukrit Singh, Gregory R. Bowman. Institute for Molecular Science, Okazaki, Japan. Biochemistry and Mol. Biophysics, Washington Univ. in St. Louis, St. Louis, The molecular mechanism of enzyme catalysis has been of great interest in pro- MO, USA. tein studies. While the catalytic role of amino acids in enzymes at the transition Biology frequently exploits allosteric (i.e. long range) communication in regula- states has been of great interest, and have been studied by various experimental tory processes, such as activation of transcription factors. However, it is often and theoretical techniques, the dynamic picture of enzyme reactions, e.g. how difficult to explain the physical basis of this communication, especially when the protein environment changes during the reaction, has been largely unclear the allosteric signal does not alter a protein’s preferred structure. This lack of a and controversial. Proline isomerization is a typical enzymatic reaction which clear physical underpinning makes it even more difficult to predict what regions plays a key role in folding and regulating the function of proteins. The mech- are communicating with one another. Past methods have focused on the role of anism of such isomerization reactions have been investigated extensively, e.g. concerted structural changes in allostery, but recent work has demonstrated multiple residues which play important roles in stabilizing the reactant and that entropy and disorder are also important factors. To capture these elements, transition state has been discussed. In addition, a few residues which may tran- we introduce the Correlation of All Rotamer Dihedral States (CARDS) frame- siently play a role about the transition state has been proposed, yet the mecha- work for quantifying correlations between both structure and disorder of different nism and dynamics remains unclear. Here we study the proline isomerization regions of a protein. To quantify disorder, we draw inspiration from the physics of reaction of Pin1, a prototypical peptidyl-prolyl isomerase protein, from a theo- glasses to classify segments of dihedral motion as being either rigid or disordered. retical perspective to reveal the dynamic picture of an enzyme catalysis. A mo- Measuring correlations between these properties reflects how well one can pre- lecular dynamics simulation and transition path sampling approach is applied to dict the structure and disorder of one region given knowledge of the structure sample and analyze the transition events at molecular detail. The residues and disorder of another region. We apply CARDS to the Catabolite Activator which are statically and dynamically important are identified, and the changes Protein (CAP), a transcriptional activator regulated by cAMP binding. Wild- in the hydrogen bonding network and protein-ligand interactions about the type CAP undergoes a concerted structural change upon cAMP binding, but active site as the reaction proceeds is discussed. The current study provides a the S62F variant loses this conformational transition while retaining ligand- dynamic view of protein, in addition to a static role of stabilizing the transition dependent DNA-binding activity, suggesting an important role for entropy in state, in a proline isomerization reaction. It is expected that the dynamics also this allosteric process. Using CARDS, we observe roles for both structure and plays a fundamental role in broader enzymatic reactions in order to achieve disorder in communication between known functional sites in both wild-type high efficiency and selectivity. and S62F CAP. Furthermore, the role of entropy is enhanced by the S62F muta- tion. CARDS also discerns key residues and mechanistic details about the mode 2454-Pos Board B61 of communication, and identifies hubs of communication centered around func- Mechanism of Allosteric Communication in GPCR Activation from Micro- tional sites without prior knowledge of their location. Thus CARDS presents a second Scale Molecular Dynamics Simulations powerful means to both dissect the mechanisms of known allosteric interactions Supriyo Bhattacharya, Nagarajan Vaidehi. and to predict previously unknown communication. Immunology, City of Hope National Medical Center, Duarte, CA, USA. G protein coupled receptors (GPCRs) are membrane proteins that signal 2451-Pos Board B58 through effector proteins bound at the intracellular (IC) interface. Under phys- Allosteric Regulation of Vinculin Activation by Alpha-Actinin iological conditions, GPCRs adopt multiple inactive and active conformations, Hengameh Shams, Mohammad R.K. Mofrad. thereby signaling through diverse signaling pathways. Binding of agonists at Bioengineering, University of California, Berkeley, Berkeley, CA, USA. the extracellular (EC) domain initiates a conformational change in the receptor The mechanical behavior of proteins is central to understanding force transmis- leading to activation. Antagonists and inverse agonists suppress activation by sion mechanisms in cell-cell and cell-matrix adhesions. It is essential to relate stabilizing one of the inactive states. The communication between the protein dynamics including mechanical modes and times scales of protein mo- agonist/antagonist binding site and the IC interface takes place through allo- tions and deformations to its structural features, in order to explain key events of steric coupling of distant receptor domains. However, the detailed mechanism mechanosensing and mechanotransduction. Vinculin is a marker of FA matura- of allosteric communication in GPCR signaling is not well understood. Using tion and its concentration correlates with both the adhesion size and forces ex- ensembles of molecular dynamics (MD) simulations on eight class A GPCRs,

BPJ 7891_7895 Wednesday, February 15, 2017 499a we showed that allosteric communication between the antagonist binding site ADP to adenylate kinase, an enzyme that plays an important role in the process and the IC interface stabilizes the inactive state, and disruption of this allosteric of energy homeostasis, we construct a hinge angle distribution consistent with communication due to agonist binding leads to activation. Furthermore, the alchemical binding free energy calculations. fundamental framework and mechanism of allosteric communication are 2457-Pos Board B64 conserved among multiple GPCRs, as is evident from the conserved allosteric 2D hubs (residues that mediate multiple allosteric pathways) in these receptors. Opposing Intermolecular Tuning of Ca Affinity for Calmodulin by Mutating the allosteric hubs either suppress activation or impart constitutive ac- Target Peptides tivity, suggesting the key role of these residues in GPCR function. We have Margaret S. Cheung, Pengzhi Zhang, Swarnendu Tripathi. shown that, besides stabilizing GPCR functional states, allosteric communica- University of Houston, Houston, TX, USA. We investigated the impact of bound calmodulin (CaM)-target compound tion is also responsible for transitions among functional states. Analysis of the 2þ deactivation dynamics of two class A GPCRs (b2 adrenergic receptor and neu- structure on the affinity of calcium (Ca ) by integrating coarse-grained models and all-atomistic simulations with non-equilibrium physics. We focused on rotensin receptor 1) shows that several allosteric hubs in the middle of the trans- 2þ membrane domain undergo concerted dihedral changes during the transition binding between CaM and two specific targets, Ca /CaM-dependent protein from the active to the inactive state. These observations provide valuable in- kinase II (CaMKII) and neurogranin (Ng), as they both regulate CaM- dependent Ca2þ signaling pathways in neurons. It was shown experimentally sights into the complex mechanism of GPCR activation and membrane protein 2þ function in general. that Ca /CaM binds to the CaMKII peptide with higher affinity than the Ng peptide. The binding of CaMKII peptide to CaM in return increases the Ca2þ 2455-Pos Board B62 affinity for CaM. However, this reciprocal relation was not observed in the Deciphering General Characteristics of Residues Constituting Allosteric Ng peptide that binds to Ca2þ-free CaM or Ca2þ/CaM with similar binding af- Communication Paths finity. Unlike CaM-CaMKII peptide that renders solved crystal structures, the Girik Malik, Anirban Banerji, Andrzej Kloczkowski. structural description of CaM-Ng peptide is unknown due to low binding affin- Battelle Center for Mathematical Medicine, Nationwide Children’s Hopital, ity, therefore, we computationally generated an ensemble of CaM-Ng peptide Columbus, OH, USA. structures by matching the changes in the chemical shifts of CaM upon Ng pep- There has been great interest in studying proteins’ dynamics and structural tide binding from nuclear magnetic resonance experiments. Next, we computed changes involved in function to gain insights into the machinery of proteins. the changes in Ca2þ affinity for CaM with and without binding targets in atom- Due to difficulty in retaining atomic details in mode decomposition of large istic models using Jarzynski’s equality. We discovered the molecular underpin- system dynamics, there have been significant computational challenges, which nings of lowered affinity of Ca2þ for CaM in the presence of Ng by showing make the study of large system dynamics very complex. that the N-terminal acidic region of Ng peptide pries open the b-sheet structure Considering all the PDB annotated proteins from the Allosteric Database between the Ca2þ binding loops particularly at C-domain of CaM, enabling (ASD) [1,2] belonging to four different classes (kinases, nuclear receptors, pep- Ca2þ release. In contrast, CaMKII increases Ca2þ affinity for the C-domain tidases, transcription factors), this work has attempted to decipher certain of CaM by stabilizing the two Ca2þ binding loops. consistent patterns present in the residues constituting the allosteric communi- cation sub-system (ACSS). While the thermal fluctuations of hydrophobic res- Membrane Protein Structures II idues in ACSSs were found to be significantly higher than those present in the non-ACSS part of the same proteins, thermal fluctuations recorded for the polar 2458-Pos Board B65 residues showed the opposite trend. Comparing and Contrasting Fluorotryptophan Substitutions for 19F Mem- While the basic residues and hydroxyl residues were found to be slightly more brane Protein NMR Spectroscopy predominant than the acidic residues and amide residues in ACSSs, hydropho- Calem Kenward, Kyungsoo Shin, Muzaddid Sarker, Carley Bekkers, bic residues were found extremely frequently in kinase ACSSs. Despite having Jan K. Rainey. different sequences and different lengths of ACSS, they were found to be struc- Dalhousie University, Halifax, NS, Canada. turally quite similar to each other, suggesting a preferred structural template for The high gyromagnetic ratio and broad chemical shift range of 19F make it sen- communication. sitive and valuable as a probe for studying protein conformation, dynamics and ACSS structures recorded low RMSD and high Akaike Information Criterion intermolecular interactions by NMR spectroscopy. In this work, we are (AIC) scores among themselves. While the ACSS networks for all the groups comparing strategies for 19F NMR characterization of membrane proteins of allosteric proteins recorded low degree centrality and closeness centrality, with the goal of developing a straightforward, robust, and versatile 19F-labeling the betweenness centrality magnitudes revealed non-uniform behavior. Though scheme. One popular and frequently used approach is the incorporation of 19F- cliques and communities could be identified within the ACSS, maximal- substituted aromatic amino acids. Using a recently introduced method showing common-subgraph considering all the ACSS could not be generated, primarily efficient and cost-effective 5-fluorotryptophan protein labeling through addi- due to the diversity in the dataset. Barring one particular case, the entire ACSS tion of 5-fluoroindole to M9 minimal medium (Crowley et al. (2012) Chem for any class of allosteric proteins did not demonstrate ‘‘small world’’ behavior, Commun 48: 10681), we have extended this approach for incorporation of except for a few sub-graphs. 19F labels at the 4-, 5-, 6-, and 7-positions. Two different fragments of the ape- Such a report can be expected to benefit the protein engineering community, lin receptor (AR), a class A G-protein coupled receptor (GPCR), were em- those who attempt to decipher the general mechanism of allostery, and in ployed: AR55 (residues 1-55 including the first transmembrane (TM) general, long-distance communication within protein structures, both from segment) and AR TM1-3 (residues 1-137 including the first 3 TM segments). knowing the topological invariants of communication paths and from knowing We have previously characterized both of these fragments previously using het- the biophysical-biochemical-and-structural patterns therein. eronuclear NMR methods in a variety of membrane-mimetic conditions. AR55 [1] Huang Z, Zhu L, Cao Y, et al. ASD: a comprehensive database of allosteric has two Trp residues, each residing close to the headgroup region of micelles. proteins and modulators. Acids Res. 2011; 39 (Database issue):D663-D669. AR TM1-3 has two additional Trp residues, one of which is predicted to fall in [2] Huang Z, Mou L, Shen Q, et al. ASD v2.0: updated content and novel fea- TM2, the other in extracellular loop 1. The effectiveness of the four fluorotryp- tures focusing on allosteric regulation. Nucleic Acids Res. 2014; 42 (Database tophan substitutions is compared both in terms of chemical shift dispersion and issue):D510-D516. in terms of relaxation behavior. These are compared and contrasted both for the topological location of a given tryptophan (assigned by mutagenesis) and for 2456-Pos Board B63 micellar environments with different headgroup chemistry (i.e., zwitterionic Reweighting the Apo to the Holo Ensemble vs. anionic headgroups). On the basis of the observed differences, strategies Chen Li. are suggested for the most effective fluorotryptophan incorporation and appli- Chemisty, IIT, Chicago, IL, USA. cation for 19F NMR studies of membrane protein topology, structural change, Molecular dynamics (MD) simulations have become a powerful and popular dynamics, and ligand binding. method for the study of protein allostery, the widespread phenomenon in which a stimulus at one site on a protein influences the properties of another site. Sim- 2459-Pos Board B66 ulations can enable the discovery of allosteric binding sites and elucidate the In Situ Solid-State Nmr Spectroscopy of APP Transmembrane Domain mechanism of allostery, providing a foundation for applications including (TM) Structure and Dimerization in Native E. Coli Membranes rational drug design and protein engineering. However, performing a separate Xiaoyan Ding1, Riqiang Fu2, Fang Tian1. simulation for every ligand can be computationally expensive. Here we demon- 1Penn State College of Medicine, Hershey, PA, USA, 2National High strate a proof of principle that conformations sampled from the apo state can be Magnetic Field Laboratory, Tallahassee, FL, USA. reweighted according to the binding potential of mean force (BPMF) to predict Cleavage of the amyloid precursor protein (APP) by a-, b- and g-secretases ensemble averages in the holo state. Using BPMFs for the binding of AMP and leads to generation of amyloid peptide (Ab), which is the primary constituent

BPJ 7891_7895 500a Wednesday, February 15, 2017 of Alzheimer’s disease (AD) plaques in the brain. Three consecutive GXXXG dependent proteins likely occurs at the lateral gate of SecY, the channel- motifs and one GXXXA motif on the transmembrane domain (TM) of APP forming subunit of the bacterial Sec translocon. On the other hand, YidC cat- have been reported to be one or two important sites for mediating APP homo- alyzes insertion by possessing a hydrophilic groove, which can accommodate dimerization in different experimental conditions, the difference are likely due the hydrophilic region of the substrate that inserts across the membrane. to the membrane mimetic. We and several other groups recently demonstrated Recently, Soman et al (2014) have reported that increasing the charge of the feasibility to directly characterize recombinant proteins in native Escheri- the translocated region of procoat can route substrates from the YidC pathway chia coli (E. coli) membranes using magic-angle spinning (MAS) solid-state into the Sec pathway. Here, we have further tested this polarity/charge hy- nuclear magnetic resonance (ssNMR)(2, 3). Here, we will present several stra- pothesis by showing that the major coat protein of bacteriophage M13 can tegies to further improve spectral sensitivity and resolution, and suppress back- become increasingly YidC/Sec dependent by making the periplasmic loop ground signals of E. coli proteins and lipids, including preparation of E. coli highly polar in the absence of charged residues. We also show that adding hy- inner membranes, reverse 13C- and 15N-isotope labeling, and selective signal drophobic amino acids to highly polar or charged loop can decrease the Sec- filtering with the frequency-selective REDOR pulse sequence. These improve- dependence of the otherwise strictly Sec-dependent substrates. Additionally ments have allowed us to characterize the structure and dimerization of APP we demonstrate that increasing the driving force of insertion by adding four TM and its variants in a native membrane environment. leucyl residues to the transmembrane segment leads to translocation of a highly charged region that was not inserted by native TM segment. Finally, 2460-Pos Board B67 we show that the length of procoat loop is a determinant for Sec dependent Structural and Dynamical Basis of Protein Kinase C Alpha Regulation by insertion. The combined results support the polarity/charge hypothesis and the C-Terminal Tail is consistent with the notion that membrane insertion occurs at the interface 1 2 2 Yuan Yang , Julia A. Callender , Alexandra C. Newton , of SecYEG and YidC. Tatyana I. Igumenova1. 1 Department of Biochemistry and Biophysics, Texas A&M University, 2463-Pos Board B70 2 College Station, TX, USA, Department of Pharmacology, University of Bip Binding Affects Integration of Transmembrane Domains California at San Diego, La Jolla, CA, USA. Mirjam Zimmermann1,2, Marco Janoschke2, Martin Spiess2. Protein Kinase C (PKC) isoenzymes are dynamic multi-modular proteins that 1Molecular and Membrane Biophysics, Institute of Biophysics, Linz, Austria, regulate signal transduction processes at the membrane surface. When acti- 2Biozentrum, Basel, Switzerland. 2þ vated by second messengers, such as Ca and diacylglycerol, PKC undergoes The Sec61 translocon mediates translocation of proteins into the endoplasmic a drastic conformational transition from the inactive cytosolic state to the acti- reticulum (ER) and enables integration of hydrophobic (H) segments into the vated membrane-bound state. The structure of either state of PKC is not known. lipid bilayer via its lateral gate. The luminal chaperone BiP (Kar2 in yeast) 2þ Using NMR, we demonstrate that the isolated Ca -sensing lipid-binding C2 has been shown to act as a molecular ratchet, recruited to the emerging poly- domain of PKCa interacts with the C-terminal tail of the kinase. We deter- peptide by the J-domain of Sec63, and to be involved in post- and cotransla- mined the structure of the complex between the C2 domain and the peptide cor- tional translocation. In this study, we address the question whether BiP responding to the phosphorylated hydrophobic motif of the C-terminal tail. The binding to the nascent chain affects the process of membrane integration vs. structure shows that the hydrophobic motif binds to the conserved lysine-rich translocation, i.e. whether integration is governed by thermodynamic equilib- cluster region of C2. The interface is stabilized by electrostatic interactions rium between pore and membrane. and stacking of the aromatic sidechains. We validated our structural model To determine the hydrophobicity threshold of membrane integration, a series of by mutating the interacting residues in full-length PKC, and characterizing model proteins based on the sequence of dipeptidylaminopeptidase B (DPAPB) these variants using in vitro FRET experiments and FRET-detected membrane with oligo-alanine H-segments containing increasing numbers of leucines. The translocation experiments in live cells. In addition, NMR-detected binding translocated loop preceding the H-segments was replaced by a sequence of 2þ studies revealed that the hydrophobic motif and Ca synergistically enhance similar length containing multiple copies of segments known to bind BiP each other’s affinities to C2. We propose a model where the C-terminal tail with high affinity or not to interact with BiP. While H-segments preceded by plays a dual role in PKC regulation: auto-inhibitory, through its interaction the DPAPB sequence required ~3.7 Leu for 50% integration, this threshold with the lysine-rich cluster of the C2 domain; and activating, through sensitiza- was reduced to ~2.1 Leu with the BiP-binding sequence and increased 2þ tion of PKC to intracellular Ca oscillations. Support: Welch Foundation grant to ~5.2 Leu with the non-binding sequence. These results show that mem- A-1784 (TII), NSF CAREER award CHE-1151435 (TII), NIH GM108998 brane integration of an H-segment is influenced by the already translocated (TII), and NIH GM43154 (ACN). sequence preceding it and suggest that BiP binding promotes membrane inte- gration. We observed changes of the apparent free energy in integration be- 2461-Pos Board B68 tween 1 to 3 kcal/mol. We propose a model in which Brownian motion of Site-Directed Spin Labeling EPR Spectroscopy of the Cytoplasmic Tail of the translocated loop with bound chaperones preferentially promotes release Influenza a M2 of the H-segment into the lipid bilayer. The work was supported by grant Alice L. Herneisen, Grace Kim, Kathleen P. Howard. W0125 of the Austrian science fund (FWF). Swarthmore College, Swarthmore, PA, USA. The M2 protein is a 97 residue homotetrameric, multifunctional ion channel 2464-Pos Board B71 that plays critical roles during the influenza infection cycle. While a variety D Elucidating the Uncoupling of ATP Hydrolysis and Ca2 Transport in of high-resolution biophysical techniques have been used to characterize the SERCA by Sarcolipin transmembrane domain (residues 22-46) and the juxtamembrane C-terminal Erin Birdsall1, Alysha Dicke1, Gianluigi Veglia1,2. region (46-62), less is known about the conformation and dynamics of the re- 1 2 maining residues of the C-terminal cytoplasmic tail. Here, we use site-directed BMBB, University of Minnesota, Minneapolis, MN, USA, Chemistry, University of Minnesota, Minneapolis, MN, USA. spin labeling electron paramagnetic spectroscopy (SDSL-EPR) experiments to 2þ probe the secondary structure and membrane topology of cytoplasmic tail res- The activity of the sarco(endo)plasmic reticulum Ca -ATPase (SERCA), which plays a key role in muscle contraction and relaxation, is regulated by sar- idues 60-80 when the protein is reconstituted into lipid bilayers. Cholesterol is colipin (SLN), a 31-residue transmembrane peptide. Sarcolipin has been shown essential for the role the C-terminal domain of the M2 protein plays in viral 2þ budding. SDSL-EPR data is collected in both in the presence and absence of to uncouple ATP hydrolysis and Ca transport in SERCA and is largely found cholesterol. in skeletal muscle and the atria of the heart. The interaction between SLN and SERCA is thought to play an important role in energy metabolism. SLN is 2462-Pos Board B69 composed of a transmembrane helix flanked by two short, unstructured N- Polarity and Charge as Determinants for Translocase Requirement for and C-termini. The N-terminus is hypothesized to play a role in the uncoupling Membrane Protein Insertion of SERCA, while the role of the C-terminus has been shown to be largely Balasubramani Hariharan1, Raunak Soman2, Ross E. Dalbey3. responsible for SERCA inhibition. In this study, we made two constructs of 1Biophysics Graduate program, The Ohio State University, Columbus, OH, SLN, with either the C- or N-terminus deleted. We then used coupled- USA, 2OSBP, The Ohio State University, Columbus, OH, USA, 3The Ohio enzyme activity assays to measure the effect of the SLN constructs on SERCA State University, Columbus, OH, USA. ATPase activity and isothermal titration calorimetry (ITC) to monitor the Membrane protein biogenesis in bacteria is a well-studied but poorly change in heat release due to ATP hydrolysis. We found through these methods understood area in the field of membrane proteins. In bacteria, the Sec that the deletion of the termini led effected the uncoupling function in SLN. translocon and the YidC insertase promote the membrane insertion process. The results of this study, in combination with forthcoming solid-state NMR Both proteins are essential, and universally conserved, and together insert studies, will help to establish the mechanism by which SLN regulates SERCA about 95% of the proteins in E.coli. Membrane insertion of SecYEG- activity.

BPJ 7891_7895 Wednesday, February 15, 2017 501a

2465-Pos Board B72 NMR are being used to document that our hypothesis is correct and to charac- NMR Resolves ’s Allosteric Regulation of the Sarco- terize the hydrogen bonding pattern. Initial results support our claim and raise D plasmic Reticulum Ca2 -ATPase an even more interesting question as to how FtsZ binds: does it bind using the Sarah E.D. Nelson, Vitaly V. Vostrikov, Tata Gopinath, Gianluigi Veglia. intrinsically disordered domain of the N-terminus or does it bind to the b-sheet? Biochemistry, Molecuar Biology, and Biophysics, University of Minnesota, Minneapolis, MN, USA. 2468-Pos Board B75 E. Coli Disruption of cardiac calcium cycling due to dysregulation of the sarco(endo) Molecular Dynamics Simulations of the Predicted FtsB-FtsL Tetra- plasmic reticulum Ca2þ-ATPase (SERCA) by the small transmembrane pro- meric Complex Gladys Diaz Vazquez1, Samson Condon2, Qiang Cui3, Alessandro Senes2. tein, phospholamban (PLN) leads to severe phenotypes including arrhythmia 1 and dilated cardiomyopathy (DCM). In order to understand how inherited mu- Biophysics, University of Wisconsin-Madison, Madison, WI, USA, 2Biochemistry, University of Wisconsin-Madison, Madison, WI, USA, tations in PLN lead to these severe phenotypes it is essential to map PLN’s 3 binding interaction with SERCA and how this binding interaction is modulated Chemistry, University of Wisconsin-Madison, Madison, WI, USA. by post-translational modifications in PLN. Division is one of the most important processes in the life of a cell. In bacteria Here we have applied both magic angle spinning (MAS) and oriented solid- division is accomplished by a complex of proteins that form what is call the di- state NMR techniques to characterize the structure, dynamics, and topology visome. FtsB and FtsL are two single-pass transmembrane proteins that form an of monomeric constructs of PLN in complex with SERCA to better understand essential part of the divisome. These two proteins interact with each other form- how post-translational modifications and mutations in PLN alter SERCA’s ac- ing a tetrameric complex, but the structure of their complex nor their function is tivity. By combining chemical shift perturbations, dipolar couplings, and other completely understood. In this work we aim to elucidate the structural organi- structural restraints with molecular dynamics simulations, we have built a zation of the FtsB and FtsL complex by using computational and experimental model of the PLN-SERCA complex that outlines a mechanism for PLN’s allo- techniques, in order to get a better understanding on how FtsB and FtsL help steric regulation of SERCA via its cytoplasmic domain. direct cell division in E. coli. We are working on a predicted tetrameric com- plex of FtsB and FtsL, obtained using co-evolutionary distance restraints. To 2466-Pos Board B73 get a better understanding of the validity of the model and the possible confor- Characterizing the Complex of Sarcolipin and SERCA using Solid-State mations and interactions that are present between the protein, we have been NMR Spectroscopy performing full-atom molecular dynamics simulations in explicit lipids. The re- Alysha A. Dicke1, Tata Gopinath1, Gianluigi Veglia1,2. sults indicate that the transmembrane region as well as the predicted coiled-coil 1BMBB, University of Minnesota, Minneapolis, MN, USA, 2Chemistry, of the complex stay together throughout the 150 ns simulation, whereas flexi- University of Minnesota, Minneapolis, MN, USA. bility is observed in the glycine rich linker region between the transmembrane Solid-state NMR (ssNMR) spectroscopy is a powerful tool to understand the and coiled-coil domains of FtsB. structure and function of membrane proteins. Oriented ssNMR allows for the 2469-Pos Board B76 determination of the topology of proteins in lipid bilayers, whereas magic angle Characterization of the FtsBL Membrane Protein Complex by Single spinning (MAS) NMR provides information more similar to solution-state. Us- Molecule TIRF Microscopy ing these two methods, we are working to better understand the interactions be- 1 1 2 1 2þ Claire R. Armstrong , Ambalika Khadria , Rahul Chadda , Aaron Hoskins , tween two proteins: sarcolipin and the sarco(endo)plasmic reticulum Ca Janice L. Robertson2, Alessandro Senes1. ATPase (SERCA). SERCA uses energy from ATP hydrolysis to transport 1 2þ Biochemistry, University of Wisconsin- Madison, Madison, WI, USA, Ca ions to cause muscle relaxation. Sarcolipin inhibits SERCA as well as al- 2 2þ Molecular Physiology and Biophysics, The University of Iowa, Iowa City, ters the ratio between ATP hydrolyzed and Ca transported. This interaction IA, USA. has significant implications for energy metabolism in the body. Using oriented The divisome is a multi-protein complex required for bacterial cell division. ssNMR, we have fully assigned the spectra of the transmembrane helix of sar- The divisome has been extensively characterized genetically, but its structural þ 2þ colipin in complex with SERCA in two states: AMPPCP /- Ca . Further- organization and the oligomeric state of its components remain mostly un- more, we have used MAS to further refine our structure of the complex and known. An essential part of the divisome is an intermediary sub-complex identify interactions in the terminal residues of sarcolipin with SERCA. formed by two bitopic membrane proteins, FtsB and FtsL, whose interaction Detailed structural information will lead to better insights as to how to modu- is mediated by their transmembrane domain, and their juxta-membrane coiled late this interaction and correct aberrations in SERCA’s activity that lead to coil domain. Our previous bulk Forster resonance energy transfer analysis of disease. the transmembrane regions of FtsB and FtsL showed that the FtsBL complex forms a stable higher order oligomer with equal number of FtsB and FtsL mol- 2467-Pos Board B74 ecules (i.e. 2:2 tetramer, 3:3 hexamer, or higher). Utilizing single molecule total Understanding the Role of the N-Terminal CrgA Interactions with FtsZ in reflection fluorescence microscopy (sm-TIRFM), we have analyzed the stoichi- Mycobacterium Tuberculosis the Divisome ometry of the full-length FtsBL complex in POPE/POPG bilayers. By moni- 1,2 2 1,2 1 Yiseul Shin , Riqiang Fu , Huajun Qin , Joshua Taylor , toring the photobleaching steps of fluorescently labeled FtsB and FtsL, we Malini R. Rajagopalan3, Timothy A. Cross1,2. 1 have acquired data that supports the fact that the FtsBL complex forms a stable Chemistry and Biochemistry, Florida State University, Tallahassee, FL, 2:2 tetramer. This work sets the stage for quantitative thermodynamic analysis USA, 2The National High Magnetic Field Laboratory, Tallahassee, FL, USA, 3 of the stability of the FtsBL wild type complex, as well as an array of know Biomedical Research Branch, The University of Texas Health Science functionally impaired mutants. The method also has the potential to be Center at Tyler, Tyler, TX, USA. extended to study the interactions of several other divisomal membrane pro- CrgA is a 93-residue helical membrane protein found in Mycobacterium Tuber- teins, which remain still biophysically uncharacterized. culosis (Mtb) divisome, the cell division apparatus, where it has been argued that it recruits other Mtb proteins to the divisome. Bacterial two-hybrid assays 2470-Pos Board B77 and pull down assays have documented 5 binding partners including FtsZ, Assessment of Protein-Protein Interactions in the FtsBLQ Complex using FtsQ, FtsI (PBPB), PBPA, and CwsA and have also shown that CrgA is a dimer. Fluorescence In Vitro A structure of the transmembrane domain of the monomer has been published Elizabeth Caselle, Samuel Craven, Alessandro Senes. (Das et al., 2015, PNAS 112:E119-E126) showing a pair of TM helices with a Biochemistry, University of Wisconsin-Madison, Madison, WI, USA. 30 residue N-terminus and an insignificant 2 residue C-terminus, both on the Cell division is an essential and challenging process in the bacteria life cycle. cytoplasmic side of the membrane while an interhelical loop of 20 residues Bacterial cell division is performed by the divisome, a complex consisting pri- is on the periplasmic side. A sampling of the first 18 residues show little anisot- marily of membrane proteins. Three of such membrane proteins, FtsB, FtsL and ropy in the solid state NMR spectra indicating that it is intrinsically disordered, FtsQ form a complex that is recruited during the middle stage of the assembly while a sampling of residues 19-30 show considerable anisotropy and are there- of the divisome. The FtsBLQ complex is essential for the recruitment of the fore thought to be structured. Since FtsZ is cytoplasmic it is thought to bind to downstream components that are responsible for the remodeling of the cell this N-terminus. A combination of mutagenesis and solid-state NMR of CrgA wall during cell division. The specific functional role of the FtsBLQ complex samples in liquid crystalline bilayer preparations of POPC/POPG bilayers is and the structural organization of the complex, however, are not yet well under- underway to characterize the structured domain of the N-terminus. The Ca stood. To address this, we have developed a fluorescence-based assay to assess isotropic chemical shifts for this region suggest that it is a b-strand conforma- the interactions that stabilize the association of FtsQ to the preformed FtsBL tion, and since there is little evidence for a loop in this 12 residue structured complex in vitro. With such assay, we have been investigating how specific domain the presumably this strand is stabilized by binding to the adjacent mutations affect the formation of the complex, as well as the possibility to monomer, i.e. an interprotein b-sheet. Isotopic labeling strategies for solid state disrupt the interaction by competition. Such approach could potentially lead

BPJ 7891_7895 502a Wednesday, February 15, 2017 to the developments of molecules that specifically target the FtsBLQ complex function result in congenital long-QT syndrome (LQTS), which predisposes in- and control bacterial division. dividuals to cardiac arrhythmia and can result in sudden death. We have previ- ously described the high level expression and purification of the S1-S4 voltage 2471-Pos Board B78 2D sensor domain (VSD) of human KCNQ1 (Peng-D et al., Biochemistry53:2032- Contribution of Individual Pore Residues to Mg Permeation and Diva- 42, 2014). Here we describe the results of NMR-based structural studies of this lent Cation Selectivity in Cora protein and of efforts to map its drug binding sites. This work was supported by Nicolaus Schmandt, Margaret Milewski, Eduardo Perozo. RO1 HL122010 and by F32 GM117770. Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, USA. 2474-Pos Board B81 Magnesium uptake in prokaryotes and mitochondria is mostly driven by elec- Exploring the Movement of S4 by Intermolecular FRET of Genetically- trophoretic influx through CorA, a homo-pentameric channel selective to diva- Encoded Voltage Indicators lent cations. Initial studies of macroscopic CorA currents in Xenopus oocytes Lee Min Leong, Bok Eum Kang, Bradley Baker. expressing CorA showed that Mg2þ-binding to the cytoplasmic side acts as a KIST, Seoul, Korea, Republic of. gating factor, defining CorA as a Mg2þ-deactivated, Mg2þ-channel. This gating Fusion of a voltage-sensing domain to the fluorescent protein, Super Ecliptic mechanism allows CorA to regulate intracellular Mg2þ concentration through a pHluorin A227D, transforms changes in membrane potential into an optical negative feedback loop. Cryo-EM studies have demonstrated that CorA gates signal. Exploration of the mechanism that couples the conformational change through an unusual mechanism whereby the release of intracellular bound of the voltage-sensing domain to the change in fluorescence suggests that the Mg2þ breaks channel symmetry, with one subunit moving away from the other genetically-encoded voltage indicator (GEVI) needs to dimerize via the cyto- four in the conducting state. We have expanded our analysis of the molecular plasmic fluorescent . This dimer configuration enables the basis of CorA permeability and selectivity by carrying out an alanine scan movement of S4 to drag a negative charge along the beta-can of a neighboring along TM1, the alpha helix that putatively lines the permeation path of the fluorescent protein resulting in a change in the environment of the chromophore channel. Individual mutants were expressed in Xenopus oocytes and evaluated and ultimately altering the fluorescent output. A process we call ‘optical from an analysis of CorA selectivity against other divalents and the properties etching’. To further investigate the dimerization of the GEVI, novel constructs of the anomalous mole fraction effect (AMFE) at various Mg2þ concentrations. fusing YFP or CFP to the voltage sensing domain were created and co- We identified Alanine mutants that either reduce the overall channel perme- transfected into HEK-293 cells. Robust voltage-dependent Fo¨rster Resonance ability while maintaining a strong AMFE, or that eliminate the AMFE and Energy Transfer signals were seen which indicate that the movement of S4 Mg2þ no longer blocks the inward sodium current. Using a combination of upon depolarization of the plasma membrane brings the chromophore dipoles functional, spectroscopic and computational methods, the study of these mu- of the fluorescent proteins closer together. This experimental paradigm allows tants should help address some of the fundamental principles associated to us to examine the biophysical consequences on the movement of S4 when CorA ion selectivity and permeability. changes to the linker segment and/or mutations to the voltage sensing domain are made. 2472-Pos Board B79 Research reported in this publication was supported by the National Institute Of Effect of GABRA1 Cytoplasmic Peptide on a Regulatory Protein Neurological Disorders And Stroke of the National Institutes of Health under Norbert W. Seidler. Award Number U01NS099691. This project is also funded by the Korea Insti- Basic Sciences, Kansas City University of Medicine and Biosciences, Kansas tute of Science and Technology (KIST) grants 2E26190 and 2E26170. City, MO, USA. The pentameric neurotransmitter-gated , GABA (type A) re- 2475-Pos Board B82 ceptor, is well-recognized for phasic and tonic neuronal inhibition. The phasic Dynamic Studies of Sweet Family Sugar Transporters through NMR Spec- response at the synapse is due to receptors typically containing two alpha, two troscopy beta, and one gamma subunit. Half of all GABA (type A) receptors in adult hu- Ampon Sae Her, Maureen Leninger, James Banigan. man brain have the alpha (type 1), or alpha-1, subunit (gene ID: GABRA1). New York University, new york, NY, USA. Each consists of an extracellular ligand-binding domain and four transmem- Sugar is a primary carbon source for prokaryotes, plants, and humans. brane helices (TM1-4), both of which are highly conserved, as well as a large Recently, a new family of sugar transporters called SWEETs was discovered intracellular loop domain (ILD), whose structure is unknown. The human re- and found to transport a variety of mono- and disaccharides across the cell ceptor is crystallized without the ILD, which connects TM3 and TM4, that is membrane. These proteins exist in all eukaryotic kingdoms including plants, engineered out. The topology and function of the 86-residue alpha-1 ILD re- fungi, and animals. SWEETs have ubiquitous expression throughout human mains to be fully elucidated. GAPDH (d-glyceraldehyde-3-phosphate dehydro- cell lines and have been proposed to help mediate vesicular glucose efflux genase) phosphorylates specific ILD residues, regulating receptor function. In from intestine and liver cells. Mutations in sugar transporters lead to a variety this study, we examined the effect of a synthesized ILD peptide (residues of diseases related to homeostatic imbalance of sugars. Therefore, elucidating 335 to 364 from GABRA1) on the oxidoreductase activity of GAPDH. The the sugar transport mechanism of this family of proteins is essential for devel- custom peptide, containing the first 30 cytosolic residues, was pre-incubated oping therapeutic drugs for treatment of diseases related to the homeostatic with GAPDH, and enzyme activity measured by NADH absorbance (340nm) imbalance of sugars. To date, the transport mechanism has been challenging upon oxidation of the substrate d-glyceraldehyde. When pre-incubated for to study due to the relatively weak binding affinity and difficulties associated 1hr at 20degC, the ILD peptide inhibited enzyme activity in a concentration- with capturing a substrate bound structure of SWEET transporters. To provide dependent manner showing an exponential non-linear function (y=a*e ^ (b/ atomic scale insight under functional conditions, a combination of solid-state xþc)). The half-maximum inhibition occurred at a [peptide] = 8.7micromolar. and solution nuclear magnetic resonance (NMR) spectroscopy was used to When pre-incubated for 48hr at 4degC, the ILD peptide further enhanced inhi- probe conformational dynamics of this family of transporters upon substrate bition of oxidoreductase activity, suggesting that hydrophobic forces play a role binding. Solid-state NMR experiments in native E. coli lipid bilayers were uti- in the interaction between the ILD peptide and GAPDH. Molecular modeling lized to study the interactions between sugar substrates and the binding pocket experiments indicate that the ILD binding site on GAPDH is in close proximity of SWEETs. In addition, solution NMR in native-like lipid nanodiscs were used to the phosphatidylserine-binding site on GAPDH. The findings may contribute for analyzing conformational dynamics in this family of membrane proteins. to our understanding of receptor function and point to possible treatments in The site-specific resolution available from NMR provided insights into protein conditions, such as Alzheimer’s dementia, where GABA receptor pharma- dynamics, the nature of protein-substrate specificity, and the transport mecha- cology is shown to play a role. nism of the SWEET family. 2473-Pos Board B80 2476-Pos Board B83 Structural Characterization of the Human KCNQ1 Voltage-Sensing Structural Snapshots of Eff-1 Mediated Membrane Fusion Domain by NMR Mariano Dellarole1, Annalisa Meola1,Ge´rard Pehau-Arnaudet2, Keenan C. Taylor, Hui Huang, Charles R. Sanders. Franc¸ois Bontems1, Mario J. Borgnia3, Thomas Krey1, Niels Volkmann4, Biochemistry, Vanderbilt, Nashville, TN, USA. Dorit Hanein4,Fe´lix A. Rey1. Voltage-gated ion channels are integral membrane proteins that mediate ion 1Structural Virology, Institut Pasteur, Paris, France, 2Ultrapole and UMR transport across membranes. KCNQ1 is a human voltage-gated potassium 3528, Institut Pasteur, Paris, France, 3National Cancer Institute, Bethesda, channel that is expressed in both epithelial and cardiac tissues. KCNQ1 associ- MD, USA, 4Sanford-Burnham Research Institute, La Jolla, CA, USA. ates with KCNE1 and mediates the Iks current responsible for the repolarization The mechanism of Eukaryotic cell-cell fusion remains unknown despite its of the . Mutations of KCNQ1 that cause a loss-of- relevance to various developmental pathways. This well regulated process

BPJ 7891_7895 Wednesday, February 15, 2017 503a was expected to require complex interaction systems and multiprotein machin- the oligomerization process of SP-D, the pathway and type of interactions eries. The discovery of the C. elegans EFF-1 protein as a single and direct involved in the formation of large oligomers, are not clearly understood. In fusing agent has challenged this concept. X-ray crystallography revealed that the current study, a structural characterization of recombinant human SP-D the ectodomain of EFF-1 is structurally homologous to class-II virus membrane (rhSP-D), produced in mammalian CHO cells, has been carried out by Atomic fusion proteins. In these viruses, acidification drives a change in the oligomer- Force Microscopy (AFM) and electrophoresis. The goal has been the determi- ization state of the fusion protein and exposes a hydrophobic loop. Both events nation of the distribution of oligomeric forms, the exploration of the possible concomitantly target and merge the viral membrane to the cellular membrane. oligomerization pathway and the description of the conformational diversity In contrast, EFF-1 needs to be located in both fusing membranes and is pro- of rhSP-D. AFM experiments revealed that rhSP-D is a mixture of trimers, hex- posed to fuse without a hydrophobic targeting loop. In consequence, several amers, dodecamers, and larger oligomeric species identified as ‘‘fuzzy balls’’, opposed hypotheses explaining how EFF-1 fuses cells coexist, whether based with the most abundant structure being the dodecamer under the conditions of or not on the class-II viral membrane fusion protein paradigm. To clarify the these experiments. We have developed a cross-linking protocol to detect the mechanism, EFF-1 driven fusion has to be studied in a lipid environment. presence of SP-D dodecameric forms by PAGE-SDS, in which dodecamer is We reconstituted recombinant EFF-1 produced in Drosophila Schneider 2 cells only visualized after chemical crosslinking and in the presence of denaturing into nanodiscs and liposomes and immunoisolated EFF-1 enriched extracellular agents, indicating the importance of hydrophobic interactions in dodecamer vesicles. A combination of cryo-electron microscopy (single particle, tomogra- formation. phy and subtomogram averaging) and spectroscopic techniques (dynamic light scattering) allowed us to assess the EFF-1 oligomerization state, its local con- 2479-Pos Board B86 centration and its spatial organization; and to correlate it to its activity require- Permeability of Pulmonary Surfactant Membranes is Modulated by Pro- ments. Our data suggests that EFF-1 mediated membrane fusion deviates from teins SP-B and SP-C 1 1,2 1 1 the mechanism described for class-II viral fusion proteins. Here, we present Marta Martı´nez-Calle , Elisa Parra ,Ba´rbara Olmeda , Jesu´sPe´rez-Gil . 1Biochemistry & Molecular Biology I, Complutense University of Madrid, EFF-1 fusogen in its natural milieu at near nanometer resolution, deepening 2 structural signatures of a powerful architecture adapted to work homotypically Madrid, Spain, Center for Single Particle Science and Engineering (SPSE), or heterotypically. University of Southern Denmark, Odense, Denmark. Pulmonary surfactant is a lipoprotein, membrane-based complex, which is 2477-Pos Board B84 required to stabilize the gas exchange surface at the mammalian alveoli. 3D Topological Structural Studies of Antimicrobial Peptides by Solid_ Hydrophobic proteins SP-B and SP-C are essential to promote a rapid adsorp- State NMR Spectroscopy tion of phospholipids at the air-liquid interface and to stabilize the surface Yongae Kim, Ji-Ho Jeong, Ji-Sun Kim. active films along inspiration-expiration cycles during breathing. Our aim is Hankuk University of Foreign Studies, Yong-In, Korea, Republic of. to further study the structure and functional properties of supramolecular as- The availability of antibiotics has allowed for the successful treatment of many semblies involving surfactant proteins SP-B and SP-C in the pulmonary surfac- bacterial infections as well as the ability to perform invasive medical proced- tant system. In the present work, we have studied the effect of SP-B and SP-C ures including surgery and chemotherapy. However, their wide use has led to on membrane permeability. Changes on the permeability of giant vesicles of pathogens’ increased drug resistance and the need to find novel classes of anti- different lipid-protein composition towards a set of polar fluorescent probes microbial peptides as alternatives to antibiotics. Lactophoricin (LPcin), a have been directly visualized by fluorescence microscopy and quantified by cationic amphipathic peptide consists of 23-mer peptide, corresponds to the image analysis. Surfactant proteins SP-B and SP-C seem to be associated carboxy terminal 113-135 region of component-3 of proteose-peptone. LPcin forming pores of a defined size in surfactant membranes. Specific blockage is a good candidate as a peptide antibiotic because it has an antibacterial activ- with antibodies against SP-B or SP-C prevents the diffusion of molecules ity but no hemolytic activity. Three different analogs of LPcin, LPcin-yk2 through the pores. Functional analysis suggest that pores formed by SP-B/ which has mutant amino acids, LPcin-yk1 and LPcin-yk3 that has shorter SP-C complexes might be part of a structure optimized to transfer surface- mutant amino acids are recently developed by using peptide engineering te- active lipid species along surfactant membranous assemblies until reaching chinques in our laboratory. These three LPcin analogs show better antibioitic the air-liquid interphase. activities than wild-type LPcin and no toxicity at all. In order to understand the structural correlation between LPcin analogs 2480-Pos Board B87 structure and antimicrobial activity under the membrane environments, we Toxic and Non-Toxic Forms of Membrane Poration are Mediated by tried to express and purify as large as amounts of LPcin and three different Structurally Distinct States of Islet Amyloid Polypeptide 1,2 3 2 1 LPcin analogs. We finally optimized and succeed to overexpress in the Melissa Birol , Sunil Kumar , Elizabeth Rhoades , Andrew Miranker . 1Yale University, New Haven, CT, USA, 2University of Pennsylvania, form of fusion protein in Escherichia coli and purified with biophysical tech- 3 niques like Ni-affinity chromatography, dialysis, centrifuge, chemical cleav- Philadelphia, PA, USA, New York University, New York, NY, USA. age, and reversed-phase semiprep HPLC. In here, we will present the Amyloid formation is a heterogenous process with multiple oligomeric species optimizing processes with high-yield expression and purification of three LPcin populated on the pathway to deposited fibrillar structures. These oligomers are analogs and solid- state NMR structural studies to figure out antibacterial highly diverse in size and structure, playing a key role in a wide range of protein killing mechanisms. misfolding diseases, including type 2 diabetes (T2D), Alzheimer’s disease and systemic amyloidosis. Here, we have investigated mechanisms of toxicity using 2478-Pos Board B85 islet amyloid polypeptide (IAPP), a peptide central to b-cell pathology whose Structural Characterization of Human Pulmonary Surfactant Protein misfolding and deposition is associated with T2D. IAPP toxicity is though to SP-D by Atomic Force Microscopy arise from oligomer-mediated damage of phospholipid membranes. The mech- Raquel Arroyo1, Alejandro Martin-Gonzalez2, Mercedes Echaide3, anism of membrane disruption is still debated, and the species of IAPP involved Jan Rosenbaum4, Fernando Moreno-Herrero2, Jesus Perez-Gil1. in its pathology remain unknown. In order to gain insight into toxic mecha- 1Biochemistry and Molecular Biology I, University Complutense Madrid, nisms of IAPP, we developed two new approaches: (i) in the first, we determine Madrid, Spain, 2Macromolecular Structures, National Center of size of IAPP-mediated membrane pores by measuring membrane disruption of Biotechnology, CSIC, Madrid, Spain, 3Biochemistry and Molecular Biology giant plasma membrane vesicles (GPMVs); and (ii) in the second, we deter- I, University Complutense Madrid, Madrid, Spain, 4Research & mine the structural properties of oligomers associated to protein dysfunction us- Development, Airway Therapeutics LLC, Madrid, OH, USA. ing an in vitro and in vivo ‘diluted-FRET’ approach in islet-derived cells. In the Pulmonary surfactant protein D (SP-D), a C-type (Ca2þ-dependent) lectin, GPMVs, we observe two kinetics phases of membrane leakage on biological belongs to the collectin family. It is found mainly in lung surfactant, partici- membranes, corresponding to the formation of small and large pores. The phys- pating in the innate immune defense of the lungs.SP-D monomer contains iological relevance of these pores are validated with mitochondrial depolariza- four structural domains: an N-terminus domain, a collagen region, an a-helical tion measurements. With FRET, we identify six distinct conformational IAPP coiled-coil neck and a C-terminus carbohydrate recognition domain (CRD). oligomers in islet cells. By varying peptide concentration to titrate toxic effects, Monomers form trimers through folding of the collagenous region into triple we can assign these oligomers to the porous states found in the membrane helices and the assembly of a coiled-coil bundle of a-helices in the neck region. disruption measurements on GPMVs. Moreover, we show that a recently re- These trimers are stabilized by two disulfide bonds in the cysteine-rich N-ter- ported small molecule inhibitor of intracellular IAPP toxicity has no effect minal domain. Trimers associate into higher order oligomers whose size and on the first kinetic phase of leakage but appears to inhibit the second revealing conformation is sensitive to environmental factors and the conditions during the significance and correlation of this kinetic phase of membrane leakage and purification and storage. Despite extensive studies carried out to characterize toxicity in cells.

BPJ 7891_7895 504a Wednesday, February 15, 2017

Membrane Protein Dynamics a significant role played by the C-terminal kink feature in modulating the bar- rier to strand separation. Finally, equilibrium simulations of NgBamA demon- 2481-Pos Board B88 strate lateral gate opening at 340K in a native lipopolysaccharide bilayer for the Parameterization of Cysteine Palmitoyl, Cysteine Farnesyl, Cysteine first time. Geranylgeranyl and Glycine Myristoyl for the Martini Forcefield 2484-Pos Board B91 Yoav Atsmon Raz1, Peter D. Tieleman2. 1 2 Hexameric E5 Protein of Human Papillomavirus Type 16 Forms a Low University of Calgary, Calgary, AB, Canada, University of Biological Selective Ion Channel - a Computational Analysis Science, University of Calgary, Calgary, AB, Canada. Dhani R. Mahato, Wolfgang B. Fischer. Peripheral membrane proteins such as the Ras and Src superfamilies go through Institute of Biophotonics, National Yang Ming University, Taipei, Taiwan. various post-translational modifications that attach specific amino acids with High risk type of Human papillomavirus type 16 (HPV 16) is the causative fatty acid tails. A post-transnationally modified protein can then bind to the agent of precancerous lesions that leads to cervical cancer. Cellular transforma- bilayer by anchoring itself to it. In our current study, we have used all-atom tion is caused by the early (E) oncogenes named E5, E6 & E7, out of which E5 simulations in the CHARMM36 force field to parameterize four of the more is the least understood. The E5 protein is active during viral infection. E5 pro- common post-translational modifications into the martini force field - palmitoy- tein when assembled into a hexamer allows the passage of small molecules or lated cysteine, farnesylated cysteine, geranylated cysteine and myristoylated ions to pass through the pore. glycine. A docking approach of E5 with its three transmembrane domains (TMDs) has Our process has been designed to reproduce the all-atom bonded parameters been used to generate a bundle model. Molecular dynamics (MD) simulations and the water-octanol partitioning free energies of these amino acids for the of the bundle embedded in a fully hydrated lipid bilayer allow water pockets martini force field. Free energy values were obtained through an alchemical re- to be generated due to the hydrophilic residues at the C-terminal side of the action coordinate via thermodynamic integration (TI) with the multiple state pore-lining second TMD. These residues are responsible to draw water mol- Bennet acceptance ratio method (MBAR). ecules into the pore and thus allow easy access of ions to pass through the pore. A Full Correlation Analysis (FCA) suggests asymmetric dynamic of 2482-Pos Board B89 the helices within the monomers of the hexameric bundle. The number of Binding, Folding, and Insertion of a b-Hairpin Peptide at a Lipid Bilayer Cl-ions passing through the pore is higher than that of the Na-ions under Surface: Molecular Dynamics Simulations various voltage conditions. Potential of mean force (PMF) calculations have Keon Reid, James Kindt. been done for a series of physiological ions crossing the pore to identify Department of Chemistry, Emory University, Atlanta, GA, USA. the specific selectivity of the bundle. In open pore structure ions experience Antimicrobial peptides (AMPs) are common in many biological systems and almost undiscriminating potentials, while in a narrow pore Ca- and Cl-ions act as host defenses against microbial pathogens. Existing theories on how face large energy barriers. AMPs permeabilize lipid membranes still lack mechanistic detail. In this study, we used atomistic molecular dynamics (MD) simulations to investigate 2485-Pos Board B92 D L the interactions of SVS-1 (KVKVKVKVP P TKVKVKVK), an anti-cancer Transport of Vitamin B12 and its Conjugate with Peptide Nucleic Acid b-hairpin peptide, with a fully hydrated neutral POPC bilayer and a mixture Through the E. Coli BtuB Outer Membrane Protein Explored with Steered of 1:1 anionic POPC/POPG bilayer. In agreement with experiment, peptides Molecular Dynamics in simulation bind more strongly to anionic bilayers than to neutral bilayers. Tomasz Pienko 1,2, Joanna Trylska1. Unfolded peptides at the anionic surface were observed to undergo several 1Centre of New Technologies, Warsaw, Poland, 2Department of Drug folding and mis-folding pathways. Folded peptides at the surface did not Chemistry, Faculty of Pharmacy with the Laboratory Medicine Division of spontaneously insert their hydrophobic faces into the bilayer on the 1 ms Medical University of Warsaw, Warsaw, Poland. timescale, but kept their Lys-rich face downward toward the lipid head Poor delivery of peptide nucleic acids (PNA) to bacterial cells is the main groups. Upon the application of sufficient surface tension, the peptides were reason for precluding their application as antigene or antisense antibacterials. observed to ‘‘flip and dip’’ into the bilayer within ~100 ns. Pre-folded However, some bacteria need to uptake such ingredients as vitamin B12 from SVS-1 peptides were observed to insert in neutral membranes with ample sur- the environment. Therefore, based on experiments performed in our group, we face tension, but remained at the surface or in solution if unfolded. We asso- proposed vitamin B12 as a potential transporter of PNA to bacterial cells. ciate this insertion phenomenon to increased hydrophobic exposure of lipid Vitamin B12 uptake by bacterial cells is receptor dependent but detailed mo- tails due to tension. Our simulations suggest electrostatics interactions and hy- lecular mechanism of this transport is still uncertain. Studies on Gram nega- drophobic exposure are key factors in witnessing an insertion event. The tive bacteria revealed energetic coupling between inner and outer membrane apparent absence of a pathway for a single peptide to produce a defect or proteins. The most probable model is that vitamin B12 first associates and pore in the bilayer suggests that the mechanism involves cooperative action binds to the outer membrane protein BtuB. Second, the BtuB receptor inter- of multiple peptides. acts with the inner membrane protein TonB which results in unfolding of the luminal domain of BtuB and enables the release of vitamin B12 to the 2483-Pos Board B90 periplasmic space. Next, captured by another protein BtuF, vitamin B12 is Simulations of Bama in a Native Outer-Membrane Model and Energetics delivered to the BtuCD complex which is further transferred to cytoplasm of Lateral Opening through the inner membrane in an ATP-dependent pathway. We focus on Karl Lundquist, James C. Gumbart. the second phase of this process. With steered molecular dynamics, we simu- Physics, Georgia Institute of Technology, Atlanta, GA, USA. lated the transport of vitamin B12 and its conjugate with PNA through the Gram-negative bacteria possess two cell membranes. The outer membrane is BtuB protein. The system was constructed based on the crystal structure of host to almost exclusively b-barrel transmembrane proteins. The b-barrel as- BtuB-TonB protein complex (PDB code: 2GSK) which we embedded in an sembly machinery (BAM) is a five-protein complex responsible for the inser- asymmetric and heterogeneous E.coli outer membrane supplied with explicit tion and assembly of outer membrane b-barrel proteins. The first crystal water and ions. The simulations were performed using NAMD 2.11 and structures of the transmembrane b-barrel domain of BamA were released in AMBER force field parameters. Trajectories have shown that the transport 2013. In these initial crystal structures, the BamA b-barrel possessed a weak of vitamin B12-PNA conjugate through the BtuB protein is mechanically interface between its first (1) and last (16) b strands, which led to strand sepa- possible and requires full unfolding of the BtuB luminal domain. Therefore, ration within 1 ms in our equilibrium simulations. This strand separation has together with our experimental evidence, we suggest that vitamin B12-PNA been proposed to act as a lateral gate for substrate passage into the membrane. conjugates may be transported via the BtuB receptor through the E. coli outer In addition, a hydrophobic mismatch near the barrel seam was shown to desta- membrane. bilize the membrane, potentially acting to aid integration of the substrate. Furthermore, full BAM complex crystal structures were recently released, 2486-Pos Board B93 showing accessory proteins rallying around this putative insertion region using Augmentation of Interactions between Dimers Stabilizes Higher Order the periplasmic domains of BamA as a scaffold. In order to address remaining Oligomers of Membrane-Bound K-Ras: A Single Molecule Perspective questions surrounding the role that BamA plays in the insertion and assembly Suparna Sarkar-Banerjee, Abdallah Sayyed-Ahmad, Priyanka Prakash, process, we have carried out additional equilibrium simulations of BamA in Kwang-Jin Cho, M.N. Waxham, John F. Hancock, Alemayehu A. Gorfe. several membrane bilayers. We also performed a calculation of energetic land- Integrative Biology and Pharmacology, McGovern Medical School at scapes associated with lateral gate formation under various conditions. These UTHealth, Houston, TX, USA. calculations reveal a lower energetic barrier to strand separation for BamA Ras proteins are small GTPases that control cell growth, differentiation and as compared to FhaC, a member of the same (Omp85) family. They also reveal proliferation. They primarily reside on the inner leaflet of the plasma membrane

BPJ 7896_7899 Wednesday, February 15, 2017 505a and are crucial anticancer drug targets. The structural organization of Ras pro- the propensity of lipids to form membranes of differing curvature dictates teins on the plasma membrane has attracted major interest in recent years. the relative population of these states. Multicomponent simulation of CW- Computational work by our group predicted three interfaces that are variously EPR spectra further show lipid bilayers of the highly curved cubic phases involved in the oligomerization of K-Ras, the most frequently mutated isoform stabilize an immobilized conformation of M2, hypothesized to be relevant of Ras proteins. In the current study, we examined a charge reversal K-Ras for curvature generation. We have also characterized the size distribution of mutant K101E and a double cysteine mutant K101C/E107C that represent con- large unilamellar vesicles of lipids of different curvature generation propen- structs with predicted weak and strong potential for oligomerization, respec- sities alone and in the presence of full-length M2 using dynamic light scattering tively relative to the oncogenic G12V control (WT). We used fluorescence (DLS). recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and image correlation spectroscopy (ICS) to monitor the distribution 2489-Pos Board B96 of dimers and higher-order oligomers of these proteins. Our data show that Pulsed EPR Studies of Bidirectional Allosteric Signaling in the TonB the distribution of monomers, dimers and higher-order oligomers is such that Dependent Transporter, BtuB the K101E mutant is mostly monomeric, K-Ras G12V exists as both dimer Arthur K. Sikora1, Benesh Joseph2, Morgan Matson3, Jacob Staley3, and higher-order oligomer whereas the percentage of dimers and larger oligo- David Cafiso3. mers is much greater in K101C/E107C, consistent with our prediction and data 1Chemsitry, LaGrange College, Lagrange, GA, USA, 2Chemsitry, Johann from signaling assays. These results establish that K101 and E107 play a key Wolfgang Goethe University, Frankfurt, Germany, 3Chemsitry, University of role in K-Ras oligomerization and establish the co-existence of diverse Virginia, Charlottesville, VA, USA. K-Ras oligomeric states. Outer-membrane TonB-dependent transporters function in the uptake of essen- tial nutrients, and are important for the success of many pathogenic bacteria. 2487-Pos Board B94 During transport, these proteins undergo a cycle of binding and unbinding to Molecular Modeling and Solution NMR Studies of the Structure and Dy- namics of K-Ras at a Lipid Membrane Containing PIP2 the inner membrane protein TonB, through an interaction that is mediated by Matthias Buck, ZhenLu Li, Shufen Cao. the Ton box, an energy-coupling segment near the transporter N-terminus. Physiology & Biophysics, Case Western Reserve University, Cleveland, Over 50 high-resolution crystal structures have been obtained for 12 different OH, USA. TonB-dependent transporters, however the mechanisms of substrate transport Due to its vital role in regulating the cellular signal transduction and a high remain unclear. Determination of membrane protein structure or dynamics with high resolution using highly heterogeneous native systems is an attractive occurrence in human tumors, the small GTPase K-Ras has been the object of intense experimental and computational studies recently. In particular, way to solve transport mechanisms. research has focused on the role of membrane anchor on the structure and In this work, the cobalamin transporter BtuB was overexpressed and spin function of K-Ras. In this research, we investigate the structure and dynamics labelled in outer membranes, interspin distances were measured to a spin of K-Ras (GTP bound state) interacting with lipid membranes containing PIP2 labelled cobalamin and between outer loop positions using pulse EPR. TonB (Phosphatidylinositol 4, 5-bisphosphate) by performing long time scale all binding on the periplasmic surface of the protein alters extracellular loop conformation allosterically. This TonB binding also partially dissociates a atom molecular dynamics simulation with the CHAMM36 force field. The spin labeled substrate analog likely by lowering the affinity of the transporter simulations show that that the K-Ras catalytic domain binds to the membrane within 100 ns, mainly due to the electrostatic attraction between membrane for the substrate. Continuous wave EPR measurements show that transport defi- and K-Ras catalytic domain. PIP2 lipids, which possess four negative charges, cient mutations in the Ton Box alter the vitamin B12 binding site. Taken form persistent (up to several hundreds of nanoseconds) contacts with residues together these data indicate a clear allosteric coupling between the Ton Box (Arginine and Lysine) from the GTPase catalytic domain. Further, the lipid and the extracellular substrate binding site, and that TonB binding may initiate bound K-Ras presents several different membrane orientations with respect a partial round of transport. This work was supported by NIGMS, GM035215 (DSC) and Deutsche For- to the membrane. We find that the competitive interaction between the attrac- tion of membrane with the positively charged protein residues and repulsion of schungsgemeinschaft SFB 807 (BJ). membrane with the negatively charged residues likely determines the K-Ras4A orientation, but this also influenced by the extent of the K-Ras sur- 2490-Pos Board B97 faces (in relation to the proteins shape)The simulation results are validated by Oligomerization and Raft Partitioning Increase Plasma Membrane Local- an NMR solution study of K-Ras with nanodiscs and liposomes. Both the full ization of Transmembrane Proteins length and hypervariable region truncated GTPase interact transiently with Joseph H. Lorent, Ilya Levental. PIP2 doped membrane model systems through its catalytic domain. Similarly Integrative Biology and Pharmacology, UT Health Science Center at to another experimental and simulation study of K-Ras4B (reviewed by us in Houston, Houston, TX, USA. [1]), the results imply that membrane orientations could be correlated with Transmembrane proteins (TMPs) comprise ~30% of the mammalian proteome K-Ras function by hindering or exposing the effector binding domain of the and mediate nearly all functions of the cell. Signalling and transport of GTPase to solvent. Furthermore, the effect of membrane binding on the flex- transmembrane proteins are often modulated by oligomerization and associa- ibility and conformation of nucleotide binding region, especially with the tion with lipid-driven lateral membrane domains known as lipid rafts. switch loops of K-Ras will be discussed. Reference: [1] Li, Z. L.; Cao S.; We recently identified structural features that determine raft affinity of Buck, M.; New & Notable: K-RAS at Anionic Membrane: Orientation, Orien- TMPs, namely transmembrane domain (TMD) length, palmitoylation and tation/Orientation. Recent Simulations and Experiments. Biophys. J. 2016, 5, the TMD accessible surface area. Since oligomerization and raft partitioning 1033-1035. depend on the TMD, we investigated the relationship of structural determi- nants guiding oligomerization, raft affinity and plasma membrane localization 2488-Pos Board B95 of TMPs by combining experimental and computational approaches. Rational Site-Directed Spin-Label EPR Spectroscopy of M2 Protein in Lipid Bila- mutagenesis of the TMD of LAT (Linker for activation of T-cells) led to the yers of Different Curvature Generation Propensity discovery of a motif (PxxxGxxxxP) that is necessary and sufficient for raft Douglas S. Arbuckle1, Kathleen P. Howard1, Tyler Alexander2, phase partitioning in Giant Plasma Membrane Vesicles (GPMVs) and very Catherine Crouch2. similar to a known dimerization motif in transmembrane proteins (GxxxG). 1Department of Chemistry and Biochemistry, Swarthmore College, Molecular modelling showed that dimerization reduces effectively the acces- Swarthmore, PA, USA, 2Department of Physics, Swarthmore College, sible surface area of the TMDs in contact with the surrounding lipid raft phase Swarthmore, PA, USA. and thereby increases raft association. We confirmed that this motif drives M2 is a homotetrameric membrane protein critical to generation of membrane LAT TMD oligomerization by FLIM-FRET in GPMVs and live cells. Bioin- curvature in the budding stage of the influenza A virus life cycle. We have de- formatics analysis revealed the physiological relevance behind these observa- signed and interpreted site-directed spin-label electron paramagnetic resonance tions and showed that dimerization motifs are over-represented in plasma spectroscopy (SDSL-EPR) experiments on the conformation and dynamics of membrane proteins compared to other cellular organelles in the human prote- M2 in lipid bilayers of different propensities for membrane curvature genera- ome. We confirmed this finding in cells and showed that TMPs that efficiently tion. We have obtained data for full length M2 protein spin labelled at multiple partition to raft domains also localize to the plasma membrane. Thus, our sites in the C-terminal juxtamembrane region, which forms a membrane asso- study shows that dimerization plays a key role in raft partitioning, which in ciated amphipathic helix. Continuous wave (CW) and pulsed EPR spectra pro- turn leads to effective transport of transmembrane proteins to the plasma vide evidence that M2 adopts two conformational states in bilayers, and that membrane.

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2491-Pos Board B98 2493-Pos Board B100 Importance of Dimerization in Facilitating the Functional Dynamics of Temperature-Sensitive Gating of TRPV1 Channel as Probed by Atomistic Neurotransmitter: Sodium Symporters Simulations Mert Gur1,2, Mary H. Cheng2, Elia Zomot3, Ivet Bahar2. Anton O. Chugunov1, Pavel E. Volynsky1, Nikolay A. Krylov1,2, 1Mechanical Engineering Department, Istanbul Technical University, Dmitry E. Nolde1, Roman G. Efremov1,3. Istanbul, Turkey, 2Computational and Systems Biology Department, 1Lab. of Biomolecular Modeling, Shemyakin-Ovchinnikov Institute of University of Pittsburgh, Pittsburgh, PA, USA, 3Department of Biomolecular Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Sciences, Weizmann Institute of Science, Reovot, Israel. Federation, 2Joint Supercomputer Center, Russian Academy of Sciences, Neurotransmitter:sodium symporters (NSS) constitute a large family that in- Moscow, Russian Federation, 3National Research University Higher School cludes many transporters involved in excitatory signaling which serve as of Economics, Moscow, Russian Federation. important drug targets. A prominent example is dopamine transporter Heat-activated transient receptor potential channel TRPV1 is one of the most (DAT). DAT is essential to mediating dopamine signaling and binding drugs studied eukaryotic proteins involved in temperature sensation. Upon heating, of abuse such as amphetamine and cocaine. Understanding the mechanism of it exhibits rapid reversible pore gating, which depolarizes neurons and gener- function of NSS, and in particular DAT, is crucial for designing methods for ates action potentials. Underlying molecular details of such effects in the modulating NSS function. NSS are characterized by the so-called bacterial pore region of TRPV1 is of a crucial importance to control temperature re- leucine transporter (LeuT) fold, the first structurally resolved member of the sponses of the organism. Despite the spatial structure of the channel in both family. LeuT and a few other members of the family have been crystallized open (O) and closed (C) states is known, microscopic nature of channel gating in dimeric state. However, it is still not clear whether NSS function as mono- and mechanism of thermal sensitivity are still poorly understood. In this work, mers or dimers, or both. The effect of dimerization on the dynamics of these we used unrestrained atomistic molecular dynamics simulations of TRPV1 transporters, which may directly define their structure-encoded mechanism of (without N- and C-terminal cytoplasmic domains) embedded into explicit lipid function, remain to be elucidated. Our study is the first, computational, bilayer in its O- and C-states [1]. We found that the pore domain with its neigh- exploring the change in the collective dynamics of LeuT and DAT upon boring loops undergoes large temperature-dependent conformational transi- dimerization. We focused on the effect of dimerization on the intrinsic ability tions in an asymmetric way, when fragments only one monomer move with of these transporters to transition between outward-facing (OF) and inward- large amplitude, freeing the pore upon heating. Such an asymmetrical gating facing (IF) states - a cooperative structural change essential to substrate- looks rather biologically relevant because it is faster and more reliable than and ion-reuptake from the extracellular region, and release to the intracellular traditionally proposed ‘‘iris-like’’ symmetric scheme of channel opening. Anal- region. To this aim, we analyzed the soft modes of motion accessible to the ysis of structural, dynamic, and hydrophobic organization of the pore domain dimer and monomer (obtained separately for DAT and LeuT using the aniso- revealed entropy growth upon TRPV1 gating, which is in line with current con- tropic network model) and evaluated their overlap with the structural changes cepts of thermal sensitivity. involved in the OF4 IF transitions. The dimeric state of the transporters is Acknowledgements: Authors thank Russian Science Foundation (14-14- clearly shown to have a significantly higher tendency to undergo the func- 00871), Russian Foundation for Basic Research (16-04-00578), RAS MCB tional transition OF4 IF (in both directions), compared to the monomeric Program. Access to computational facilities of the Supercomputer Center state. This is a very important result, lending support to the physical signifi- ‘‘Polytechnical’’ at the St. Petersburg Polytecnic University is gratefully cance of dimerization. acknowledged. Reference: 2492-Pos Board B99 [1] A.O. Chugunov et al., Scientific Reports 6 (2016) 33112. Mapping Ceacam 1-4L’s Nanoscale Spatial Distribution, Structure, 2494-Pos Board B101 Dynamics, and Association with Lipid Ordered Domains by Super- Molecular Modelling of Hexamer and Tetramer Forms of the Orai Resolution Microscopy Calcium Channel 1 2 3 Amine Driouchi , Scott Gray-Owen , Christopher M. Yip . 1,2 2,3 1 2 Tugba N. Ozturk , Guillaume Lamoureux . Biochemistry, University of Toronto, Toronto, ON, Canada, Molecular 1 3 Department of Physics, Concordia University, Montreal, QC, Canada, Genetics, University of Toronto, Toronto, ON, Canada, Chemical 2Centre for Research in Molecular Modeling (CERMM), Montreal, QC, Engineering, University of Toronto, Toronto, ON, Canada. Canada, 3Department of Chemistry and Biochemistry, Concordia University, Super-resolution microscopy has provided novel quantitative insights into Montreal, QC, Canada. the nanoscale spatial distribution of membrane proteins, including the obser- In many cell types, calcium influx is carried out by the activation of calcium vation that some systems form micro- and nano-sized clusters. However, de- release-activated calcium (CRAC) channels in the plasma membrane. CRAC tails of the protein’s self-association state (monomer / dimer / oligomer) channels are assembled from Orai proteins and stromal interaction molecules within the different cluster types are often missing. Since it has been shown (STIM). Orai proteins form the transmembrane subunit of CRAC channels, that a protein’s oligomeric form might be a crucial determinant of its function, and STIM proteins play a role in their activation. Although recent crystal struc- it is important to have a technique capable of measuring both spatial distribu- ture of Orai exhibits a hexameric form with a threefold, quasi-sixfold axial sym- tion and association state. To that end, we have applied a correlated metry around the central axis of the channel [Hou et al., Science 338, 1308 dSTORM/homo-FRET approach to characterize CEACAM 1-4L, the progen- (2012)], it has been suggested that only the tetrameric form of Orai achieves itor isoform of the carcinoembryonic antigen-related cellular adhesion calcium selectivity, whereas the hexameric complexes of Orai produce nonse- molecule (CEACAMs) family. CEACAMs are cell surface glycoproteins lective cationic currents [Thompson and Shuttleworth, Sci. Rep. 3, 1961 involved in homo- and hetero-philic intercellular interactions that control (2013)]. In this study, we examine the stability of the hexameric Orai structure cellular growth, differentiation, tumourigenesis, inflammation and infection. and of a model of the tetrameric structure built using the hexamer as a template While CEACAM1-4L is known to exist as a monomer or oligomer with by performing all-atom molecular dynamics simulations. To explore the func- the former being hypothesized to be involved in activatory intercellular tional significance of both forms of the Orai channel (hexamer and tetramer), interactions, the basis for its spatial distribution and association state as a we analyze the contacts between the channel subunits, the change in pore radius function of distribution remain poorly understood. Our correlated approach during the simulation, and the occupancy of the pore by water and ions. To involved live cell homo-FRET studies of eYFP-CEACAM1-4L in HeLa investigate ion selectivity, we also calculate the free energy profiles for cal- cells followed by fixed cell dSTORM imaging performed using nanobody la- cium, potassium and sodium ions permeating through the pore. beling with Alexa Fluor 647 (AF647) conjugated to an anti-eYFP single domain antibody. Cluster analysis of the dSTORM datasets using Voronoi 2495-Pos Board B102 tessellation revealed that the CEACAM1-4L nanoclusters were preferentially Time-Resolved Wide-Angle X-Ray Scattering Reveals Protein Quake in comprised of monomeric CEACAM1-4L while microclusters contained a het- Rhodopsin Activation erogeneous distribution of CEACAM1-4L monomers and oligomers. To S. M.D.C. Perera1,X.Xu2, A.V. Struts1,3, U. Chawla1, S. Boutet4, further characterize these clusters, super-resolution imaging using Alexa- S. Carbajo8, M.D. Seaberg4, M.S. Hunter4, J.M. Martin-Garcia6, J.D. Coe5,6, 647 labelled cholera toxin was performed to examine the co-localization of M.O. Wiedorn7,8, G. Nelson9, S. Chamberlain10, D.P. Deponte4, lipid ordered domains, which are known to drive membrane protein lateral R. Fromme5,6, T.D. Grant10,11, R.A. Kirian5,9, P. Fromme5,6, M.F. Brown1,2. distribution and clustering, with CEACAM1-4L. Finally, live-cell single par- 1Department of Chemistry and Biochemistry, University of Arizona, Tucson, ticle tracking (SPT) of CEACAM1-4L was performed to investigate the puta- AZ, USA, 2Department of Physics, University of Arizona, Tucson, AZ, USA, tive confined, obstructed, free diffusion, and directed motion dynamics of 3Laboratory of Biomolecular NMR, St. Petersburg State University, St. CEACAM1-4L. Petersburg, Russian Federation, 4Linac Coherent Light Source, SLAC

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National Accelerator Laboratory, Menlo Park, CA, USA, 5Center for Applied the proton donor E108, and membrane environment on the dynamics of PR Structural Discovery at the Biodesign Institute, Arizona State University, during the early stages of its photocycle. Our findings show that hydration Tempe, AZ, USA, 6Department of Chemistry and Biochemistry, Arizona of the protein interior directly affects the dynamics of the transmembrane he- State University, Tempe, AZ, USA, 7Department of Physics, University of lices. Specially, helices F and G and the EF loop are highly dynamic, in agree- Hamburg, Hamburg, Germany, 8Center for Free Electron Laser Science, ment with experimental observations for PR and bacteriorhodopsin [2]. Deutsches Elektronen-Synchrotron DESY, Hamburg, Germany, 9Department Residues that play an important role in the photocycle are generally more of Physics, Arizona State University, Tempe, AZ, USA, 10Department of hydrated. In addition, hydration of E108 in the deprotonated state is Structural Biology, State University of New York, Buffalo, NY, USA, considerably larger than in the protonated state. Finally, solubilization with 11Hauptman-Woodward Medical Research Institute, Buffalo, NY, USA. detergent micelles remarkably alters the stability and mobility of different Rhodopsin is the canonical prototype of class A G-protein-coupled receptors components of PR compared to a bilayer environment. The complex of PR (GPCRs) that are the targets of many human pharmaceuticals. Here we with dodecylphosphocholine (DPC) detergent micelle is highly dynamic, report the use of X-ray free electron laser (XFEL) technology in a pump- such that the helices are displaced from their position in the initial structure probe, time-resolved wide-angle X-ray scattering (TR-WAXS) experiment and the EF loop is partially unfolded, while PR complexes with n-dodecyl- to study the early activation events of the visual receptor rhodopsin. The b-D-maltoside (DDM) are more stable. XFEL beam generated by the Linac Coherent Light Source (LCLS) at [1] Bamann, C. et. al., 2014 Biochemica et Biophysica Acta 1837:614. SLAC National Accelerator Laboratory has a very high peak X-ray bright- [2] Andersson, C. et. al., 2009 Structure 17:1265. ness, which is key for probing elusive ultra-fast structural changes that occur during biological function of protein molecules. A highly sophisticated pump- 2498-Pos Board B105 probe experiment with femtosecond time resolution based on the diffraction- The Blue-Light Quenching Effect of Proteorhodopsin Carl E. Eckert1, Jagdeep Kaur2, Clemens Glaubitz2, Josef Wachtveitl1. before-destruction principle was carried out using a 525-nm pump laser and 1 2 LCLS as the probe with 40-fs X-ray pulses. Rhodopsin was purified in Institute of Physical Chemistry, Frankfurt am Main, Germany, Institute of CHAPS detergent from bovine retinas, and was delivered to the X-ray Biophysical Chemistry, Frankfurt am Main, Germany. beam using micro-jet technology involving a gas-dynamic virtual nozzle From bacteriorhodopsin as well as proteorhodopsin (PR), it is known that (GDVN) of 50-mm diameter. The 2D images of the scattered X-rays were re- excitation of the M intermediate which contains a 13-cis retinal with deproto- corded with an ultra-fast Cornell-SLAC Pixel Array Detector (CSPAD) with nated Schiff base disrupts the photocycle significantly. This so called blue- pump laser on-versus-off frequency of 60 Hz (X-ray pulse frequency 120 Hz) light quenching effect (BLQ) results in an immediate halt of proton pumping generating light minus dark-state data for specified time points after rhodopsin activity and the protein returns to its ground state or a state spectroscopically photoactivation. The 2D-scattered X-ray intensities were radially integrated to very similar to the ground state. Thus, the BLQ effect offers a way for light generate 1D-scattering profiles (scattered X-ray intensity versus momentum control of microbial retinal proteins. However, almost 40 years after its dis- transfer vector, Q). On-the-fly data analysis using the OnDA software package covery, still little is known about the molecular mechanism. The particular revealed the light-triggered ‘‘protein quake’’ in rhodopsin during very early processes that are triggered by the BLQ effect and the time scale of which stages of its activation (within 4 ps). The data collected from 200 fs to they occur are unclear. We investigate the BLQ effect on PR with time- 2400 ps showed changes in the difference profiles indicative of conforma- resolved spectroscopy. Background illumination of the PR proton donor tional changes of rhodopsin during its activation process. Pump-probe exper- mutant E108Q leads to an increased accumulation of the M intermediate in iments involving an XFEL bring structural biology to a whole new level by the photostationary equilibrium. Subsequently, fs pump-probe experiments enabling scientists to record high-resolution molecular movies of biomole- in the visible and mid-IR range are conducted and provide insight into the cules in action. isomerization dynamics of the photoexcited M state together with the nano- second reprotonation step of the Schiff base. Based on our findings of the 2496-Pos Board B103 BLQ effect in PR, we currently extend our experiments on other microbial Dynamic Hydrogen-Bonding Networks in Photosystem II rhodopsins, like channelrhodopsin. Federico Guerra, Ana-Nicoleta Bondar. 2499-Pos Board B106 € Physics, Freie Universitat, Berlin, Germany. Characterization of Rhodopsin’s Activation Mechanism using Multi-Basin Photosystem II is a large membrane-embedded complex that uses cofactor mol- Structure-Based Models ecules to split water molecules into molecular oxygen, electrons, and protons. Letty Salas, Tod D. Romo, Alan Grossfield. The dynamics of protein/water hydrogen-bonded networks that could serve as University of Rochester, Rochester, NY, USA. proton transfer pathways are poorly understood. A particularly important issue G protein-coupled receptors (GPCRs) are integral membrane proteins that can is whether dynamic carboxylate clusters contributed by the extrinsic PsbO transduce extrinsic information across lipid membranes. This allosteric process, subunit of photosystem II could participate in proton transfers. As a first step which involves large conformational changes upon receptor activation, is not towards addressing these questions, and relying in part on force-field parame- well understood yet. Crystal structures of the inactive and active conformations ters derived by us, we carry out all-atom molecular dynamics simulations of several GPCRs showing these activation-induced structural differences have of wild-type and mutant photosystem II embedded in a hydrated lipid mem- been successfully solved in the last decade. However, to fully understand the brane. The simulations provide the foundation for us to characterize the dy- molecular basis of GPCR activation and deactivation, it is also necessary to namics of protein/water hydrogen-bond networks that can connect the elucidate the nature of the pathway or pathways linking these conformations. reaction site to the bulk, and the response of the networks to mutations and To date, the intrinsic timescales of such processes still poses a substantial dif- changes in protonation. ficulty for capturing state transitions using unbiased molecular dynamics sim- This work was supported by the DFG Collaborative Research Center SFB ulations with standard force fields at high molecular resolutions. Here, we use 1078 ‘Protonation dynamics in Protein Function’ Project C4 (to A-N.B.), simpler structure-based (Go-like) potentials to model the energy landscape of and by the HLRN, The NorthGerman Supercomputing Alliance (bec00063, GPCR rhodopsin. This approach allows for extensive sampling of the recep- to A-N.B.). tor’s transition pathways with relatively inexpensive all-heavy atom simula- tions. We also incorporate a potential mixing strategy that facilitates the 2497-Pos Board B104 inclusion of multiple protein states and the study of state interconversion in Proteorhodopsin Hydration and Dynamics is Governed by Surrounding equilibrium. Environment Sadegh Faramarzi, Jun Feng, Blake Mertz. 2500-Pos Board B107 West Virginia University, Morgantown, WV, USA. Energy Landscape Model and Spatial Motion Models in Rhodopsin Proteorhodopsin (PR) is a light-activated retinal protein with the ability to Activation pump protons from the cytoplasmic to the extracellular side of the cell [1]. Suchithranga M.D.C. Perera1, Udeep Chawla1, Andrey V. Struts1,2, The proton pumping process is highly efficient and can be reversed depending Michael F. Brown1,3. on environmental pH. These properties make PR a promising candidate in 1Department of Chemistry and Biochemistry, University of Arizona, Tucson, design of applications involving harvesting of solar energy. Although the pho- AZ, USA, 2Laboratory of Biomolecular NMR, St. Petersburg State tointermediates in the photocycle are well-defined spectroscopically, the role University, St. Petersburg, Russian Federation, 3Department of Physics, of dynamics and internal hydration of PR on the process of the proton pump- University of Arizona, Tucson, AZ, USA. ing is poorly understood. In this study, we have used molecular dynamics G-protein-coupled receptor (GPCR) function is associated with significant fluc- (MD) simulations to determine the role of hydration, protonation state of tuations of the protein structure [1]. Activation of the visual GPCR rhodopsin

BPJ 7896_7899 508a Wednesday, February 15, 2017 leads to changes in protein dynamics, followed by binding of the cognate (OPN1LW) has a much higher affinity for dimerization than that of human G-protein (transducin) initiating the biological signaling. Present X-ray crystal- green (OPN1MW) and blue (OPN1LW) cone opsins. We then identified three lographic structures do not reveal changes in protein dynamics which are the amino acid positions in transmembrane helix 5 for which OPN1MW-OPN1LW key for understanding the activation mechanism. Here we compare an energy swap mutations disrupted dimerization in red cone opsin and induced dimeriza- landscape model (ELM) and spatial motion model (SMM) analysis of both tion in green cone opsin. These same amino acids were found to be responsible elastic and quasielastic neutron scattering (QENS) data to explain regulation for 20% of the spectral distinction between red and green cone opsins. Identi- of integral membrane protein mobility by the retinal cofactor of rhodopsin. fication of amino acids that regulate cone opsin dimerization affinity provides Mean-square displacements calculated from elastic incoherent neutron scat- unique insight into the larger problem of GPCR dimerization, for which there is tering (EINS) are consistent with a dynamical transition as observed for glob- little quantitative data to test hypothetical dimerization interfaces. It also has ular proteins. In the SMM analysis the quasielastic spectrum is dissected into an potential applications in understanding the molecular basis of color vision. elastic peak due to quasistatic atoms, and quasielastic wings due to homoge- We will also present recent data on analogous mutations in rhodopsin as well nous line broadening from the mobile atoms in the protein. By contrast, the as the potential role of transmembrane helix 6 in red cone opsin dimerization. ELM adopts a wave-mechanical approach and describes the QENS spectrum in terms of inhomogeneous lines due to the various conformational substates 2503-Pos Board B110 of the protein [2]. Application of mode-coupling theory as developed for Investigating the Gating Mechanism of G Protein-Activated Inward Recti- glass-forming liquids to SMM analysis of the QENS spectra reveals a slowing fying Potassium Channels down of picosecond-nanosecond dynamics in the b-relaxation region for Harald Bernsteiner, Anna Stary-Weinzinger. ligand-free opsin versus dark-state rhodopsin. Alternatively, ELM analysis re- Pharmacology and Toxicology, University of Vienna, Vienna, Austria. veals that the ensemble of conformational substates in opsin is smaller versus Opening of inward rectifying Kir3 channels leads to hyperpolarization of neu- the dark state. The results are consistent with increased local crowding due to a rons and as a result to inhibitory control of excitability. It is known that Kir more collapsed protein structure in ligand-free opsin versus the dark-state channels open after PIP2 binding. Additionally to PIP2, the Kir3.x family is € rhodopsin. A novel powdered GPCR preparation method together with the also gated by binding of Gbg subunits of G proteins (Luscher and Slesinger, QENS technique uncovers changes in structural fluctuations governed by 2010). There are crystal structures in closed and open states with or without retinal cofactor of rhodopsin. PIP2-analogues bound available (Whorton and MacKinnon, 2011). A G protein [1] A.V. Struts et al. (2011) PNAS 108, 8263-8268. bound state of Kir3.2 has also been provided (Whorton and MacKinnon, 2013). [2] H. Frauenfelder et al. (2014) PNAS 111, 12764-12768. Currently we are investigating the conductance mechanism of this channel by performing molecular dynamics (MD) simulations. By applying a driving force 2501-Pos Board B108 we can observe conductance events in both inward and outward directions. Our Activation of GPCR Rhodopsin Investigated by Solid-State NMR simulations suggest that residue E152, which is unique to the Kir3.x family (all Spectroscopy other families have a GLN residue at this position) seems to play a critical role Andrey V. Struts1,2, Xiaolin Xu3, Trivikram R. Molugu1, Mike C. Pitman1, for gating of this channel. Further our simulations reveal that the ion concen- Samira Faylough4, Charitha Guruge4, Carolina L. Nascimento4, tration in the channel cavity appears to surprisingly high (4-6 ions). This is Nasri Nesnas4, Michael F. Brown1,3. in contrast to a recent study on Kv1.2 (Sumikama and Oiki, 2016). Summari- 1Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA, zing our simulations provide insights into the conduction mechanism of inward 2Laboratory of Biomolecular NMR, St. Petersburg State University, rectifier potassium channels. St. Petersburg, Russian Federation, 3Physics, University of Arizona, Tucson, 4 2504-Pos Board B111 AZ, USA, Chemistry, Florida Institute of Technology, Melbourne, FL, USA. Single Protein Tracking of P450-Reductase in an Endoplasmic Reticulum Rhodopsin is the G-protein-coupled receptor (GPCR) responsible for scotopic Biomimetic Reveals a NADPH Dependent Interaction with the Membrane vision in vertebrates. Although published X-ray structures are similar, the James A. Brozik, Carlo Barnaba, Adam O. Barden, Linda Agyen, conformation and orientation of the retinal chromophore of active rhodopsin Sean L. Sheridan. are not established unambiguously. Under debate is a proposed flip of all-trans Chemistry, Washington State University, Pullman, WA, USA. b retinal in the active state, due to a change of the roll angle of the -ionone ring, Cytochrome P450-Reductase (CPR) is the redox partner for most human P450 as well as the central part of the polyene chain that includes the C9-Me group 2 enzymes. These membrane proteins are found in the endoplasmic reticulum of [1]. Here we applied solid-state H NMR spectroscopy to study the structure eukaryotic cells. They bind NADPH, which can reduce the enzyme by trans- and orientation of the retinylidene ligand in the active state of rhodopsin in 2 ferring electrons to both the FAD and FMN domains within the protein. The aligned membranes [2]. Rhodopsin was regenerated with retinal H-labeled NADPH/FMN domain is connected to the FAD domain by a hinge region that at the C5-, C9-, or C13-Me groups, and was recombined into phospholipid opens up when the enzyme is fully reduced. We will present the results of a membranes, then trapped in the light-actived state [3]. By fitting the solid- series of single protein tracking studies that directly measure the mobility of state 2H NMR spectra, we determined the orientations of the 2H-labeled methyl 2 CPR in an endoplasmic reticulum biomimetic (ER) as a function of NADPH groups to the local membrane normal. The H NMR data together with the elec- concentration. These results show two distinct states associated with CPR in tronic transition dipole moment from linear dichroism data provided the orien- the ER membrane. In the absence of NADPH, CPR diffuses rapidly with a tational restraints for calculating retinal structures using a three-plane model. diffusion coefficient consistent with a peripheral membrane protein. In the We discovered two structures without any steric clashes in the binding pocket present of excess NADPH, the mobility of CPR is drastically reduced and of the active rhodopsin. One of the two structures has a very similar conforma- comparable to the diffusion coefficients observed in transmembrane proteins. tion to retinal in the ‘‘flipped’’ X-ray structures [1]. The other structure has the At stoichiometric concentrations of NADPH a distribution of the two states b b same position of the -ionone ring, but the polyene chain and -ionone ring are (fast and slow) is observed. These experimental observations have led to a not flipped, and the chromophore orientation is similar to the dark state. Lastly model in which CPR exists as a peripheral membrane protein in its neutral we discuss the implications of our findings for understanding of the rhodopsin and closed state and as a transmembrane protein in its fully reduced open activation mechanism. state. We will also present evidence that ‘peripheral CPR’ is in equilibrium [1] J. Feng et al. (2015) Biophys.J. 108, 2754. with a soluble CPR state. This equilibrium favors partitioning into the ER [2] A.V. Struts and M.F. Brown (2014) in Adv. Biol. Solid-State NMR, Royal but in the absence of NADPH CPR is not permanently attached to the Soc. Chem., pp.320–352. membrane. [3] A.V. Struts et al. (2011) PNAS 108,8263. 2505-Pos Board B112 2502-Pos Board B109 A Eukaryotic Sensor for Membrane Lipid Saturation Manipulating the Dimeric Interface of Human Cone Opsins Roberto Covino1, Stephanie Ballweg2, Claudius Stordeur2, William D. Comar. Jonas B. Michaelis2, Kristina Puth2, Florian Wernig2, Amir Bahrami1, Chemistry, The University of Akron, Akron, OH, USA. Andreas M. Ernst3, Gerhard Hummer1, Robert Ernst2. G protein-coupled receptors (GPCRs) are responsible for relaying a wide vari- 1Department of Theoretical Biophysics, Max-Planck-Institute of Biophysics, ety of biological signals across the cell plasma membrane. Dimerization of Frankfurt am Main, Germany, 2Institute of Biochemistry and Buchmann several class A GPCRs is proposed to modulate their respective signal trans- Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt duction processes, but identifying the structural features of dimerization has am Main, Germany, 3Department of Cell Biology, School of Medicine, Yale been difficult. In this study we investigated dimerization of human cone opsins, University, New Haven, CT, USA. which are responsible for trichromatic photopic vision. Using a time-resolved The degree of lipid saturation is a key feature of membranes that determines fluorescence technique, PIE-FCCS, we determined that human red cone opsin lipid packing and membrane fluidity. It must be tightly regulated to guarantee

BPJ 7896_7899 Wednesday, February 15, 2017 509a the correct functioning and identity of organelles, being thus essential for examined the aggregation state of these nAChR-DCs using Electron Micro- cell signaling and survival. Here, we describe a eukaryotic mechanism scopy (EM). In the EM analysis, the nAChR-LFC16 sample shows distinct of lipid saturation sensing. We show that Mga2, a transcription factor pentameric complexes with a size of ~9nm with no visible aggregation. In conserved among fungi, operates as a sensor for lipid packing in the ER contrast, the nAChR-FC12 showed a remarkable degree of aggregation. The membrane and can control the production of unsaturated fatty acids. We nAChR-FC14 also showed distinct pentameric complexes, however, a signif- employ extensive in vivo mutagenesis, molecular dynamics simulations, and icant degree of aggregation of this complex was observed. Both MCGE and electron paramagnetic resonance spectroscopy to show that the membrane EM analysis indicates that the best candidate for structural studies is the environment stabilizes different configurations of dimers of Mga2 trans- nAChR-LFC16. In order to improve the quality of the nAChR-LFC16 com- membrane helices. Such configurations are defined by alternative relative plex we evaluated its functionality after a mild and severe alkaline treatment rotations of the helices. In this way, altered membrane properties can trigger to remove intrinsic proteins using the two electrode voltage clamp (TEVC) the proteolytic activation of Mga2. This work establishes a novel eukaryotic and its aggregation state using (EM). The results of this study provide a strategy of lipid saturation sensing that differs significantly from the analo- detailed biophysical characterization behind the preparation of functional, sta- gous bacterial mechanism of sensing the hydrophobic thickness of the ble and pure nAChR-DCs for structural studies. membrane. 2506-Pos Board B113 Intrinsically Disordered Proteins (IDP) and Analysis of the Dynamics of a Multi-Drug Exporter AcrB in the Absence and Presence of Substrates Aggregates III Tomoki Matsuda, Seiji Yamasaki, Kunihiko Nishino, Takeharu Nagai, Akihito Yamaguchi. 2508-Pos Board B115 The Institute of Scientific and Industrial Research, Osaka University, Ibaraki Multiply-Phosphorylated Intrinsically Disordered Signaling Molecules Osaka, Japan. Can Exhibit Emergent Cooperativity and Sequential Binding Lara Clemens1, Omer Dushek2, Jun Allard1,3. RND-type multidrug efflux exporter AcrAB-TolC tripartite complex is a major 1 drug exporter in Gram-negative bacteria. The crystal structures of each compo- Center for Complex Biological Systems, University of California - Irvine, Irvine, CA, USA, 2Sir William Dunn School of Pathology, University of nent have been determined and recently their tripartite structures were also 3 visualized by single-particle cryo-electron microscopy. In this way tripartite Oxford, Oxford, United Kingdom, Department of Mathematics, Department complex formation is gradually being unraveled from the structural point of of Physics, University of California - Irvine, Irvine, CA, USA. view. However, their dynamic property on cell membrane is not yet been Many proteins involved in cell signaling have intrinsically disordered re- well-investigated. Here we analyzed diffusion of AcrB on the plasma mem- gions, including some with no cytoplasmic structure at all. Despite this, un- brane of living E. coli cells to assess the possibility of the substrate- structured signaling molecules have been found to exhibit complex nonlinear dependent mode change from ‘free diffusion for searching substrate’ to behavior, including cooperativity and sequential binding. For example, the ‘restricted diffusion by forming tripartite for substrate efflux’. Diffusion was intrinsically disordered T Cell Receptor zeta chain has six tyrosines and ex- measured under the microscope by FDAP (fluorescence decay after photocon- hibits an approximately 100-fold enhancement from first to sixth phosphor- version) analysis using PA-GFP (photoactivatable-GFP)-labeled AcrB. In ylations. Here, we model the zeta chain with a simplified theta-solvent order to facilitate observation of fluorescence diffusion in the intracellular freely-jointed chain model coupled to idealized spherical binding enzymes. space, cells were extended by treatment of ceftazidime. Diffusion of AcrB Following experimental and theoretical evidence, we explore three model as- was slowed when it was expressed in the acrB-deficient cells than in the sumptions: (1) phosphorylation stiffens nearby peptide bonds, (2) phosphor- acrB/tolC-deficient cells. When different substrates were added to the acrB- ylation changes the electrostatic interaction of the chain with the inner leaflet deficient cells, diffusion of AcrB differed depending on the type of the sub- of the membrane, and (3) phosphorylation allows enzymes to remain bound strates. In the case where small size substrates that are bound at the distal to the phospho-tyrosine, as has been suggested for Zap-70. We find that, first, pocket in the crystal structures were added, the diffusion speed was similar entropic flexibility alone leads to cooperativity or anti-cooperativity in ki- to that in the absence of substrate. On the other hand, diffusion is decelerated nase activity, and, second, that sequential binding emerges naturally from when large size substrates that bound at the proximal pocket is added. Introduc- entropic effects. In all cases, additional steric exclusion due to the presence tion of mutations into AcrB to disturb transport of proximal binding substrates of a nearby membrane further enhances these effects. Some of this enhance- eliminated this substrate depended deceleration. These results suggest that as- ment is due to the specific locations of tyrosines along the chain. For sociation and dissociation of AcrAB-TolC tripartite complex is in dynamic example, the membrane-proximal tyrosine on the zeta chain is further equilibrium even if there is no substrate and the complex formation is more sta- from the membrane than the distal-most tyrosine is to the distal end. This bilized when proximal binding larger substrates are added. Residence time of introduces the possibility that receptor function could be tuned simply by substrates in the proximal pocket might have correlation with life time of the changing the distance between tyrosines in the disordered sequence. Our re- AcrAB-TolC complex. sults demonstrate that protein flexibility alone can lead to cooperativity, anti- cooperativity and sequential binding, even in the absence of structure. The 2507-Pos Board B114 disordered regions themselves may therefore act as modules in signal trans- Preparation of Nicotinic Acetylcholine Receptor Detergent Complexes for duction cascades. Structural Studies: Assessment of Purity, Functionality and Aggregation Analysis 2509-Pos Board B116 Bianca N. Valde´s Ferna´ndez1, Hui Wei2, Luis Lo´pez Cruz1, Phosphorylation Induces Sequence-Specific Conformational Switches in Francis Reilly Andujar1, Rafael Cordero Villamil3, Bryan Cobo Torres1, the RNA Polymerase II C-Terminal Domain Orlando Gonzalez Martinez1, Jose A. Lasalde Dominicci1. Scott A. Showalter, Eric B. Gibbs. 1Biology, University of Puerto Rico, Rio Piedras, Puerto Rico, 2New York Department of Chemistry, The Pennsylvania State University, University Structural Biology Center, New York, NY, USA, 3Biology, University of Park, PA, USA. Puerto Rico, Mayaguez,€ Puerto Rico. The Carboxy-Terminal Domain (CTD) of the RNA polymerase II (Pol II) The torpedo californica (Tc) nicotinic acetylcholine receptor (nAChR) is a large subunit cycles through multiple phosphorylation states that correlate large (~290 kDa) integral membrane protein composed of four homologous with progression through the transcription cycle and regulate nascent subunits. The main obstacle towards the achieving of high resolution structure mRNA processing. Structural analyses of yeast and mammalian CTD have of the nAChR is the preparation of milligram amounts of pure, functional and been hampered by their repetitive sequences. Here we identify a region of stable nAChR-detergent complex (nAChR-DC). A crucial aspect in the prep- the Drosophila melanogaster CTD that is essential for Pol II function aration nAChR-DCs is the functionality, purity and aggregation state of the in vivo and capitalize on natural sequence variations within it to facilitate solubilized protein. In the present study we performed an in-depth analysis structural analysis. Mass spectrometry and NMR spectroscopy reveal that of the purity, functionality and stability of nAChR-DCs prepared with lipid hyper-Ser5 phosphorylation transforms the local structure of this essential re- analog detergents FC12, LFC14 and LFC16. The main contaminants present gion via proline isomerization. The sequence context of this switch tunes the in the crude Torpedo californica (Tc) membranes are the peripheral nAChR- apparent activity of the CTD phosphatase Ssu72, suggesting a mechanism for associated proteins, mainly the 43-kDa rapsyn protein and the V-type ATPase. the selective recruitment of cis-proline specific regulatory factors that may We evaluated the purity of nAChR-FC12, nAChR-LFC14 and nAChR-LFC16 synergize with CTD phosphorylation to augment gene regulation in develop- using microfluidic capillary gel electrophoresis (MCGE). In addition, we also mentally complex organisms.

BPJ 7896_7899 510a Wednesday, February 15, 2017

2510-Pos Board B117 charge per residue above ~0.10e can impart enough electrostatic force Interplay Among Binding, Phosphorylation and Denaturation in Disor- to bias the folding equilibrium at low salt to an unfolded structure. We dered 4E-BP2 as Probed by Single Molecule Fluorescence demonstrate that this behavior can be modulated as a function of pH and so- Zhenfu Zhang1, Alaji Bah2, Julie D. Forman-Kay2, Claudiu C. Gradinaru1. lution ionic strength, providing a wide functional dynamic range of folding 1Physics, University of Toronto, Mississauga, ON, Canada, 2The Hospital for energies. At the correct pH and salt concentration the proteins exhibit Sick Children, Toronto, ON, Canada. ligand-induced folding, and we have shown that this behavior can manifest Intrinsically disordered proteins (IDPs) are a class of proteins that lack as cooperative ligand binding. Furthermore, we are extending this supercharg- well-defined 3D structures while still carry out their biological functions. ing to natural biopolymers, starting with green fluorescence protein, and we They play a crucial role in mediating interactions with multiple partners demonstrate that this behavior can be implanted on this fold. These adjustable and often function as protein interaction hubs. Cap-dependent initiation characteristics have inspired a biosensing project in which we attach super- of translation is regulated by the interaction of the eukaryotic initiation charged IDPs to a gold surface and sense conformational changes using sur- factor 4E (eIF4E) with disordered eIF4E binding proteins (4E-BPs) in a face plasmons. This conformational change will increase the refractive index phosphorylation-dependent manner. Single molecule fluorescence resonance at the gold surface, which will shift the angle of minimum reflectance. We energy transfer (smFRET), fluorescence correlation spectroscopy (FCS) and calculate that the shift in the resonance angle caused by the ligand induced fluorescence anisotropy decay (FAD) were used to study the conformations, folding of an IDP is almost two orders of magnitude more than simple ligand dynamics and binding of 4E-BP2. FAD was employed to obtain the local binding to an already folded protein. Continuing reflectometry studies will chain flexibility at various points within the 4E-BP2. A heterogeneous local provide practical insight into the use of our model as conformational switches flexibility behavior was observed throughout the protein sequence and for biosensing devices. regional amino acid composition was found to play an essential role in determining the rigidity map of the chain. The segmental flexibility is 2513-Pos Board B120 hindered in the folded region upon phosphorylation, whereas the rest of the Like Charge Regions (LCRs) are One of the Key Regulators of Nucleocy- chain becomes more flexible. The local segments become more flexible toplasmic Transport upon denaturation, which is a prominent sign of deviation from random coils. Mohaddeseh Peyro, Mohammad Soheilypour, Mohammad R.K. Mofrad. Segmental rotational correlation times and wobbling cone angles extracted for Bioengineering and Mechanical Engineering, UC Berkeley, Berkeley, different sites along the chain provide a rigidity map of 4E-BP2 and was used CA, USA. to evaluate its binding mode to eIF4E. Intra-chain and inter-chain kinetics Nuclear pore complex (NPC) is the only gateway that mediates bidirectional were accessed by FCS whereby heterogeneous local quenching kinetics transport of cargos into and out of the nucleus. The NPC is made up of pro- were observed. Multi-site phosphorylation of the protein slows down the prox- teins named nucleoporins (Nups) that can be categorized into two major imal chain motions and modulates the kinetics of distal regions. smFRET groups, FG Nups and non FG Nups. FG Nups are rich in Phe-Gly repeats, analysis reveals changes in the conformational ensemble in response to are intrinsically disordered and are known to be key role players in nucleocy- phosphorylation, denaturation, salt and pH. Our results demonstrate that toplasmic transport (NCT). Translocation of cargos is mediated via the tran- electrostatics play a critical role in modulating the dimensions and compact- sient interactions between transporters and FG Nups. Despite extensive ness of IDPs. research, the underlying mechanism of NCT through the NPC is still under much debate. 2511-Pos Board B118 Using bioinformatics techniques, we have recently discovered interesting Sequence Determinants of the Conformational Properties of an Intrinsi- evolutionarily conserved features in the sequences of FG Nups. One of these cally Disordered Protein Prior to and Upon Multisite Phosphorylation features is long sequences of uninterrupted positively charged residues located Erik W. Martin1, Alex S. Holehouse2, Rohit V. Pappu2, Tanja Mittag1. at the N-terminus of FG Nups that are of low charge density, which we named 1Structural Biology, St Jude Children’s Research Hospital, Memphis, TN, positive ‘‘like charge regions’’ (LCRs). Our further analysis on the biophysical USA, 2Biomedical Engineering and Center for Biological Systems role of LCRs showed that LCRs are key regulators of spatial distribution and Engineering, Washington University, St. Louis, MO, USA. interaction of FG Nups inside the pore. For this stage of our work, a previously Multisite phosphorylation is critical for signaling, regulation and other cellular developed one-bead-per-amino-acid coarse-grained molecular dynamics model functions and typically maps to intrinsically disordered protein regions. The was used. accessibility of phosphorylation sites by writers, readers and erasers is presum- To further extend the model, a representation of the cargo complex was ably governed by the sequence-encoded conformational properties of the disor- added to investigate how sequence patterns affect the interaction between dered regions. Expanded coil behavior, which would provide unhindered cargo complex and FG Nups and how the transport process would be access to binding partners, has been correlated with a high fraction of charged affected. Our results show that presence of LCRs lead to a more dynamic residues. Here we interrogate the interplay of proline and charged residues in behavior of cargo complex. Besides, cargo complex would be able to determining the global dimensions of an archetypal protein sequence undergo- make more interactions with different FG Nups rather than binding to one ing multisite phosphorylation. Small-angle X-Ray scattering (SAXS), NMR FG Nup for a longer time. This behavior would act in favor of faster trans- and atomistic simulations were used to examine the C-terminal disordered re- port, which is desirable for NPC as a high throughput macromolecular gion of the transcription factor Ash1. Despite its low FCR, unphosphorylated machine. Ash1 is expanded and conformationally heterogeneous. Phosphorylation adds considerable charge density, but SAXS data show no perturbation to the global 2514-Pos Board B121 dimensions. However, atomistic simulations and NMR spectroscopy reveal The Intrinsically Disordered Tail of FtsZ Impacts Polymerization and Bac- phosphorylation-induced shifts in conformational biasing that lead to mutually terial Cell Division Through Sequence-Encoded Charge Patterning compensatory local expansion and contraction, leaving the global dimensions Megan C. Cohan, Ammon Posey, Steven Grigsby, Alex S. Holehouse, unperturbed. Simulations of amino acid sequence variants reveal the signifi- Anuradha Mittal, Paul J. Buske, Petra Levin, Rohit V. Pappu. cance of proline residues for tuning the conformational landscape. We propose Washington University in St. Louis, Saint Louis, MO, USA. a mechanism wherein synergy between proline and charged residues maintains The sequence patterning of oppositely charged residues determines the confor- expanded, coil-like ensembles in proteins undergoing multisite phosphoryla- mational preferences polyampholytic IDRs (intrinsically disordered regions). tion in all phosphorylation states. The sequence features of Ash1 are shared We have used the C-terminal linker (CTL) of the FtsZ protein B. subtilis as by other proteins; it remains to be tested whether their global dimensions are an archetypal polyampholytic IDR to uncover the relationships between also insensitive to phosphorylation. sequence-encoded interactions of IDRs and bacterial cell division. FtsZ has a tubulin-like GTPase core with a C-terminal tail (CTT) that includes a hyper- 2512-Pos Board B119 variable CTL. Despite poor sequence conservation of the CTL, the parameter Design of Supercharged Proteins to Impart Allosteric Behavior and their k that quantifies the extent segregation / mixing of oppositely charged residues Use in Biosensing is bounded between 0.15 and 0.4. Using de novo sequence design, we examined Peter Schnatz1, Joseph Brisendine1, Derek Kosciolek2, David Crouse2, the impact of CTT k on FtsZ ring formation in vivo and the mechanisms of as- Ronald Koder1. sembly in vitro. We find that FtsZ variants with a CTT k value within the afore- 1City College of New York, New York, NY, USA, 2Clarkson University, mentioned bounds support robust ring -formation through a GTP-dependent Potsdam, NY, USA. assembly mechanism. As k is increased further, ring formation is impaired We have designed a series of supercharged single-chain four-helix bundles as and the CTTs promote an alternative GTP-independent assembly. Our findings maquettes of intrinsically disordered proteins (IDPs). We show that a net provide a physical explanation for the observed bounds on k, and suggest that

BPJ 7896_7899 Wednesday, February 15, 2017 511a charge patterning is conserved generally, influencing both protein function and hypothesis of a sharp conformational transition within monomeric HttEx1 cellular phenotype. as a function of increased polyQ length. Instead, increasing the polyQ length shifts concentration thresholds for crossing phase boundaries toward lower 2515-Pos Board B122 values. The effects of ligand binding can counter these shifts in concen- Dissecting Conformational Features and Binding Mechanisms of tration thresholds. Accordingly, we also present a conceptual framework Phenylalanine-Glycine Rich Nucleoporins for how an endogenous ligand can suppress HttEx1 aggregation and cellular Piau Siong Tan, Iker Valle Aramburu, Swati Tyagi, Aritra Chowdhury, toxicity. Edward A. Lemke. EMBL, Heidelberg, Germany. 2517-Pos Board B124 Phenylalanine-glycine-rich nucleoporins (FG-Nups) are intrinsically disor- Molecular Dynamics Study of Early Stage Kinetics of Polyglutamine dered proteins (IPDs), constituting the selective barrier of the nuclear pore Aggregation complex (NPC), which can be crossed by nuclear transport receptors Jason Haaga1, C. Nadia Buckles2, James D. Gunton1. (NTRs). How interaction of NTRs affects conformational features of FG- 1Physics, Lehigh University, Bethlehem, PA, USA, 2Physics and Astronomy, Nups to orchestrate nucleocytoplasmic transport remainsunclear. We previ- University of Oklahoma, Norman, OK, USA. ously showed that NTRs interact with FG-Nups with remarkably fast Huntington’s disease is one of a set of nine progressive, neurodegenerative diffusion-limited kinetics, employing a set of multivalent interactions diseases caused by the expansion of CAG sequence repeats. This results in requiring no large-scale conformation changes. This ensures rapid yet specific affected proteins with abnormally long polyglutamine tracts, which, beyond nucleocytoplasmic transport via formation of a dynamic ‘‘fuzzy’’ complexes. a pathological threshold, results in aggregation. These diseases show a Here we demonstrate that the binding of CRM1, an export NTR, can induce a dependence on this repeat length in both age of onset and progression speed. conformational change in FG-Nup214. The affinity of this complex can be Evidence suggests the toxic species in these diseases may be small, interme- further modulated by RanGTP, which binds to CRM1. Kinetic measurements diate oligomers, rather than the equilibrium fibril structures. Using the show that the binding of CRM1 with FG-Nup214 is very fast but slower than PLUM coarse-grain protein model, we computationally study the kinetics of most other FG-Nups,NTRs interactions. These results suggest the formation this early stage of aggregation to gain insight into how repeat length and su- of a structurally stable complex, which is different from the dynamic multiva- persaturation affect properties such as induction time and the distribution of lent FG-Nup,NTR ‘‘fuzzy’’ complexes previously seen. However, a major cluster sizes. This work is supported by the G. Harold and Leila Y. Mathers question remains, if those studies are relevant to physiological conditions, Foundation. since metazoan NPCs are highly glycosylated. We thus provide insight into the FG-Nup,NTR interaction mechanism of O-linked-N-acetylglycosamine 2518-Pos Board B125 modified FG-Nup214. We observed that glycosylation modulates the valency Crowding Induced Coil-Globule Transitions of Intrinsically Disordered and interaction strength of Nup214,CRM1 complex, while retaining the bind- Proteins ing mode of Nup214 and CRM1,RanGTP. Our results indicate the possibility Youngchan Kim1, Jeetain Mittal2. that post-translational modifications of FG-Nups may add another layer of sub- 1Center for Computational Materials Science, Naval Research Laboratory, tle regulation of FG-Nup functions. In summary, our findings highlight the Washington, DC, USA, 2Department of Chemical and Bimolecular layers of complexity involved in FG-Nup,NTR interactions. This brings us Engineering, Lehigh University, Bethlehem, PA, USA. a step forward to decipher transport across the NPC from a molecular stand- Macromolecular crowding effects on intrinsically disordered proteins (IDPs) point by directly interrogating details like their diversity, spatial localizations have been studied via molecular dynamics simulations of a coarse-grained and dynamics. IDP model developed by Ashbaugh and Hatch [J. Am. Chem. Soc., 130, 9536, 2008]. This model can successfully reproduce the empirical Uversky 2516-Pos Board B123 diagram which is used to distinguish amino acid content of IDPs from their Polyglutamine Length Dependent Structural Properties and Phase folded counterparts [Proteins: Struc. Funct. Genet., 41, 415, 2000] in the Behavior of Huntingtin Exon 1 charge-hydrophobicity parameter space. Using a relatively simple and Kiersten M. Ruff1, John B. Warner2, Ammon E. Posey3, Piau Siong Tan4, commonly employed approximation of crowding agents as spherical parti- Edward A. Lemke4, Rohit V. Pappu3, Hilal A. Lashuel2. cles, we first investigate the excluded-volume effects on the phase boundary 1Computational and Systems Biology, Washington University in St. Louis, of the coil-globule transitions as a function of protein charge, hydrophobic- Saint Louis, MO, USA, 2Laboratory of Molecular and Chemical Biology of ity, and crowder volume fraction. Crowding induces coil to globule Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fe´de´rale de transition at a lower hydrophobicity and higher net charge than in bulk, Lausanne (EPFL), Lausanne, Switzerland, 3Department of Biomedical which is consistent with previous models of IDP crowding. The phase Engineering and Center for Biological Systems Engineering, Washington boundary follows a simple scaling relationship combining three length University in St. Louis, Saint Louis, MO, USA, 4Structural and scales available in the problem, i.e., the crowder size, protein’s bulk radius Computational Biology Unit and Cell Biology and Biophysics Unit, of gyration and monomer bead size. It was also found that the largest European Molecular Biology Laboratory (EMBL), Heidelberg, Germany. compaction induced by crowders is found for IDPs that exist near the phase Huntington’s disease is caused by a mutational CAG repeat expansion in boundary in the sequence space. Furthermore, the compaction of an IDP in exon 1 of the huntingtin (Htt) gene. Post-transcriptional processing leads the presence of crowders is governed by a universal scaling function to fragments of the Htt protein that span exon 1 (HttEx1). Within HttEx1, involving the aforementioned three length scales and a universal polymer the CAG-repeat encoded polyglutamine (polyQ) tract is flanked by an N-ter- scaling exponent. Together our studies help build a comprehensive frame- minal 17-residue amphipathic stretch and a C-terminal 50-residue proline- work to understand the structure and function of IDPs in a crowded cellular rich stretch (C50). The existence of a pathogenic polyQ length threshold environment. has led to the hypothesis that there should be a sharp, length-dependent change in the structure of HttEx1. We test this hypothesis by character- 2519-Pos Board B126 izing the conformational properties of monomeric HttEx1 as a function of Thermodynamic Properties of SynB1-Cys-ELP-Drug Carrier and the Role polyQ length. Previous studies showed that the phase diagram for HttEx1 of Osmolytes is complex and oligomers can form at nanomolar concentrations. This Valeria Zai-Rose, John J. Correia. means that experiments have to be performed in the sub-nanomolar range Biochemistry, University of Mississippi Medical Center, Jackson, MS, USA. to guard against confounding contributions from oligomerization, aggrega- Macromolecular crowding modulates the association and aggregation tion, and phase separation. Accordingly, single molecule Fo¨rster reso- behavior of IDPs in the physiological environment of serum and intracellular nance energy transfer (smFRET) experiments were deployed to examine space. Here we show the biophysical consequences of protecting and dena- the conformational properties of HttEx1 as a function of polyQ length. turing osmolytes on an engineered IDP, SynB1-Cys-ELP comprised of We have combined experimental measurements and simulation results to [VPG(V5G3A2)G]150, developed as a thermally targeted protein-drug carrier extract conformational ensembles of HttEx1 that are consistent with the for treatment of solid tumors. Density and preferential interaction measure- smFRET data. We observe a continuous global compaction of HttEx1 as ments are used to investigate the effect of molecular crowding on ELP aggre- polyQ length increases. This arises from the increased prominence of the gation. The mechanism of interaction of ELP in the presence of osmolytes is globular polyQ region and does not reflect any special intramolecular determined by calculating the preferential interaction parameter x3 =(vg3 / interactions amongst the three regions. Our results do not support the vg2)Tm1,m3 the osmolytes in the presence of the protein at equilibrium dialysis

BPJ 7896_7899 512a Wednesday, February 15, 2017 in terms of its partial specific volume. A negative preferential interaction 2522-Pos Board B129 coefficient shows that protecting osmolytes are preferentially excluded from Exploring the Effects on the Conformational Propensity of Alanine in the ELP’s surface and enhance its aggregation out of solution, while a positive Unblocked Tripeptide Glycyl-Analyl-Glycine in Water/Ethanol Mixtures preferential interaction coefficient shows that denaturants bind to the ELP David DiGuiseppi1, Nina Kubatova2, Gabrielle Lewis3, Harald Schwalbe2, backbone and destabilize its aggregation. The results show that protecting Reinhard Schweitzer-Stenner1. osmolytes like TMAO and Betaine preferentially hydrate the ELP out of 1Chemistry, Drexel University, Philadelphia, PA, USA, 2Institut fur€ solution as the ELP’s TT linearly decreases with increasing osmolyte concen- Organische Chemie und Chemische Biologie, Johann Wolfgang Goethe- trations. Arginine, however, a weak denaturant, preferentially, yet weakly Universit€at, Frankfurt am Main, Germany, 3Biology, Drexel University, interacts with the ELP backbone at lower concentrations and suppresses Philadelphia, PA, USA. aggregation by increasing its TT and solubility in solution. Using different Short peptides have been shown to be model systems for obtaining the confor- molecular weights PEG we show that inert additives enhance ELP aggrega- mational propensities of individual amino acids. Recent studies on cationic tion consistent with an excluded volume effect. These results show that GxG (x: different guest amino acid residues) peptides in water revealed the ELP aggregation is driven by solvent interactions and that changes in the sol- Ramachandran plots for 16 amino acid residues. Out of the natural amino vent environment upon addition of different osmolytes enhance or diminish acids, alanine in GAG has been shown to stand out owing to its unusually the aggregation of ELP. Future experiments will use complimentary VPO high propensity for polyproline II (pPII) conformations (0.72). Thus, its measurements to look at the changes in water activity in the presence of conformational distribution differs substantially from the classical ones of osmolytes. Ramachandran and Flory. Peptide-water interactions have been proposed as the major determinant of alanine’s preference for pPII. To elucidate how pep- 2520-Pos Board B127 tide hydration affects conformational preferences of alanine we used FTIR, Tardigrade Intrinsically Disordered Proteins as Potential Excipients for vibrational circular dichroism, polarized Raman and NMR spectroscopy to Biologics determine Ramachandran plots of GAG in aqueous solutions with 3, 12, Samantha Piszkiewicz1, Aakash Mehta1, Thomas C. Boothby1, and 42mol% ethanol. We observed a modest decrease of the pPII fraction William Daniel1, Sergei Sheiko1, Bob Goldstein2, Gary J. Pielak1. from 0.72 to 0.65 and an increase in b-strand population from 0.15 to 0.20 1Chemistry, UNC Chapel Hill, Chapel Hill, NC, USA, 2Biology, UNC from pure water to 12 mol% ethanol. Interestingly, the corresponding changes Chapel Hill, Chapel Hill, NC, USA. of the enthalpic and entropic differences between pPII and b -strand by far Protein-based ‘biologics’ — drugs derived from living organisms — are exceed changes in Gibbs energy at room temperature owing to enthalpy- among the most effective therapeutic treatments on the market. However, entropy compensation effects. Our thermodynamic data reveal that the cosol- protein-based biologics are unstable, have short half-lives and require low vent stabilizes pPII enthalpically ( 10 kJ/mol in water to 18 kJ/mol in temperature storage, which often makes them prohibitively expensive. 12 mol% ethanol) but destabilizes it even more entropically ( 23 to 50 Although some drugs can be stabilized by formulation with excipients, J/mol*K). Comparisons of IR and NMR spectra of GAG and N- methylace- most still require low temperature storage. In our search for new, more robust tamide in the same ethanol/water mixtures reveal changes of peptide hydra- excipients, we turned to the tardigrade, a microscopic animal that synthesizes tion which causes the observed destabilization of pPII of alanine and the a unique family of intrinsically disordered proteins (IDPs) to protect its concomitant increase of its conformational entropy. cellular components during desiccation. Oscillatory shear experiments demon- strate that at high concentrations and low temperatures, these tardigrade IDPs DNA Replication, Recombination, and Repair form reversible hydrogels. Encapsulating a globular test protein in this IDP 2523-Pos Board B130 hydrogel stabilizes the folded state of that protein. Additionally, these IDPs Near-Atomic Structural Model for Bacterial DNA Replication Initiation are an order of magnitude better than the FDA-approved excipient trehalose Complex and its Functional Insights at protecting enzyme activity during desiccation and rehydration. These pro- Masahiro Shimizu1, Yasunori Noguchi2, Yukari Sakiyama2, tective properties show the potential of tardigrade IDPs to serve as novel ex- Hironori Kawakami2, Tsutomu Katayama2, Shoji Takada1. cipients for stabilizing biologics. 1Kyoto University, Kyoto, Japan, 2Kyushu University, Fukuoka, Japan. Formation of the DNA replication initiation complex precedes chromosomal 2521-Pos Board B128 DNA replication. In Escherichia coli, this complex forms on the replication Exploring the Unexpected Gelation of Tripeptides in a Binary Mixture of origin oriC, and is composed of about 11 DnaA molecules and one IHF dimer. Water and Ethanol This complex initiates DNA replication by unwinding specific DNA sequence David DiGuiseppi1, Stefanie Farrell1, Nicolas Alvarez2, in oriC and by loading DNA helicases there. While the researches on this Reinhard Schweitzer-Stenner1. 1 2 initiation process have been accumulating, the entire structure of the oriC- Chemistry, Drexel University, Philadelphia, PA, USA, Chemical and DnaA-IHF complex has not been elucidated. In order to reveal the structure Biological Engineering, Drexel University, Philadelphia, PA, USA. of the replication initiation complex, we employed hybrid approaches of Hydrogels formed by peptides of different length are a special class of mate- computational modeling and biochemical assay. By coarse-grained molecular rials that have been heavily researched in the past due to their inherent biode- dynamics simulations, we tested several possibilities of the oriC-DnaA-IHF gradability. In this context, some low molecular weight di- or tripeptides with complex. Obtained structural models were further tested by biochemical as- aromatic residues and terminal groups have been shown to form gels after says using reconstituted system. Coarse-grained structures were back- self-assembling into large supramolecular structures above critical concentra- transferred into atomistic model. Finally we obtained the near-atomic model tions in the centimolar range. Contrary to expectations, our group recently of crucial core part of E. coli replication initiation complex. The complex discovered that cationic glycylanalylglycine (GAG), a tripeptide of rather is composed of three parts. Left subcomplex contains 5 DnaA molecules limited hydrophobicity, forms a gel in 55 mol% ethanol/45 mol% water at and IHF, middle subcomplex is a single DnaA molecule, and right subcom- room temperature if the concentration exceeds 200 mM. The underlying struc- 5 plex contains 5 DnaA molecules. In the left and right subcomplexes, ture is comprised of unusually long crystalline fibrils (in the 10 m range), AAAþ domain of DnaA formed homo pentamer in a head-to-tail manner. which do not exhibit the canonical b-sheet structure. Rheological measure- 0 00 We also investigated the significance of the spaces between protein binding ments revealed a particularly strong gel with G and G values in the upper sites by similar hybrid approaches. The space between IHF binding site and 104 Pa range. Still, the gel melts if the temperature is increased above adjacent DnaA box was crucial for oriC-DnaA-IHF complex formation. In 36 C. Melting curves measured by increasing and decreasing temperature re- addition, the space between the left subcomplex and the right subcomplex vealed a significant hysteresis. Kinetic experiments revealed that the gelation was important for proper DNA helicase loading. of GAG is a multi-step mechanism that can be disentangled by spectroscopic means. By using UV circular dichroism as a novel indicator of gelation we are 2524-Pos Board B131 currently performing measurements that are aimed at constructing a three- Upf1-Like Helicaes - Same Subfamily, Yet so Different Behavior dimensional phase diagram of the peptide with regard to its melting Saurabh Raj1, Joanne Kanaan2, Herve´ Le Hir2, Vincent Croquette1. temperature, peptide concentration and cosolvent fraction. Rheological mea- 1CNRS, Paris, France, 2IBENS, CNRS, Paris, France. surements and optical microscopy will allow us to identify and characterize Helicases are ubiquitous enzymes that are involved in all kinds of DNA/RNA different gel phases. Thus, we will identify conditions which make the gel us- metabolisms including transcription, recombination and replication. Although able for practical applications such as ointments, sensors, and drug release the most basic function of most helicases is to unwind a double stranded nucleic systems. acid substrate, their properties vary a lot from one helicase family to another.

BPJ 7896_7899 Wednesday, February 15, 2017 513a

Sometimes even within the same subfamily, the mechanistic properties of two Proper cell function requires preservation of the spatial organization of helicases can be very different. S. cerevisiae Upf1 (yUpf1) and human chromatin modifications. Maintenance of this epigenetic landscape necessi- Smubp-2 are two helicases from Upf1-like family that belong to SF1B. In tates the transfer of parental nucleosomes to newly replicated DNA, a this work we show that despite having a similar helicase core, the helicase process which is stringently regulated and intrinsically linked to replication domain (HD) of these two enzymes work in a very different manner. yUpf1- fork dynamics. The complex and dynamic environment in which nucleosome HD has a strong binding affinity to ssDNA and a very high processivity, transfer occurs is a challenging setting to define the essential components that whereas Smubp-2-HD has week binding to ssDNA and almost unrecognizable underlie the transfer mechanism. To examine the role of DNA mechanics processivity. Helicase cores of Smubp2 and Upf1 have similar folds: they and identify the minimum system requirements for mediating nucleosome contain two RecA-like domains (1A and 2A) and two sub-domains, 1B and transfer, we utilized a DNA template with a single nucleosome and incremen- 1C, inserted in domain 1A. Rec-A like domains are believed to act as a motor tally added complexity by first displacing the nucleosome mechanically, in a helicase. Hence it is puzzling why the two helicases behave so differently then with an isolated replicative helicase, and finally with a simplified repli- despite having highly conserved domains 1A and 2A. We try to identify the role cation complex. We found that the nucleosome is passively transferred to of sub-domains 1B and 1C in this respect. We have utilized a magnetic twee- available dsDNA as predicted by a simple physical model of DNA loop for- zers based single molecule assay, acting in parallel on tens of molecules at the mation. These results demonstrate a fundamental role for DNA mechanics in same time, which allows detecting the unwinding of DNA hairpin substrates. mediating nucleosome transfer and preserving epigenetic integrity during Using this system, we have studied various chimeras of yUpf1-HD and replication. Smubp-2-HD. We show that domains 1B and 1C play an equally important role in processivity of these helicases. 2528-Pos Board B135 The Interplay between a Bacterial Chromatin Protein and DNA 2525-Pos Board B132 Polymerase Szu-Ning Lin1, Remus T. Dame2, Gijs J.L. Wuite1. Investigating the Enhancement of XPD Helicase Processivity by Single- 1 2 Stranded Binding Protein RPA2 Vrije University Amsterdam, Amsterdam, Netherlands, Leiden University, Barbara Stekas1, Masayoshi Honda2, Maria Spies2, Yann Chemla1. Leiden, Netherlands. 1Physics, University of Illinois - Urbana-Champaign, Urbana, IL, USA, In all domains of life the replication of DNA is carried out by functionally 2Biochemistry, University of Iowa, Iowa City, IA, USA. conserved DNA polymerase. Most DNA polymerases can perform both Ferroplasma acidarmanus helicase FacXPD is a Superfamily 2B helicase DNA replication and proofreading (via exonucleolysis) functions. In the involved in transcription initiation and nucleotide excision repair. This archaeal cell DNA polymerase operates in a crowded environment, with multiple other protein serves as a model for understanding the molecular mechanisms of yeast proteins binding along DNA. However it is unknown how the binding of such Rad3 and human xeroderma pigmentosum group D protein (XPD). Previous proteins affects the activity of DNA polymerase.In this study, we use T7 DNA work has shown that the unwinding of double-stranded DNA by FacXPD is polymerase as model system to investigate polymerisation (pol) and exonu- enhanced by the single-stranded DNA binding protein FacRPA2. However, clease (exo), activities in the presence of DNA bound proteins as potential the mechanism by which unwinding enhancement occurs remains unknown. roadblocks. The bacterial chromatin protein HU is one of the most abundant Here, we monitored the unwinding of a DNA hairpin by XPD in the presence proteins in E. coli. HU binds non-specifically to DNA and is involved in of RPA2 using a single-molecule optical trap assay. By loading XPD and RPA2 genome organization. We investigated the impact of this physiologically rele- onto DNA in a controlled sequence and by analyzing helicase unwinding dy- vant roadblock protein on T7 DNA polymerase activities. We use optical namics, we distinguish between different potential mechanisms of regulation. tweezers with concurrent fluorescence imaging to directly observe fluores- Our data point toward a model of processivity enhancement in which RPA2 cently labeled DNA-bound HU and to determine its interplay with T7 DNA transiently interacts with the XPD-DNA complex and stabilizes a faster, polymerase. more processive state of the helicase. 2529-Pos Board B136 Real-Time Fluorescence Assays to Monitor DNA Polymerase Activities 2526-Pos Board B133 Etienne Henry, Ghislaine Henneke, Didier Flament. Probing the Nucleic Acid Binding Properties of the Single-Stranded DNA CNRS, Ifremer, UBO, Laboratoire de Microbiologie des Environnements Binding Protein of Bacteriophage T4 Replication Complex at Single Nucle- Extreˆmes UMR 6197, Plouzane´, France. otide Resolution We report fluorescence-based methods to study the polymerase and the exonu- Benjamin R. Camel, Katherine Meze, Davis Jose, Peter H. von Hippel. clease activities of thermostable DNA polymerases in real time. The methods Institute of Molecular Biology, University of Oregon, Eugene, OR, USA. are based on the use of a fluorescent reporter which become fluorescent upon Previous studies have mapped the structural details and assembly properties binding to double-stranded DNA (method #1), the use of a quencher reporter of the single-stranded (ss)DNA binding protein (gp32) of bacteriophage T4 strand displacement from a tripartite substrate (method #2) or the use of fluo- as it binds to various ssDNA lattices, both as isolated monomers and as rescence fading of a nucleic acid stain concomitantly to DNA degradation cooperatively bound gp32 clusters. Building on previous studies, our work (method #3). These methods are compatible with standard spectrofluorimeters, seeks to understand these binding interactions at single nucleotide resolution. plate-readers or Real-Time PCR instruments. Here, we follow the efficiency of We have utilized site-specifically positioned 2-aminopurine (2-AP) fluores- primer extension and degradation reactions by Pyrococcus abyssi thermostable cent base analogs of adenine incorporated into ssDNA lattices, as either family B DNA polymerases at 55C. In addition, the size of extended products monomer or dimer-pair probes, to map the detailed interactions of gp32 is systematically examined by gel electrophoresis followed by fluorescence vis- with ssDNA lattices of various lengths. To this end we have employed ualization.These real-time methods are very sensitive, quantitative, and well changes in the fluorescent and circular dichroism (CD) spectra of these suited for the screening of extension and exonuclease activity by DNA poly- probes in order to determine how the binding sites of the proteins interact merases. Moreover, these assays could be used to characterize other DNA en- with these site-specifically positioned probes. Our results demonstrate that zymes like helicases or nucleases. gp32 binds at random at low concentrations, and then shifts to preferential Based on operational properties of different DNA pols, these results could raise binding at the 5’-end of the lattice as the proteins shift into cooperative, specific features with possible evolutionary scenario for the origin of families cluster-bound forms at higher gp32 concentrations. We have also used acryl- DNA polymerases. amide quenching to monitor solvent exposure of the ssDNA bases at various lattice positions, indicating greater ‘shielding’ of the ssDNA bases exists at 2530-Pos Board B137 the center of the binding site under saturating conditions. These results pro- Dynamic Proofreading in the Bacterial DNA Polymerase vide new insights into the molecular mechanisms of the gp32-ssDNA interac- Hailey L. Gahlon1, Gengjing Zhao2, Rafael Fernandez-Leiro2, tions that are involved in controlling the functions of the T4 DNA replication Meindert H. Lamers2, David Rueda1. complex. 1Medicine, Imperial College London, London, United Kingdom, 2MRC Laboratory of Molecular Biology, Cambridge, United Kingdom. 2527-Pos Board B134 Ensuring DNA is replicated with high fidelity is important for maintaining DNA Looping Mediates Nucleosome Transfer genome stability, since the introduction of errors can lead to mutations and Lucy D. Brennan1, Robert A. Forties2, Smita S. Patel3, Michelle D. Wang1. potentially cancer. For this reason, high fidelity polymerases contain 30 to 50 1Cornell University, Ithaca, NY, USA, 2Advion Inc, Ithaca, NY, USA, proofreading activity that can remove incorrectly inserted nucleotides from 3Rutgers-Robert Wood Johnson Medical School, Piscataway, NJ, USA. the primer terminus. DNA polymerase III (DNA Pol III) is the replicative

BPJ 7896_7899 514a Wednesday, February 15, 2017 polymerase in E. coli. It can synthesize DNA at a rate of 1000 bp/s. During to their destabilization, and allow us to formulate the complete mechano- proofreading, the primer strand migrates from the polymerase domain to the chemical cycle of RT’s polymerization reaction. In addition, the data shows exonuclease domain, a distance of 60 A˚ for DNA Pol III. As the dynamics of that the polymerization reaction is governed by the kinetics of the enzyme’s this process is not well understood, the goal of this work is to evaluate active pausing, which takes place in competition with productive elongation. site switching dynamics during proofreading at a single-molecule level. For this, the catalytic core of the DNA Pol III holoenzyme comprising the polymer- 2533-Pos Board B140 An Enhanced, in Vivo Assay for DNA Mismatch Repair Reveals Different ase a subunit, the sliding clamp b2 subunit, the exonuclease ε subunit and a q subunit was tested. We also examined proofreading with a and ε subunit mu- Repair Mechanisms of Lagging Strand Mismatches tants with altered exonuclease activity. In this study, active site switching was Eric A. Josephs, Piotr E. Marszalek. monitored with varying errors on primer-template termini including several Mechanical Engineering and Materials Science, Duke University, Durham, mismatches and an abasic site analog paired opposite A. Single-molecule NC, USA. FRET data will be presented that measured the kinetics of primer strand trans- In E. coli, a DNA mismatch repair (MMR) pathway corrects errors that occur fer as well as the distribution of the DNA in the polymerase and exonuclease during replication, such as the mis-pairing of nucleotides, by coordinating the domains. This, along with primer extension assays performed in bulk, reveal excision and re-synthesis of a long tract of DNA between an epigenetic signal that the dynamics of proofreading vary as a function of the terminal DNA on the newly-replicated strand and the replication error after the error is iden- mismatch. Also, single-molecule protein induced fluorescence assays will be tified by protein MutS. Recent evidence is suggestive that this ‘‘long-patch presented for the binding kinetics of the a and aε subunits. Overall, our repair’’ between these sites is coordinated in the same direction of replication work provides unique insight into the mechanism that ensures high fidelity by the replisome. Using a novel replication-coupled assay to quantify the DNA synthesis and preserves the integrity of the information stored in the nucleotide-dependence, strand-dependence, and directionality of MMR on genomic DNA. genomic mismatches in vivo, we find that repair of lagging strand mismatches occurs bidirectionally, and that directional bias in the origin of strand excision 2531-Pos Board B138 and effect of mutations to the MMR proteins is dependent on the molecular spe- Single-Molecule Characterization of E. Coli Pol III Core Catalytic Activity cies of the mismatch. is dependent on the molecular species of the mismatch. M. Nabuan Naufer1, David A. Murison2, Ioulia Rouzina3, Furthermore, in combination with atomic force microscopy and other single Penny J. Beuning2, Mark C. Williams1. molecule techniques, we are able to identify an important contributing role 1Department of Physics, Northeastern University, Boston, MA, USA, in the cryptic ability for MutS to tetramerize on the repair of some lagging 2Department of Chemistry and Chemical Biology, Northeastern University, strand mismatches. While all mismatches recognized by MutS are thought to Boston, MA, USA, 3Department of Chemistry and Biochemistry, Ohio State be repaired consistently—albeit with differing efficiencies—our results for University, Columbus, OH, USA. the first time provide direct evidence that the repair of different mismatches Antibiotic resistance is a growing health problem. Hence new antibiotics and on the lagging strand might be coordinated by differential mechanisms in a mis- new antibiotic targets are critically needed. Bacterial DNA polymerases are matched nucleotide-dependent manner. potentially attractive targets for the development of new antibiotics because, 2534-Pos Board B141 while the process of DNA replication is conserved, the structures and interac- Elucidation of the Structure-Function Relationship of S. cerevisiae Muts tions of the proteins involved vary considerably between prokaryotes and eu- Homolog Msh4 and Msh5 with the Holliday Junction karyotes. DNA polymerase III (Pol III) is the replicative DNA polymerase in Sudipta Lahiri. E. coli and is composed of 10 subunits that tightly coordinate leading and lag- Molecular Biology & Biochemistry, Wesleyan University, Middletown, ging strand synthesis. The core of the polymerase (Pol III core) contains the cat- CT, USA. 0 / 0 ε alytic polymerase subunit, a, the 3 5 proofreading exonuclease, , and a The MutSg protein complex plays a significant role in the process of meiotic subunit of unknown function, q. Here we employ optical tweezers to charac- recombination and double-strand break repair (DSBR) and exhibits a high bind- terize Pol III core activity on a single DNA molecule. We quantify the transi- ing affinity for DNA Holliday junctions (HJ). We have recently overexpressed tions between single-stranded DNA (ssDNA) and double-stranded DNA and purified from E. coli the S. cerevisiae MutSg protein complex, Msh4- (dsDNA) during Pol III core activity via constant force measurements, in which Msh5. We find that Msh4-Msh5 binds DNA HJ with high affinity (Kd = the change in extension can be used to determine the rate of polymerization or 0.3 nM1), while binding to double-stranded DNA (dsDNA) is almost a 100- exonucleolysis at forces below the melting transition force. These experiments fold weaker. We have combined our experimental investigations with molecu- are performed at forces greater than the force at which dsDNA and ssDNA lar dynamic simulations to determine the role of Msh4-Msh5 conformational stretching curves cross; therefore, while polymerization is inhibited, exonu- dynamics in DNA binding and identify the features of the Msh4-Msh5- cleolysis is favored by force. We show that the application of tension facilitates junction interaction that lead to specific recognition relative to dsDNA. Fo¨rster the inter-molecular transfer of the primer between the polymerase and exonu- resonance energy transfer measurements demonstrate that Msh4-Msh5 prefer- clease subunits of the Pol III core assembly. Here we report the dependence of entially binds to stacked junctions and does not further alter junction conforma- these catalytic rates on template tension and find the zero-force polymerization tion upon binding, suggesting that Msh4-Msh5 stabilizes the stacked 5 rate of Pol III core to be 38 9 nts/s. We show that the dwell times of proc- conformation during the processes of homologous recombination and DSBR. 5 essive events between pauses for polymerization and exonucleolysis are 0.7 We used homology modelling to generate an ATP bound structural model of 5 0.1 s and 0.3 0.1 s, respectively, suggesting distinct DNA binding dynamics the Msh4-Msh5 heterodimer. We successfully docked HJ and dsDNA onto during the polymerase and exonuclease activities. this model and performed all-atom molecular dynamics simulations for 100 ns. These simulations yielded an average Msh4-Msh5 heterodimeric struc- 2532-Pos Board B139 ture in complex with the DNA substrates that allows us to identify a putative The Mechano-Chemistry of Reverse Transcriptase DNA binding region in the Msh4-Msh5 heterodimer. These models have also Omri Malik1, Hadeel Kahamis2, Sergei Rudnizky3, Ariel Kaplan3. 1 revealed specific and non-specific interactions of Msh4-Msh5 with Holliday The Russell Berrie Nanotechnology Institute, Technion- Israel Institute of Junction and dsDNA and suggest a mechanism of specificity and differentiation Technology, Haifa, Israel, 2Department of Physics, Technion- Israel Institute 3 between the two substrates. Finally, principal component analysis of the MD of Technology, Haifa, Israel, Department of Biology, Technion- Israel trajectories has uncovered protein conformational dynamics related to binding. Institute of Technology, Haifa, Israel. These analyses suggest that Msh4-Msh5 binding to the DNA substrates is facil- Retroviral Reverse Transcriptase (RT) is a multi-functional DNA polymerase itated by breathing motions of the DNA binding regions and clamp domains, that catalyzes the synthesis of an integration-competent dsDNA molecule, us- suggesting an induced fit model of binding and recognition. ing the viral RNA as a template. During polymerization, RT is able to over- come the structural barriers imposed by its template’s secondary structures, 2535-Pos Board B142 without the need for assisting helicases. Although the structured architecture Mismatch Repair on the Go: Extending the Mechanical Model of DNA of the retroviral genome suggests that such strand-displacement polymerization Mismatch Repair through Single Molecule Study activity may play an important role in reverse transcription, its mechanism Pengyu Hao1, Ruoyi Qiu2, Dorothy Erie3, Keith Weninger1. remain poorly understood. In this work, we used single-molecule high-resolu- 1Physics, North Carolina State University, Raleigh, NC, USA, 2Molecular & tion optical tweezers to follow strand-displacement polymerization by Molo- Cellular Physiology, Stanford University, Stanford, CA, USA, 3Chemistry, ney Murine Leukemia RT under a broad range of chemical and mechanical The University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. conditions. Our findings indicate that RT unwinds duplex nucleic acids by DNA polymerase has a small but non-zero rate of making single base mis- rectifying their spontaneous thermal opening, with only a small contribution match errors during DNA replication. To preserve the integrity of genomic

BPJ 7896_7899 Wednesday, February 15, 2017 515a information, a cellular system comprised of DNA mismatch repair (MMR) pro- ment sites for two DNA double-strands to specifically mimic a DNA DSB. tein machinery is present that is conserved in all organisms. Among the proteins Following assembly and purification, transmission electron microscopy in this system, MutS and MutL are crucial for mismatch recognition and (TEM) images as well as atomic force microscopy (AFM) experiments demon- discrimination of parent and daughter strand following replication. MutS pro- strated the correct folding of our DNA origami. In a proof of principle exper- tein initiates the MMR cascade by recognizing and binding onto the base-base iment, two fluorescently labeled DNA double-strands were bound to the DNA mismatches on the hybrid daughter-parent dsDNA; MutL follows and creates a origami and their complementary overhangs of four nucleotides ‘‘repaired’’ nick on the newly synthesized DNA strand to guide the downstream repair with the T4 DNA ligase. The successful ligation reaction was monitored by processes. single-molecule Fo¨rster Resonance Energy Transfer (FRET) both in solution We have used single-molecule methods to monitor the interaction between and on the surface. Thus, the presented DNA origami structure provides a use- MutS/MutL and mismatched DNA in various nucleotide conditions. The dy- ful platform for further single-molecule fluorescence studies of DNA DSB namics of MutS and MutL interacting with mismatched DNA is reported by repair. single molecule Fluorescence Resonance Energy Transfer (smFRET) measure- ments, which are sensitive to nanoscale molecular motions. In addition to 2538-Pos Board B145 RecA FRET, experiments using the Tethered Particle Motion (TPM) technique are Guiding the Self-Assembly of Proteinfilaments on DNA Scaffolds to conducted to estimate the dynamic changes in the end-to-end length of a Create Rationally Designed Nanostructures DNA molecule when interacting with MMR proteins. In this method, one Daniel R. Schiffels, Veronika Szalai, James A. Liddle. end of the DNA is anchored on microscope slide and the other attached to a CNST, NIST, Gaithersburg, MD, USA. micrometer-sized bead. Imaging the excursions of the bead position as a func- Over the past three decades DNA self-assembly has been used to create increas- tion of time reveals the length of the DNA tether length. Our observations ingly complex nanoscale structures. DNA nanotubes with tunable persistence combine to reveal a picture of the molecular behaviors of MMR proteins inter- length have provided an attractive model system for protein filaments and struc- acting with mismatched DNA that supports models of the key processes under- tures with rationally defined nanoscale shapes, made using the ‘‘DNA origami’’ lying the MMR pathway. technique are promising for applications such as drug delivery and nano- plasmonics. However, the scale-up necessary for many DNA origami applica- 2536-Pos Board B143 tions remains prohibited by cost and less than perfect yields. We seek to Dissecting the Mechanism of the HerA NurA DNA Break Resection Com- combine the ability of rational design afforded by DNA origami with the plex using Native Mass Spectrometry cost-efficient, high yield assembly of protein filaments to enable the construc- Zainab Ahdash1, Robert Byrne2, Karl-Peter Hopfner2, Argyris Politis1. tion of nanostructures with new functionalities. For a protein to be used in com- 1King’s College London, London, United Kingdom, 2Ludwig-Maximilians- bination with DNA nanostructures, it must interact with DNA in a controllable University of Munich, Munich, Germany. way. RecA protein is an ideal choice because of its ability to form rigid fila- DNA double-stranded breaks (DSBs) are one of the most the cytotoxic and ments on DNA. Here we demonstrate the construction of a nanostructures deleterious forms of DNA damage that can result in genetic instability and including tetrahedra, rectangular shapes as well as micrometer scale filaments the development of cancer in humans. DSBs can be repaired by homologous from DNA origami and RecA protein. We first fold specific sections of M13 recombination, during which the DNA ends are resected by a helicase- single-stranded DNA, using the DNA origami technique, to create a mechani- nuclease system to generate 3’ single strand tails. In archaea, the repair cally flexible skeleton. Unfolded sections of the M13 DNA sequence are made mechanism requires the Mre11-Rad50 complex and the ATP-dependent heli- double-stranded using a DNA polymerase gap-filling reaction. This skeleton is case-nuclease complex HerA-NurA. In the HerA-NurA, dsDNA enters the hex- then rigidified by the addition of RecA, which self-assembles into filaments of americ HerA ATPase channel and subsequently unwound by the narrower defined length at specific double-stranded DNA target sites. Our method greatly dimeric NurA, which acts as a ploughshare. The processes of DNA transloca- improves upon conventional DNA origami in several ways. By reducing the tion and unwinding are ‘‘fueled’’ by the presence of six ATPs binding to the number of distinct input components, we not only reduce cost but also the num- nucleotide-binding sites on HerA monomers. Despite the importance of this ber of possible mis-folding events, allowing for higher yield nanostructures. system in DNA damage repair, the mechanism of ATP binding and its implica- Furthermore, the direct incorporation of proteins with origami adds a new level tions in the complex assembly and conformations remain unknown. of functionality, thus broadening the scope of potential origami applications. Here, by combining native mass spectrometry with functional assays and 2539-Pos Board B146 modelling we propose a model of HerA-NurA DNA assembly and ATP bind- Insights into Damaged Base Detection by DNA Glycosylases: A Computa- ing. In this model we highlight the stabilizing role of NurA, the presence of tional Study of AlkD which imposes oligomeric selectivity on the HerA within the complex. Binding Kevin Votaw, Martin McCullagh. of dsDNA increases the stability of the complex and enables conformational Chemistry, Colorado State University, Fort Collins, CO, USA. changes that freeing four ATP binding sites. Our findings support a novel In order to protect the cellular genome from damage, a wide array of proteins model of ordered ATP binding through a mechanism of alternating conforma- known as DNA repair enzymes must be able to rapidly locate nucleic acid mod- tional ‘‘opening’’ and ‘‘tightening’’ of the HerA-NurA channel. Overall, this ifications. The first step of the base excision repair pathway is the removal of work leads to an improved understanding of the molecular basis of HerA- modified nucleotides, which requires that DNA glycosylases be able to effi- NurA complex as it offers a link between conformational changes, stability ciently detect their target. Once located, most DNA glycosylases utilize the and nucleotide binding. base-flipping mechanism to flip damaged nucleobases into a binding pocket on the protein. Recent crystallization studies show that there is at least one 2537-Pos Board B144 exception, AlkD, which repairs methylated DNA without forcing the damaged Establishing a DNA Origami Platform for Single-Molecule Fluorescence nucleobase out of its base pair. The unique nature of AlkD allows us to inves- Studies of DNA Double-Strand Break Repair tigate the ways in which DNA glycosylases locate modified DNA. Using 1 1 2 2 Kira Bartnik , Alvaro H. Crevenna , Mauricio Pilo-Pais , Tim Liedl , microsecond MD simulations, we demonstrate that AlkD is able to stabilize Don C. Lamb1. 1 a dramatically contorted DNA structure by promoting localized B-to-A-DNA Physical Chemistry, Department of Chemistry, Ludwig-Maximilians- transitions in the methylated DNA. On the contrary, non-methylated DNA is € € 2 Universitat Munchen, Munich, Germany, Faculty of Physics, Ludwig- found to unbind from the putative binding region in order to form a more linear € € Maximilians-Universitat Munchen, Munich, Germany. B-DNA type conformation. These observations are further supported by Custom-designed DNA nanostructures (DNA origami) are a useful platform for computing the free energy of binding for methylated and non-methylated precisely arranging molecules on the nanometer scale. Thus, it is possible to DNA sequences. create a locally high concentration of biomolecules and to investigate interac- tions with low affinity, while maintaining the overall concentration low enough 2540-Pos Board B147 to perform single-molecule experiments. We use DNA origami as a model sys- Direct Single Molecule Imaging Reveals Heterogeneity in Nucleotide Exci- tem to explore the mechanisms and dynamics of macromolecular complexes, sion Repair e.g. during the repair of DNA double-strand-breaks (DSBs). DNA DSBs are Luke Springall1, Michelle Simons2, Craig D. Hughes3, Bennet Van Houten4, considered the most cytotoxic form of DNA damage and efficient repair is Neil M. Kad1. crucial to maintain genomic integrity. Although the overall process of non- 1School of Biosciences, University of Kent, Canterbury, United Kingdom, homologous end joining (NHEJ) - the major pathway to repair DNA DSBs 2School of Biological Sciences, University of Essex, Colchester, United in higher organism - is well documented, little is currently known about the dy- Kingdom, 3Cambridge Veterinary School, University of Cambridge, namics during the assembly of the repair complex. To investigate the molecular Cambridge, United Kingdom, 4Hillman Cancer Center, University of mechanism during NHEJ, we designed a DNA origami structure with attach- Pittsburgh, Pittsburgh, PA, USA.

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Defects in human nucleotide excision repair (NER) cause xeroderma pigmen- bulge loop and the conserved 6nt apical hairpin loop of SLII. Mutations tosum and cockayne’s syndrome. Prokaryotic NER is highly mechanistically that alter the sequence and/or structure of these elements are highly delete- conserved and therefore provides an excellent model for the more complex rious to viral translationand impair viral replication. Conversely, in a novel mammalian system. NER can be separated into two phases; pre- and post- RNA recognition mechanism, SLII binding to AUF1 promotes folding of incision. In this study we examined pre-incision complex formation initiated the RRM1-2 domains of AUF1. To assess how SLII structure may be playing by damage detection. Classic models describe a linear process of repair begin- a role in these two distinct functional outcomes, the high resolution solution WT CCC ning with a UvrA2B2 complex locating damage, releasing UvrA2 before re- structure of SLII and a SLII mutant, wherein the conserved UAG motif cruiting UvrC to cut either side of the lesion. How these protein complexes in the bulge loop is mutated to a CCC, were determined using a hybrid NMR- negotiate the excess of undamaged DNA to locate and verify damage is still SAXs based approach. Structural studies indicate the presence of continuous poorly understood. Single molecule real-time fluorescence imaging provides a stacking in SLIIWT that leads to the adoption of an L-shape in solution, while direct and quantitative description of damage detection. To shed light on these the SLIICCC mutant adopts a more rodlike shape in solution. Thus, these mechanisms we fluorescently tagged each of the pre-incision proteins and studies indicate that global solution architecture of SLII is vital to transla- studied them together on DNA tightropes. This unique method involves sus- tional control in EV71. pending DNA between silica beads immobilized on a microscope slide, thus permitting access to the DNA free from surface artifacts. By introducing 2543-Pos Board B150 labelled DNA damage we were able to assess which complexes were capable Investigating Transient Events in Nucleotide Excision Repair using Single- Molecule Dark Field Imaging of detecting lesions. 46 56% of UvrA2B2 complexes were found to be dam- Jamie Barnett, Jingyu Wang, Neil M. Kad. age bound, greater than that of UvrA2 alone (29 53%). The previously iden- tified UvrBC complex showed surprisingly high levels of DNA damage Biosciences, University of Kent, Canterbury, United Kingdom. colocalization (52 55%), revealing a previously unrealized lesion detection Nucleotide excision repair (NER) is a DNA repair pathway that can detect and property that may offer a back-door to NER. Furthermore, we demonstrate remove a wide array of adducts and lesions detrimental to genomic integrity. Successful repair requires the rapid coalescence of multiple repair enzymes that UvrA2 shows tension-dependent damage preference suggesting it alters DNA structure upon binding, distinct from when in complex with UvrB. onto a specific damage site that resides amongst millions of undamaged Finally, using multi-color imaging we detect the formation of a UvrA-B-C DNA bases. To understand how this is possible, we image individual fluores- containing complex scanning the DNA, suggesting the existence of a prokary- cently tagged prokaryotic NER proteins interacting with DNA suspended be- otic NER repairosome. tween silica pedestals. Such studies of the repair complexes UvrA2 and UvrA2B2 reveal that DNA binding and damage localization occurs within the ~100 fps temporal resolution for fluorescence microscopy. Therefore, to Protein: DNA Interactions: Dynamics understand how NER proteins locate DNA damage, it is imperative to image with faster time resolution. Here, we use light scattering of gold nanoparticles 2541-Pos Board B148 conjugated onto the same NER complexes to investigate how damage is Human Sex Determination at the Edge of Ambiguity: Biophysical Studies located. The use of gold nanoparticles in combination with dark field imaging of Clinical Mutations in a Master Transcription Factor enables our time resolution to be improved. Dark field scattering from gold Michael A. Weiss, Yen-Shan Chen, Joseph Racca, Nelson B. Phillips. nanoparticle tagged proteins provides much greater photon flux than fluores- Biochemistry, Case Western Reserve University, Cleveland, OH, USA. cence. This enables particle detection at speeds only limited by the camera Male sex determination in therian mammals is initiated by an architectural tran- acquisition time. Using this approach we have achieved up to 1x106 fps in scription factor encoded by the sex-determining region of the Y chromosome. burst mode, and 1.4x104 fps in continuous mode (within a limited field of Designated Sry, this factor contains a sequence-specific high-mobility-group view of 19.2 mm by 150 nm), and 1x103 fps full frame (19.2 mmx (HMG) box, a conserved motif of sharp DNA bending. Sry binds to specific 19.2 mm). UvrA2 is seen to statically bind DNA using fluorescence detection, DNA sites within a testis-specific super-enhancer in its principal autosomal with <5% diffusing along DNA. However at 1x103 fps, we observe transient target gene Sox9, which in turns regulates the expression of a male-specific binding to and diffusion of UvrA2 on DNA. This combination of techniques gene-regulatory network in the embryonic gonadal ridge. Our overall program offers the opportunity to investigate transient kinetic events not yet seen of research exploits a growing genetic database of clinical mutations in human that may be critical to our understanding of protein-DNA interactions in SRY leading to XY somatic sex reversal (Swyer syndrome). Such mutations general. cluster in the HMG box. Here, we focus on a clinical mutation of the conserved ‘‘cantilever’’ residue 2544-Pos Board B151 of human SRY: an Ile that inserts between DNA base pairs to disrupt Exploring Mechanisms of Site Specific DNA Cleavage with Single Mole- base stacking but not base pairing at a site of sharp DNA bending. Our cule Sensitivity studies combine NMR spectroscopy, single-molecule flourescence methods, Raquel M. Ferreira1, Sadie C. Piatt2, Allen C. Price2. stopped-flow FRET assays, and biological assays in embryonic gonadal cells 1Biology, Emmanuel College, Boston, MA, USA, 2Chemistry and Physics, to investigate the function of the SRY cantilever. Together, these studies Emmanuel College, Boston, MA, USA. demonstrate that partial DNA intercalation by the cantilever side chain We have developed an assay for measuring site specific dsDNA cleavage with provides a ‘‘kinetic anchor’’ controlling the lifetime of the bent protein- single molecule sensitivity. In our technique, micron sized beads are tethered DNA complex. Transcriptional assays in cell culture suggest that a threshold with single DNAs in a microfluidic flow cell and imaged using video micro- lifetime is required for SRY-directed activation of Sox9 and in turn male scopy. The exact time of cleavage of each DNA is recorded as the time of disap- development. pearance of its tethered bead. We are using our assay to study facilitated diffusion and the role of divalent cations in restriction endonuclease (RE) 2542-Pos Board B149 mechanism. The theory of facilitated diffusion predicts RE cleavage rates Structural Regulation of Cap-Independent Translation in Enterovirus 71 should show a peak as salt concentration is varied as the one dimensional 1 1 2 Michele Tolbert , Christopher E. Morgan , Mei-Ling Li , sliding length depends strongly on salt. We show that NdeI exhibits a peak 1 Blanton S. Tolbert . in cleavage rates near 80 mM NaCl. We are currently designing experiments 1Chemistry, Case Western Reserve University, Cleveland, OH, USA, 2 to verify that this effect is due to DNA sliding. In order to isolate the role of Biochemistry and Molecular Biology, Rutgers, Robert Wood Johnson Mg2þ in the mechanism, we must eliminate the effects of diffusion to the cleav- Medical School, Piscataway, NJ, USA. age site. We are doing this by designing an assay in which REs are pre-bound to Enterovirus 71 (EV71) is the major causative agent of neurovirulent hand, DNA in the absence of Mg2þ. Cleavage is then activated by introducing Mg2þ foot and mouth disease. Infection is acute and may culminate in severe neuro- to the prebound enzyme-DNA complex. We report our current efforts to vali- logical and/or cardiovascular complications that have been linked to severe date this new technique. morbidity and/or death. There currently are no approved vaccines or antivirals to help prevent infection or spread of disease, underscoring an urgent need to 2545-Pos Board B152 better understand the molecular determinants behind this serious threat to pub- Dynamic Studies of MutS-MutL-DNA Complexes in Mismatch Repair lic health. EV71 utilizes a type I IRES to promote viral translation via a Sharonda LeBlanc1, Jacob Gauer1, Pengyu Hao2, T’ Yasah Walser2, poorly understood cap-independent mechanism. Two proteins shown to modu- Keith Weninger2, Dorothy Erie1. late this process are the positive ITAF hnRNP A1 and the negative ITAF 1UNC Chapel Hill, Chapel Hill, NC, USA, 2NC State University, Raleigh, hnRNP D/AUF1. Both have been shown to interact with the highly conserved NC, USA. Stem Loop II (SLII) domain of the IRES. Work from our group has shown DNA mismatch repair (MMR) is a post-replicative system of proteins that cor- that A1 binds via a thermodynamic two-state transition to the conserved 5nt rects rare mistakes in the genome of all organisms. In the human genome of

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6 billion bases, there are ~ 600 errors per round of replication, per cell. If left order to compare them with a previously studied sister molecule DD-P. All uncorrected, errors accumulate as permanent mutations in a genome, and can three molecules have the same middle intercalating dppz component that in- lead to a disease state in the organism. MutS and MutL homologs are tasked teracts with the DNA and only their ancillary side chains are varied. DD-P and with recognizing a mismatch in 107 correctly paired bases, discriminating be- LL-P have the same side chain with different chirality, while on the other tween parent and daughter strand, then initiating repair. Single amino acid mu- hand DD-P and DD-B have the same chirality but vary in the size of the tations in MutS and MutL proteins have been linked to hereditary and sporadic side chain. These intercalators are introduced to the system at different con- colorectal cancer, the third most common cancer worldwide. Although these centrations, while a single DNA molecule is held at a constant force. Mea- mutations, mostly associated with MutL, have been identified in cancer cases, surements of DNA extension as a function of time provide the DNA it is unclear how MMR deficiencies initiate and advance the disease. Failures in equilibrium binding affinity and binding kinetics for these molecules. Prelim- the mismatch repair pathway likely initiate tumorigenesis, but we lack a funda- inary data analysis suggests that LL-P and DD-B exhibit noticeably fast bind- mental understanding of the MMR process. ing kinetics compared to the very similar DD-P. The equilibrium binding MutS and MutL are ATPases that undergo conformational changes upon ATP profile of the LL-P and DD-B complexes reveals that the dissociation con- binding and hydrolysis. We seek to understand how these conformations are stants are on the same order of magnitude as DD-P. These complexes have functional in coordinating repair. On the molecular level, we know that the same chemical structure in the middle intercalating part, which explains MutS initially recognizes a DNA mismatch, and undergoes ATP-dependent the similar affinity for all three of them and the difference in their chirality conformational changes to slide along the DNA. MutL is recruited to the and size explains the difference in the kinetics of their approach to the final site, and interacts with MutS on DNA to coordinate repair with PCNA, threaded intercalation state. EXO1, DNA polymerase, RFC clamp loader, RPA single strand binding pro- tein, and DNA ligase. The formation of the ternary MutS-MutL-DNA complex 2548-Pos Board B155 is a key step in this pathway that involves multiple transient protein-protein and Design of Novel Magnetic Tweezers and its use for Studying DNA- protein-DNA interactions. In these studies, we used in vitro single molecule Compacting Proteins 1,2 2,3 4 fluorescence resonance energy transfer (smFRET) to investigate the dynamic Roberto Jr Fabian , Tyson Christopher , Anneliese Striz , 4 1,2 1,2 molecular mechanism of MMR. Pamela L. Tuma , Ian L. Pegg , Abhijit Sarkar . 1Physics Department, The Catholic University of America, Washington, DC, 2 2546-Pos Board B153 USA, Vitreous State Laboratory, The Catholic University of America, Washington, DC, USA, 3Biomedical Engineering Department, The Resolving Structural Dynamics and DNA-Binding of APOBEC3G Protein 4 Suresh Gorle, Lela Vukovic. Catholic University of America, Washington, DC, USA, Biology Department of Chemistry, The University of Texas at El Paso, El Paso, Department, The Catholic University of America, Washington, DC, USA. TX, USA. We report on the development of a modified version of horizontal magnetic APOBEC3G (A3G) protein, implicated in natural defense against HIV-1, is tweezers used to study DNA-protein interactions at the single molecule level a two-domain protein that catalyzes deamination of deoxycytidine on the that can apply a force to the single DNA in the horizontal focal plane. We viral single-stranded DNA (ssDNA) during reverse transcription. Its two use a 16.4 mm l- DNA attached to a 2.8 mm superparamagnetic bead on both N-terminal (NTD) and C-terminal (CTD) domains bind to ssDNA and cata- ends. The DNA constructs are introduced into a special made sample cell under lyze its deamination, respectively. While structural information about A3G physiological buffer condition. One end of the superparamagnetic beads is im- and A3G:DNA complexes would be useful for development of antiviral ther- mobilized in a glass surface while the other end is near the magnet. We describe apies, A3G oligomerization and precipitation at low concentrations pose the tweezers in detail and present data validating its performance. We show that major challenges for structure determination. Here, we use molecular dock- using a simple design complemented with image processing techniques, we can ing and molecular dynamics (MD) simulations to reconstruct the complete reliably measure changes in the DNA’s extension suitable for studying the structure of A3G monomer and examine its interactions with ssDNA. An binding of proteins. We conclude with a discussion of our experiments on ensemble of A3G structures is prepared by probing mutual orientations of the binding mechanism of the protein mIHF that plays an important role in two experimentally determined A3G domains, and ensuring that DNA-, the infection pathway of tuberculosis. RNA-, and partner protein-interacting residues of A3G are solvent exposed. 2549-Pos Board B156 The obtained ensemble of A3G structures was found to be in good agree- Single-Molecule Picometer Resolution Nanopore Tweezers Resolution ment with results of high speed AFM experiments. Finally, A3G-DNA Jens H. Gundlach1, Ian M. Derrington1, Andrew H. Laszlo1, nucleotide interactions were examined with molecular docking approaches, Jonathan M. Craig1, Ian C. Nova1, Henry Brinkerhoff1, Mathew T. Noakes1, identifying high-resolution structures of A3G:DNA complexes and extended Richard H. Ebright2, Abhishek Mazumder2. regions on A3G surfaces with favorable DNA binding. Our results lay the 1Physics, University of Washington, Seattle, WA, USA, 2Waksman Institute foundation towards designing new ways for controlling the anti-HIV activity of Microbiology, Rutgers University, Piscataway, NJ, USA. of A3G. Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT) is a new technique for observing the motion of single motor enzymes on nucleic acid 2547-Pos Board B154 tracks with more than an order of magnitude improvement in spatiotemporal Role of Threading Moiety Size and Chirality in Determining the DNA resolution over existing single-molecule technologies such as optical tweezers. Binding Characteristics of Threading Intercalators Because SPRNT is derived from nanopore DNA sequencing, it also provides a 1,2 2 1 Thayaparan Paramanathan , Andrew Clark , Nicholas Bryden , readout of the DNA sequence moving through the enzyme, allowing us to query 3 4 2 5 Fredrik Westerlund , Per Lincoln , Micah J. McCauley , Ioulia Rouzina , how nucleic acid sequence regulates the motion of motor enzymes. We discuss 2 Mark C. Williams . how SPRNT works in the context of helicase and RNA polymerase systems to 1Department of Physics, Bridgewater State University, Bridgewater, MA, 2 which we have applied SPRNT, and present results including a full kinetic USA, Department of Physics, Northeastern University, Boston, MA, USA, model relating ATP-hydrolysis steps to sub-nucleotide helicase motion and 3Department of Biology and Biological Engineering, Chalmers University of 4 observation of RNA polymerase pausing and termination at unprecedented Technology, Gothenburg, Sweden, Department of Chemical and Biological resolution. Engineering, Chalmers University of Technology, Gothenburg, Sweden, 5Department of Chemistry and Biochemistry, Ohio State University, 2550-Pos Board B157 Columbus, OH, USA. Interaction of Anti-DNA Antibody MRL4 with DNA Studied at the Single- Small molecules that bind to DNA by threading their ancillary motifs through Molecule Level the DNA bases to intercalate their middle planar section between the DNA Rustem I. Litvinov1, Tatiana A. Nevzorova1,2, Qingze Zhao1, base pairs are known as threading intercalators. These molecules in general Yakov A. Lomakin3, Valerie Tutwiler1,4, Prashant K. Purohit1, exhibit incredibly slow kinetics and high binding affinity, which puts them John W. Weisel1. in the class of prospective anti-cancer drugs. We have been exploring a 1University of Pennsylvania, Philadelphia, PA, USA, 2Kazan Federal variety of ruthenium based threading intercalators using optical tweezers. In University, Kazan, Russian Federation, 3Institute of Bioorganic Chemistry, an optical tweezers set-up a single DNA molecule is attached between Russian Academy of Sciences, Moscow, Russian Federation, 4Drexel two polystyrene beads and manipulated in the presence of various concentra- University, Philadelphia, PA, USA. tions of intercalators to characterize their DNA binding properties. In this Anti-DNA antibodies are produced in autoimmune diseases, such as sys- study we explore the binding of two binuclear ruthenium complexes LL- temic lupus erythematosus. Despite theoretical and clinical importance, mo- 4þ 4þ [m-bidppz(phen)4Ru2] (LL-P) and DD-[m-bidppz(bipy)4Ru2] (DD-B) in lecular properties of pathogenic anti-DNA antibodies and mechanisms of

BPJ 7896_7899 518a Wednesday, February 15, 2017 their interactions with DNA antigens remain largely unknown. The goal of Antimicrobial peptides (AMPs) are small, cationic peptides that have been this work was to study the interactions between a monoclonal anti-DNA recognized for their potential to combat antibiotic-resistant bacteria. While IgG antibody MRL4 from a lupus erythematosus mouse with DNA at the many biophysical studies focus on considering AMP properties in dilute single-molecule level. We applied optical trap-based rupture force spectros- buffers, these buffers do not represent crowded cellular environments. This copy of surface-attached DNA and immobilized antibody that was used is particularly true for AMPs that have intracellular targets, such as the earlier to study the nanomechnics of various bimolecular interactions. We histone-derived antimicrobial peptide (HDAP) buforin II (BF2). BF2 utilizes found that the DNA-MRL4 rupture force spectra displayed two regimes, a translocation-based method of action whereby it binds with nucleic acids one ranging from 20 to 40 pN and another at the forces >40 pN, reflecting inside the cell and interferes with intercellular processes. Experimental formation of at least two bound states of immune complexes with different and computational binding studies in our lab have considered BF2-DNA affinities. We determined two-dimensional kinetic parameters of binding interactions in the absence of crowders. Here we expand on that previous and unbinding of antibody MRL4 with two types of DNA with distinct mo- work using molecular dynamics simulations to study BF2-DNA binding in lecular weights. The average force-free off-rate for the DNA-MRL4 interac- lysozyme-crowded environments. Analyses of protein crowder volume frac- tions was 2.2103 s1 which corresponds to a moderately stable complex. tions at 10% suggest a change in the dynamics of the BF2 - DNA complex The average transition state distance was relatively long (0.94 nm), implying from that seen in uncrowded, dilute conditions. Quicker equilibration and that the DNA-antibody complex is not brittle and the forced dissociation oc- less fluctuation of the complex are also apparent in the crowded environment, curs gradually. The apparent on-rate was near 5.26 s1 and the spring suggesting more restrained movement. Further, the presence of crowders constant of the entire complex 0.0021 pN/nm, indicating a soft and deform- appears to affect hydrogen bonding between BF2 and DNA. These results able macromolecular complex. The nanomechanical parameters of the DNA are being compared to ongoing experimental binding measurements under stretching elasticity extracted from the rupture force distribution histograms analogous conditions in our laboratory and will form the basis for future were characteristic of single-stranded DNA, suggesting that the anti-DNA computational studies to probe how crowding alters the energetic contribu- monoclonal MRL4 antibody binds preferentially to one strand of DNA. tions toward binding of particular chemical moieties. Together, these results The results provide fundamental characteristics of DNA-antibody interac- support the need to consider intracellular AMP interactions under crowded tions that are useful to understand mechanisms of DNA-protein interactions conditions. and molecular basics of formation and dissociation of immune DNA- containing complexes formed in autoimmune diseases. 2553-Pos Board B160 Computational Investigation of Proton Transfer, pKa Shifts and pH- 2551-Pos Board B158 Optimum of Protein-Dna and Protein-RNA Complexes Beyond Diffusion-Controlled Kinetics: How a Protein Implements a Fast Yunhui Peng, Emil Alexov. Search on DNA to Get to its Specific Binding Site Department of Physics and Astronomy, Clemson University, Clemson, Milagros Castellanos1,2, Victor Mun˜oz1,3. SC, USA. 1Macromolecular Structure, National Biotechnology Center (CNB-CSIC), Protein-nucleic acid interaction plays crucial roles in many biological processes Madrid, Spain, 2IMDEA Nanoscience, Madrid, Spain, 3School of in cell life. This work focused on how the changes of pKa’s and protonation Engineering, University of California Merced, Merced, CA, USA. states of the ionizable groups may accompany the protein-nucleic acid interac- There are a wide number of proteins that initiate and regulate the transcrip- tion. Taking advantages of recently developed pKa’s calculation tool tion of genes. All have DNA-binding domains that give them the ability (DelphiPka), we statistically studied the proton transfers and pKa shifts in to bind specific sequences. Classical binding reactions are controlled by the protein-DNA/RNA interactions using a large protein-nucleic acid interac- diffusion limit. In those cases, association rate for binding doesn’t exceed tion database, NPIDB database. It was revealed that the protein’s interfacial 108 M1S1 and then, for high affinity binding (nM), dissociation rate would basic residues experience favorable electrostatic interactions while the protein expands dozens of seconds. Find a short target sequence within the enor- acidic residues undergo proton uptake to reduce the energy cost upon the bind- mous pool provided by genomic DNA requires protein moving fast towards ing. Both base group and phosphate group of nucleotides were found to partic- the specific binding site (fast association and dissociation while searching). ipate into the binding and the base group binding introduces the most Before reaching their targets, DNA-binding proteins encounter nonspecific significant pKa shifts or proton transfers in nucleic acids. This work also clas- DNA first and bind to it, although with weaker affinity than DNA specific sified different pKa shift origins for all ionizable groups based on different sequence. As observed first in 1970, DNA binding proteins binds its target chemical and physical properties and binding modes. Furthermore, the opti- ~100 times faster than allowed by the 3D diffusion limit. This faster-than- mum pH of the binding and their protein components were determined in diffusion binding was explained by the facilitated-diffusion model, in which this work and their optimum pHs tend to be correlated. a DNA-binding protein interacts with nonspecific DNA before reaching its target by three main mechanism: sliding, where protein slides in 1D along 2554-Pos Board B161 nonspecific DNA; hopping, where the protein dissociates from DNA briefly, Thermodynamic Additivity for Impacts of Base-Pair Substitutions on performing free 3D diffusion, and lands back on DNA at other close loca- Association of the Egr-1 Zinc-Finger Protein with DNA tion; and jumping, where the protein’s DNA landing location is not corre- Abhijnan Chattopadhyay, Levani Zandarashvili, Ross H. Luu, lated to the dissociation site. Facilitate diffusion has been studied Junji Iwahara. theoretically, computationally and experimentally. However, very little is Biochemistry & Molecular Biology, University of Texas Medical Branch, known from the protein viewpoint, and how protein and DNA dialogue in Galveston, TX, USA. an efficient manner. We are using the DNA-binding engrailed homeodomain The transcription factor Egr-1 specifically binds as a monomer to the 9-bp module that binds with high affinity to DNA sequence TAATTA/G. In the target DNA sequence, GCGTGGGCG, via three zinc fingers and plays impor- present work we will analyze how DNA sequence seems to have evolved tant roles in the brain and cardiovascular systems. Using fluorescence-based towards an organization that ensures biologically precise gene expression. competitive binding assays, we systematically analyzed the impacts of all Combination of specific sequence binding, degenerated sequence binding possible single nucleotide substitutions in the target DNA sequence and deter- and nonspecific binding could promote an antenna effect for guiding the mined the change in binding free energy for each. Then, we measured the protein to the specific site. Mechanical statistical models help us to confirm changes in binding free energy for sequences with multiple substitutions and the experimental data and shed light on this fundamental and multifactorial compared them with the sum of the changes in binding free energy for each biological problem. substitution. For the DNA variants with 2 or 3 nucleotide substitutions in the target sequence, we found excellent agreement between the measured and pre- 2552-Pos Board B159 dicted changes in binding free energy. Interestingly, however, we found that Investigating Buforin II Interactions with Nucleic Acids under Crowded this thermodynamic additivity broke down with a larger number of substitu- Conditions tions. For DNA sequences with 4 or more substitutions, the measured changes Carla Perez1, Mala L. Radhakrishnan2, Donald E. Elmore2. in binding free energy were significantly larger than predicted. Based on these 1Department of Chemistry, Wellesley College, Wellesley, MA, USA, results, we analyzed the occurrences of high-affinity sequences in the genome 2Department of Chemistry and Biochemistry Program, Wellesley College, and found that the genome contains millions of high-affinity sequences that Wellesley, MA, USA. might serve as natural decoys and sequester Egr-1.

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Membrane Physical Chemistry II [4] Benedetto, Ballone ACS Sustainable Chem. Eng. 4, 392 (2016). [5] Benedetto, et al. J. Phys. Chem. B 118, 12192 (2014). 2555-Pos Board B162 [6] Benedetto, Ballone private-communication. Membranes Matter: Predicting Drug Toxicity 2557-Pos Board B164 R. Lea Sanford1, Wesley Chao1, Jeanne Chiaravalli-Giganti2, Antonio Luz2, 2 1 The Propensity of Ceramides to Segregate into Ceramides-Rich Phase in J. Fraser Glickman , Olaf S. Andersen . Fluid Phosphatidylcholine Bilayer is Markedly Influenced by their Long- 1Biophysics and Physiology, Weill Cornell Medicine, New York, NY, USA, 2 Chain Sphingoid Base The Rockefeller University, New York, NY, USA. Md. Abdullah Al Sazzad1, Tomokazu Yasuda2, Michio Murata2, It remains a challenge to predict whether a new drug candidate will have undesir- J. Peter Slotte1. able side effects. Many biologically active molecules, including drugs and drug- 1Science and Engineering, A˚ bo Akademi University, Turku, Finland, leads, are amphiphiles that partition into lipid bilayers, which will tend to alter 2Organic Chemistry, University of Osaka, Osaka, Japan. bilayer physical properties, thereby modulating membrane protein function. Ceramides contain a sphingoid long-chain base to which mostly saturated acyl Such bilayer-modifying molecules may be promiscuous modifiers of membrane chains are N-linked. The length of both the sphingoid long-chain base and N- protein function, raising the possibility that they have off-target effects. Conse- linked acyl chain may vary in ceramides from various tissues and organism. quently, it may be possible to predict whether a compound will have off-target The biophysical properties of ceramides in membrane bilayers are known to effects based on quantitative studies on the compound’s bilayer-modifying poten- be affected by the nature and length of both the sphingoid base and the acyl tial. To address this question, we developed an assay to quantify the bilayer- chains. We have examined how the lateral segregation of ceramides in 1-palmi- modifying potential of large numbers of compounds using a gramicidin-based toyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was affected by the length fluorescence assay (GBFA), which reports how a compound alters the gramicidin 4 of both the sphingoid base and the N-linked acyl chain. We performed differ- monomer dimer equilibrium. Using this assay, we have shown that many drug ential scanning calorimetry and various fluorescence based measurements to and drug-leads alter lipid bilayer properties at the concentrations where these study the lateral segregation and formation of ceramide-rich phases. The lateral compounds become indiscriminate modifiers of membrane protein function. segregation of the ceramide-rich phase occurred at increasingly higher bilayer Such indiscriminate modifiers of membrane protein function are likely to have concentration, as the long chain base chain was shortened (from d20:1 to off target effects; we pursued this question in a study on libraries of compounds d16:1). A similar but much weaker trend was observed when the N-linked that had been tested for toxicity in ‘‘high-content’’ screening assays that quanti- acyl chain length was shortened (from 20:0 to 14:0). We also prepared two cer- fied cellular ATP levels, nuclear morphology, nuclear membrane integrity, and amide analogs which were mirror images to each other with d16:1/17:0 or apoptosis in immortalized liver, lung, and neuronal cell lines. We tested 524 total d20:1/13:0 chains. We observed that the ceramide analog with longer sphingoid compounds comprising four libraries (one non-toxic library and three mixed li- base d20:1/13:0 was able to segregate into ceramide-rich phase at lower bilayer braries of non-toxic and toxic compounds; the libraries were ‘‘blinded’’ until concentration as compared with shorter sphingoid base analog d16:1/17:0. Our the results of the GBFA were known) and found that the GBFA predicts cellular results indicate that the length of the long-chain base of ceramide affected cer- toxicity, with minimal assignment of false positives. These results suggest that in amide segregation in fluid bilayers more than the N-linked acyl chain length. vitro measurement could be used as a warning sign for off-target biological ef- fects in drug discovery efforts and, further, provides a mechanism by which am- 2558-Pos Board B165 phiphiles exert their toxicity, namely by altering lipid bilayer physical properties. Graphene Oxide and Phospholipids at the Air-Water Interface Kassidy W. Rodriguez1, Cain Valtierrez2, Joan C. Kunz1, 2556-Pos Board B163 Benjamin L. Stottrup2. Biomolecules, Water and Room-Temperature Ionic Liquids: Challenges 1Chemistry, Augsburg College, Minneapolis, MN, USA, 2Physics, Augsburg and Opportunities in Basic Science and Applications College, Minneapolis, MN, USA. Antonio Benedetto. Graphene Oxide (GO) has many potential and realized applications as a novel School of Physics, University College Dublin (UCD), Dublin (IE), and material, additive, and thin film coating. However, like other nanoparticles, the Laboratory for Neutron Scattering and Imaging, Paul Scherrer Institut (PSI), interactions of graphene oxide with biological membranes and tissue is not Villigen (CH), Switzerland. fully understood and therefore it is important to determine modes of interaction. In 560 BC, the Greek philosopher Thales conjectured that water is the primary GO has been shown to be surface active under a variety of experimental con- essence of life [1]. Thales’ hypothesis is now a primary scientific paradigm, and ditions and preparations. Further, GO has been shown to insert into lipid mono- has also found credit in the mesoscopic and microscopic world of cells, proteins layers based on lipid head group affinity. We test the competitive absorption of and biomembranes. It is well known, for instance, that in living organisms, wa- graphene oxide with phospholipids at the air-water interface using Langmuir ter molecules actively interact with and support the biochemistry of a panoply film-balance techniques and Brewster angle microscopy. We determine how of biomolecules, such as proteins, enzymes, DNA, cell membranes, and carbo- monolayer phase behavior, GO concentration, and subphase pH impact these hydrates [2,3]. nanoparticle/phospholipid interactions. Brewster angle microscopy was also In this context, water is a shorthand for ‘‘water electrolyte solution’’, since used to observe the morphology of GO packing with phospholipids at the almost without exception a variety of ions dissolved in water are needed to air-water interface as well as estimating the height of GO when in the lipid ensure the stability of bio-systems, greatly contributing to their complexity. monolayer. In recent years, the development of organic ionic compounds of the so-called room-temperature ionic liquid (RTIL) family has enormously expanded the 2559-Pos Board B166 number of ionic systems that could be used to modify the properties of the wa- Dithiothreitol Raises Transition Temperatures in Giant Plasma Mem- ter at bio-interfaces, and thus to affect the behaviour of bio-systems [4]. brane Vesicles Experimental techniques, in primis X-ray crystallography and NMR, allowed Ruth Gerstle, Rohan Desai, Sarah Veatch. us to shed light on the microscopic structure of these biosystems solvated by University of Michigan, Ann Arbor, MI, USA. their hydration water, and one of the major results is that the knowledge of Giant plasma membrane vesicles (GPMVs) can be isolated from adherent cells structures is not sufficient to understand their bio-function, since dynamical as- using a variety of biochemical treatments. GPMVs prepared using dithiothreitol pects seem to play a major role [3]. (DTT) produce GPMVs that have systematically higher miscibility transition Our research activity is focus on such dynamical aspects, it primarily concerns temperatures than vesicles produced using other methods (Levental et al. the microscopic mechanisms underlying RTIL effects on biosystems through PNAS 108(28):11411-6 (2011)). Past work has proposed that this difference their hydration water, and relies on the combination of neutron scattering arises from the removal of post-translational lipid modifications from plasma and molecular dynamics simulations. membrane proteins by this chemical. Here we present results suggesting that In our contribution we will briefly review the state of the art in this topic [4]. Then an increase in transition temperature is a direct result of the presence of this we will focus on two specific cases. The first one concerns their interaction with small molecule in solution, presumably due to its incorporation into the mem- biomembranes: we will show how RTILs diffuse into the hydrophobic portion of brane. Here we show that GPMVs prepared through incubation with DTT have biomembranes, enhancing the penetration of water into the bilayer [5]. The sec- lower transition temperatures when DTT is removed from solution through ond one concerns the RTIL interaction with proteins [4]. We will show how these dialysis, and that higher transition temperatures are restored when this chemical organic salts can either help or prevent amyloidogenesis [6]. Implications in bio- is added back to GPMV suspensions. We also find that GPMVs can be pro- medicine, sensing technology, and material science will be presented. duced from cells briefly washed with DTT containing buffers, and that these [1] Aristotle, Metaphysics 983 b6 8-11,17-21. GPMVs also have low transition temperatures. Several implications of these [2] Billeter, et al. Mol. Biol. 234, 1084 (1993). findings will be discussed, including the likelihood that plasma membrane tran- [3] Rasmussen, et al. Nature 357, 423 (1992). sition temperatures likely reside close to 0C in the absence of DTT.

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2560-Pos Board B167 mixture of lipids that exhibits liquid-ordered and liquid-disordered coexistence, Protonation of Long Chain Fatty Acids at the Membrane-Water Interface and contain a small amount of monosialoganglioside (GM1), which binds fluores- Alina A. Pashkovskaya1, Mario Vazdar2, Olga Jovanovic1, Peter Pohl3, cent cholera toxin subunit B (CTB). When shear flow is used to form a concen- Elena E. Pohl1. tration gradient in CTB anchored to the bilayer, a corresponding lipid 1Univ Vet Medicine Vienna, Vienna, Austria, 2Rudjer Boskovic Inst, Zagreb, concentration gradient is created. Similar gradients of GM1 and associated lipids Croatia, 3Johannes Kepler Univ, Linz, Austria. have been formed in supported bilayers previously using electrophoresis [1]. Long-chain free fatty acids (FA) play an important role in several physiological Since lipid composition in the two leaflets has been shown to be tightly coupled and pathological processes such as lipid fusion, alteration of membrane perme- [2,3], a flow-induced lipid gradient provides a physical mechanism for transmit- ability and fluidity, as well as regulation of enzyme and protein activity. FA- ting a signal from the outer leaflet to the inner one. facilitated membrane proton transport (flip-flop) and FA-dependent proteina- [1] Dynamic Reorganization and Correlation Among Lipid Raft Components. ceous proton transport (as in the case of mitochondrial uncoupling proteins) Mo´nica M Lozano, Jennifer S. Hovis, Frank R. Moss III, and Steven G. Boxer, are governed by the difference between FA’s pK and the pH in the immediate Journal of the American Chemical Society, 138, 9996-10001(2016). membrane vicinity1,2. So far, a quantitative understanding of the process is [2] Tuning lipid mixtures to induce or suppress domain formation across leaf- hampered by the shift in the pK value of carboxylic groups that occurs upon lets of unsupported asymmetric bilayers. Marcus D. Collins and Sarah L. Kel- moving the FA from the aqueous solutions into the membrane. Its molecular ler. Proceedings of the National Academy of the Sciences of the United States of origin is not understood, conceivably because the reported pKa values for the America 105 (1) 124-128 (2007). same FA are scattered between 5 and 10.5. Here we systematically obtained [3] Transbilayer Colocalization of Lipid Domains Explained via Measurement the dependence of apparent pKa values on chain length, number and position of Strong Coupling Parameters. Matthew C. Blosser, Aurelia R. Honerkamp- of double bonds by measuring the zeta-potential of liposomes reconstituted Smith, Tao Han, Mikko Haataja, Sarah L. Keller. Biophysical Journal 109 with FA at different pH. pKa ranged from 6.4 to 7.6. Combining the zeta poten- (11) p2317-2327 (2015). tial measurements with molecular dynamic simulations, we found that pK is 2563-Pos Board B170 determined by the interplay between the energetic costs for burying a charged Electrostatic Charging of Membranes by Adenosine Triphosphate moiety into the bilayer and the hydrophobic interactions between the free and Ryan Z. Lybarger, Daniel J. Bose, Bruce D. Ray, Horia I. Petrache. lipid-bound fatty acid chains. Department of Physics, Indiana University Purdue University Indianapolis, 1. Pohl, E. E., Voltchenko, A. M. & Rupprecht, A. Flip-flop of hydroxy fatty Indianapolis, IN, USA. acids across the membrane as monitored by proton-sensitive microelectrodes. Biological processes rely on energy transfers from chemical sources to molecular Biochim.Biophys.Acta 1778, 1292-1297 (2008). machines such as ion pumps, mechanical motors, and molecular switches. This 2. Rupprecht, A. et al. Role of the transmembrane potential in the membrane energy transfer often takes place in the vicinity of lipid membranes, however, proton leak. Biophys.J. 98, 1503-1511 (2010). the interaction of ATP with simple lipid bilayers is yet unknown. We investigate 2561-Pos Board B168 this interaction by NMR spectroscopy, small-angle x-ray scattering, and dynamic The Changes of Physical Parameters of Lipid Membrane caused by Lipid light scattering. We find that ATP molecules bind to phospholipid bilayers as Peroxidation-Derived Aldehydes measured by a restriction of phosphate motion as well as by a net charging effect Ksenia Chekashkina1, Olga Jovanovic2, Piotr Kuzmin1, Elena Pohl2, of membranes. Interestingly, the membrane charging due to ATP gives rise to an Bashkirov Pavel3. unbinding transition of phosphatidylcholine multilayers which is not seen in the 1A.N. Frumkin Institute of Physical Chemistry and Electrochemistry, presence of monovalent ionic salts. We quantify this particular electrostatic effect Moscow, Russian Federation, 2Institute of Physiology, Pathophysiology and of ATP in terms of surface charge distributions and interbilayer interactions. This Biophysics, University of Veterinary Medicine, Vienna, Austria, 3Federal study can help descriptions of energy transfers in biomembrane processes. Scientific Clinical Center of Physical-Chemical Medicine of FMBA of 2564-Pos Board B171 Russia, Moscow, Russian Federation. Bending Modulus of Multicomponent Lipid Membranes Reactive oxygen species (ROS) are very important biological signaling mole- Pavel Bashkirov1,2, Ksenia Chekashkina1,2, Piotr Kuzmin2, Vadim Frolov3,4. cules that play a pivotal role in cellular processes. Imbalance in ROS produc- 1Federal Scientific Clinical Center of Physical-Chemical Medicine of FMBA tion and antioxidant protection results in oxidative stress, under which crucial of Russia, Moscow, Russian Federation, 2A.N. Frumkin Institute of Physical biomolecules (DNA, proteins, lipids) can get damaged by ROS and their deriv- Chemistry and Electrochemistry of RAS, Moscow, Russian Federation, atives. Reactive aldehydes (RAs) are among various products of ROS interac- 3Biophysics Unit (CSIC-UPV/EHU) and Department of Biochemistry and tion with unsaturated lipids. RAs are implicated in disfunctioning of various Molecular Biology, University of the Basque Country, Leioa, Spain, proteins. The molecular mechanisms underlying these interactions are still 4IKERBASQUE, Basque Foundation for Science, Bilbao, Spain. poorly understood. Recently a novel lipid-mediated mechanism was proposed, Cellular membranes contain substantial amounts of lipids with high intrinsic according to which biologically important RAs (4-hydroxy-2-nonenal, 4-hy- curvature, such as dioleoylphosphatidylcholine (DOPE). Such lipids tend to droxy-2-hexenal and 4-oxo-2-nonenal) modified the activity of several self-assemble into highly curved non-bilayer structures often linked to interme- membrane transporters. These RAs were shown to covalently modify the mem- diates of membrane remodeling, fusion and fission. We found that membrane de- brane lipid phosphatidylethanolamine (PE), causing formation of different formations caused demixing of such lipids from the lipid species of low intrinsic membrane-active adducts. curvature and revealed how this demixing contributes to the effective elastic Here we study the changes of physical properties of lipid membranes induced compliance of the membrane. We discriminated the entropic elasticity due to by interaction of aldehydes with the lipid bilayer containing a large amount of lipid demixing from the instantaneous elastic response associated with molecu- PE molecules. We used lipid nanotubes pulled from the flat lipid bilayer to lar deformations, the ‘‘material’’ rigidity. We derived the analytical expression measure membrane bending rigidity and spontaneous curvature of membrane describing the interplay between the material and demixing components in terms compounds. We showed that RAs caused significant reduction of the mem- of the corresponding elastic moduli. We found experimentally that the material brane bending rigidity. The observed decrement of the elasticity was associated and demixing contributions to the elastic compliance could be comparable for with pronounced changes of the lipid molecular geometry and with the changes physiologically relevant lipid mixtures. Furthermore, the effects of phospho- of packing density of lipids in the bilayer. The magnitude of the effect of lipids species with high intrinsic curvature and cholesterol on the effective mem- different RAs on the transporter activity correlated with their effect on the lipid brane elasticity were profoundly different in different lipid backgrounds, bilayer properties. revealing molecular organization of multicomponent membrane monolayers. 2562-Pos Board B169 2565-Pos Board B172 Lipid Concentration Gradient Resulting from Shear Flow Advection of Quantifying the Relationship between Monovalent Cation Size and Lipid Membrane-Bound Proteins Domain Formation in Anionic-Zwitterionic Mixed Lipid Bilayers Aurelia R. Honerkamp-Smith1, Raymond E. Goldstein2. Sai J. Ganesan, Hongcheng Xu, Silvina Matysiak. 1Physics, Lehigh University, Bethlehem, PA, USA, 2Applied Mathematics Fischell Department of Bioengineering, University of Maryland, College and Theoretical Physics, University of Cambridge, Cambridge, United Park, MD, USA. Kingdom. Phosphatidylserine (PS) and phosphatidylcholine (PC) are two of the major Flow mechanosensation sets many essential biological processes in motion. Un- anionic and zwitterionic phospholipids in mammalian cell membranes. Ion-PS der the right conditions, fluid flow can advect membrane-associated proteins with interaction is hypothesized to play a crucial role in a range of biological events large extracellular domains. To explore these conditions we prepare a model sys- including membrane fusion, lipid phase modulation, membrane protein insertion tem consisting of a discrete patch of supported lipid bilayer formed by bursting a and translocation. Here we use the coarse-grained (CG) WEPMEM model to þ giant unilamellar vesicle onto a glass coverslip. The vesicles are composed of a investigate the role of salt addition, with different monovalent cation (M ) sizes,

BPJ 7900_7903 Wednesday, February 15, 2017 521a in lipid headgroup-driven nanodomain formation in PC-PS lipid mixtures. Our effect on lipid membranes. Using solution atomic force microscopy study shows a dependency of monovalent cation size Mþ on lipid domain forma- (AFM), we observe polyQ oligomers coexisting with short fibrils. Fourier tion of anionic PS and zwitterionic PC in mixed bilayers. The formation of these transform infrared spectroscopy reveals that the content of b-sheet enriched domains are dependent on lipid-ion binding modes and partition free energy aggregates increases with incubation time. PolyQ aggregates-induced of ions in the water-bilayer interface. Certain binding modes lead to growth membrane disruption is inferred from time-dependent calcein leakage of ion-mediated PS lipid clusters. A coupled relationship between lipid curva- of lipid vesicles. Detailed structural and mechanical perturbations of lipid ture and asymmetry is observed in highly demixed PC/PS mixed bilayers. This membranes are revealed by solution AFM. We find that membrane dis- is the first molecular-level simulation study on the monovalent cation size- ruption by polyQ aggregates proceeds by a two-step process, involving dependent lipid domain formation in zwitterionic-anionic mixed lipid bilayers. partial and full disruption. In addition to height contrast, the resulting partially and fully disrupted bilayers have distinct rigidity and adhesion 2566-Pos Board B173 force properties compared to the intact bilayer. Specifically, the bilayer Examining the Translocation of Amphiphiles across Lipid Bilayers using a rigidity increases as the intact bilayer becomes partially and fully disrupted. Gramicidin Channel-Based Fluorescent Assay Surprisingly, the adhesion force first decreases and then increases dur- Thasin Peyear, Radda Rusinova, Olaf S. Andersen. ing the disruption process. By resolving individual fibrils deposited on Physiology and Biophysics, Weill Cornell Medical College, New York, NY, bilayer surface, we show that both the length and the number of fibrils USA. can increase with incubation time. Our results highlight that membrane Detergents are common tools in molecular biology and biochemistry, including disruption could be the molecular basis of polyQ aggregates induced the solubilization and purification of membrane proteins. Detergents have long cytotoxicity. been known to alter the lateral organization of lipid bilayers, more recently they have been shown to increase lipid bilayer elasticity (Lundbæk et al., 2569-Pos Board B176 PNAS 2010). Some are able to cross lipid bilayers, others not (le Maire et al, Ceramide-Induced Lamellar Gel Phases in Fluid Cell Lipid Extracts Biochemistry, 1987; Heerklotz, Biophys J, 2001), but less is known about this Felix M. Goni, Aritz Garcia-Arribas, Hasna Ahyayauch, Jesus Sot, aspect of detergent-bilayer interactions. We explored this question using grami- Pablo L. Lopez-Gonzalez, Alicia Alonso. cidin channels to detect amphiphile-induced changes in bilayer elasticity with a Instituto Biofisika (CSIC-UPV/EHU), Leioa, Spain. sequential-mixing fluorescence quench assay, which allows for determining the The effects of increasing amounts of palmitoylceramide (pCer) on human time course of detergent interaction with the outer leaflet of a large unilamellar red blood cell lipid membranes have been studied using atomic force vesicle (LUV), including the time is takes to cross the bilayer to intercalate microscopy of supported lipid bilayers, in both imaging (bilayer thickness) into the intravesicular leaflet. Three common detergents were investigated: and force-spectroscopy (nanomechanical resistance) modes. Membranes CHAPS; SDS; and Triton X-100. All three produced a fast (complete within appeared homogeneous with pCer concentrations up to 10 mol % because 15 ms) increase in fluorescence quench rate, indicative of a shift in the gramicidin of the high concentration of cholesterol (Chol) present in the membrane 4 monomer dimer equilibrium toward the conducting dimers. In the case of (about 45 mol %). However, the presence of pCer at 30 mol % gave rise Triton X-100, this initial phase was followed by a second phase, complete within to a clearly distinguishable segregated phase with a nanomechanical resis- 500 ms, which is indicative of Triton translocation to the intravesicular leaflet. tance 7-fold higher than the continuous phase. These experiments were The same general pattern was observed with SDS, though the slow phase was validated using differential scanning calorimetry. Furthermore, Chol deple- complete only after ~1 min, whereas CHAPS appeared to reside in the extrave- tion of the bilayers caused lipid domain generation in the originally sicular leaflet only. We tested the implications of these results using LUVs pre- homogeneous samples, and Chol-depleted domain stiffness significantly pared in the presence of detergent, where Triton X-100 and SDS again increased with higher amounts of pCer. These results point to the possibility appeared to be membrane permeant, whereas CHAPS appeared to be imperme- of different kinds of transient and non-compositionally constant, complex ant. The LUV size distributions were determined dynamic light scattering; though gel-like phases present in RBC lipid membranes rich in both pCer and the size distributions differed when the LUVs were prepared in the presence of Chol, in contrast to the widespread opinion about the displacements be- detergent, these differences could not account for the changes in fluorescence tween pCer-enriched ‘‘gel-like’’ domains and liquid-ordered ‘‘raft-like’’ quench rates (or our conclusions). Chol-enriched phases. Changes in the biophysical properties of these 2567-Pos Board B174 complex gel-like phases governed by local modulation of pCer:Chol ratios could be a cell mechanism for fine-tuning the properties of membranes as Calcium Ion-Mediated PIP2-Cluster Formation from All-Atom Molecular Dynamics Simulations required. Kyungreem Han, Richard M. Venable, Richard W. Pastor. Laboratory of Computational Biology, NIH/NHLBI, Rockville, MD, USA. 2570-Pos Board B177 Effects of Carotenoids on Membrane Bending Rigidity Phosphatidylinositol (4,5)–bisphosphate (PIP2), a phosphorylated derivative of Rudy M. Me´ndez Reina1, Maria I. Pe´rez Lo´pez1, Chad Leidy2, phosphatidylinositol, is a critical lipid in cell membranes. Not only PIP2 itself Manu Forero-Shelton1. is the precursor of the essential second messengers for the cellular signal trans- 1 duction such as inositol (1,4,5)–trisphosphate (IP ) and diacylglycerol (DAG), Department of Physics, Universidad de los Andes, Bogota´, Colombia, 3 2Student Academic Success Center, University of California, Davis, CA, but also the cooperative action from PIP2–clusters plays indispensable roles in the onset and progression of various human diseases, including cancer, neurode- USA. generation, metabolic disorder, and inflammation. In the present study, we have Carotenoids are a group of pigments found in plants and some photosyn- performed all–atom molecular dynamics simulations to discover the context– thetic organisms that play several important physiological functions. For example, carotenoids have been shown to promote virulence by stabilizing dependent nature of the PIP2–cluster formation. Special attention has been paid to the hydrogen bond network formation depending on local ion and/or lipid com- themembraneofStaphylococcus aureus during infection. In this work we positions. We examine synergistic, additive, and inhibitory effects between Kþ focus on how carotenoids modulate the properties of the cell membrane and Ca2þ in clustering based on a series of molecular dynamics simulations on of S. aureus as well as model membranes. We observed a dramatic decrease hydrated mono- and dimethylphosphate anions, molecular fragments which in the pigmentation of S. aureus biofilms when compared to planktonic cells. We quantified the carotenoid content of membranes isolated from bio- model properties of the PIP2 head–group. Furthermore, hypotheses which address the roles of cholesterol in the hydrogen bond network formation are tested by sim- films and planktonic cultures of S. aureus by absorption spectroscopy and confirmed this is due to decreased carotenoid content. We also characterized ulations of pure PIP2 and PIP2–rich mixed monolayers. These results are applied to the interpretation of Langmuir trough experiments. This study provides a the lipid composition in S. aureus membranes and found that the most abun- computational framework for better understanding the fundamentals of the dant lipid was C14. For this reason, we chose a mixture of two C14 lipids cation–mediated PIP –pair, –cluster, and –network formation. (DMPC:DMPG), in order to model both the mechanical and electrostatic 2 properties of the membrane of S. aureus. In order to understand how carot- 2568-Pos Board B175 enoids affect the membrane, we first measured the phase transition of Lipid Membrane Structural and Mechanical Properties Modulated by DMPC with carotenoids by monitoring GP Laurdan as a function of temper- Polyglutamine Aggregates ature. We observed that carotenoids lower the cooperativity of the lipid Nawal K. Khadka1, Fengyu She2, Jianfeng Cai2, Jianjun Pan1. phase transition. We also measured the bending rigidity for DMPC:DMPG 1Physics, University of South Florida, Tampa, FL, USA, 2Chemistry, vesicles in the presence of carotenoids with Vesicle Fluctuation Analysis University of South Florida, Tampa, FL, USA. (VFA) and saw an increase in rigidity. These changes could be important Lipid membranes are potential targets by protein aggregates. We study in understanding the mechanism of S. aureus0 resistance to antimicrobial aggregates formed by a polyglutamine (polyQ) peptide, and their disruptive peptides.

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2571-Pos Board B178 2574-Pos Board B181 Stereochemistry and Phase Behavior in Hydroxycholesterol/Phospholipid Hallmarks of Reversible Phase Separation in Living, Unperturbed Cell Monolayers Membranes Vision B. Bagonza1, Blair E. Stewig1, Caroline Wochnik1, Joan C. Kunz2, Scott Rayermann, Glennis Rayermann, Alex Merz, Sarah Keller. Benjamin L. Stottrup3. University of Washington, Seattle, WA, USA. 1Augsburg College, Minneapolis, MN, USA, 2Chemistry, Augsburg College, Controversy has long surrounded the question of whether micron-scale Minneapolis, MN, USA, 3Physics, Augsburg College, Minneapolis, MN, lateral phase separation can organize proteins and lipids within the mem- USA. branes of unperturbed living cells. A clear answer hinges on observation Hydroxycholesterols are cholesterol analogs formed either enzymatically or of hallmarks of a reversible phase transition. Here, by directly imaging non-enzyamatically to have a second hydroxyl function group. In this work micron-scale membrane domains of yeast vacuoles in vivo, we demonstrate we study compositions of 24(R), 24(S), and 27 hydroxycholesterol mono- that the domains arise through a phase separation mechanism. The domains layers mixed with 1,2-Dimysristoyl-sn-glycero-3-phosphocholine (DMPC). disappear above a distinct miscibility transition temperature, Tmix, and reap- These sterols induce liquid-liquid phase separation which share similarities pear below Tmix over multiple heating and cooling cycles. The domains are and distinct differences from commonly studied lipid raft model composi- large, have smooth boundaries, and can merge, consistent with fluid phases. tions with cholesterol. Previous work has identified these differences and Hence, large-scale membrane organization in living cells under physiologi- the role that the position of the second hydroxyl group plays in determining cally relevant conditions can be controlled by tuning a single thermodynamic phase behavior. Less well studied is the observation that unlike other liquid- parameter. liquid phase separated lipid monolayers, hydroxycholesterols domains display distinct nucleation and growth behavior. We use fluorescence micro- 2575-Pos Board B182 scopy and Langmuir isotherms to test how the observed thermodynamic Small-Reservoir Electrostatics behavior matches with theory. A recently developed method of measuring Joel A. Cohen. line tension using domain size distribution is applied and results are Physics, Univ of Massachusetts, Amherst, MA, USA. compared to well-studied cholesterol/DMPC compositions. We have previ- The solution to the Poisson-Boltzmann (PB) equation for the mean-field ously identified the important role optical activity plays in the differences be- electrostatic potential and ion distributions surrounding an isolated charged tween 22(R) and 22(S) hydroxycholesterol phase behavior. We attempt to particle generally employs a boundary condition of thermodynamic equilib- develop a general model for these differences and present the results from rium with a reservoir whose properties are specified, such as its bulk ion studies of 24(R) and 24(S). concentrations. The reservoir, by definition infinitely large, provides the reference state for the system. However no experimental reservoir is truly 2572-Pos Board B179 infinite. Here we consider a single macroion suspended in an electrolyte Surfactant pKa Calculations using Molecular Dynamics Simulations equilibrated with a finite reservoir of small enough volume that establish- W.F. Drew Bennett, Joan-Emma Shea, Frank L. Brown. ment of the macroion’s screening cloud depletes the reservoir’s concentra- Chemistry, University of California, Santa Barbara, Santa Barbara, CA, tion of counterions and enhances its concentration of coions. Such a USA. ‘‘reservoir’’ no longer provides a useful reference, as its state depends on The protonation state for ionizable surfactants is crucial for self-assembly, the macroion charge. In a semi-confined volume, such as inside an ion chan- phase behavior, and morphology. For example, oleic acid forms vesicles at nel, a large-enough and/or close-enough reservoir may not be available, in intermediate pH conditions (~7-9), micelles at high pH, and oil phases at which case reservoir-depletion effects may occur. An extreme example is low pH. The pKa for oleic acid shifts from ~4.8 as monomers in water to the colloidal crystal, where no bulk phase exists and there is no bulk ion ~7.5 in model phospholipid vesicles. It remains challenging to accurately reservoir at all. For this system the usual solution of the PB equation does model surfactant self-assembly and protonation behavior with molecular dy- not apply, as electrostatic potentials are nowhere constant, hence there is namics simulations. We use constant pH molecular dynamics simulations no bulk phase by which to establish reference ion concentrations. In such and thermodynamic integration calculations for protonating a single fatty a closed system the conserved quantities are the total ion numbers rather acid in different chemical environments. Long time scale Martini coarse- than bulk ion concentrations. Reformulation of the PB solution referenced grained simulations show the two methods produce very similar titration to total ion numbers yields an expression having the form of a Fermi- curves for oleic acid in micelles. We use CHARMM36 atomistic simulations Dirac distribution. Comparisons between PB solutions referenced to reser- to calculate titration curves for oleic acid in different micelle environments voir ion concentrations and PB solutions referenced to total ion numbers and bilayers. The pKa for oleic acid in a DOPC bilayer is 7.2, which com- are illustrated and compared to experimental data for colloidal-crystalline pares well to experiments. Cholesterol content is shown to decrease the liposome suspensions. pKa for oleic acid to 5.9 compared to a pure DOPC bilayer. Our calculations provide insight into the mechanism of oleic acid self-assembly and the effect 2576-Pos Board B183 of the local chemical environment on its pKa. The Pathway of Singlet Oxygen Diffusion through the Membrane Governs Whether Double Bonds or Aromatic Rings of a Molecule are Damaged 2573-Pos Board B180 Valery Sokolov1, Oleg Batischev1, Sergey Akimov1, Anna Gavrilchik1, Membrane Permeability of Ascorbic Acid Arsenij Shcherbakov1, Vsevolod Tashkin1, Denus Knyazev2, Peter Oihl2. Christof Hannesschlaeger, Peter Pohl. 1Frumkin Institute of Physical Chemistry and Electrochemistry RAS, Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria. Moscow, Russian Federation, 2Institute of Biophysics, Johannes Kepler Vitamin C (ascorbic acid, AscH) is an essential nutrient for humans. Published University, Linz, Austria. values of the passive membrane permeability to AscH range over several orders Photosensitizers (PS) are used to treat cancers and various skin diseases. of magnitude and hence appear to be contradictory. Since the characterization Upon illumination, PS generate reactive oxygen species, e.g. singlet oxygen of AscH-carriers requires knowledge of AscH’s background membrane perme- (SO). In turn, SO targets double bonds and aromatic residues of proteins, ability, we used scanning electrochemical microscopy for its determination. lipids and DNA, thereby damaging the adjacent cells. Although SO lives Addition of AscH to only one of the compartments separated by a free standing long enough to diffuse about 100 nm in a lipid membrane, damage does planar lipid bilayer resulted in a transmembrane flux of the neutral (protonated) not occur randomly. The mechanism that governs the susceptibility of form of the acid. In the receiving compartment, the acid dissociated, giving rise different chemical moieties is not known. Here we adsorbed aluminum phtha- to an increase in proton concentration within the unstirred layers in the imme- locyanines to only one interface of planar lipid bilayers (Sokolov, V. S. and diate membrane vicinity [1]. We monitored the local pH distribution by moving P. Pohl. 2009. BJ: 96:77-85). Photo-excited SO damaged the aromatic ring a custom built pH-sensitive micro-electrode in micrometer steps towards and of membrane-embedded dipolar molecules (ANEPPS) in a concentration- away from the membrane. By numerically solving the system of reaction- dependent manner as reported by changes of the membrane boundary poten- 8 diffusion-equations [2] we calculated the membrane permeability PAscH=10 tial 4b (Sokolov, V. S., / Y. G. Gorbunova. 2016. J Photochem Photobiol B: cm/s from the experimental pH profiles. We used this value to validate a new 161:162-169). However, 4b changed at a higher rate when the target and PS assay that is based on time lapse measurements of scattered light intensity of a were added to different sides of the bilayer instead of being added to the same vesicle suspension during AscH efflux. The new label-free approach calculates interface. This observation suggests that part of the SO must have been membrane permeability based on changes of (i) vesicle size and (ii) refractive quenched by ANEPPS’s double bonds which in the latter case were closer index of the vesicle interior. to the PS than the aromatic ring. We confirmed the model by using PS [1] Saparov et al. Biophys.l J. 90.11 (2006): L86-L88. molecules that release SO at various penetration depths from the membrane [2] Antonenko et al. Biophys. J. 64.6 (1993): 1701. surface due to differences in their number of sulfonate groups. In all cases,

BPJ 7900_7903 Wednesday, February 15, 2017 523a we controlled the abundance of both PS and target molecules by fluorescence 2579-Pos Board B186 correlation spectroscopy. We conclude that the probability of a reactive moi- Monitoring of Bacillus Thuringiensis Cry3Aa Toxin Pore Formation using ety being oxidized is mainly governed by its distance to the SO source rather Artificial Bilayer Array with Fused Brush Border Membrane Vesicles than by its chemical nature. Supported by Russian Scientific Fund N 14-13- from Colorado Potato Beetle Larvae 01373. Ekaterina Zaitseva1,2, Gerhard Baaken2, Victor M. Ruiz-Arroyo3, Inmaculada Garcı´a-Robles3, Camila Ochoa-Campuzano3, Galo A. Goig3, 2577-Pos Board B184 Amparo C. Martı´nez-Ramı´rez4, Carolina Rausell3, Jan Behrends1, Sphingomyelin - Cerebroside Exchange in Lipid Membrane Lateral Dolores Real3. Domain Segregation 1Institute of Physiology II, University of Freiburg, Freiburg, Germany, Emilio Gonza´lez-Ramı´rez, Alicia Alonso, Felix M. Goni. 2Ionera Technologies, Freiburg, Germany, 3Department of Genetics, Instituto Biofisika (CSIC-UPV/EHU), Leioa, Spain. University of Valencia, Valencia, Spain, 4SCSIE, University of Valencia, Galactocerebrosides, commonly referred to as cerebrosides (Ceb), are a fam- Valencia, Spain. ily of glycosphingolipids, which contain a saccharide head group (galactose, Bacillus thuringiensis Cry proteins are insecticidal toxins that bind to specific glucose) covalently linked to a double-tailed ceramide via a glycosidic link- receptors on the brush border membrane of the susceptible insect midgut cells age through the primary hydroxyl. They can be found in neuronal tissues in and induce cell lysis by inserting into the membrane and forming pores. Here the , in the epithelial cells of the small intestine and we report a novel Cry3Aa toxin pore formation assay utilizingparallel lipid colon and in the granular layer of the skin epidermis. Ceb, like sphingomye- bilayer platform based on microelectrode cavity array (MECA) chip. The lins (SM), are found to be highly saturated in natural sources and both share a Cry 3Aa pore-forming activity has been tested and characterized in lipid bila- sphingoid backbone but with a different headgroup, a phosphorylcholine yers fused with Brush Border Membrane Vesicles (BBMVs) as well as in (SM) or a mono/oligosaccharide (Ceb). Their hydrogen bonding capability receptor-free synthetic lipid membranes. is due to the interaction between the saccharide group and the hydroxyl First we developed protocols for efficient fusion of BBMVs isolated from and amide groups of the sphingosine base. The aim of this study is to explore the coleopteran pest Colorado potato beetle (CPB) (Leptinotarsa de- the effect of the exchange between sphingomyelin and cerebroside in the for- cemlineata) larvae with lipid membranes on the MECA chip. BBMVs fusion mation of segregated lateral domains. Natural origin lipids (ox brain) were led to the incorporation of different types of membrane proteins and used. The samples were analyzed by differential scanning calorimetry, ion channels presented in the midgut epithelial membrane into the lipid confocal microscopy and atomic force microscopy in order to characterize bilayers. Addition of the Cry3Aa toxin to the bilayers fused with lipid domains in supported planar bilayers and giant unilamellar vesicles. BBMVs from CPB larvae induced in approximately 70 % of the membranes In general Ceb exhibit a much smaller tendency than SM to form bilayers pronounced changes in the membrane currents due to the toxin pore forma- at 37 C, either in the form of liposomes or as supported lipid bilayers. Their tion. The presence of receptor in the fused BBMVs allowed for reliable bilayer forming capacity increases notoriously when mixed with typical detection of Cry3Aa channel activity at toxin concentrations as low as lamellar-phase lipids, e.g. dioleoyl phosphatidylcholine. Even under these 0.5 nM. conditions bilayer formation is extremely difficult at Ceb proportions above In contrast to the native membrane preparations Cry3Aa pore forming abil- 25 mol%. ity in synthetic lipid bilayers was significantly lower. In receptor-free lipid membranes Cry3Aa pores were only observed at toxin concentrations above Membrane Active Peptides and Toxins II 14.5 nM. Most of the pores displayed a variable kinetic behavior with low open probability, complex activity patterns and multiple subconductance 2578-Pos Board B185 states. The present combination of the MECA-chip electrophysiology with Calcium Tightly Regulates Disorder-To-Order Transitions Involved in the native membrane preparations allows for rapid functional characterization Secretion, Folding and Functions of the CyaA Toxin of Bordetella of the insecticidal toxin pores and receptor-toxin interactions. Pertussis, the Causative Agent of Whooping Cough Darragh P. O’Brien1, Sara E. Cannella1, Dominique Durand2, Ve´ronique Y. Ntsogo Engue´ne´1, Belen Hernandez3, Mahmoud Ghomi3, 2580-Pos Board B187 Orso Subrini1, Audrey Hessel1, Christian Malosse1,Ve´ronique Hourdel1, Impact of Dendrimer Surface Chemistry on Anthrax Toxin Channel Patrice Vachette2, Julia Chamot-Rooke1,Se´bastien Brier1, Daniel Ladant1, Blockage: A Single Molecule Study Alexandre Chenal1. Goli Yamini, Ekaterina M. Nestorovich. 1Structural Biology and Chemistry, Institut Pasteur, Paris, France, 2Institut de Biology, The Catholic University of America, Washington, DC, USA. Biologie Inte´grative de la Cellule, UMR 9198, CNRS, ORSAY, France, Multivalent ligands often possess an additive or cooperative affinity for a 3Groupe de Biophysique Mole´culaire, Universite´ Paris 13, Paris, France. multivalent target which is significantly greater than that of a single func- The adenylate cyclase toxin (CyaA) plays an essential role in the early stages tional group interacting with a monovalent target. This effect was recently of respiratory tract colonization by Bordetella pertussis, the causative agent investigated on a number of the anthrax toxin inhibitors assembled on cyclo- of whooping cough. Once secreted, CyaA invades eukaryotic cells, leading dextrin and dendrimer multivalent scaffolds. Multivalent binding can be to cell death. The cell intoxication process involves a unique mechanism strong despite individual bonds being relatively weak, yet from a drug of translocation of the CyaA catalytic domain directly across the plasma design perspective, we typically strive to design multivalent compounds membrane of the target cell. Our results show that calcium is involved in with high individual functional group affinity toward the respective binding several steps of this intoxication process. In the low calcium environment site on a multivalent target. of the bacterial cytosol, the C-terminal, calcium-binding RTX domain of Keeping this requirement in mind, here we perform a single-channel/single- CyaA, RD, is an elongated, intrinsically disordered coil, appropriately sized molecule study to investigate kinetic parameters of the anthrax toxin PA63 for transport through the narrow secretion machinery. Upon secretion, the channel blockage by generation 2 poly(amido amine) (PAMAM) dendrimers high calcium concentration in the extracellular milieu induces the refolding (G2) functionalized with different surface ligands, including G2-NH2, of RD, which likely acts as a scaffold to favour the refolding of the upstream G2-OH, G2- succinamate, and G2-COONa. We found that the earlier re- domains of the protein. Due to its hydrophobic character, CyaA is known for ported difference in IC50 values of the G2-OH/PA63 and G2-NH2/PA63 bind- its propensity to aggregate into multimeric forms in the absence of a chaot- ing reactions is determined by both on- and off-rates of the reversible ropic agent in vitro. We have recently defined the experimental conditions dendrimer/channel binding reaction. In 1M KCl, we observed about 10 times required for CyaA folding, that is, calcium binding and molecular confine- decrease in both kon and toff with G2-OH compared to G2-NH2, whereas for ment (which may mimic the secretion channel constraints). Both parameters both blockers kon and toff increased dramatically with the increase of trans- are critical for CyaA folding into a stable, monomeric and functional form. membrane voltage. PAMAM dendrimers functionalized with negatively The monomeric CyaA toxin exhibits efficient permeabilization in vitro and charged succinamate, but not carboxyl surface groups, were still reported haemolytic activities in cellula; both activities are conserved even in a fully to have some residual activity in inhibiting the anthrax toxin channels. calcium-free environment. In contrast, the toxin requires sub-millimolar cal- The on-rate of the G2-succinamate binding was comparable to that of G2- cium concentrations in solution to translocate its catalytic domain across the OH, however the off-time showed opposite voltage sensitivity. We also plasma membrane of the target cell, indicating that free calcium is actively describe kinetics of the PA63 ion current modulation by two different types involved in the CyaA toxin translocation process. Overall, this data demon- of the ‘‘imperfect’’ PAMAM dendrimers, - the mixed surface G2 75% OH/ strates the adaptation of bacterial RTX toxins to the great variations in cal- 25% NH2 and G3-NH2 dendron. Both the blockers showed weaker voltage cium concentrations encountered in the successive environments during the dependence compared to G2-NH2,howevertheirKDsin1MKClwere intoxication process. comparable.

BPJ 7900_7903 524a Wednesday, February 15, 2017

2581-Pos Board B188 Pore forming toxins (PFT) belong to a class of bacterial toxin proteins that form Effects of Erythrocytes Treated with Alpha Hemolysin of E.Coli on Endo- nano-scaled pores on target cell’s membrane and cause unregulated efflux of ions thelial Cells and biomolecules leading to cell death. They are released in a water-soluble Vanesa Herlax1, Marı´a Florencia Leal Denis2, Cora Alvarez2, conformation which upon membrane exposure undergoes large structural rear- Sophie Denise Lefevre3, Nicola´s Enrique4, Sabina Mate´1, Vero´nica Milesi4, rangement. This membrane bound monomer further oligomerizes and forms of Mariano Ostuni3, Pablo Schwarzbaum2. a complete pore. While high resolution structural information of complete pores 1INIBIOLP, Facultad Ciencias Me´dicas, UNLP, La Plata, Argentina, are available, our understanding of early events of PFT binding and assembly is 2IQUIFIB-CONICET/UBA, Ciudad Auto´noma de Buenos Aires, Argentina, incomplete owing to the highly dynamic nature of the aforementioned processes. 3INSERM. UMR_S1134-Universite´ Paris Diderot, Paris, France, 4IIFP- In this study, we use single molecule tracking and spectroscopy to understand the CONICET/UNLP, La Plata, Argentina. dynamics of Cytolysin A (ClyA), a prototypical a-PFT from E. coli, on lipid Uropathogenic strains of E. coli deliver the toxin alpha-hemolysin (HlyA) to bilayer membranes. Binding of ClyA to PEG-cushioned supported bilayer was optimize the host environment for the spread of infection. It was reported that rapid and reached saturation within a few seconds. Diffusional analysis of particle at high concentrations, the toxin forms pores in eukaryotic membranes, leading trajectories showed existence of two discrete mobility states exhibiting ‘fast’ and to cell lysis, while lower concentrations might interfere with host-cell-signaling ‘slow’ motions. Binding of PFT proteins was invariably in the ‘fast’ mobility state pathways, causing apoptosis. In the present investigation we demonstrate that a that was followed by transitions of the single monomer between the two states. relatively low concentration of HlyA induces morphological changes and phos- The slow moving population was significantly enhanced in cholesterol containing phatidylserine (PS) externalization of human erythrocytes. On the other hand, bilayers. We argue that the change in mobility is a consequence of structural tran- the unacylated nonhemolytic form of HlyA, ProHlyA induces similar morpho- sition to an assembly competent intermediate, the protomer state. Preliminary logical changes but no PS externalization. We performed osmoscan experiments analysis indicates that cholesterol enhances conformational transition by direct to test the effect of both proteins on erythrocytes structure. HlyA treated eryth- binding to the N-terminus of ClyA leading to stabilization of the protomer-like rocytes show increased membrane fragility and cell volume as well as dimin- state. This stabilization directly translates to increased formation of higher order ished cytoplasmic viscosity and S/V ration. ProHlyA-treated erythrocyte are structures at high concentrations of protein as measured by analysis of single not different from control ones. Since PS exposure of erythrocytes is known molecule photobleaching trajectories of pre-formed ClyA pores. Therefore, we to induce cell adhesion, we used a dynamic cell adhesion platform to study propose a molecular mechanism for selective pore formation in eukaryotic mem- the consequences of HlyA vs ProHlya exposure of erythrocytes on their adhesion branes driven by conformational selectivity in the presence of cholesterol. to human endothelial cells (HMEC). Results indicate that HlyA-treated erythro- cytes adhere more to endothelial cells than Pro-treated erythrocytes at low flux 2584-Pos Board B191 (0.5 din). At higher fluxes (1 and 2 din), however, HlyA-treated erythrocytes de- Role of the Tryptophan-Rich Motif of Listeriolysin O in Membrane tached easily than control ones, indicating that the adherence is weak. We also Binding 1 2 1 study the efflux of ATP from erythrocytes treated with both toxins by luciferin- Frances Separovic , Miriam Kozorog , Marc-Antoine Sani , 2 luciferase luminescence. Results demonstrate that HlyA induces the efflux of Gregor Anderluh . 1School of Chemistry, University of Melbourne, Melbourne VIC, Australia, ATP while ProHlyA does not. Since PS exposure was suggested to simulta- 2 neously increase extracellular ATP and adhesion to the vascular endothelium, National Institute of Chemistry, Ljubljana, Slovenia. and erythrocyte derived ATPe can alter the caliber of the vascular lumen, future Listeriolysin O (LLO) is a major virulence factor of the Gram-positive patho- experiments will be designed to relate HlyA induced efflux of ATP of erythro- genic bacteria, Listeria monocytogenes. It is secreted as a soluble monomer that cytes with their adhesion and interaction with endothelial cells. binds to cholesterol rich membranes, oligomerizes and forms pores. LLO be- longs to a group of cholesterol-dependent citolysins (CDCs) that are composed 2582-Pos Board B189 of four structural domains. Mutations in tryptophan-rich domain 4 identified Reversible Permeabilization of Cell Membranes via Lysenin Channels several amino acids as neccessary for binding to cholesterol-rich lipid mem- Nisha Shrestha1, Christopher A. Thomas1, Devon Richtsmeier1, branes. However, the exact mechanism at the molecular level is still unclear. Raquel Brown2, Juliette Tinker3, Daniel Fologea1. LLO was expressed with fluorine labelled tryptophan to obtain greater insight 1Biomolecular Sciences Graduate Program/Physics, Boise State University, into the role of the tryptophans in LLO binding to cholesterol. The presence of Boise, ID, USA, 2Biomolecular Research Center, Boise State University, cholesterol-rich membrane bilayers induced significant 19F chemical shift Boise, ID, USA, 3Biomolecular Sciences Graduate Program/Biology, Boise change in LLO, indicating that the toxin bound to the membranes. Moreover, State University, Boise, ID, USA. the relatively narrow resonance is likely from a single population, which indi- The selective nature of the cell-membrane hinders the permeability of drugs, cates that LLO is fully partitioned into the lipid bilayer. 31P NMR spectra of fluorescent probes, and other macromolecules into living cells. To introduce d31-POPC/cholesterol vesicles with and without LLO were similar, indicating foreign molecules as well as preserve cell viability, reversible permeabilization no loss of lipid bilayer integrity but the 2H spectra showed that LLO had an of the cell membrane is required. To achieve this aim, several techniques have impact on the order of the lipid acyl chains. Finally, 13C CPMAS revealed a 13 been proposed including microinjection, electroporation, optoporation, and greater broadening of the 3,4- C2 cholesterol than the lipid carbons in the pres- lipophilic and peptide carriers. These methods are either highly invasive, inef- ence of LLO, suggesting that LLO was preferentially bound to cholesterol. ficient, limited to single-cell, require sophisticated instruments, or need tedious conjugation process. Therefore, we proposed a simple yet efficient method of 2585-Pos Board B192 controlled transport of exogenous molecules into viable cells using the pore- Dimerization, a Key Step for Pore Formation of Fragaceatoxin C, an Ac- forming toxin lysenin. Lysenin inserts a large conducting pathway into the lipid tinoporin from the Sea Anemone Actinia Fragacea 1 1 2 bilayer membrane containing sphingomyelin, hence allowing cell-impermeable Haydee Mesa Galloso , Karelia H. Delgado-Magnero , Uris Ros , Pedro A. Valiente3, D. Peter Tieleman1. molecules to cross the membrane barrier. In addition, the lysenin pore is irre- 1 versibly blocked by biologically inert chitosan molecules. In this way, the Biological Sciences, University of Calgary, Calgary, AB, Canada, 2Interfaculty Institute of Biochemistry, University of Tuebingen, Tuebingen, lysenin-induced pore formation and the activity act as a nano-valve. In our 3 work, we temporarily permeabilized mammalian cells ATDC5 with lysenin Germany, Centre for Protein Studies, University of Havana, Havana, Cuba. channels and loaded the membrane-impermeable fluorescent dye propidium io- Actinoporins are produced by sea anemones, and are excellent models of eukary- dide. The process was blocked by the addition of chitosan and the viability of otic a-pore forming toxins. Recent research on actinoporins has mainly focused the cells was assessed by using viable-cell indicators. Similarly, we employed on the oligomerization step on the membrane and the detachment of the N-termi- lysenin channels to introduce the cell-impermeant actin marker phalloidin into nal region. Crystallographic data points suggested the key role of a small hydro- the cells. These results indicate that lysenin channel can be used as a simple and phobic protein-protein interaction surface for actinoporins oligomerization and efficient tool to deliver bioactive molecules into living cells. pore formation in membranes. Here, we demonstrated that disrupting the key hy- drophobic interaction between V60 and F163 (fragaceatoxin C (FraC) numbering 2583-Pos Board B190 scheme) in the oligomerization interface of FraC, equinatoxin II (EqtII) and sti- Studying Binding, Conformational Transition and Assembly of E. Coli cholysin II (StII) impairs the pore formation activity of these proteins. We com- Cytolysin a Pore Forming Toxin by Single Molecule Fluorescence bined molecular dynamics simulations with biochemical and biophysical tools to Pradeep Sathyanarayana1, Satyaghosh Maurya2, Ganapathy Ayappa2, design, obtain and characterize the double mutants FraCV60D/F163D, Eq- Sandhya S. Visweswariah3, Rahul Roy2. tIIV60D/F163D and StIII58D/I161D which contain mutations in the oligomeri- 1Centre for Biosystems Science and Engineering, Indian Institute of Science, zation interface. We predicted that these mutations in the oligomerization Bangalore, India, 2Department of Chemical Engineering, Indian Institute of interface would disrupt dimerization and therefore pore formation. Consequently, Science, Bangalore, India, 3Department of Molecular reproduction, the double mutants completely lacked the activity of the wild type proteins, Development and Genetics, Indian Institute of Science, Bangalore, India. although they maintained the main structural properties of actinoporins and the

BPJ 7900_7903 Wednesday, February 15, 2017 525a capacity to bind to membranes. We also performed atomistic molecular dynamics thermodynamics-based computational method for fast prediction of passive simulations to determine whether the first thirty N-terminal residues are able to permeability of structurally diverse molecules across different membranes. detach from the core of the protein in its monomeric and dimeric forms and their The method operates with atomic 3D structures of molecules and represents role in the mechanism. Moreover, our findings support the importance of dimer anisotropic properties of the lipid bilayer by transbilayer profiles of dielectric formation, which seems to be a functional intermediate in the assembly pathway and hydrogen-bonding capacity parameters. These profiles have been derived of different pore-forming proteins. These findings support the hybrid pore pro- for several artificial and natural membranes from distributions of groups in posal mechanism as a universal model of actinoporin pore formation. lipids and membrane protein structures along the bilayer normal [1]. The optimal translocation pathway of a solute was defined by calculating the lowest 2586-Pos Board B193 energy rotational orientation and conformer of the molecule in every point of Evaluation of the Hybrid Resolution Pace Model for the Study of Folding, the transmembrane trajectory. The method calculates: (1) the equilibrium Insertion and Pore Formation of Membrane Associated Peptides spatial arrangement and binding energy of the solute in membrane, including Michael D. Ward, Shivangi Nangia, Eric May. selective accumulation of amphiphilic compounds at the lipid-water interface; Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA. (2) free energy profile of the solute along the translocation pathway; and (3) the PACE is a hybrid model, in which lipids and solvents are coarse-grained consis- permeability coefficient. The results of calculations are consistent with pub- tent with the MARTINI mapping, while proteins are described by a united-atom lished experimental binding affinities, spatial positions in membranes, and model. However, given PACE is inherently linked to MARTINI, which is permeability data for small molecules and peptides with known three- widely used to study membranes, the behavior of proteins interacting with mem- dimensional structure. Testing on Caco-2 cell permeability data for ~150 branes has only been limitedly examined in PACE. In this work the PACE drug-like compounds allows parameterization of the method to reproduce model is employed to examine the behavior of several well studied peptides membrane properties of the gut epithelium. in membrane environments, namely the designed WALP peptides, the antimi- [1] Pogozheva ID, Tristram-Nagle S, Mosberg HI, Lomize AL. Structural ad- crobial peptide melittin and the influenza hemagglutinin fusion peptide aptations of proteins to different biological membranes. Biochim Biophys Acta. (HAfp). The results for the simple WALP peptides are mostly encouraging. 2013; 1828: 2592-2608. Folding and tilting angles of WALP19 and WALP23 are consistent with previ- ous all-atom studies. However, the transmembrane stability of WALP16 is 2589-Pos Board B196 underestimated due to the mismatch of hydrophobic thickness from the lack Mechanism of Action of pH-Triggered Membrane Active Peptides of compressibility of the lipids. The melittin study examines the dynamics of Sarah Y. Kim. a tetrametric transmembrane pore, and the results are in good agreement with Molecular Biophysics, Johns Hopkins University, Baltimore, MD, USA. previous all-atom simulations. We also simulated the melittin pore in MARTINI The plasma membrane insulates a cell from its outside environment, serving and while dynamic water flux was observed in the PACE simulation, the MAR- as a selectively impermeable barrier across which only small or hydrophobic TINI pore essentially remained devoid of water. Lastly, in the studies of HAfp, molecules can pass. Although the membrane is necessary for life, it is also PACE and MARTINI both failed to maintain the kinked hairpin structure problematic when useful macromolecules such as antibodies, peptides, poly- observed in NMR experiments. Overall, the PACE model performed well in saccharides, and imaging agents are blocked from entry. Most macromole- the study of simple helices interacting with membranes, and offers advantages cules can easily be uptaken by the cell through endocytosis, but remain over MARTINI from the increased peptide flexibility. However, more complex trapped and eventually degraded within endosomes, which mature into lyso- topologies, such as the HAfp hairpin structure, were not well modeled, which somes. To promote the escape of macromolecules from endosomes prior to may point toward an over stabilization of helices in the PACE model. their maturation into lysosomes, we have used a high throughput screen to 2587-Pos Board B194 design pH triggered, pore-forming peptides. To determine their mechanism Molecular Dynamics Simulation of Bioactive b-Casein Peptides in a POPC of action, we measured the peptides’ circular dichroism, binding, and leakage. Lipid Bilayer. Evaluation of the Casein Hydrolysates Hydrophobicity at At a P/L of 1/200, the pKa of binding and leakage is 5.5 but the pKa of helix Membrane Interfaces to coil transition is 5.75, implying that the peptide can cause significant Eduardo Jardo´n-Valadez1, Derik Castillo-Guajardo2, leakage even when the majority of the peptide is in an unstructured, bound Judith Jime´nez-Guzma´n3, Mariano Garcı´a-Garibay3,4. state. We explore two mechanisms that can explain large-scale membrane 1Earth Resources, Universidad Auto´noma Metropolitana, Unidad Lerma, destabilization; a detergent like effect of unstructured, membrane bound pep- Lerma, Mexico, 2Environmental Sciences, Universidad Auto´noma tides, and an exchange of unstructured, solution-state peptides with helical, Metropolitana, Unidad Lerma, Lerma, Mexico, 3Food Sciences, Universidad actively-pore-forming peptides. Auto´noma Metropolitana, Unidad Lerma, Lerma, Mexico, 4Biotechnology, Universidad Auto´noma Metropolitana, Unidad Iztapalapa, Mexico, Mexico. 2590-Pos Board B197 Consumption of dairy products may prevent the onset of metabolic syndrome, Synthetic Vesicles as a Predictive Tool for Permeabilizing Activity of Pep- obesity, and/or type 2 diabetes mellitus. Previous In vitro assays on enteroen- tides in Biological Membranes docrine cells suggest that exogenous source of bovine b-casein (b-CN) hydro- Cameron Morris, William C. Wimley. lysates favor cell proliferation, and expression of glucagon-like peptide-1. A Biochemistry and Molecular Biology, Tulane, New Orleans, LA, USA. description of the dynamics of peptides in a lipid bilayer interface could To better understand the basis for selective membrane-permeabilizing activity shed light into the molecular mechanisms that govern the biological activity by cationic, amphipathic peptides, the bee venom lytic peptide melittin and of b-CN hydrolysates. The objective of this work was to generate and analyze various antimicrobial peptides were assayed for their ability to cause leakage molecular dynamics simulation trajectories (0.5 ms) for two systems containing of entrapped contents from synthetic vesicles of varying lipid compositions. a bioactive peptide and a lipid bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3- The vesicle compositions were selected on the basis of cell type and consist phosphocholine (POPC) molecules. The b-CN(94-123) and b-CN(114-119) of common membrane models for eukaryotic cells as well as Gram-negative fragments were set up in our simulations because they have been identified and Gram-positive bacteria. Membrane active peptides are shown to display as bioactive peptides. We hypothesized that bioactive peptides display favor- a variety of membrane composition, sequence, and concentration-dependent able interactions in the lipid bilayer interface and that computational ap- leakage-inducing activity. The various membrane models are good predictors proaches combining sequence analysis of bioactive peptides, calculation of of antimicrobial and cytolytic potency. In order to differentiate between the peptide hydrophobicity, and molecular dynamics simulations would allow leakage mechanisms that conserve vesicle structural integrity and activity us to predict peptides with emulsifier and bioactive functions in dairy products. due to vesicle aggregation and fusion, a common artifact of membrane- active peptides, 5 mol percent DOPE-PEG(2000) lipids were introduced into 2588-Pos Board B195 each membrane model to inhibit aggregation. Vesicles with POPE rather Prediction of Passive Membrane Permeability and Translocation Path- than POPC as the primary zwitterionic lipid were the most impacted by the ways of Biologically Active Molecules addition of PEGylated lipids, showing an increase in stability as well as the Andrei L. Lomize, Irina D. Pogozheva. largest increases in peptide concentration required to induce 50% leakage of College of Pharmacy, University of Michigan, Ann Arbor, MI, USA. entrapped contents (LIC50). In POPC vesicle, the melittin derived peptides Libraries of naturally-sourced compounds that serve as structural scaffolds showed the largest increases in LIC50, while the inverse was true of POPE ves- in the search for new drug leads are constantly growing. A significant num- icles with library derived antimicrobial peptides being inactive at peptide to ber of natural products with relatively high molecular weights have been lipid ratios lower than 1:25 in vesicles with PEGylated lipid. The results shown to passively penetrate through membranes. Partitioning and perme- show both the utility and the limitations of synthetic vesicles as predictors of ability of the molecules depend on their conformation and orientation in cytolytic activity as well as the impact of PEGylated lipids beyond limiting membranes and on their interactions with lipids. We have developed a novel aggregation.

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2591-Pos Board B198 sample of antiphospholipid antibody present within their serum that doubles by Identification of Novel Peptide Sequences with Improved Nuclear Delivery two weeks post booster injection as determined by APS ELISA. However, the of Peptide Nucleic Acid (PNA) Sequences levels of actual antibody generated are low and without subsequent booster injec- William B. Kauffman, William C. Wimley. tions, the rats present with significant antibody binding remaining only in the ven- Department of Biochemistry and Molecular Biology, Tulane University, New tricles of the heart by one month post booster injection. Our data collectively Orleans, LA, USA. demonstrates that the 10F5 epitope is responsible for the generation of antiphos- Peptide Nucleic Acids, DNA mimics featuring a peptide-like backbone and nu- pholipid antibodies and that these antibodies may interact within the heart and cleobases that bind nucleic acids, are capable of impacting gene expression, are brain as is seen in antiphospholipid syndrome pathology. resistant to degradation, and show promise as therapeutic agents. PNAs are cell-impermeant and intracellular delivery has been a challenge, however 2594-Pos Board B201 cell-penetrating peptides show some promise as delivery vehicles. To identify Drug Lipidation in Membranes sequences that improve nuclear PNA delivery we constructed a library of 8192 Hannah M. Britt, Jackie A. Mosely, John M. Sanderson. unique peptide sequences based on the well-known cell penetrating peptides Dept. of Chemistry, Durham University, Durham, United Kingdom. Tat and Penetratin. These sequences are covalently linked to the N-terminus Drugs undergo intrinsic lipidation reactions in lipid membranes, involving acyl of an 18-residue PNA sequence (PNA705) that blocks an aberrant splice site transfer from a lipid to a reactive group on the drug. Of the low molecular weight in a recombinant luciferase gene (pLuc705) stably expressed in Hela cells, compounds examined, some have been found to have little or no reactivity, whereas thereby correcting its function. A live cell screen of the library peptides has others have reactivities comparable to peptides, despite having a lower membrane- generated several PNA Delivery Peptides (PDEPs) capable of delivering water partition coefficient. Ongoing work to understand the factors that control this PNA705 to the nucleus of Hela705 cells with much greater efficiency than reactivity, and its consequences for membrane properties and the development of the well-known cell penetrating peptides used as a basis for the screen. As vary- adverse drug activities such as phospholipidosis, will be presented. ing the PNA sequence will change the nucleic acid target without changing its physicochemical properties significantly, we envision these PDEP sequences 2595-Pos Board B202 serving as generic delivery vectors for useful PNA sequences in cell based sys- Selective Delivery of Auristatins to Cancer Cells and Tumors using pHLIP 1 2 2 tems in the laboratory. PDEPs, which may also enable the use of therapeutically Kelly E. Burns , Harvey Hensley , Matthew K. Robinson , Damien The´venin1. beneficial PNAs in vivo, will provide researchers with a valuable tool moving 1 2 forward and have the potential for significant translational applications. Chemistry, Lehigh University, Bethlehem, PA, USA, Molecular Therapeutics Program, Fox Chase Cancer Center, Philadelphia, PA, USA. 2592-Pos Board B199 The targeting of therapeutics specifically to diseased tissue is crucial for the How do Peptides and Nanoparticles Interact with the Membranes of development of successful cancer treatments. The approach herein is based E. Coli? Insights from Molecular Dynamics on the pH(Low) Insertion Peptide (pHLIP) for the delivery of a potent mitotic Pin-Chia Hsu, Damien Jefferies, Syma Khalid. inhibitor monomethyl auristatin F (MMAF). We investigated six pHLIP vari- University of Southampton, Southampton, United Kingdom. ants conjugated to MMAF to compare their efficacy in vitro against cultured The development of bacterial resistance to current antibiotics is a major prob- cancer cells. While all pHLIP-MMAF conjugates exhibit potent pH- and lem for healthcare worldwide. The rational development of new therapeutics concentration-dependent killing, their cytotoxicity profiles are remarkably relies upon a detailed knowledge of how drugs and potential drugs interact different. We also show that the lead conjugate exhibits significant therapeutic efficacy in mouse models without overt toxicities. This study confirms with the membranes that surround and protect bacteria. To this end, here we employ molecular dynamics simulations to explore at the molecular level the pHLIP monomethyl auristatin conjugates as possible new therapeutic options for cancer treatment and supports their further development. interaction of pristine carbon fullerenes (C60) and the honey bee toxin, melittin with realistic models of E. coli membranes. 2596-Pos Board B203 We find that pristine C60 has a limited tendency to penetrate lipopolysaccharide Spontaneous Membrane Translocating Peptides: Mechanisms and Motifs (LPS) leaflets in the presence of calcium ions at 310 K, but more readily pen- Taylor Fuselier, William C. Wimley. etrates LPS leaflets at higher temperatures, in the presence of sodium ions, or Tulane University School of Medicine, New Orleans, LA, USA. when small patches of palmitoyloleoylphosphoethanolamine (POPE) lipids We previously used an orthogonal high-throughput screen to select peptides that are present within the LPS membranes. spontaneously cross synthetic lipid bilayers without permeabilization. Interest- Melittin has a higher mobility in the inner membrane compared to the outer ingly, many of the 12-residue spontaneous membrane translocating peptides membrane, as in the latter the LPS molecules of the outer leaflet act as a (SMTPs) selected from the library contained a consensus LRLLR motif. We hy- ‘clamp’ to restrict movement. We highlight important differences between pothesized that the conserved LRLLR motif could be a minimal motif for sponta- the E.coli inner and outer membranes and show that models of the two mem- neous membrane translocation. To test this and to explore the mechanism of branes must incorporate the biochemical differences of their compositions, spontaneous membrane translocation, we synthesized six arginine spacing vari- for accurate representation of the in vivo membranes. ants of LRLLR and compared their membrane partitioning, translocation, and perturbation to one of the parent SMTPs, called TP2. Several motif variant pep- 2593-Pos Board B200 tides translocate into synthetic vesicles with rates that are similar to TP2. Howev- Streptococcal M Protein Epitope 10F5 Generates Antibodies that Remain er, the actual motif peptide, LRLLR, was not the fastest, showing that sequence Significantly Bound in the Heart Ventricles context is important for translocation efficiency. While none of these peptides per- Marie Kelly-Worden1, Victoria Cuebas1, Morenci Manning1, meabilize bilayers, the motif peptides translocate faster at higher peptide to lipid Robin Gebhard2, Mathew Osborne3. ratios, suggesting that bilayer perturbation and/or cooperative interactions are 1Ball State University, Muncie, IN, USA, 2Ohio State University, Columbus, 3 important for their translocation. On the other hand, TP2, which was actually OH, USA, Idaho State University, Pacotello, ID, USA. selected in the screen, translocates dramatically faster as its concentration is A link between the development of streptococcal pharyngitis and the develop- decreased, suggesting that TP2 translocates as a monomer and is inhibited by ment of acute rheumatic fever has long been understood. The tie that binds these lateral interactions in the membrane. We tested variants with shorter and longer conditions together is the presence of autoimmune antibodies generated in arginine sidechain analogs and found that neither translocate as fast as those response to the bacterial infection that possess molecular mimicry. This molecu- with arginine. Finally, we tested concatenated motifs and measured slow translo- lar mimicry being between antibodies against group A streptococcus (GAS) M cation compared to the motifs alone. In summary, multiple variants of the protein and human proteins such as cardiac myosin, tropomyosin, vimentin, conserved motif translocate as well as TP2, showing that amino acid composition and laminin. Streptococcal M protein antibody probe mAb10F5 recognizes a is an important factor, but one that is influenced by sequence context. These re- 15 amino acid sequence within the C repeat region of the type 6 M protein and sults also show that rational engineering of SMTPs is not yet possible, providing it is this sequence that is associated with the majority of acute rheumatic fever out- even more reason to pursue SMTP discovery with synthetic molecular evolution. breaks (Quinn et al, 1998). Injection of mAb10F5 into Lewis rats results in heart manifestations with significant binding in the heart, especially in the valve region, 2597-Pos Board B204 even three days after injection. The presence of the antibody may also be detected Cell-Penetrating Peptide for Transcellular Transport: Membrane Binding within the caudate and putamen of the brain as demonstrated by significant immu- and Uptake in Live Cells nofluorescence. When mAb10F5 interactions are analyzed, the antibody is found Alexander Komin1, Michael D. Paul2, Alexander M. Luera3, to directly interact with membrane phospholipids and results in a positive anti- Peter C. Searson1, Kalina Hristova1. phospholipid ELISA test. When the Lewis rats are allowed to generate their 1Materials Science and Engineering Department, Johns Hopkins University, own antibody against just the 10F5 epitope, the result is a significant detectable Baltimore, MD, USA, 2Program in Molecular Biophysics, Johns Hopkins

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3 University, Baltimore, MD, USA, Program in Molecular and Cellular the two assays using the model detergents CHAPS and C12EO8, causing all-or- Biology, Johns Hopkins University, Baltimore, MD, USA. none and graded type leakage, respectively. The onset concentration of leakage Cell-penetrating peptides (CPP) are a potential drug delivery vehicle into cells is the same in GUVs and in LUVs. However, at the same detergent concentration, and across biological barriers. We discovered that a CPP, called the CL peptide, all-or-none type leakage induces much more leakage in GUVS compared to delivered a conjugated fluorophore into different cell lines and enhanced ten- LUVs. Oppositely, the graded type leakage is more harmful in LUVs as compared fold the permeability of the fluorophore across an epithelium. We synthesized to GUVs. I.e., either the entrapped volume opened per pore or the surface to vol- CL peptide variants to identify the important parameters that determine the ume ratio governs the flux-efficiency of the two leakage mechanisms. While cell- and barrier-penetrating properties. We also used Fully-quantified Spectral GUV-microscopy is the gold standard for obtaining the leakage distribution Imaging (FSI) method to investigate the binding of the CL-fluorophore conju- explicitly, LUV-TCSPC has a much higher throughput, and is more flexible for gate and its variants to live cell membranes and correlate it to the intracellular testing various conditions. Practically, it shall be ideal to perform systematic delivery efficiency. LUV-TCSPC experiments and validate or refine leakage distributions of a few prominent samples by GUV microscopy. 2598-Pos Board B205 Cell Penetrating Peptide Mediated Transport across Membranes General Protein-Lipid Interactions IV Xin Li, Matthew Holden, Min Chen. Chemistry, UMASS-Amherst, Amherst, MA, USA. 2601-Pos Board B208 We investigate how a small cell-penetrating peptide called Pep-1 is able to carry a Membrane Lipid Asymmetry, a Key Property of the Mammalian Plasma large object, such as a protein, across a lipid bilayer. To create a membrane, two Membrane, Alters the Insertion of the pHLIP Peptide sub-microliter, lipid-encased aqueous droplets are contacted - termed a droplet Haden Scott. interface bilayer (DIB). The peptides adsorb to the protein cargo non- BCMB, University of Tennessee, Knoxville, TN, USA. covalently and somehow ‘‘carry’’ the protein from one droplet to the other The plasma membrane has an asymmetric distribution of lipids between the inner through the membrane. We then quantitate the translocated cargo through a fluo- and outer leaflets of the bilayer. Two types of phospholipid asymmetry can occur rogenic assay in real-time. We found that transport is dependent membrane between the leaflets: head group asymmetry and acyl chain asymmetry. One lipid charge, and that the symmetry of the bilayer membrane also play a role in Pep- head group of interest is phosphatidylserine (PS), which carries a net negative 1-mediated protein translocation. Proteins as large as b-gal (465 kDa) can be charge. In healthy cells, PS is actively sequestered to the inner leaflet of the mem- translocated using this method and we can detect as few as several thousand mol- brane but can redistribute to the outer leaflet in situations such as apoptosis and ecules. By charactering Pep-1/protein complex with DLS and gel electrophoresis, cancer. Not only do membranes contain lipids, they also contain proteins. we could get more information about the active form for effective translocation. Marginally hydrophobic membrane proteins can contain acidic residues in their We expect this new method combing translocation experiments with the size transmembrane sequence and can have topological transitions after membrane as- characterization will give some hints on the transportation driving force and sociation. What is unclear is how lipid asymmetry affects these topological tran- make deciphering direct translocation mechanisms possible. sitions. Here, we propose to use the pH low insertion peptide (pHLIP) as a model 2599-Pos Board B206 to study topological transitions in relation to membrane head group asymmetry in Synthetic Molecular Evolution of Membrane-Active Peptides free floating asymmetric phosphatidylcholine (PC)/PS vesicles. Based on envi- William C. Wimley. ronmental pH, pHLIP undergoes a topological reorientation and inserts into the Tulane University School of Medicine, New Orleans, LA, USA. membrane. The inclusion of PS in symmetric vesicles affects the insertion pro- Membrane-active peptides have potential utility in many areas, including in the cess by lowering the pH midpoint of insertio n. However, we do not know how cellular delivery of polar compounds, cancer therapy, biosensor design, and in PS asymmetry will affect insertion. We propose that having an asymmetric dis- antibacterial, antiviral and antifungal therapies. Yet, despite decades of tribution of PS (more in the inner leaflet) will affect the insertion of pHLIP. Fluo- research on thousands of known examples, useful sequence-structure- rescence spectroscopy data show that the pH midpoint of pHLIP insertion into function relationships are essentially unknown. Because peptide-membrane in- asymmetr ic vesicles is higher compared to symmetric vesicles containing PS. teractions within the highly fluid bilayer are dynamic and heterogeneous, ac- Acrylamide quenching data show that the two tryptophans in pHLIP are more sol- counts of mechanism are necessarily vague and descriptive, and have little vent exposed in the presence of asymmetry. We hypothesize that pHLIP insertion predictive power. This creates a significant roadblock to advances in the field. is affected by a change in the transmembrane potential induced by the asymmetric We are bypassing that roadblock with synthetic molecular evolution: iterative distribution of charged lipids between the leaflets. peptide library design and orthogonal high-throughput screening. We start with template sequences that have at least some useful activity, and create small, 2602-Pos Board B209 focused libraries using structural and biophysical principles to design the Lipid Headgroups Modulate Conformational Switching during Membrane sequence space around the template. Orthogonal high-throughput screening Insertion of Cancer-Targeting pHLIP Peptides 1 2 2 is used to identify gain-of-function peptides by simultaneously selecting for Victor Vasquez-Montes , Kelly E. Burns , Damien Thevenin , 1 several different properties (e.g. solubility, activity and toxicity). Multiple gen- Alexey S. Ladokhin . 1Department of Biochemistry and Molecular Biology, University of Kansas erations of iterative library design and screening have enabled the identification 2 of membrane-active sequences with heretofore unknown properties, including Medical Center, Kansas City, KS, USA, Department of Chemistry, Lehigh clinically relevant, broad-spectrum activity against drug-resistant bacteria and University, Bethlehem, PA, USA. enveloped viruses as well as pH-triggered macromolecular poration. pHLIP (pH-Low Insertion Peptide) is a highly charged peptide that has been tradi- tionally described as undergoing three different states during its membrane inter- 2600-Pos Board B207 action. State I, unstructured and soluble at neutral pH; State II, unstructured in the GUV and LUV Leakage: How All-Or-None and Graded Leakage Scale membrane interface at neutral pH; and State III, as a transmembrane a-helix after with Vesicle Size acidification. In a previous publication we showed that pHLIP only forms the pre- Stefan Braun1, Sa´rka Pokorna´2, Radek Sachl 2, Martin Hof2, viously observed State II in membranes composed solely of phosphatidylcholine Heiko Heerklotz1,3, Maria Hoernke1,3. (PC). In contrast, it was found to transition directly to its inserted form in all other 1Pharmaceutical Technology and Biopharmacy, Albert-Ludwigs-Universit€at, membranes tested. Here we follow on this work using the pHLIP P20G variant Freiburg i.Br., Germany, 2J. Heyrovsky Institute of Physical Chemistry, and show that its insertion pathway is heavily dependent on membrane composi- Academy of Sciences, Prague, Czech Republic, 3BIOSS Centre for tion; undergoing different intermediate states depending on the used lipids. Biological Signalling Studies, Albert-Ludwigs-Universit€at, Freiburg i.Br., pHLIP P20G behaves in the same manner as wt pHLIP and was initially thought Germany. to insert directly into all tested membranes except for 100% PC bilayers. Using Many antimicrobial compounds, (anti-cancer) drugs, and drug delivery vehicles different spectroscopic techniques we confirm here the formation of an interfacial harm their target cells by permeabilizing the cell membrane. For more than three unstructured state at neutral pH in vesicles with mixed lipid compositions. How- decades, the efficiency of membrane permeabilization is studied as leakage of ever, the formed state (State II’) is a very superficial state that is not observable by model membrane vesicles and is assumed to correlate with the biological activity. either CD or Trp fluorescence. Additionally, pHLIP P20G in the presence of In the more elaborate methods, leakage is assigned to the ideal mechanisms: membranes at neutral pH was found to be present in an intermediate form be- graded (all vesicles leak some content) or all-or-none (some vesicles are fully tween State II and State III. In this State IIb pHLIP has a higher helical content permeable). The most recently developed leakage assays use giant unilamellar (while remaining mostly unstructured) and have deeper membrane penetration vesicles (GUVs) for microscopy or large unilamellar vesicles (LUVs) in TCSPC than State II as observed by Trp fluorescence and quenching experiments. fluorescence measurements. They provide the distribution of degrees of leakage Furthermore, the protonation-dependent membrane insertion pK50 of both pep- over the vesicle population, i.e., a more detailed mechanism. Here, we compare tides was affected similarly by changes to interfacial physiochemical properties;

BPJ 7900_7903 528a Wednesday, February 15, 2017 confirming our previous observations with wt pHLIP. These results show that pH- R2,22 WALP23. The pH dependence of the tilt then suggests an overall pKa of dependent peptide-membrane insertion is more complex than previously sug- about 4 for H2,22 WALP23 in DLPC. The combined results suggest that the tilt gested; requiring more than a simple 3-state system to describe it and strongly of the core peptide increases when the distance between two histidines increases, modulated by the lipid composition. and the extent of dynamic averaging remains low. Further investigations on pH dependence and possibilities for multistate properties of different His-anchored 2603-Pos Board B210 peptides are underway. Model Assessment and Simulation of Lipid-Protein Interactions Ronald D. Hills Jr, Jacob Fosso-Tande, Cody Black. 2606-Pos Board B213 Pharmaceutical Sciences, University of New England, Portland, ME, USA. Interfacial Tryptophan Residues Govern Transmembrane Helix Dynamics Annular lipid solvation and specific lipid binding events modulate membrane Matthew J. McKay1, Ashley N. Martfeld1, Anna A. De Angelis2, protein function and catalytic mechanisms in ways we are only beginning to Stanley J. Opella2, Denise V. Greathouse1, Roger E. Koeppe II1. understand. Multiscale coarse-graining was used to develop models for study- 1Chemistry & Biochemistry, University of Arkansas, Fayetteville, AR, USA, ing molecular level interactions between proteins and the lipid bilayer. Interac- 2Chemistry & Biochemistry, University of California at San Diego, La Jolla, tion potentials constructed for common physiological lipids were found to be CA, USA. transferable in representative membrane mixtures. Permeation profiles for side- Transmembrane protein domains often contain interfacial aromatic anchor res- chains in the bilayer were used to further refine the model, obtaining attractive idues which may play roles for the insertion and stability of membrane helices. interfacial interactions expected for positive charged sidechains. The utility of Residues such as Trp or Tyr therefore may be found situated at the lipid-water the force field for membrane protein simulation is demonstrated by application interface. This work aims to examine the extent to which the precise locations to multidrug ABC transporters. The role of conformational change and annular of Trp residues can influence peptide helix orientation and dynamics. To lipid solvation is investigated in the efflux proteins MsbA and P-glycoprotein. address these questions, we have modified the GWALP23 ((acetyl-GGALW5(- 19 LA)6LW LAGA-[ethanol]amide)) model peptide framework. Peptide orienta- 2604-Pos Board B211 tion and dynamics were analyzed by means of solid-state NMR spectroscopy to Solid-State NMR Investigations of Transmembrane Helix Interactions monitor specific 2H and 15N labeled residues. GWALP23 adopts a defined tilted Kelsey Knobbe, Venkatesan Rajagopalan, Ashley Martfeld, orientation within lipid bilayers with minimal dynamic averaging of NMR ob- Denise Greathouse, Roger Koeppe II. servables such as 2H quadrupolar splittings or 15N-1H dipolar couplings. Here University of Arkansas, Fayetteville, AR, USA. we examine how peptide dynamics are impacted by relocating the anchoring Transmembrane proteins are vital to numerous biological processes, including Trp (W) residues on both ends by 100 outward to positions 4 and 20, on signal transduction and ion conduction. In many cases, the behavior of potentially opposing faces of the helix. In contrast to GWALP23, GW4,20 ALP23 experi- ionizable residues in the hydrophobic interior of the membrane is crucial to un- ences extensive dynamic averaging of the NMR observables in several lipid bi- derstanding protein function. We have employed a low-dynamic model a-helical layers of varying thickness. Individual and combined Gaussian analyses of 2H transmembrane peptide, GWALP23 (acetyl-GGALWLALALALALALALW- and 15N signals confirm that the extent of dynamic averaging, particularly rota- þ LAGA-amide) as a framework to investigate the possibility of ( /-) ion-pair in- tional ‘‘slippage’’ about the helix axis, is strongly coupled to the locations of teractions between arginine and glutamic acid side chains located on different and 2 2 the interfacial Trp residues. H labels on A3 and A21 indicate partial fraying independent (separately diffusing) transmembrane helices, by H NMR spectros- of the helix ends. Even within the context of partial unwinding, the locations copy. One peptide with an Arg residue, GWALP23-R14 (acetyl-GGALWLALA- of the Trp residues are prominent factors that influence transmembrane helix LALARALALWLAGA-amide), and another with a Glu residue, GWALP23- orientation and dynamics within the lipid membrane environment. E14 (acetyl-GGALWLALALALAEALALWLAGA-amide), were incorporated into DOPC bilayers together (1:67 peptide:lipid ratio). Past experiments have 2607-Pos Board B214 shown that GWALP23-R14 alone is well-aligned in DOPC bilayers, gives narrow Response of GWALP23 Transmembrane Peptides to Incorporation of resonances for 2H-labeled alanines and remains charged, even under alkaline pH Specific Pairs of Buried Charged Arginine Residues conditions (JACS 2010, 132(16):5803). By contrast, 2H-Ala-labeled GWALP23- Karli A. Lipinski, Ashley N. Martfeld, Denise V. Greathouse, E14 exhibits much broader 2H NMR spectra. We now find that the spectra of Roger E. Koeppe II. labeled GWALP23-R14 become noticeably broadened and weakened, without Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. changing the quadrupolar splittings, when even 16 mol% GWALP23-E14 is Membrane proteins are essential components for many cell processes yet are un- included with GWALP23-R14 in the DOPC bilayer samples. A control peptide, derstood less than soluble proteins. Charged residues, such as arginine, may GWALP23, has little effect on GWALP23-R14 at 16 mol%, but spectral broad- contribute significantly to the function of membrane proteins. To characterize ening and weakening occur with at 50 mol%. Conversely, the addition of the effect of these residues on transmembrane proteins, it is useful to employ a GWALP-R14 (16 or 50 mol%), has no effect on the already broad spectra of model peptide system such as, for example, GWALP23 (acetyl-GGALW5LA- labeled GWALP-E14 in DOPC bilayers. Preliminary results, therefore, suggest L8ALALALAL16ALW19LAGA-amide), a designed transmembrane peptide that while GWALP-E14 causes concentration-dependent effects on the spectra with interfacial tryptophan anchors. We have substituted R8 and R16 in place of GWALP-R14, the complex or possibly multi-state behavior of GWALP- of L8 and L16 in GWALP23, equidistant from the center, and incorporated spe- E14 is not ‘rescued’ by the addition of GWALP-R14 in DOPC bilayers. cific 2H-labeled alanine residues for detection by means of solid-state 2HNMR. The –R8,16 peptide folds into alpha helical secondary structure in DLPC, DMPC, 2605-Pos Board B212 and DOPC lipid vesicles, as confirmed by circular dichroism. Solid-state 31P Influence of Paired Histidine Residues on Transmembrane Helix Orienta- NMR spectra of oriented samples confirm intact bilayers in the presence of the tion and Dynamics peptide. The pattern of 2H-Ala quadrupolar splitting magnitudes along the helix Fahmida Afrose, Denise V. Greathouse, Roger E. Koeppe II. indicates a significantly tilted transmembrane orientation that we currently are Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. refining for the GWALP23-R8,16 helix in several oriented lipid membranes. Interfacial charged or aromatic amino acids are significant for defining the orien- Reciprocal experiments in other labs make use of double-electron-electron reso- tations and dynamics of membrane-spanning alpha-helices. The synthetic neutral 2 5 19 22 nance (DEER) to measure the distance between pairs of charged functional model peptide GWALP23 (acetyl-GG ALW (LA)6 LW LAG A-amide) is a groups similar to arginine. The combined results from complementary experi- useful host framework for investigating the influence of single as well as multiple mental techniques will enhance our fundamental understanding of membrane ionizable amino acids in modulating the protein-lipid interactions. To understand protein structure and function, helix orientation and side-chain ‘‘snorkeling,’’ the ‘‘anchor’’ residue dependence, we have substituted either both of the trypto- properties which are crucial for living cells. This work will be expanded with in- phan anchors, W5 and W19, or the glycine residues located at the N and C termini, 5,19 2,22 vestigations of additional peptide sequences including, for example, comparisons G2 and G22, with two histidine residues (GH ALP and H WALP respec- to the properties of the -R2,22 and -R5,19 transmembrane helices. tively). Solid-state 2H NMR experiments, with deuterated alanine residues in the core of peptide, show well defined 2H quadrupolar splittings for GH5,19ALP23 2608-Pos Board B215 over a pH range from 2.0 to 8.0. Analysis of the tilt and rotation of this helix in Microsecond Simulations of Amyloid Beta Fibril Nucleation in Reverse DLPC and DOPC lipid bilayers reveals similar results compared to GWALP23, Micelles although in DOPC, GH5,19ALP23 seems to exhibit multistate behavior at pH Gozde Eskici1, Paul Axelsen2. above 8.0. The H2,22 WALP23 peptide, by contrast, shows well defined 2Hquad- 1Biochemistry, and Biophysics, University of Pennsylvania, Philadelphia, rupolar splittings in DLPC, DMPC and DOPC bilayers at both pH 4 and 8, sug- PA, USA, 2Pharmacology, Biochemistry, and Biophysics, University of gesting that the peptide helix is well oriented in this pH range. Tilt analysis in Pennsylvania, Philadelphia, PA, USA. DLPC lipid bilayers over a pH range of 2.5-8.5 indicates a similar tilt at low The 40-residue amyloid beta protein (Ab) is the unstructured cleavage product pH for H2,22 WALP23, when compared to GWALP23, K2,22 WALP23 and of a common membrane protein that is produced in large quantities, but

BPJ 7900_7903 Wednesday, February 15, 2017 529a normally cleared from the brain before it exerts any apparent toxicity. Under organelle membranes can vary, preferential membrane-ligand association may some conditions, however, it undergoes a conformational change and aggre- regulate the rate of apelin/apela-GPCR binding (potency & efficacy), signaling gates into fibrils with cross-beta structure. These fibrils then coalesce into am- pathways (Gai vs. b-arrestin), and signaling mechanisms (endocrine vs. auto- yloid plaques,which are the pathognomonic brain lesions of Alzheimer’s crine). Characterizing ligands of the apelin receptor presents a rare opportunity disease. The plaques are centers of active oxidative stress and neuronal death, to test for membrane catalysis as a method to control and diversify hormonal so the conditions under which the conformational change of Ab is of high in- signaling mechanisms both in physiological conditions and for therapeutic terest. When Ab is encapsulated in a reverse micelle under laboratory condi- targeting. tions, infrared spectroscopy indicates that it spontaneous adopts an extended beta-sheet conformation, which is remarkable because only one Ab strand is 2611-Pos Board B218 present in each reverse micelle. That observation suggests that some aspect Molecular Basis of Ligand Binding by the Endosomal Adaptor Protein of the reverse micelle environment such as crowding, dehydration, proximity Tom1 to a membrane, or high ionic strength can induce Ab to nucleate amyloid fibril Wen Xiong1, Ji Woong Choi1, Xiaolin Zhao1, Jeff F. Ellena2, formation. Therefore, an understanding of the factors that induce Ab to adopt Daniel G.S. Capelluto1. this conformation in reverse micelles may reveal how it is induced to form am- 1Protein Signaling Domains Laboratory, Department of Biological Sciences, yloid fibrils in Alzheimer’s disease. ms lond Molecular dynamics simulations of Biocomplexity Institute, Virginia Tech, Blacksburg, VA, USA, 2Department Ab in reverse micelles have been performed on supercomputer Anton to iden- of Chemistry, University of Virginia, Charlottesville, VA, USA. tify and understand these factors. Results indicate that Ab side chain interac- Tom1 (target of Myb 1) plays a role in membrane trafficking by serving as an tions with the reverse micelle surface help stabilize intrachain hydrogen alternative endosomal sorting complex required for transport (ESCRT)- bond formation and secondary structure formation. These results also represent 0 component. Tom1 possesses an N-terminal VHS domain followed by a important sequence-specific details within an increasingly large body of evi- central GAT domain. Tom1 has been shown to serve as a new phosphatidy- dence suggesting that Ab-membrane interactions are important for the forma- linositol 5-phosphate (PI(5)P) effector at signaling endosomes through its tion of amyloid fibrils in Alzheimer’s disease. VHS domain, delaying cargo degradation in a bacterial infection model. The Tom1 VHS domain also binds ubiquitin moieties in cargo for endosomal 2609-Pos Board B216 transport and degradation; therefore, we hypothesize that the ubiquitin and Lipid-Peptide Interaction Dynamics with Reaction-Diffusion Fluorescence PI(5)P compete each other for Tom1 VHS binding. In order to address Correlation Spectroscopy this question, the backbone NMR resonances of Tom1 VHS were assigned. Xiaosi Li, Xiaojun Shi, Adam W. Smith. The Tom1 VHS secondary structure prediction scores, using TALOSþ,are University of Akron, Chem Dept, Akron, OH, USA. in good agreement with the secondary structural elements reported for the Peptides containing positively charged residues interact with anionic lipids in crystal structure of the protein. Our heteronuclear single quantum coherence the plasma membrane. Phosphatidylinositol 4,5-bisphophate (PIP2), an impor- data revealed that Tom1 VHS interacts with PI(5)P following a fast- tant anionic lipid in the cytoplasmic leaflet of the plasma membrane, is re- exchange regime, with the PI(5)P binding site predicted to be at a region cruited to membrane-associated proteins that contain basic domains. Despite spanning a-helices 6 and 8. In contrast, we found that the ubiquitin- the biological importance of PIP2 lipids and peripheral proteins in cell binding site in Tom1 VHS is located at the a-helices 2, 5 and 7 of Tom1 signaling, we know little about their affinity and interaction kinetics. Here, VHS. Despite the binding sites are not overlapped, the ubiquitin and PI(5) we used pulsed interleaved excitation fluorescence cross-correlation spectros- P may compete each other by inducing conformational changes in the copy (PIE-FCCS) to assess the mobility and correlated diffusion of two periph- Tom1 VHS domain upon binding. Also, we identified a conserved central eral peptides with PIP2 lipids on asymmetric supported lipid bilayers. Two hydrophobic patch at the ubiquitin surface to be the binding site for the histidine-tagged peptides containing basic residues were designed to investi- Tom1 VHS domain. The ubiquitin hydrophobic patch is also involved in gate this electrostatic interaction, the first peptide had eight lysine residues Tom1 GAT domain binding, suggesting that Tom1 can bind ubiquitin mol- (His8-Lys8) and the second one corresponding to the basic domain of protein ecules through two independent sites. By providing the molecular basis of MARCKS (His8-MARCKS(152-176)). We use a reaction-diffusion model to the Tom1 interactions, we will generate cargo sorting mechanistic insights, analyze the FCS data, from which we are able to extract lipid-peptide associa- create functionally specific mutations, and precisely manipulate alternative tion and dissociation rates. Our results provide direct evidence for the forma- ESCRT-0 proteins. tion of a stable peptide-lipid complex. The experimentally determined equilibrium constant reports on the binding energy of PIP2 with the His8- 2612-Pos Board B219 Lys8 and His8-MARCKS(152-176) peptides. By altering the buffer conditions, Quantifying the Ability of Clathrin Triskelia to Sense Membrane the binding affinity and kinetics was modified for each peptide. Overall, our re- Curvature sults provide unique insight into the dynamics of lipid-peptide interactions. Avinash Gadok, Jeanne Stachowiak. University of Texas, Austin, TX, USA. 2610-Pos Board B217 Clathrin-mediated endocytosis is a primary pathway of entry into the cell. Apelin and Apela, Ligands for the Same GPCR, Differ in their Isoform- Therefore, understanding the molecular mechanisms that drive the assembly and Headgroup-Dependent Micelle Interaction of the clathrin coat is a fundamental physical problem in biology. Adaptor pro- Kyungsoo Shin, Muzaddid Sarker, Shuya K. Huang, Jan K. Rainey. teins, which bind to the membrane and to clathrin, are responsible for the Biochemistry & Molecular Biology, Dalhousie University, Halifax, NS, biochemical recruitment of clathrin to endocytic structures. However, the Canada. physical cues that influence clathrin’s recruitment remain debated. In partic- The membrane catalysis theory states that ligands associate with membranes to ular, while adaptor proteins such as epsin and amphiphysin bind preferentially enhance its rate of binding to cell surface receptors. The initial association in- to regions of high membrane curvature, it remains unknown whether clathrin creases local concentration of ligand, reduces diffusion from a 3D to a 2D pro- triskelia themselves possess the ability to sense membrane curvature and bind cess, and/or induces conformational change for receptor recognition. Variations preferentially to curved structures. To address this question, we isolated cla- in membrane composition mean that ligands may encounter a variety of envi- thrin’s curvature sensing ability in the absence of adaptor proteins by using ronments, with potential for lipid-dependent preferences in both binding and recombinant, histidine-tagged clathrin that is engineered to bind directly to conformation. We tested for evidence of membrane catalysis with apelin and Ni-NTA-containing membranes. To examine clathrin binding to these mem- apela, two peptide hormones of a single Class A GPCR (the apelin receptor). branes, we used a quantitative fluorescence intensity-based approach and Both hormones can be processed into multiple isoforms. Apelin exists as 55, quantified the bound triskelia to lipid ratio for individual vesicles spanning 36, 17, or 13-residue isoforms, apela as 32, 22, or 11-residue isoforms. All iso- a broad range of diameters. Our results demonstrate that clathrin binds pref- forms retain the C-terminal residues to bind to and activate the receptor, in turn erentially to membranes of higher curvature. Specifically, when incubated regulating a variety of physiological systems. Far-UV CD and solution-state with the same concentration of clathrin triskelia, vesicles with an average NMR spectroscopy demonstrate that all apelin isoforms exhibit b-turn charac- diameter of 30 nm recruit twice as many clathrin triskelia per membrane sur- teristics in the presence of anionic, but not zwitterionic, micelles, suggestive of face area in comparison to vesicles with an average diameter of 400 nm. Our a preferential lipid interaction. Conversely, apela-32 exhibited a similar level of ongoing work is mapping the distribution of bound triskelia over vesicles with conformational change with both zwitterionic and anionic micelles, but a range of different curvatures. This data is being used to make a statistical removal of the N-terminal region led to disproportionate level of micelle- model from which the curvature dependence of clathrin-lipid binding energy mediated changes, as observed in apela-11. Thus, membrane-association af- can be derived. These findings will provide fundamental insight into the pro- fects apelin and apela isoforms differently in response to membrane composi- cess by which clathrin is recruited to highly curved membranes during tion although they are ligands of a single GPCR. Since composition of cell and clathrin-mediated endocytosis.

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Membrane Receptors and Signal Transduction II mutation completely obliterated the allosteric modulation. Nevertheless, given our previously identified divergence between D2R and D3R in the SBP, it is 2.65 2613-Pos Board B220 not clear if the observed phenotype of E95 A is due to either the loss of a direct interaction between SB269652 and E952.65, or changes in the shape and size of Evaluation of High Affinity Scaffolds from High-Throughput Discovery 2.65 and Targeting DR5 and TNFR1 the SBP in the E95 A construct, or both. We carried out comparative molec- 1 2 2 ular dynamics simulations of both D2R and D3R wild-type (WT) and their Tiffany L. Senkow , Daniel R. Woldring , Max Kruziki , 2.65 þ Benjamin J. Hackel2, Jonathan N. Sachs1. E95 A mutants bound with SB269652, in the presence or absence of Na . 1Biomedical Engineering, The University of Minnesota, Minneapolis, MN, Based on Markov State Model analysis of our trajectories, we identified receptor USA, 2Chemical Engineering and Materials Science, The University of conformational states associated with the various ligand binding modes and char- acterized different conformational equilibria between D2R and D3R. Our results Minnesota, Minneapolis, MN, USA. 2.65 þ Misregulation of Death Receptor 5 (DR5) and Tumor Necrosis Factor Receptor 1 suggest both E95 A mutation and the presence or absence of Na has profound (TNFR1) is associated with diseases and disorders in autoimmunity (such as effects on the receptor conformations as well as on ligand binding modes: whereas E952.65A has significant effect on binding of secondary- rheumatoid arthritis and ulcerative colitis) and cancer. Currently, clinical avail- þ able therapeutics function by sequestering the receptors’ ligands or by blocking pharmacophore of SB269652 in the SBP, presence or absence of Na signifi- signaling with antibodies. These approaches have unintended consequences and cantly affects the overall binding mode. Additionally, free energy calculations dangerous side effects. Thus, there is a desperate need for alternate therapeutic were carried out to quantitate the effect of these elements on ligand binding. strategies for inhibiting members of the TNFR superfamily. Here, we used Our findings provide us insights on the complex interaction network involved directed-evolution of small protein scaffolds, namely Affibody and Gp2, to spe- in allostery mechanism in D2R-like receptors. cifically target DR5 and TNFR1. These small protein scaffolds can provide improved stability, production, and physiological distribution compared with an- 2616-Pos Board B223 tibodies, and avoid the problems associated with current treatments. Combinato- Ionic Lock: Functional Role in Activation of Metabotropic Glutamate rial protein libraries were designed for both affibody and Gp2 using a sitewise- Receptor 2 designed gradient of diversity to promote efficient evolution. Each protein li- Yu Xu1, Amr Ellaithy1, Takeharu Kawano1, Javier Gonzalez-Maeso2, brary, consisting of >109 unique clones, was sorted in a high-throughput manner Diomedes Logothetis1. 1 2 via yeast surface display to achieve high-affinity binding. The discovery of lead Northeastern University, Boston, MA, USA, Virginia Commonwealth molecules was evaluated based on (i) high thermal stability, (ii) strength and University, Richmond, VA, USA. specificity of binding to mammalian cell surface receptors, as well as (iii) effi- Background: G protein coupled receptors (GPCRs) are a large superfamily of cacy of modulating receptor function with and without the receptors’ native li- membrane proteins that, upon activation by extracellular ligands, transduce sig- gands. The robust peptides discovered in this work are ideal candidates for nals to the cell interior. Several stabilizing interactions within the GPCR trans- novel cancer and autoimmunity disease therapeutics. membrane domain (TMD) helices, referred to as ‘‘molecular switches,’’ regulate receptor activation. An example is a salt bridge between 2 highly 2614-Pos Board B221 conserved amino acids at the bottoms of TM3 and TM6 that has been charac- terized for a number of class A GPCRs. This ‘‘ionic lock’’ seems to restrain the Selective Chemical Labeling of Type II Receptor CB2 for 19F-NMR and EPR Studies receptor in an inactive state since ionic lock disruption leads to receptor activa- Alexei Yeliseev, Lioudmila Zoubak, Kirk G. Hines, Walter E. Teague Jr., tion. Recently, crystal structures have suggested similar ionic locks for the class Klaus Gawrisch. C GPCRs, metabotropic glutamate receptors mGlu1 and mGlu5. It is unknown Lab Membrane Biochem/Biophys, NIH/NIAAA, Bethesda, MD, USA. whether other class C GPCRs utilize the TM3-TM6 ionic lock to stabilize their inactive state and whether its disruption contributes to receptor activation. Human cannabinoid receptor CB2, a G protein-coupled receptor (GPCR) is implicated in an array of health related systemic body functions including the Methods: We docked several mGlu2 positive allosteric modulators (PAMs) immune response, inflammation, and pain sensing, and it is an important target into an mGlu2-TMD model based on the mGlu1 crystal structure and per- for pharmaceutical drug development. Structural and functional studies of formed 300 ns molecular dynamics (MD) simulations on receptor-ligand com- GPCR by NMR, EPR and fluorescence techniques require non-intrusive, site- plexes. Mutagenesis and voltage-clamp experiments were used to test the effect specific labeling of the protein. This can be achieved by chemical labeling of of disruption of the predicted K653-E758 ionic lock. WT and mutant receptors cysteines of the receptor. In this study we explored feasibility of labeling of were expressed in the Xenopus laevis oocyte heterologous expression system, selected cysteines by minimizing the number of reactive SH groups through sys- and G protein sensitive inwardly-rectifier potassium (GIRK) channels were tematic replacement of the majority of the 13 naturally-occurring cysteines of used as reporters of receptor activation. Results and conclusions: Our results indicate that in the single mutants K653E CB2. The replacement of the six water-exposed cysteines resulted in a well- expressed, fully functional receptor as evidenced by ligand binding- and G pro- and E758K, agonist-induced activation of mGlu2 was significantly reduced. tein activation studies. While the replacement of four additional cysteines in the The effect was partially rescued in the double mutant K653E/E758K. ‘‘Partial’’ transmembrane domain of the protein lowered ligand binding affinity several rescue suggests that the mutations could have affected other local interactions fold, the receptor could still be activated by an excess of a high affinity agonists that likely also play a role in receptor activation. Such additional interactions of and performed competently in an in vitro G protein activation assay. The reduced K653 and E758 were predicted by our computational model and functionally supported by mutations of the interacting residues. How these interactions cysteine templates of CB2 were either labeled directly or used to introduce new cysteines in selected positions at the extracellular and intracellular surfaces of the within mGlu2 lead to G protein activation is a future focus of this work. protein. The cysteine mutants of CB2 were chemically labeled with 2,2,2-trifluor- oethanethiol (TET) and their spectra recorded by 19F NMR in micelles composed 2617-Pos Board B224 of dodecyl maltoside and CHAPS or in particles formed by the NVoy polymer. Contribution of Mutations and the C-Terminal Tail to Adenosine A2A The latter was advantageous since it yielded monomeric receptor suitable for Receptor Activity and Stability recording solution-state 19F NMR spectra with decent resolution. This study is Kirsten N. Swonger, Annie Tir, Anne S. Robinson. a precursor for exploring the structure-function relationship of CB2 by NMR Chemical Engineering, Tulane University, New Orleans, LA, USA. and EPR. Adenosine A2A receptor (A2AR) is a family A GPCR, which is expressed widely in the body, and is one of the main drug targets for the treatment of 2615-Pos Board B222 neurodegenerative diseases, diabetes, inflammatory diseases, cancer and heart Molecular Determinants of SB268652 Mediated Allosterism in D2R and disease. While there are a number of crystal structures for truncated (D316) hu- D3R man A2AR, and a number of biophysical studies of the full-length A2AR, it re- Ravi K. Verma, Lei Shi. mains unclear how various stabilizing mutations contribute to its activity in Computational Chemistry and Molecular Biophysics Unit, National Institute various conditions. Here, we investigate A2AR stability by measuring ligand of Drug Abuse, Baltimore, MD, USA. binding to full-length A2AR, A2AR with a C-terminal truncation at amino The dopamine D2R-like receptors (D2R, D3R, and D4R) are important targets acid 316 (A2AD316R), and Rag23, an A2AD316R variant with five point muta- for antipsychotics and for the treatment of drug abuse. Bitopic ligands that simul- tions, identified for favorable agonist binding by Tate and colleagues (2008). taneously target both the orthosteric binding site (OBS) and the secondary bind- Ligand binding was determined by purifying receptors in micelles and ing pocket (SBP) in these receptors have improved sub-type specificity and may measuring binding via fluorescence anisotropy for the fluorescent agonist lead to a reduction in the side effects. SB269652, an allosteric bitopic ligand, has FITC-APEC. By determining equilibrium binding, temperature stability, ki- been found to be a negative allosteric modulator of both D2R and D3R. Previous netic rates, and competition with agonists and antagonists, we describe the 2.65 2.65 studies identified Glu95 in the SBP to be critical for allostery, and E95 A contribution of mutations and the C-terminal truncation on A2AR stability.

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2618-Pos Board B225 ing the release of neurotransmitters. Time courses of light intensity-dependent Effect of Bilayer Thickness on Rate of Receptor-G Protein Coupling photoresponses; changes in concentrations of phosphorylated VPs and activated Drake C. Mitchell1, Michael P. Bennett2. Trs/PDEs, as well as CNG currents, both in rods and cones from carps were re- 1Physics, Portland State University, Portland, OR, USA, 2Envision Pharma ported from Kawamura’s group. Although the chemical processes through the vi- Group, Southport, CT, USA. sual transduction cascades in rods and cones are similar, their light senilities and We investigated the effects of hydrophobic thickness on rates of G-protein bind- temporal resolutions of the flash responses are quite different. These differences ing in membranes consisting of 14:0,14:1 PC (hydrophobic thickness = 21.4 ang- are attributed to distinct reaction rates throughout the visual phototransduction stroms) and 18:0,18:1 PC (hydrophobic thickness = 29.2 angstroms). Purified systems in these photoreceptor cells, however, have not been systematically bovine rhodopsin was reconstituted with and without transducin (Gt) in large uni- investigated since none of the former models could reconstruct quantitative lamellar liposomes consisting of 14:0,14:1 PC and 18:0,18:1 PC at a lipid:protein changes in molecular reactions during photoresponses. On the basis of the Hamer ratio of 200. Kinetics of formation of the signaling competent state MII and MII- model, here we developed a detailed mathematical model of visual signal trans- Gt complex formation where measured with flash photolysis, and bilayer proper- duction system in order to quantitatively investigate how photoresponses are ties were assessed via time-resolved fluorescence anisotropy decay measure- divergently regulated in rods and cones. The current models successfully recon- ments of diphenylhexatriene (DPH). All samples were examined from 20 to structed time courses of light intensity-, ATP- and GTP-dependent changes in 40C. The equilibrium concentration of MII was higher in 18:0, 18:1 PC by about concentrations of phosphorylated VPs and activated Trs/PDEs, as well as a factor of 2 at all temperatures. MII formation kinetics was analyzed in terms of a CNG currents in rods and cones reported by Kawamura’s group. This study re- i model with 2 pathways to MII. In the absence of Gt the rate of MII formation was vealed that the assumption of the inactive state of VPs (MII ) and RGS9- higher in 18:0,18:1 PC at all temperatures by a factor of 2 to 3. Analysis of the mediated inactivation of Trs in vivo are indispensable to simulate the CNG cur- temperature dependence of the kinetics in terms of reaction rate theory showed rents, which temporal resolutions are significantly higher than that for the this was chiefly due to increased activation enthalpy for two of the forward rates; changes in PDE activities in vitro. Lumi to MI-380 and MI-480 to MII. All 6 of the microscopic rate constants for the conversion of lumirhodopsin to MII were accelerated in the thicker bilayer. At 2621-Pos Board B228 30C in 18:0,18:1 PC, MII formed with a time constant of 1.2 ms and the MII-G Cardiac Gene Therapy with Phosphodiesterases PDE2A and PDE4B Ame- t liorates Cardiac Function in a Mouse Model of Heart Failure Induced by complex formed in 1.4 ms. Formation of MII-Gt rapidly after MII formation has also been observed for rhodopsin in the native membrane. In 14:0,14:1 PC at Chronic Isoproterenol Infusion 30C MII formed in 2.9 ms and MII-G complex formed in 19.2 ms. This long Jerome Leroy. Faculty of Pharmacy, Inserm, UMR-S 1180, Univ. Paris-Sud, Universite lag between appearance of MII and Gt binding demonstrates that hydrophobic mismatch slows the time course of G protein-coupled signaling. Paris-Saclay, Chaˆtenay-Malabry, France. Abstract 2619-Pos Board B226 While acute stimulation of b-adrenergic receptors (b-ARs) increases cAMP and A Photoreceptor Model Considering Regulation of Ionic Homeostasis improves cardiac function, their chronic activation in heart failure (HF) is detri- Saya Ito, Kazuma Sato, Yukiko Himeno, Yukari Takeda, Akira Amano. mental to the heart, by promoting deregulation of intracellular calcium handling Ritsumeikan Unversity, Kusatsu, Japan. and maladaptive remodeling. Multiple phosphodiesterases (PDEs) finely tune Detailed photoreceptor model would be a powerful diagnostic tool for identifi- b-AR responses by degrading and compartmentalizing cAMP. PDE2A is cation of pathological states in retina. Kamiyama model (1996) may reconstruct increased in HF and blunts b-AR responses and the pro-hypertrophic effect of 2þ 1 2 timecourse of the light-induced membrane current (Iphoto) and Ca dynamics noradrenaline. In contrast, PDE4B is decreased in cardiac hypertrophy and in photoreceptors. However, the model does not consider ionic homeostasis, knock-out mice for PDE4B are more susceptible to develop cardiac arrhyth- þ except for Ca2 , and is incapable of simulating changes in concentrations of mias.3 Since chronic treatment with PDE inhibitors increases mortality in HF, þ þ K ,Na , and Cl-. Calculating these ionic concentrations is critically important we hypothesized that decreasing cAMP levels may have therapeutic effects. þ since they indirectly control intracellular Ca2 , and thus ATP. On the basis of To do so, we explored whether AAV-9 mediated cardiac overexpression of the Kamiyama model (1996), we propose a new model by elaborating ion chan- PDE2A or PDE4B could prevent maladaptive hypertrophy in a mouse model þ þ nels and transporters. Specifically, Na -K -Cl- co-exchanger current was of chronic isoproterenol (Iso) infusion (60mg/g/day during 2 weeks). Male þ þ þ developed referring to Terashima (2006). Na /Ca2 -K exchanger current C57BL/6 mice were injected in the tail vein with serotype 9 adeno-associated þ þ was constructed based on Na /Ca2 exchanger current (Luo, 1994). Some cur- viral vectors (AAV9, 1x1012 viral particles (vp)) to increase PDE2A and þ rent models in Kamiyama (1993), e.g. voltage dependent Ca2 current, devel- PDE4B in the heart. Eight weeks after inoculation, mice showed a five-fold in- oped based on lizard or salamander photoreceptor were modified to mammalian crease in PDE2A and PDE4B protein levels. In control mice injected with a þ þ cell-based models. For concentrations of K ,Na and Cl-, one compartment of Luciferase-AAV9 (1x1012 vp), chronic Iso treatment induced cardiac hypertro- þ each ion were assumed through the cell, whereas Ca2 concentrations at outer phy (N=10, p<0.01), fibrosis (N=9, p<0.01), and decreased ejection fraction (OS) and inner segment (IS) were separately calculated due to relatively slower (EF) measured by echocardiography by 31.653,5% (N=10, p<0.001). Overex- þ diffusion property of Ca2 . Using the model, timecourses of the light-induced pression of each enzymes did not prevent cardiac hypertrophy (N=10) but abol- þ þ þ change in concentrations of Ca2 ,K ,Na and Cl- were simulated. Upon light ished the increase in fibrosis (N=9, p<0.05). More importantly, EF was 2þ þ stimulation, decrease in Ca -and Na -permeable Iphoto at the outer segment preserved when either PDE2A (N=10, p<0.001) or PDE4B (N=10, p<0.001) þ 2þ caused hyperpolarization and simultaneous decrease in [Na ]i and [Ca ]OS. were overexpressed in this pathological model. Altogether, these results suggest þ - [K ]i and [Cl ]i were also reduced due to subsequently decrases in activities that gene therapy with AAV-9 encoding PDEs is a potential therapeutic þ þ þ - 2þ of Na /K pump and K /Cl exchanger, respectively. On the contrary, [Ca ]IS approach for cardiac maladaptive hypertrophy. þ was increased due to a rise in driving force for Ca2 entry through leak chan- 1.Mehel H, et al. J Am Coll Cardiol. 2013;62:1596-606. nels duing hyperpolarization. However, membrane potential as well as ionic 2.Abi-Gerges A, et al. Circ Res. 2009;105:784-92. concentrations were all returned to the resting state when Iphoto was recovered 3.Leroy J, et al. J Clin Invest. 2011;121:2651-61. from inhibition. The proposed model thus well maintained ionic homeostasis before and after light stimulations. 2622-Pos Board B229 Extremely Rapid Palmitoylation of Signaling Proteins Downstream of 2620-Pos Board B227 b-Adrenergic Stimulation in Cardiomyocytes Simulation Analysis of Phototransduction Systems in Rods and Cones Jessica Jie Chen, Askar Akimzhanov, Darren Boehning. Kazuma Sato1, Saya Ito1, Yukari Hosoki1, Yukari Takeda1, Chieko Koike2, Biochemistry and Molecular Biology, UT Health Science Center, Houston, Akira Amano1. TX, USA. 1Department of Bioinformatics, Ritsumeikan University, Kusatsu, Japan, S-palmitoylation is a reversible posttranslational modification that plays an 2Department of Pharmaceutical Science, Ritsumeikan University, Kusatsu, important role in regulating protein localization, trafficking, and stability. Previ- Japan. ous work from our group showed that rapid and transient palmitoylation of the Retinal photoreceptor cells, rods and cones, are the key players in visual system, tyrosine kinase Lck after death receptor stimulation is essential for downstream which convert photons of light into chemical and electrical signals. When light signaling events and apoptosis. Here we investigated whether b-adrenergic stim- stimulate visual pigments (VPs) on membranous disks of photoreceptors, heter- ulation of neonatal rat cardiomyocytes led to palmitoylation of key signaling pro- otrimeric G-proteins, transducins (Trs), are activated. Stimulated VPs are simul- teins. We found that b-adrenergic stimulation led to rapid Gas palmitoylation. taneously desensitized through phosphorylation and inactivation processes. The kinetics of palmitoylation was temporally consistent with the downstream GTP-bound a subunits of the Trs subsequently activate phosphodiesterases activation of and subsequent cAMP generation. Transient inhi- (PDEs), which in turn, hydrolyzes cGMP. Decreasing in [cGMP]i deactivates bition of the palmitoylation enzymatic machinery led to a decreased inotropic the CNG channels and subsequently hyperpolarizes the photoreceptor, prevent- response to b-adrenergic stimulation, establishing physiologic relevance. Our

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results reveal unprecedented palmitoylation kinetics of Gas that mediates cardiac T cells screen the surfaces of antigen presenting cells (APC) for peptides orig- function downstream of b-adrenergic receptors. inating from potentially harmful foreign matter in order to trigger an immuno- logical response if necessary. The pivotal interaction in this process takes place 2623-Pos Board B230 between the T cell receptor (TCR) and peptide-presenting MHC (pMHC) com- Regulation of Protein Kinase D1 Activity and Translocation by plexes. It enables the T cell to detect even very subtle differences in the anti- Nitrosylation genic peptides while at the same time only very low densities of agonistic Maura Ferrero, Ian P. Palmer, Matthew L. Stein, Lisa J. Gilardoni, pMHCs are required to cause T cell signaling. Little is known about the precise Julie Bossuyt. mechanisms behind this exceptional sensitivity and specificity, but recent find- Pharmacology, University of California Davis, Davis, CA, USA. ings suggest that mechanical forces play an important role. Protein kinase D (PKD) signaling plays a pivotal role in key physiological In order to gain further insights, we develop a force sensor for insertion in the processes such as protein trafficking, gene expression, cell survival and immunological synapse, the site of interaction between T cells and APCs. A excitation-contraction coupling. PKD1 has also been linked to pathological car- short peptide forms the core of this sensor, acting as a molecular spring. De- diac remodeling. We previously showed that PKD is activated by direct oxidation pending on the amount of force exerted by the cells, the end-to-end distance of a juxtaposed cysteine/methionine pair (CM653/54) located within a highly of the peptide will change. The peptide carries one fluorophore at each end, conserved region of the catalytic domain. The effect was primarily dependent chosen such that the fluorophores constitute a FRET pair. Therefore the change on C653, introducing the possibility that PKD could also be regulated by reactive in distance can be detected with nanometer precision by means of fluorescence nitrogen species. Here we show that PKD1 interacts with endothelial and neuronal microscopy. nitric oxide synthases (eNOS and nNOS) in adult cardiomyocytes using coimmu- After calibration of the sensor, we will have a tool that will enable us, among noprecipitations. Confocal fluorescence microscopy imaging of GFP-tagged other things, to measure molecular forces between T cells and APCs, to corre- PKD1 found minimal nuclear translocation of PKD1 in response to the NO donor late forces and T cell activation, and to create high resolution force maps of the Nitrosoglutathione (150 mM GSNO): after 2hrs the Fnuc/Fcyto increased from immunological synapse. Preliminary data demonstrating the validity of the 0.62 5 0.02 to 0.74 5 0.02. Likewise nuclear PKD activity measured by nls- approach will be presented. DKAR (a FRET-based biosensor for PKD activity) increased by 7.5 5 0.8% after 5 40 min (vs. 7.7 1% for PDBu). Valine substitution of the CM653/54 residues 2626-Pos Board B233 prevented NO-induced nuclear translocation of PKD induced by GSNO (and 1 In Vitro Formation of a Multi-Protein Complex Involved in TCR Signaling mMH2O2), suggesting a direct effect of NO. Probing immunoprecipitated PKD Asit K. Manna. with an S-nitrosocysteine antibody verified that PKD can be nitrosylated. More- Laboratory of Cellular and Molecular Biology, National Institute of Health, over in vitro kinase assay indicate GSNO inhibits baseline PKD activity and pre- Bethesda, MD, USA. vents H2O2-induced PKD activity. Valine substitution of C653, deletion of the PH The T cell antigen receptor (TCR) binds a ligand consisting of antigenic peptide domain or alanine substitution of cysteines within the PH domain all reduced the bound to a cell surface molecule encoded by the major histocompatibility com- nitrosylation signal, suggesting C653Vis not the only nitrosylation site. Our results plex (pMHC) to begin the process of T cell activation. TCR engagement leads demonstrate a novel regulatory mechanism of PKD signaling by nitrosylation. to protein tyrosine kinase (PTK) recruitment to the TCR and PTK activation, 2624-Pos Board B231 and activated PTKs phosphorylate the membrane-bound adapter protein LAT MG53 Negatively Regulates NLRP3 to Inhibit Inflammation Associated and the cytosolic adapter protein SLP-76. Phosphorylated LAT and SLP-76 re- with Tissue Injury cruit a number of other adapters and enzymes including GADS, PLCg, GRB2, Xinxin Wang1,2, Junwei Wu1,2, Xinyu Zhou1, Pei-hui Lin1, Haichang Li1, NCK, VAV and ITK. The protein complexes nucleating at LAT are highly het- Hua Zhu1, Tao Tan1, Matthew Sermersheim1, Renzhi Han1, Jianguo Wen2, erogeneous, and the formation of these complexes is highly co-operative. Fangxia Guan2, Jianjie Ma1. Different LAT complexes activate different effector function. For example, 1 the LAT/GADS/SLP-76 complex recruits the PTK ITK to activate PLCg, lead- Department of Surgery, Davis Heart and Lung Research Institute, The Ohio 2þ State University, Columbus, OH, USA, 2The First Affiliated Hospital of ing to Ca flux and MAPK activation. Here we study the LAT-GADS-SLP-76 Zhengzhou University, Zhengzhou, China. tri-molecular complex by generating it in vitro from purified component pro- MG53 is a member of the tripartite motif family with E3 ubiquitin ligase activity, teins and a LAT phospho-peptide. Isolation of this complex and others should and plays important roles in cell membrane repair, tissue regeneration and wound enable generation of structural information, which would be required to design healing. We previously showed that topical or systemic administration of recom- specific inhibitors for modulation of signaling. binant human MG53 (rhMG53) protein protected against tissue injury and reduced the level of inflammatory cytokines (e.g. IL-1b) in circulation. 2627-Pos Board B234 NLRP3 is a key member of the inflammasome complex that mediates the cleav- The HIV gp41 Conserved Pocket Binding Domain is Bifunctional, Alterna- age and activation of caspase-1 for control of IL-1b release into circulation. As tively Mediating both Immunosuppression and Membrane Fusion tissue injury is often associated with inflammation, we hypothesize that MG53 Gal Kapach, Yoel A. Klug, Etai Rotem, Benjamin Dubreuil, Yechiel Shai. interacts with NLRP3 to control inflammasome signaling associated with tissue The Weizmann Institute of Science, Rehovot, Israel. injury. With the mg53-/- mice, we found the spleen organ was significantly The human immunodeficiency virus (HIV) utilizes its envelope glycoprotein enlarged compared with the wild type littermates. The protein level of NLRP3 gp160, specifically the gp41 subunit, to enter its host cell. During this process, in the spleen was up-regulated in the mg53-/- mice. In cultured HEK293 cells, a gp41-central coiled coil is formed from three N- and three C-terminal heptad co-expression of MG53 with NLRP3 led to down-regulation of NLRP3 in a repeats (NHR and CHR, respectively). This structure is termed the six-helix proteasome-dependent manner. Down-regulation of NLRP3 was not observed bundle (SHB) and it drives membrane fusion. Situated at the CHR N-terminus in cells co-transfected with a mutant MG53 lacking E3-ligase activity. Co- is the conserved pocket binding domain (PBD) that stabilizes SHB formation. immunpprecipitation studies revealed a physical interaction between MG53 A Recent study reported that a region housing the PBD acts as an immune sup- and NLRP3. With human monocytes (THP-1 cells) transfected with RFP- pressor by binding the T-cell receptor (TCR) in the membrane. Since the PBD MG53 and GFP-NLRP3, we observed the trafficking of MG53 and NLRP3 asso- is highly conserved, we hypothesized that it might mediate gp41 based immune ciated with inflammasome formation. Upon treatment with LPS and ATP, THP-1 suppression. We used independent cell culture models along with biophysical cells responded with formation of inflammasomes and co-segregation of NLRP3 and biochemical techniques such as fluorescence resonance energy transfer and and MG53. The level of NLRP3 in THP-1 cells co-expressing MG53 was signif- circular dichroism to investigate the PBD functions. We revealed that the PBD icantly lower compared with cells do not express MG53. Together, these findings is bifunctional, alternating between fusion facilitation and immunosuppression. suggest that MG53 act as a negative regulator of NLRP3 to control inflamma- Since SHB formation is proposed to occur in the virological synapse, the PBD some signaling. More in vivo studies are required to dissect the physiological would need to alternate between being buried in the SHB to binding the TCR in role of MG53 in modulating the immune response associated with tissue injury. the membrane for the immune suppression activity. Interestingly, we found that the PBD stabilizes a TCR binding conformation in the membrane resulting in 2625-Pos Board B232 signaling inhibition. Moreover, successful membrane fusion doesn’t entirely Measurement of Forces in the Immunological Synapse rely on the PBD. Our data suggests that immunosuppression may be a possible Lukas Schrangl1, Janett Go¨hring2, Florian Kellner2, Melanie Ko¨hler3, driving force for the high degree of PBD conservation. Through this multidis- Peter Hinterdorfer3, Johannes Huppa2, Gerhard J. Schutz€ 1. ciplinary approach we shed light on the mechanisms behind many gp41 func- 1Institute for Applied Physics, TU Wien, Wien, Austria, 2Institute for tions and detail its complex relationship with the membrane and the proteins Hygiene and Applied Immunology, Medical University of Vienna, Wien, residing within. While structural data at atomic resolution detailing membrane Austria, 3Institute for Biophysics, Johannes Kepler University Linz, Linz, proteins is limited, our approach may be implicative to other membrane pro- Austria. teins in various biological systems.

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Mechanosensation regarding theplasma membrane localization and organization of Piezo1, but previous work suggests that membrane cholesterol content is a key determinant 2628-Pos Board B235 of Piezo1 function. Using a previously characterised Piezo1-GFP fusion protein From Force to Function - Investigating Mechanosensitive Piezo Receptors (hP1-1591-GFP[1]), we investigated the effect of the cholesterol depleting by AFM agent methyl-b-Cyclodextrin (mbCD) on the membrane organization and the Benjamin M. Gaub, Daniel J. Mueller. response of Piezo1 to mechanical force in HEK-293 cells. STORM super- D-BSSE, ETH Zurich,€ Basel, Switzerland. resolution imaging revealed at the nanoscale that Piezo channels associate in Mechanosensation is the process by which cells sense mechanical forces and the membrane as clusters, as previously inferred by electrophysiological data translate them into electrical and chemical signals. Important physiological [2]. Both cluster size and diffusion rates, as determined using TIRF micro- functions including sensation of touch, sensation of sound and proprioception scopy, were modulated by treatment with mbCD (5 mM). In addition, electro- are based on mechanosensation. Recent studies identified the molecular iden- physiological recordings in the cell-attached configuration revealed that mbCD tity of a highly conserved group of mechanosensitive receptors, called Piezo re- caused a right-ward shift in the Piezo1 pressure-response curve and a delay in ceptors, which are both necessary and sufficient for cells to sense force. the initial response (i.e. increased latency). We suggest that cholesterol rich Currently, structure-function information of Piezo receptors is limited, and micro-domains host Piezo1 clusters and that this nanoscale membrane organi- the mechanism how these receptors sense force, which conformational changes zation is essential for efficient Piezo1-mediated mechanotransduction. This is they undergo, and which precise mechanical stimuli cause these channels to consistent with cholesterol rich domains acting as ‘‘membrane force foci’’. open, remains poorly understood. [1] Cox et al., (2016) Nat Commun 20;7:10366 Studies to date have used rather brute forces reaching several hundrets of nano- [2] Bae et al., (2013) PNAS 110(12): E1162-E1168 Newton to mechanically activate Piezo channels, which are likely too high to be of specific nature. To manipulate Piezo receptors at high precision and accuracy 2631-Pos Board B238 in a cellular context, new techniques have to be applied. We are currently devel- Enantiomeric Forms of Abeta Peptides Inhibit the Shear Stress Response oping assays to investigate Piezo receptors at the molecular level with delicate of PIEZO1 force control. Using a combination of high-resolution atomic force microscopy Philip A. Gottlieb1, Mohammad M. Maneshi2, (AFM) and time-lapse fluorescent calcium imaging, we aim to find more effi- Radhakrishnan Gnanasambandam1, Susan Z. Hua2, Frederick Sachs1. 1 cient ways to functionally control Piezo receptors by externally applied forces. Physiology and Bophysics, SUNY at Buffalo, Buffalo, NY, USA, 2 Importantly, the tip of the AFM cantilever can be conjugated with small mole- Department of Mechanical & Aerospace Engineering, SUNY at Buffalo, cules, including ligands and antibodies, which enables manipulation of mem- Buffalo, NY, USA. brane proteins at nanometer precision with picoNewton sensitivity. PIEZO1 is a eukaryotic mechanosensitive ion channel that is cation selective and We will use this assay to i) get a more quantitative understanding of Piezo rapidly inactivates. Using fluid shear stress as a stimulus generated in a microflui- gating by mechanical forces, ii) directly compare Piezo receptor responses to dic chamber, we have monitored PIEZO activity in the presence of various pep- different modes of mechanical stimulation including pressure, stretch and vi- tides. To do so, we first developed a cell line that overexpresses the PIEZO1 bration, and iii) screen native extracellular matrix (ECM) derived ligands for channel in HEK293T cells. We estimate that there are about 5000 channels per their ability to bind and functionally modulate the Piezo channel. Our results cell. These cells are seeded in the microfluidic chamber and loaded with calcium will help shed light on the force sensing mechanism of mammalian mechano- indicator. A pressure servo is fitted to the chamber and allows control of the sensitive receptors. applied shear stress stimulus. The calcium change is monitored as an increase in fluorescence. Applying a 10 ms pulse at 13 dyn/cm2, we observed a robust 2629-Pos Board B236 response for the PIEZO1 cell line well above the background observed for Molecular Dynamics Analysis on the Force Transmission Pathway via HEK293T cells only. Peak response is achieved within a few seconds and is fol- Inter-Subunit Pathway for Mechano-Gating of Bacterial Mechanosensi- lowed by adaptation. Disrupters of the cytoskeleton applied to the cell line, such tive Channel MscL as cytochalasinD and cholchicine, rendered the PIEZO1 channel unresponsive to Yasuyuki Sawada1, Takeshi Nomura2, Masahiro Sokabe1. the stimulus. The peptide GsMTx4 inhibits PIEZO1 currents. We measured the 1Mechanobiology Lab, University Graduate School of Medicine, effect of GsMTx4 on PIEZO1 activity as a way to compare the microfluidic Nagoya, Japan, 2Physical Therapy, Grad Sch Health Sciences Kyushu approach to previous work. We applied various concentrations and measure Nutrition Welfare Univ, Kitakyushu, Japan. the peak response. The inhibition curve showed a Ki of around 250 nM similar The bacterial mechanosensitive channel MscL is constituted of homopentamer to the value measured previously by patch clamp. Next, we examined the effect of a subunit with TM1 inner and TM2 outer transmembrane helix. The major of Abeta peptides on PIEZO1 activity. A known attribute of these peptides is their issue on MscL is to understand the gating mechanism driven by membrane ten- ability to bind membranes. Given PIEZO1 is modulated by membrane tension we sion. Upon membrane stretch, the helices are dragged by lipids at the tension wanted to determine how the channel behaved in the presence of Abeta peptides. sensor F78 and tilted, accompanied by outward sliding, leading to a gate expan- We prepared the monomer form of the peptide and measured the peak response as sion. To get insights into the relationship between F78 and the gate including a function of peptide concentration. At the same time we prepared the D form of G22, we performed MD simulations of G22N GOF and F78N LOF MscLs these peptides and measured them as well. Our data show that both the L and D and G22N showed spontaneous opening without membrane stretch, while form of Abeta 1-40 and 1-42 inhibit channel function in the fM to pM range. The F78N could not be opened even under strong membrane tension. To assess enantiomeric forms of each peptide were similar in their ability to inhibit PIEZO1 the role of Asn22 for the spontaneous opening, the double mutant G22N/ activity. The lack of stereospecific interaction indicates that these peptides modu- F78N MscL simulation was performed with and without membrane stretch late activity of the channel by changing the properties of the membrane. and found that G22N/F78N MscL did not begin channel opening upon mem- This work was funded by NIHLBI and NINDS. brane stretch under the both conditions. Furthermore, we found that the closed MscL can transmit resting tension to the gate via the interaction between F78 in 2632-Pos Board B239 TM2 and I32-L36-I40 in the neighboring TM1 and the transmitted force can Mechanosensitivity of Coupled Active Hair-Cell Bundles lead to slight opening of the pore. It is suggested that the substitution of F78 Tracy-Ying Zhang1, Seung Ji2, Dolores Bozovic1. with Asn loses the transmission pathway, leading to be harder to open than 1Department of Physics and Astronomy, University of California Los G22N even though G22N/F78N has Asn22 with the hydrophilic side chain. Angeles, Los Angeles, CA, USA, 2Department of Physical Science, Los Angeles Mission College, Sylmar, CA, USA. 2630-Pos Board B237 The sensitivity of the auditory system depends in part on the active response of Human Piezo1 Membrane Localization and Gating Kinetics are hair cells in the inner ear. Individual hair bundles display frequency selectivity Modulated by Cholesetrol Levels and compressive nonlinearity in response to stimuli. In many auditory and Pietro Ridone1, Charles Cox1, Massimo Vassalli2, Elvis Pandzic3, vestibular end organs, the hair bundles are coupled by overlying structures. Philip Gottlieb4, Boris Martinac1. This motivates our study on how coupling affects the hair bundle sensitivity. 1Molecular Cardiology and Biophysics, Victor Chang Cardiac Research We couple two to four spontaneously oscillating bundles with a microbead, Institute, Sydney, Australia, 2Institute of Biophysics IBF, Genova, Italy, and apply mechanical stimuli to the group. Prior work showed that under these 3Biomedical Imaging Facility (BMIF), University of New South Wales, coupling conditions, innate oscillations synchronize. Here, we show that the Sydney, Australia, 4State University of New York, Buffalo, NY, USA. synchronized bundles exhibit broad frequency selectivity, over a bandwidth en- The human mechanosensitive ion channel Piezo1 gates in response to mem- compassing each bundle’s natural frequency. The amplitude of the response brane tension and regulates essential biological processes such as vascular shows a nonlinear dependence on the applied stimulus. Experimental data development and erythrocyte volume homeostasis. Currently little is known will be presented, as well as a simple numerical model, to explain how the

BPJ 7904_7909 534a Wednesday, February 15, 2017 behavior observed in the coupled system arrives from the active motility of in- MscL-like channel. Similar to MscS and MscL of E. coli, the activation thresh- dividual hair bundles. olds of MscCG and the MscL-like channel were determined as 6 mN/m and 12 mN/m, respectively, by measuring membrane patch curvature and applying the 2633-Pos Board B240 Laplace’s law. To examine the effects of the lipid composition, we compared Generalized Fluctuation-Dissipation Theorem Applied to Active Hair the gating of MscCG in C. glutamicum and E. coli membrane and found that Bundles MscCG remained open at highly hyperpolarized membrane potential in C. glu- Janaki K. Sheth. tamicum membrane, suggesting that MscCG has suitable gating properties to Physics, University of California, Los Angeles, Los Angeles, CA, USA. release glutamate continuously. Moreover, micropipette aspiration technique The first step in auditory detection occurs through specialised sensory cells combined with molecular dynamics simulations revealed that the areal elastic- called the hair cells; mechanically sensitive ion channels open in response to ity modulus, KA, of the C. glutamicum membrane was significantly lower than the deflections induced by incoming sounds. Stereocilia of some species are that of E. coli membrane. These findings indicate that the membrane lipid com- also known to oscillate without external stimulation. The role of these oscilla- ponents greatly influence the MscCG gating properties and that effective gluta- tions is not entirely understood, but they were shown to have a basis in an un- mate transport through MscCG occurs effectively in the native lipid derlying active mechanism. A number of models have been proposed for active environment. amplification by hair bundle motility, which include myosin motor activity and calcium feedback. However, the internal variables are not readily accessible 2636-Pos Board B243 experimentally. Hence, an algorithm based on non-equilibrium steady-state Spatiotemporal Relationships for the Three-State Mechanosensitive fluctuation-dissipation theorem has been proposed to reconstruct the dynamics Channel MscS of the internal active elements in the hair cell. We will evaluate the efficacy of Ugur Cetiner1, Sergei Sukharev2. this algorithm, by comparing the reconstructed dynamics of active elements to 1Biology, University of Maryland, College Park, MD, USA, 2Biology and predictions based on numerical simulations and to experimental measurements IPST, University of Maryland, College Park, MD, USA. performed on hair cells of the bullfrog sacculus. Adaptive desensitization and inactivation is a common property of most ion 2634-Pos Board B241 channels and receptors. For channels, which act by dissipating vital gradients Chaotic Behavior of Oscillatory Hair Cells of ions and solutes, this behavior sets the necessary term limits for opening, Justin Faber. commensurate with the mode of action and metabolic resources of the cell. Physics, UCLA, Los Angeles, CA, USA. Voltage-gated channels typically inactivate from the open state to ensure the The inner ear is capable of detecting sounds that elicit motions below the sto- full spike amplitude. In contrast, the bacterial mechanosensitive channel chastic noise levels. Hair cells are specialized sensory cells essential for the MscS, acting as a low-threshold osmolyte release valve in the event of osmotic hearing process. They convert mechanical energy from incoming sound into shock, inactivates from the closed state at moderate non-saturating tensions. currents, by opening and closing mechanically sensitive ion channels in This behavior prevents futile dissipation of metabolites from small cells. It response to the induced deflections. Hair cells of certain species are also known has been shown that MscS’s two separate tension-driven transitions from the to oscillate without external stimulation. The role of these spontaneous oscilla- closed (C) into the open (O) or inactivated (I) states are associated with tions is not understood, but they are believed to be a signature of an underlying different degrees of lateral protein expansion (DA) and proceed with different active mechanism. As this active process constitutes one of the most important rates. In this work, we attempt the reconstruction of the energy landscape for open topics in auditory research, a deeper understanding of spontaneous MscS based on patch-clamp experiments utilizing pressure ramps and pulse- motility could have important implications on understanding the extreme sensi- step-pulse stimulation protocols. The three-state continuous time Markov tivity of hearing. Hair cells have been shown to flicker between the oscillatory model, where the transition rates are governed by Arrhenius-type equations, de- and quiescent states; this phenomenon was modeled with dynamic feedback scribes the tension-dependent distribution of channel population between the acting on an internal control parameter that determines the dynamic state of three states and quantitatively explains the experimentally observed inactiva- the cell. This simulation predicted a positive Lyapunov exponent for innate tion in the ramp and pulse-step-pulse stimulation regimes. Through different bundle motility, indicating the presence of chaos. We will present experimental kinetics of outflow from the C to either O or I states, MscS senses the dynamics measurements of spontaneous hair bundle oscillations, which were obtained of the stimulus by opening to an abrupt tension onset and ‘ignoring’ slowly from the sacculus of the American bullfrog. Using the delay-coordinate tech- applied stimuli. Our solution also provides an analytic derivation for the area nique, the phase space of the oscillator was reconstructed, allowing for estima- of I state relative to C and O states from the experimental parameter of tion of the number of state variables required for modeling the system. Using ‘maximum inactivation’ tension easily accessible in pulse-step-pulse mode. Poincare´ maps, we observed a quasiperiodic transition from low dimensional We present experimental data illustrating applicability of this approach to the chaos to order, as the amplitude of an applied mechanical stimulus was previously uncharacterized MscS homolog from Pseudomonas aeruginosa. increased. This transition was accompanied by a reduction in Kolmogorov en- tropy, a quantity used to determine the degree of disorder in a dynamical 2637-Pos Board B244 system. Temperature Sensitive Mutants of the MscL Mechanosensitive Channel Prevent Cell Growth at High Temperature 2635-Pos Board B242 Naoto Owada, Kohei Morita, Megumi Yoshida, Kenjiro Yoshimura. Electrophysiological Characterization of Mechanosensitive Channels in Department of Machinery and Control Systems, Shibaura Institute of the Native Membrane of Corynebacterium Glutamicum Technology, Saitama, Japan. Yoshitaka Nakayama1, Kosuke Komazawa2, Navid Bavi1,3, The MscL mechanosensitive channels of E. coli cells transiently opens on hy- Ken-ichi Hashimoto2, Hisashi Kawasaki2, Boris Martinac1,3. poosmotic shock and protect cells from lysis. MscL gain-of-function mutants, 1Victor Chang Cardiac Research Institute, Sydney, Australia, 2Tokyo Denki which are constitutionally active, cause a lethal phenotype when expressed University, Tokyo, Japan, 3University of New South Wales, Sydney, because of continuous leakage of cytoplasmic components. To gain insights Australia. into the thermodynamics of MscL gating and future use as a ‘‘heat-activated Mechanosensation is an indispensable physiological function, and mechano- nano-valve,’’ we isolated temperature sensitive mutants of MscL by screening sensitive channels are the molecular entities to sense mechanical force. Bacte- mutants that allowed cell growth at 25C but not at 42C. After the initial ria have two types of mechanosensitive channels, MscS and MscL to monitor screen, we determined the temperature dependence of the cell growth of osmotic environment. Gram-positive soil bacterium, Corynebacterium gluta- each mutant and identified two mutants that allowed normal cell growth at micum, secretes a massive amount of glutamate through an MscS homolog, 32C but not at 42C. One mutation (L86P) resided in the middle of the second MscCG, in the process of industrial glutamate production. However, how transmembrane segment (TM2) and the other mutation (D127V) resided in the MscCG gating is involved in the glutamate export has not been fully under- cytoplasmic helix. We introduced hydrophilic mutations (L86S, L86T) or a stood. Since bacterial mechanosensitive channels are gated by membrane ten- flexible mutation (L86G) at Leu-86 and found that cells expressing these mu- sion directly, it is particularly important to examine the lipid-channel tants grew at a normal rate. This finding suggests that a break in TM2 alpha he- interaction during the gating of MscCG. C. glutamicum membrane consists lix caused by proline results in lethal phenotype. We substituted Asp-127 with mostly of negatively charged lipids, phosphatidylglycerol (PG) and cardiolipin other hydrophobic amino acids (D127A, D127L) or hydrophilic amino acids (CL), whereas E. coli membrane is made largely of phosphatidylethanolamine (D127K, D127S) and found again that cells expressing these mutants grow at (PE). Here we show a successful preparation of C. glutamicum giant sphero- a normal rate. Introduction of valine into neighboring hydrophilic residues plasts that enable us to record mechanosensitive channel activities and charac- (T123V, E124V, R126V) also resulted in normal growth. This finding suggests terize their membrane mechanical properties. Using the patch-clamp technique, the importance of the bundle of cytoplasmic helices for the stability of MscL we identified two types of mechanosensitive channels, MscCG and an although the mechanism of the lethal phenotype remains to be determined.

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2638-Pos Board B245 two levels of integrin tensions respond differently to actin and myosin II inhi- Expression and Characterization of the Putative Mechanosensitive bition, serum deprivation and microtubule inhibition, indicating their indepen- Transmembrane Channel-Like Protein 1 dent tension sources and dynamics. Conclusion: Our experiments demonstrate Angela Ballesteros Morcillo, Kenton Swartz. that integrin tensions are active both inside FAs and outside FAs at separate ten- NINDS-NIH, Bethesda, MD, USA. sion levels, and these two types of tensions coexist during stable cell adhesion. Our senses of touch, hearing, balance, and proprioception depend on mechan- This result renovates the common view that FAs are the main mechanotrans- ically gated ion channels, which transduce mechanical energy into electrical duction sites by revealing the existence of substantial integrin tensions outside signals that are transmitted to the brain. Previous studies on the mechanisms FAs. We also demonstrate that mTGT provides a convenient and robust plat- of hearing have elucidated the biophysical properties of the mechanotransduc- form to comprehensively monitor the spatial distribution and dynamics of in- tion channel responsible of hearing, but the channel’s molecular identity still tegrin tensions at different levels. remains elusive. Transmembrane channel-like (TMC) proteins are conserved from worms to humans and were initially identified as essential for hearing. 2641-Pos Board B248 In addition, point mutations in TMC1 not only cause human deafness, but Ligand-Dependent Concentric Actomyosin Arcs Regulate TCR Mechano- also alter the mechanotransduction channel properties, suggesting a potential Transduction at the Immunological Synapse channel function for this protein. In an effort to enable study of the functional Jinsung Hong, John A. Hammer. properties of TMC1 in a heterologous system, we expressed the protein in National Institues of Health, Bethesda, MD, USA. HEK293 cells, COS7 cells and Xenopus oocytes. In both types of mammalian T cell receptor (TCR) mechano-transduction is an emerging but poorly under- cells, TMC1 is effectively translated, but localizes to the ER with no evidence stood component of T cell activation. To define the contribution made by the of trafficking to the plasma membrane. In contrast, TMC1 can be robustly ex- cytoskeleton to TCR mechano-transduction, we investigated the role played pressed in oocytes and surface biotinylation experiments demonstrate that it by contractile actomyosin arcs populating the pSMAC region of the immuno- traffics to the plasma membrane. We mutated eleven ER retention sites found logical synapse (IS) in ligand discrimination. Using super resolution micro- in TMC1 and generated several deletion constructs to facilitate trafficking of scopy, we show that the generation of organized actomyosin arcs depends on the protein to the plasma membrane in mammalian cells, however, each of the ligand potency and the ability of non-muscle myosin II to contract actin fil- these manipulations failed to alter robust ER localization. We also co- aments. While weak ligands induce disorganized actomyosin arcs, strong li- expressed TMC1 with other membrane proteins essential for hearing mechano- gands result in organized actomyosin arcs that correlate well with tension- transduction, but none altered the ER localization of TMC1 in mammalian sensitive CasL phosphorylation and the accumulation of ligands at the IS cen- cells. We are currently looking for channel activity in oocytes expressing ter. Furthermore, the presence of ligand-dependent actomyosin arcs generates a TMC1 and exploring the expression of TMC1 in tissues other than organ of dynamic range in the phosphorylation of the early signaling molecules. Taken Corti. Our goal is to purify TMC1 from a range of tissues to identify other pro- together, our correlative study implicates ligand-dependent actomyosin arcs are teins that form a complex with this putative mechanosensitive ion channel. a mechano-chemical feedback mechanism that amplifies the accumulation of critical signaling molecules at the IS. 2639-Pos Board B246 Towards a Complete Structural Model for the Essential Inner-Ear Protein 2642-Pos Board B249 Cadherin-23 Intracellular Local Thermogenesis Initiates Stress Granule Formation Avinash Jaiganesh, Aniket Patel, Pedro De-la-Torre, Domenic Termine, Beini Shi1, Kohki Okabe1,2, Takashi Funatsu1. Florencia Velez-Cortes, Marcos Sotomayor. 1Dept Pharmac Sci, Univ Tokyo, Tokyo, Japan, 2JST, PRESTO, Tokyo, Japan. Chemistry and Biochemistry, The Ohio State University, Columbus, OH, USA. Under stress, eukaryotic cells form stress granule (SG), responsible for transla- Inner-ear mechanoreceptors rely on long, fine ‘‘tip-link’’ filaments that directly tion regulation. Recent studies suggested that SG formation involves phase sep- pull on mechanosensitive channels to start sensory perception. Two proteins of aration and self-aggregation of SG components. The initiation mechanism, the cadherin superfamily, cadherin-23 (CDH23) and protocadherin-15 however, still remains uncertain. SG can dynamically spontaneously assemble (PCDH15) interact heterotypically through their extracellular domains to and disassemble, which is thought to be some physical parameters involved in form tip-links. These domains have multiple extracellular cadherin (EC) re- these processes. In this study, we focused on the intracellular temperature peats that are similar, but not identical in sequence and fold. CDH23 has 27 changing during SG formation, and hypothesized that the heat released from of these EC repeats that form 3/4th of the tip link. Each ~100 amino-acid mitochondria might play a critical role in triggering SG formation. The objec- long repeat contains highly conserved calcium-binding motifs at the linkers be- tive of this study was to clarify the intrinsic relationship between intracellular tween adjacent EC repeats. Multiple sites on CDH23 have been identified as thermogenesis and SG formation. susceptible to deafness-causing mutations, but little is known about the archi- Firstly, we imaged intracellular temperature in stressed COS7 cells by using a tecture of CDH23 EC repeats and the structural effects of these missense mu- fluorescent polymeric thermometer (FPT) and fluorescence lifetime imaging tations. Here, we present X-ray crystal structures of 13 EC repeats of CDH23: microscopy (FLIM). The result demonstrated that intracellular temperature in- EC6-8, EC12-13, EC17-18, EC19-21 and EC22-24. These structures cover creases during SG formation. Secondly, we found that cells containing excess over half of the entire CDH23 extracellular domain and reveal multiple varia- FPT could not assemble SG, which was due to the heat absorption of FPT. tions among EC repeats. Additionally, they show over 50% of the sites affected Moreover, this heat absorption by FPT specifically prevented SG formation by deafness-related missense mutations. Our study provides a structural frame- initiation, indicating that intracellular thermogenesis is necessary for initiating work to understand CDH23 function in hearing. SG assembly. Furthermore, to investigate the intrinsic relationship between intracellular local thermogenesis and SG formation, we introduced artificial 2640-Pos Board B247 heating system by infrared (IR) laser. The results demonstrated that this method Co-Mapping Integrin Molecular Tension Both Inside and Outside Focal could induce the transient SG assembly. The quantification of local temperature Adhesions during this experiment revealed that the threshold of local temperature in initi- Yongliang Wang, Xuefeng Wang. ating SG formation is 42 oC. Therefore, we confirmed that intracellular local Iowa State University, Ames, IA, USA. thermogenesis could facilitate SG formation initiation.In this study, we discov- Background: Integrins are mechano-sensitive receptors that regulate cell adhe- ered that intracellular thermogenesis facilitated SG formation initiation, which sion, migration and many other cellular functions. Molecular tensions on integ- may be a novel principle of temperature signaling in cell biology. rins are critical for integrin dynamics and activities. Such tensions operate in a broad range and may go up to 100 piconewtons (pN). To simultaneously map 2643-Pos Board B250 integrin tensions at different force levels, we developed multiplex tension Under Pressure: Mechanosensitivy Properties of the Bacterial Divisome gauge tether (TGT) with a detection range of 10~100 pN and applied it to Marta Dies, Javier Buceta. co-map multiple levels of integrin tensions in live cells. Methods: TGT is a Chemical and Biomolecular Engineering; Bioengineering Program, Lehigh tension sensor that sends out fluorescence signal if the TGT sustains a molec- University, Bethlehem, PA, USA. ular tension higher than its inherent rupture force (Ttol). We multiplexed TGTs Bacterial cell division starts with the formation of the prospective septum by with different Ttol values and labeled with different fluorophores. The multiplex the polymerized FtsZ protein in the mid-cell position (the so-called Z-ring). TGT (mTGT) was used to simultaneously report integrin tensions at separate In this context there is evidence that Escherichia coli division can be perturbed levels. Results: mTGT reported two-level integrin tensions during cell adhe- by mechanical pressure. Here, using time-lapse fluorescence microscopy and sion with 54~100 pN integrin tensions located inside focal adhesions (FA) microfluidics means, we show that when compressing growing E. coli cells where integrins cluster with other proteins, and 12~54 pN integrin tensions ho- up to a ~ 40% cell thickness reduction, the divisome complex becomes a mogenously distributed outside FAs. The outside-FA integrin tensions remain cutting-point scaffold that slashes the membrane as it moves, thus arguing active after FA formation and coexist with integrin tensions inside FAs. These against the current knowledge about bacterial cytokinesis.

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Previous studies regarding the Min system oscillations (responsible for select- in desmosome hyperadhesivity affects desmosome force. Lastly, we observed ing the mid-cell position during the Z-ring formation) revealed that Min pro- higher dsg-2 forces in MDCK cells grown as 3D acini compared to 2D mono- teins can oscillate in different patterns so that they are able to capture the layers. To determine the importance of desmosome forces in acini, we used domi- symmetry for several cellular shapes. It is generally agreed that the Min system nant negative desmoplakin (DP-NTP) to prevent desmosome association with the determines the cell’s shortest axis of symmetry so that the Z-ring is formed on IF cytoskeleton. DP-NTP expressing MDCK cells did not form hollow acini with that plane. However, this seems to be in disagreement with our observations, as a single lumen, but instead were a solid mass of cells. These results provide, to our the FtsZ cutting-point scaffold is assembled into a single point and its progres- knowledge, the first direct evidence of mechanical force across desmosomes, and sion is often not even confined to a plane, showing a gap of knowledge in the by extension suggest that the intermediate filament cytoskeleton is capable of interplay between Min oscillations and FtsZ under mechanical perturbations. applying tensile forces at cell-cell contacts. In order to elucidate the dynamics of the divisome cutting-edge complex under mechanical compression in single cells, we use a custom software that esti- 2646-Pos Board B253 mates the symmetry axes of cells and relates the localization of the divisome Patterned Cell Alignment in Response to Macroscale Curvature 1 2 3 1 cutting scaffold with respect to them. We speculate that this will unveil the ex- Nathan D. Bade , Randall Kamien , Richard Assoian , Kathleen Stebe . 1Chemical and Biomolecular Engineering, University of Pennsylvania, istence of geometrical constrains (if any) in the dynamics of the divisome under 2 mechanical compression. Philadelphia, PA, USA, Physics and Astronomy, University of Pennsylvania, Philadelphia, PA, USA, 3Pharmacology, University of 2644-Pos Board B251 Pennsylvania, Philadelphia, PA, USA. Prostate Cancer Cells are Reprogrammed to Normal via Ultrasound- The formation of spatial behavior patterns in tissues is a long-standing problem Induced Mechanotransduction in biology. Decades of research have focused on understanding how biochem- Hakm Y. Murad, Heng Yu, Daishen Luo, Gray Halliburton, ical signaling and morphogen gradients establish cell patterns during develop- Damir Khismatullin. ment and tissue morphogenesis. Here, we show that geometry and physical Biomedical Engineering, Tulane University, New Orleans, LA, USA. cues can drive organization and pattern formation. We find that mouse embry- Introduction: Prostate cancer is the most incident cancer in men. Since the ma- onic fibroblasts and human vascular smooth muscle cells sense curvature differ- jority of prostate cancer patients are elderly men, often not suitable for invasive ently when in monolayers than when isolated on surfaces with various amounts procedures, there is a need for minimally invasive therapies (e.g., focused ul- of Gaussian curvature. The apical and basal stress fibers within these cells align trasound). We tested the hypothesis that focused ultrasound (FUS) drastically in distinct patterns that depend on the magnitude of substrate curvature and reduces the metastatic potential of chemically-treated cancer cells via overpro- RhoA activity. By modulating the activity of RhoA, we can recapitulate the duction of heat-shock protein 70 (HSP70), death receptor Fas and its ligand F-actin orientation that is observed in blood vessels in vivo. FasL. Materials and Methods: Suspensions of cultured DU145 and PC3 pros- tate cancer cells were placed in 0.2ml tubes. The cells were left untreated or Calcium Signaling III exposed to FUS alone, 4% ethanol, or FUSþ4% ethanol. The FUS signal was generated by a 1.1 MHz transducer, with acoustic power ranging from 2647-Pos Board B254 4.1 to 20 W. Fas and FasL expression was measured by flow cytometry, and Calcium Regulation of Cardiac Bioenergetics HSP 70 protein levels were determined by Western blot analysis at 2, 24, Andrew P. Wescott, Liron Boyman, W. Jonathan Lederer, and 72 hours post-treatment. To confirm that cancer cells lose aggressiveness, George S.B. Williams. we measured the number of cancer cells adhered to TNF-a-activated endothe- University of Maryland Baltimore, Baltimore, MD, USA. lium as well as tested the ability of cells to form multi-cellular spheroids. Re- Cardiac muscle must pump blood and maintain constant perfusion of the body for sults and Discussion: Prostate cancer cells significantly increased their all of life. Such an important task requires a reliable and adaptable source of en- expression of Fas, FasL, and HSP 70 immediately after being exposed to ergy. While adenosine triphosphate (ATP) is generated continuously by mito- FUSþ4% ethanol and continued to produce these molecules at a significantly chondria through oxidative phosphorylation, ATP is also consumed by the higher amount than untreated, ethanol, or FUS alone alone-treated cells. Static myocyte at a high rate due to contractile work. Under normal conditions, the adhesion assay showed that cells in the combined treatment were less likely to vascular system provides the extremely abundant heart mitochondria (~10,000 attach to endothelium. Cells exposed to both ethanol and FUS were unable to per cardiomyocyte) with a generous delivery of oxygen, fatty acids, and carbohy- form three-dimensional tumor spheroids. We showed that FUS induces Hsp70 drates. However, if ATP production were to abruptly stop there is less than 1 min- overproduction in prostate cancer cells and promotes cell apoptosis via an in- ute of high energy phosphate reserve available within the cell. It is therefore crease in expression of Fas and its ligand FasL. Both these factors lead to necessary to regulate mitochondrial production of ATP both robustly and care- phenotypic changes in cancer cells that reduce their aggressiveness. fully, however the mechanisms that govern this process remain controversial. In combination with the availability of matrix adenosine diphosphate, the con- 2645-Pos Board B252 2þ 2þ centration of calcium (Ca ) in the mitochondrial matrix ([Ca ]m) has been pro- Desmosomes are Subject to Mechanical Load posed as a primary signaling mechanism responsible for regulating mitochondrial Sindora Baddam, Paul Arsenovic, Nicole Duggan, Vani Narayanan, ATP production. Virtually every stage of mitochondrial energy production uti- Daniel E. Conway. lizes Ca2þ-sensitive proteins, including those in the tricarboxylic acid cycle, Biomedical Engineering, Virginia Commonwealth University, Richmond, the electron transport chain, and ATP synthesis. Recent animal studies using VA, USA. mice with genetic ablation of the primary cardiac mitochondrial Ca2þ uptake Strong cell-cell junctions, formed by adherens junctions, tight junctions, and des- 2þ 2þ pathway, the mitochondrial Ca uniporter, support a critical role of [Ca ]m mosomes, are critical to the integrity of cellular tissues, including the ability to in regulating mitochondrial ATP production; these animals have impaired car- resist mechanical stress. While it is well established that tensile forces are exerted diac adaptation to increased workload. However, other studies have provided across cell-cell junctions, including actin-connected adherens junctions, it is not conflicting results; some show little to no effect of Ca2þ on ATP production. Ex- known if other components of the cell-cell junction, such as desmosomes, expe- isting measurements are not convincing, largely due to a failure to measure rience mechanical force. Desmosomes are intermediate filament (IF)-connected 2þ [Ca ]m, and thus cannot yet resolve the matter. To this end, we have developed structures found in epithelial and muscle tissues that bind cells together, which 2þ a new approach using calibrated fluorescent measurement of [Ca ]m in isolated consist of a transmembrane desmosomal cadherin (desmocollin or desmoglein) mitochondria combined with real-time Clark type electrode measurement of ox- connected to intermediate filaments by the linker proteins plakoglobin, plakophi- ygen consumption and bioluminescent measurement of ATP production. These lin, and desmoplakin. Desmosome-targeting genetic, autoimmune, and infectious assays have provided quantitative data yielding insights into the regulation of car- diseases present clinically in the skin and heart, two organs subjected to signifi- 2þ diac ATP production by [Ca ]m and energy demand. cant mechanical force. This has led to the hypothesis that a major function of des- mosomes is to resist mechanical stress at cell-cell contacts. To directly measure 2648-Pos Board B255 mechanical forces applied to desmosomes we used an existing FRET-force probe Functional BKCa Channel in Human Resident Cardiac Stem Cells (known as TSmod) to develop a force sensor for the desmosomal cadherin Expressing W8B2 desmoglein-2 (dsg-2), which is expressed in epidermal, epithelial, and cardiac Oualid Ayad1, Aurelien Chatelier1, Christophe Magaud1, Jocelyn Bescond1, muscle cells. The sensor localizes well to desmosomes (verified by electron mi- Stephane Sebille1, Christian Cognard1, Jean Francois Faivre1, Patrick Bois2. croscopy) and retains interactions with both plakoglobin and desmoplakin. We 1Universite´ de Poitiers, Signalisation et Transports Ioniques Membranaires, validated the force responsiveness of the sensor using contracted and relaxed car- Poitiers, France, 2Universite´ de Poitiers/CNRS, Signalisation et Transports diomyocytes, observing that dsg-2 forces are higher in contracted cardiomyo- Ioniques Membranaires, Poitiers, France. cytes. Using the dsg-2 sensor we also observed that desmosomes in epithelial The adult human heart harbors several populations of cardiac/progenitors stem cells (MDCK and A431) are subject to mechanical tension. In addition, changes cells that express specific markers. Recently, a new population of resident

BPJ 7904_7909 Wednesday, February 15, 2017 537a cardiac stem cells was identified. These cells are positive for W8B2 marker pathways. Here we report that intracellular Ca2þ release from the endoplasmic (called also mesenchymal stem cell antigen-1). W8B2 positive cardiac stem reticulum (ER) via the type 1 inositol 1,4,5-trisphosphate receptor (IP3R) is cells (W8B2þ CSCs) exhibit a strong therapeutic potential when transplanted required for Ca2þ-dependent eNOS activation. EC-specific IP3R1 knockout into a chronic myocardial infarction rat model. However, the functional char- (EC IP3R1-/-) mice develop severe hypertension (HTN) by 3 months of age acterization (electrophysiology and calcium signaling) of these cells has not and blunted vasodilation in response to acetylcholine (ACh). eNOS activity been studied yet. We first establish the conditions of isolation and expansion is reduced in both isolated IP3R1 deficient murine EC, and in human EC of W8B2þ CSCs from human heart biopsies by magnetic cell sorting system following IP3R1 knock-down. IP3R1 resides upstream of calcineurin, a followed by flow cytometry cell sorting. These cells have a self-renewal capac- Ca2þ/calmodulin activated serine/threonine protein phosphatase. The calci- ity demonstrated by their ability to form colonies. In addition, our preliminary neurin/NFAT pathway was less active and eNOS levels are decreased in results of RT-qPCR show an induction of transcripts encoding contractile pro- IP3R1 deficient EC. The calcineurin inhibitor cyclosporin A (CsA) reduced teins such as actin and cardiac troponin-T after in vitro differentiation of eNOS activity and vasodilation following ACh stimulation. Our results demon- W8B2þ CSCs. This differentiation is accompanied by the appearance of cyclic strate that IP3R1 provides the Ca2þ necessary for EC-mediated vasorelaxation calcium activity assessed by the calcium protein sensor GCaMP protein. The and the maintenance of normal blood pressure. analysis of calcium activity shows that calcium oscillations profile change dur- ing differentiation. Using patch-clamp in whole cell configuration, we show for 2651-Pos Board B258 D the first time the electrophysiological signing of BKCa channel. In addition, Tissue-Specific Mitochondrial Decoding of Cytoplasmic Ca2 Signals is RT-PCR analysis reveals the presence of KCNMA1 (BKCa) mRNA in Controlled by the Stoichiometry of MICU1/2 and MCU W8B2þ CSCs. Interestingly, BKCa channel inhibition by paxilline decreases Melanie Paillard1, Gyo¨rgy Csorda´s1, Gergo¨ Szanda2,Tunde€ Golena´r1, cell proliferation in a concentration-dependent manner and abolishes calcium Valentina Debattisti1,A´ da´m Barto´k1, Cynthia Moffat1, Erin L. Seifert1, activity after W8B2þ CSCs differentiation. Taken together, our results are Andra´s Sp€at2, Gyo¨rgy Hajno´czky1. consistent with an important role of BKCa channels on cell cycle progression 1MitoCare Center, Pathology, Thomas Jefferson University, Philadelphia, and on calcium activity during stem cell differentiation. PA, USA, 2Department of Physiology, Semmelweis University, Budapest, Hungary. 2649-Pos Board B256 Mitochondrial Ca2þ uptake through the uniporter is central to oxidative meta- Novel Microarchitecture Induces Functional Remodeling of the Calcium bolism and cell signaling. However, the mechanisms underlying its adaptation Signaling Mechanisms in Restructured IPSC-Cardiomyocytes to the tissue-specific differences have remained elusive. Both mitochondrial þ Nicole Silbernagel1, Benjamin Richter2, Mona Jaggy3, Sarah Bertels2, Ca2 uptake kinetics and mRNA level for the pore forming unit of the uni- þ Martin Bastmeyer3, Nina D. Ullrich1. porter (MCU) and the Ca2 sensing regulators (MICUs) are tissue-specific. 1University of Heidelberg, Heidelberg, Germany, 2Cell- and Neurobiology, We have investigated if the mitochondrial Ca2þ uptake differences are deter- Zoological Institute, Karlsruhe Institute of Technology (KIT), Karlsruhe, mined by tissue-specific stoichiometry between MICUs and MCU. In fluoro- Germany, 3Cell- and Neurobiology, Zoological Institute, Institute of metric measurements of mitochondrial Ca2þ uptake, heart mitochondria Functional Interfaces (IFG), Karlsruhe Institute of Technology (KIT), displayed a decreased threshold and lesser cooperativity compared to liver Karlsruhe, Germany. mitochondria. Additionally, NAD(P)H elevation was detectable after exposure New promising therapeutic strategies for regeneration of the diseased heart to moderate [Ca2þ] elevations only in heart mitochondria. Assessment of focus on cardiomyocytes derived from pluripotent stem cells (iPSC-CM). more direct mitochondrial Ca2þ uptake by 45Ca sequestration showed that Despite their cardiogenic properties, a well-known drawback of these cells liver mitochondria has a higher threshold and Ca2þ-dependence for uniporter is their immature structural and functional phenotype in contrast to adult car- activation than heart and skeletal muscle from the same donor mouse. Only diomyocytes. A characteristic feature of iPSC-CMs is the lack of a defined the MICU1 to MCU protein expression ratio showed a complementing pattern microarchitecture, which results in disorganized myofilament arrangement, with the mitochondrial Ca2þ uptake phenotype: lower MICU1 to MCU ratio altered electrophysiological characteristics and desynchronized calcium sig- in heart and skeletal muscle correlated with lower threshold and cooperativity nals. As a consequence, iPSC-CMs exhibit spontaneous contractile activity of the uniporter than in liver. Increasing the MICU1 protein level in HEKs and inefficient force production. The aim of this study was to test the hypoth- cells led to a higher MICU1 amount pulled down by MCU, showing that esis that a change in cell morphology influences the Ca2þ-handling properties the abundance of MICU1 relative to MCU is reflected directly in the associ- of iPSC-CMs leading to an improved functional phenotype comparable to ation between MICU1 and MCU. Overexpression of MICU1 in the heart us- adult cardiomyocytes. Using the method of direct laser writing, 3D-scaffolds ing AAV9-MICU1 tail-vein injection increased the MICU1 to MCU ratio and of different geometries were produced from photolabile polyethylene glycol caused conversion to liver-like mitochondrial Ca2þ uptake phenotype, further for single cell analysis. Cells were seeded in predesigned rectangular or hex- leading to a contractile dysfunction of the heart. In summary, our work iden- agonal shapes and compared with control cells growing without any spatial tifies the MICU1 to MCU ratio as a physiological regulator of the mitochon- 2þ 2þ limitations. Immunocytochemical investigations of the sarcomeric units re- drial Ca uptake, contributing to the tissue-specific decoding of [Ca ]c vealed strong parallel alignment of myofilaments in rectangular scaffolds, oscillations towards differentially regulating oxidative metabolism in each while hexagonal-shaped cells exhibited diffuse myofilament orientation tissue. similar to control cells. Combining the techniques of whole-cell voltage clamp and confocal Ca2þ imaging using fluo-4, we discovered that despite similar 2652-Pos Board B259 L-type Ca2þ current densities (in pA/pF: control 12.250.9, rectangle Interactions between Transmembrane Helices in Orai1 Regulate CRAC 11.751.4, hexagon 10.651.4) the fast Ca2þ-dependent current inactiva- Channel Gating tion (t1) was significantly slower in rectangular cells due to structural cellular Priscilla S.-W. Yeung, Megumi Yamashita, Murali Prakriya. reorganization compared to hexagonal and control cells (in ms: control Department of Pharmacology, Northwestern University, Chicago, IL, USA. þ þ 11.351.1, rectangle 28.657.5, hexagon 16.751.9). Furthermore, sponta- Ca2 release-activated Ca2 (CRAC) channels are a family of plasma þ neous Ca2þ transients in rectangular-shaped cells showed a reduction in the membrane-localized Ca2 channels that are activated in response to depletion þ peak-to-peak interval variation indicating enhanced signaling maturation of ER Ca2 stores, a common occurrence following stimulation of a variety of (mean variance: control 0.2150.07, rectangle 0.1750.12; range: 1.61 vs. cell surface receptors. CRAC channels are assembled from two proteins: þ 1.05, respectively). In conclusion our data demonstrate that structural remod- Orai1, the pore-forming subunit, and STIM1, the ER Ca2 sensor and CRAC eling of iPSC-CMs by distinct cell shapes is paralleled by functional adapta- channel activator. Each channel consists of six Orai1 molecules with the trans- tions resulting in maturation of the Ca2þ handling machinery and contractile membrane (TM) helices arranged in concentric rings around the pore-lining activity. TM1 segments. The current model of channel gating postulates that the Orai1 C-terminus recruits STIM1 to the channel while STIM1 binding to the Orai1 2650-Pos Board B257 N-terminus is exclusively responsible for channel gating. Mutations at the N-ter- Role of the Endothelial Inositol 1,4,5-Trisphosphate Receptor in Blood minus abolish gating, which was attributed to a defect in STIM1 binding at the Pressure Regulation N-terminus. However, several recent studies have suggested a role for the Qi Yuan. Orai1 C-terminus in gating while calling into question the relevance of the Physiology, Columbia University, New York, NY, USA. STIM1 binding site at the N-terminus. The increasingly evident role of the C-ter- Endothelial cells (EC) are critical mediators of blood pressure regulation, pri- minus in channel activation has led us explore a conformational wave that prop- marily via the generation and release of vasorelaxants including nitric oxide. agates from the Orai1 C-terminus through the TMs to open the pore. In order to Nitric oxide (NO) is produced in EC by endothelial nitric oxide synthase locate key interactions between the TMs that are involved in pore opening, we (eNOS) which is activated both by calcium (Ca2þ)-dependent and independent performed a cysteine scan of all four TMs using patch-clamp analysis and

BPJ 7904_7909 538a Wednesday, February 15, 2017 have identified more than twenty gain-of-function mutations. These mutants pro- properties. This will allow us to identify new targets for the development of vide information about specific loci in Orai1 that are crucial in keeping the chan- therapies against diseases with gain of function mutations in ORAI1. nel closed and highlight the potential importance of reversible interactions between the TMs that play a role in Orai1 channel gating. 2655-Pos Board B262 The Role of EHD2 in Orai1-STIM1 Interaction 2653-Pos Board B260 Dora Bodnar. Contribution of Orai1 to Sex-Based Differences in Cardiac Excitation- Molecular Physiology and Therapeutics Branch, NIH, NIDCR, Bethesda, Contraction Coupling MD, USA. þ Fiona Bartoli1, Baptiste Rode2, David J. Beech2, Ana Maria Gomez1, Ca2 is a second messenger whose cytoplasmic concentration is determined by þ þ Jean Pierre Benitah1, Jessica Sabourin1. Ca2 pumps and channels. Ca2 enters non-excitable cells though store oper- 1Faculty of Pharmacy, Inserm UMR-S1180, Chaˆtenay-Malabry, France, ated channels (SOC). The main SOC is Orai1, responsible for the Ca2þ- 2School of Medicine, Leeds Institute of Cardiovascular and Metabolic release-activated (CRAC) current. Orai1 is activated in response to Ca2þ Medicine, Leeds, United Kingdom. release from the ER and is gated by the ER Ca2þ sensor STIM1. Store depletion The Orai1-dependent Ca2þ entry is the major mechanism of Ca2þ entry in non- results in Orai1-STIM1 clustering in ER/PM junctions. However formation of excitable cells but its role in cardiac Ca2þ homeostasis is unclear. Although a junctions and its affect on the Orai1-STIM1 complexes is not well understood. role in cardiac pathological processes such as arrhythmias, hypertrophy and The C-terminal Eps15 homology domain-containing protein 2 (EHD2) is an heart failure is increasingly recognized, the contribution of Orai1 to normal car- ATPase that oligomerizes/localizes to caveolae in the PM inner leaflet and diac function remains elusive. Using a mouse model with cardiac specific associate with PI(4,5)P2. Caveolae regulate membrane turnover, many expression of a dominant negative Orai1R91W mutation (dn-Orai1R91W), we as- signaling processes. EHD2 confines caveolae to the plasma membrane by sessed the Orai1 contribution to cardiac function with special emphasis on sex providing a link to actin filaments. Endogenous EHD2 is enriched in ER/PM differences. junctions positive for endogenous caveolin1. Here we asked whether EHD2 At 2/3-months of age, dn-Orai1R91W Tg mice show normal cardiac function role in the ER/PM junctions affect Orai1-STIM1 clustering and current. We evaluated by echocardiography and ECG telemetry under physiological condi- used combination of techniques such as whole cell patch clamp, calcium imag- tions compared to WT littermates. ing, Co-IP, FRET. When co-expressed with Orai1 and STIM1, EHD2 markedly Using Mn2þ-quenching method, we observed on freshly isolated adult ventric- increases while the knockdown of EHD2 dramatically reduces the Orai1 cur- 2þ 2þ 2þ ular myocytes similar level of store-operated Ca entry activated by Ca rent size. Measurements of [Ca ]i corroborated the current measurements. stores depletion in male and female WT myocytes that is reduced by 50% in The EHD2 enhanced Orai1 current rapidly inactivated, but the effect of dn-Orai1R91W Tg myocytes. Interestingly, immunostaining data illustrate a EHD2 was independent of SARAF, which mediates the slow Ca2þ-dependent T-tubular network of Orai1 in female cardiomyocytes whereas Orai1 pattern inactivation of Orai1. To determine which property of EHD2 is responsible for in male is preferentially at the sarcolemma. the increased current size, different mutants have been used. The K327 mutant, 2þ Analysis of Ca homeostasis using confocal microscopy on Fluo-4 loaded my- which reduces PI(4,5)P2 interaction has no effect; the F322 mutant that elimi- ocytes did not reveal any obvious differences between male WT and dn-Or- nates localization of EHD2 at the ER/PM junctions and I157Q that forces ai1R91W Tg mice, whereas alterations were observed in female. Female WT EHD2 into puncta by increasing its ATPase activity both decreased the current 2þ myocytes have smaller [Ca ]i transients amplitude associated with lower size by acting as dominant negatives. Co-IP results showed strong interaction 2þ 2þ sarcoplasmic reticulum (SR) Ca content and prolonged [Ca ]i transients between EHD2 and STIM1 either in the presence or the absence of Orai1 decay time than male with similar cell shortening. The Ca2þ sparks frequency that is enhanced by store depletion. These findings suggest that EHD2 may is unchanged but sparks amplitude is smaller in female cardiomyocytes, which play an important role in formation of stabilization of the ER/PM junctions is consistent with lower SR Ca2þ content. In comparison, female dn-Orai1R91W in Caveolar sites to modulate the Orai1-STIM1 complexes. 2þ myocytes have increased [Ca ]i transients amplitude associated with larger þ þ SR Ca2 load, faster decay time, increased cell shortening and Ca2 sparks fre- 2656-Pos Board B263 quency compared to female WT cells. Role of Zinc Transporters in Mammalian Heart under Physiological and In conclusion, our findings highlighted the possible involvement of Orai1 in Pathological Conditions cardiac excitation-contraction coupling in sex-based differences. Aysegul Durak1, Yusuf Olgar1, Erkan Tuncay1, Verda C. Bitirim1, Evren Ozcinar2, Mustafa Bahadır Inan2, Kamil Can Akcali1, Semir Ozdemir3, 2654-Pos Board B261 Ahmet Ruchan Akar2, Belma Turan1. Orai1 Mutations with Distinct Channel Gating Defects cause Tubular 1Biophysics, Ankara University Faculty of Medicine, Ankara, Turkey, Aggregate Myopathy 2Cardiovascular Surgery, Ankara University Faculty of Medicine, Ankara, Monica Bulla Didier, Maud Frieden, Nicolas Demaurex. Turkey, 3Biophysics, Akdeniz University Faculty of Medicine, Antalya, Cell Physiology and Metabolism, Faculty of Medicine of Geneva, Geneva, Turkey. Switzerland. Cellular free Zn2þ-changes, including those in either sarco(endo)plasmic retic- Skeletal muscle contraction and differentiation is controlled by cytosolic Ca2þ ulum [S(E)R] or sarcolemma, are primarily coordinated by Zn2þ-transporters. signals relying partly on store-operated Ca2þ entry (SOCE), a Ca2þ homeostat- Both role of Zn2þ-transporters in the heart and association between defective ic mechanism mediated by STIM and ORAI proteins that links the depletion of Zn2þ-homeostasis and cardiovascular dysfunction in mammalians is not known endoplasmic/sarcoplasmic (ER/SR) Ca2þ stores to the activation of membrane yet. Since Zn2þ-homeostasis is regulated through Zn2þ-transporters, any imbal- Ca2þ-permeable channels. Gain of function mutations in STIM1 or ORAI1 ance in Zn2þ-homeostasis via these transporters maybe associated with several cause tubular aggregates myopathy (TAM), a skeletal muscle disorder with chronic diseases. Although it has been mentioned that ZIP8 and ZIP14 mostly muscular pain, weakness and cramping with exercise. Here, we characterize localize in sarcolemma while others including ZIP7, ZnT7 and ZnT8 localize to two new mutations in the ORAI1 gene associated with TAM. Mutation subcellular compartments, however, very little is known about their roles and V107M is located near the channel selectivity filter, and mutation T184M in subcellular localizations in mammalian cardiac cells. In this regard, we first the third transmembrane domain. Clinical features differed between patients examined the expression levels of these transporters in left ventricle of failing carrying the two different mutations, prompting us to test the regulation and human heart. For further confirmation, we used either rat model mimicing heart Ca2þ permeability of the mutated channels. When ectopically expressed in failure via transverse aortic constriction or rat ventricular cell line (H9c2 cells) HEK cells, both mutated channels formed plasma membrane clusters, mimicking heart failure via adriamycin incubation. Protein expression level of conveyed high basal Ca2þ entry and increased SOCE, indicative of constitutive ZIP8 and ZnT7 were decreased while ZIP14, ZIP7 and ZnT8 were increased in activation. Electrophysiological recordings revealed that currents carried by the all cardiac preparations, significantly. Additionally, high intracellular free Zn2þ V107M channel were of larger amplitude, reduced Ca2þ selectivity, and level, high ER-stress marker proteins, GRP78 and Gadd153, high apoptotic sta- increased fast Ca2þ-dependent inactivation; while slow Ca2þ-dependent inac- tus marker such as expression of Bax/Bcl-2 were observed in all failing heart tivation was preserved in both mutants. Preliminary results suggests that the samples. Our confocal imaging examinations for subcellular localizations in T184M mutation alters the sensitivity of the channel to ROS, suggesting that H9c2 cells showed that ZIP8, ZIP14 and ZnT8 were localized on the sarco- this mutation might hinder the accessibility of a reactive nearby cysteine resi- lemme as about over 60% and into the S(E)R as about 40-50% due to calculated due. Molecular dynamics simulations will explore this possibility and test Pearson’s coefficients. Overall data provide an impression on possible contri- whether substitution V107M enlarges the first portion of the channel bution of differential expression of these Zn2þ-transporters as one of the crucial pore, increasing its permeability to ion and displacing the residues responsible signs of failing heart and they may provide important information on their role for Ca2þ selectivity. By characterizing new mutations of ORAI1, we hope for the new therapeutic strategy against heart failure (Supported through Tubi- to improve our understanding of the channel permeation and regulatory tak SBAG-113S466).

BPJ 7904_7909 Wednesday, February 15, 2017 539a

2657-Pos Board B264 physiological and pathological conditions. We used fluorescence imaging Tonic Activation of Extrasynaptic NMDA Receptors Decreases Neuronal to measure mitochondrial membrane potential and intracellular calcium con- 2þ Excitability in Alzheimer’s Disease centration ([Ca ]i) in primary cultures of hippocampal or cortical neurons David Gall1, Antonio Lobo-Antunes2, Genevie`ve Dupont2. (10-15 days in vivo), using Rhodamine123 and Fura-ff respectively. Applica- 1Laboratoire de Physiologie et Pharmacologie (CP604), Universite´ Libre de tion of glutamate (50-100mM) causes a stereotypical response consisting 2 2þ Bruxelles, Bruxelles, Belgium, Unite´ de Chronobiologie The´orique of an initial transient spike in [Ca ]i followed by a secondary increase in 2þ (CP231), Universite´ Libre de Bruxelles, Bruxelles, Belgium. [Ca ]i which is coincident with a delayed mitochondrial collapse. Pre- Amyloids b (Ab) are a hallmark of Alzheimer’s disease. They affect the treatment of the neurons and astrocytes with different length of polyP signifi- communication between neurons. They can also bind to neuronal targets and cantly reduced the number of cells with a secondary delayed calcium deregu- thereby affect both intracellular signalling and neuronal electrical activity. Dur- lation induced by high concentration of glutamate. Moreover, glutamate- ing the onset of Alzheimer’s disease, a positive feedback loop between Ab40/ induced mitochondrial depolarization was also prevented by PolyP addition. 42 and cytosolic calcium is thoughtto accelerate the progression of the disease. Importantly, long chain PolyP successfully protected cells against glutamate If intracellular calcium and Ab reinforce themselves through this mechanism, induced cell death. Thus, polyP protects neurons against glutamate-induced ex- one would expect that the neurons targets of Ab may display an altered electri- citotoxicity by reduction of the calcium overload and restoring mitochondrial cal activity caused by the increase in cytoplasmic calcium as it is known that function. there is a tight coupling between calcium dynamics and the electrical excita- bilty. The aim of this work is to test this assumption when considering one Intercellular Calcium Channels and Calcium of the privileged target of Ab, the activity of extrasynaptic NMDA receptors. Sparks and Waves II Our theoretical model is a simple description of neuronal electrical activity based on the Hodgkin-Huxley like formalism, including a term that corre- 2660-Pos Board B267 sponds to the activity of the NMDA receptor and a cytosolic calcium compart- ATP Release through Hemichannels Increases Ca2D Spark ment. When the tonic activity of extrasynaptic NMDA receptors is increased, Occurrence via P2Y Purinoceptor Signaling in Rat Ventricular Myocytes neurons are less excitable. This is a counterintuive result as NMDA receptors under Shear Stress exert an excitatory effect. Further analysis show that this inhibitory effect is Jun Wang, Joon-Chul Kim, Sun-Hee Woo. due to the activation of calcium-dependent potassium channels, which hyper- College of Pharmacy, Chungnam National University, Daejeon, Korea, polarize the neurons. Activation of extrasynaptic NMDA receptors also pro- Republic of. vokes a marked increase in intracellular calcium concentration, thus Shear stress in ventricles increases under pressure/volume overload caused by reinforcing the feed-forward relation between Ab production and calcium. valve diseases, heart failure, and hypertension and enhances Ca2þ transients in 2658-Pos Board B265 ventricular myocytes. We have previously proposed that the shear-mediated 2þ 2þ Regulation of Axon Growth by Alpha 7 Nicotinic Receptor Calcium Ca transient increase is in part due to sensitization of Ca release sites through Transients at the Growth Cone ROS generation by NADPH oxidase (NOX) and NO synthase (NOS). Here, we 2þ E. Bak, J. Jedrzejewska-Szmek, J. King, K. Blackwell, N. Kabbani. investigated remaining mechanism for the activation of Ca release sites under 2 Krasnow Institute for Advanced Study, George Mason University, Fairfax, shear stress in rat ventricular myocytes. Shear stress of ~16 dyn/cm was applied 2þ VA, USA. onto single cells using micro-jet apparatus. Two-dimensional confocal Ca im- 2þ Cell surface receptors of the growth cone (GC) transmit extracellular information aging was performed at 30 Hz. The frequency of resting Ca sparks was imme- that is essential for synaptogenesis and proper brain wiring. Various external cues diately increased to ~180% and further increased to ~250% by prolonged including neurotransmitter gradients serve to modulate the turning, extension, exposure (20 s). Pretreatment of cells with inositol 1,4,5-trisphosphate receptor and retraction of the GC by targeting intracellular calcium signaling pathways. (IP3R) inhibitor 2-APB (2 mM) or protein kinase C (PKC) inhibitor chelerythrine Both the magnitude of the intracellular calcium rise as well as its source encode (2 mM) partly (about 50%) suppressed both immediate and prolonged shear ef- specific information leading to cytoskeletal remodeling during axon growth or fects on the spark occurrence. Blockade of P2 purinoceptors (30 mM suramin) retraction. We have shown that calcium conducting alpha7 nicotinic acetylcho- almost completely suppressed shear-induced spark enhancements. Inhibition of line receptor (a7nAChR) channels bind Gaq thereby promoting IP3 receptor phospholipase C (PLC) using U73122 (5 mM) significantly suppressed (~80%) 2þ mediated calcium store release during nAChR channel desensitization. Here shear-induced Ca spark increases. Pretreatment of P2Y1 receptor antagonist we examine the role of a7 nAChR-mediated calcium transients in neurite growth MRS2179 (400 nM) diminished immediate and late increases in spark occur- and retraction using a stochastic reaction-diffusion model of calcium gradients, rence during shear stimulation by 60-70%. Inhibition of gap junction hemichan- cAMP, and Gaq pathways within the GC. The model allows for prediction of nels using carbenoxolone (50 mM) or external treatment of ATP metabolizing intracellular calcium dynamics via the entry of calcium through the a7 nAChR, apyrase (2 units/ml) eliminated the stimulatory effects of shear stress on the spark activation of voltage gated channels channels, and the metabotropic signaling occurrence. Consistently, luciferin-luciferase assay revealed ATP release from properties of the a7nAChR on local ER. We show that the identity of the calcium these myocytes by shear stress within 2 s. These results suggest that shear stress 2þ source impacts the dynamics of non-linear interactions between a cohort of cal- may enhance Ca spark occurrence partly via activation of P2Y-PLC-IP3R/PKC cium sensitive effectors such as PP2B and PP1, CaMKII, PKA, and . Our signaling by hemichannel-mediated ATP release in rat ventricular my- model begins to explain experimental observations on neurite growth in cultured ocytes. Possible link between this signaling and NOX/NOS upon shear-mediated PC12 cells and hippocampal neurons suggesting non-monotonic dependence of spark enhancement in ventricular myocytes needs further investigation. structural growth on calcium levels, where both high and low calcium can inhibit growth. Elucidating the mechanisms of calcium signaling within the GC yields a 2661-Pos Board B268 better understanding of synaptic growth and plasticity, as well as an opportunity Structural Dynamics of Calmodulin in Regulation of Calcium Release in for fostering regeneration. Health and Disease Megan R. McCarthy, Robyn T. Rebeck, Razvan L. Cornea, 2659-Pos Board B266 David D. Thomas. Inorganic Polyphosphate Protects Neurons against Glutamate-Induced Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Excitotoxicity Minneapolis, MN, USA. Marta Maiolino1, Vincenzo Lariccia2, Salvatore Amoroso2, We have used time-resolved fluorescence (FRET) and EPR (DEER) spectros- Plamena R. Angelova1, Andrey Y. Abramov1. copies to study the structural changes in calmodulin (CaM) that are relevant 1Molecular Neuroscience, UCL Institute of Neurology, London, United to regulation of the muscle calcium release channel, the ryanodine receptor Kingdom, 2Biomedical Science, UNIVP Universita’ Politecnica delle (RyR). Regulation of RyR by CaM is disrupted by oxidation and disease- Marche, Ancona, Italy. causing mutations. However, the structural basis for these regulatory changes, Glutamate excitotoxicity is responsible for neuronal death in acute neurological and the role CaM plays in the development of heart failure and arrhythmias, disorders including stroke, trauma, and neurodegenerative diseases. Loss of is not well understood. Several studies suggest that the modulatory role of calcium homeostasis and mitochondrial dysfunction are the key mediators of CaM is closely tied to its conformation when bound to RyR, but the correlation glutamate induced cell death. Recently, we found that inorganic polyphosphate between structure and function in physiologically relevant conditions is largely (Poly P) can act as a calcium-dependent gliotransmitter mediating communica- unknown. To test the hypothesis that the modulatory action of CaM on RyR is tion between astrocytes, while its role in regulation of neuronal activity remains caused by structural changes in the CaM-RyR complex, we use site-directed still undefined. Considering the number of studies which demonstrate the close spectroscopy to determine the structural changes that contribute to calcium interaction between neurons and glia in physiology and pathology, we studied regulation in skeletal and cardiac muscle. The approach is to prepare CaM mu- the effect of the polyP on glutamate induced calcium signal in neurons, in tants that contain a single Cys on each of the two lobes (N and C), then attach

BPJ 7904_7909 540a Wednesday, February 15, 2017 spectroscopic probes to those sites. Ryanodine binding measurements are per- pipette þ 30-min pre-incubation of the cells in AIP2 prior to the experiment) formed to ensure functional integrity of labeled CaM constructs. Then, we mea- also abolished the effect of flecainide (6 mM, both in patch pipette and extracel- sure changes in intra-CaM distance distributions using time-resolved FRET or lular solution) on spontaneous Ca waves in voltage-clamp experiments DEER. Our previous studies employed DEER of isolated CaM. Now, we rely (vehicle: 6.651.2 waves/45s.; flecainide: 2.150.4 waves/45s.; flecainideþ on time-resolved FRET, to resolve structural changes of CaM bound to AIP2: 8.151.9 waves/45s.; p<0.05 for veh. vs. flec. and flec.þAIP2 vs. functional RyR in sarcoplasmic reticulum membranes. We will compare these flec.). Importantly, in the absence of flecainide, AIP2 had no significant effect measurements with those of CaM bound to a peptide corresponding to the on the frequency of Ca waves compared to vehicle group (5.051.7 waves/45s. CaM-binding domain of RyR (RyR1 residues: 3614-3643). This work was sup- vs. 6.651.2 waves/45s., respectively). Our data identifies CaMKII activity as a ported by NIH grants AG26160 and HL092097 (to DDT) and a Predoctoral critical determinant for inhibitory effect of flecainide on arrhythmogenic Ca Fellowship from the American Heart Association 15PRE25700131 (to MRM). waves in Casq2 -/- ventricular myocytes. 2662-Pos Board B269 2664-Pos Board B271 2D 2D Effects of Expression Levels of WT and Mutant RyR2 on Ca Cardiac Specific IP3R Over-Expression: IP3ICR Contribution in Ca Homeostasis in HEK Cells Signaling Nagomi Kurebayashi1, Takashi Murayama1, Naoyuki Tetsuo1, Joaquim Blanch i Salvador, Marcel Wullschleger, Marcel Egger. Ryosaku Ohta2, Fumiyoshi Yamashita2, Takashi Sakurai1. Physiology, University of Bern, Bern, Switzerland. 1 Department of Pharmacology, Juntendo University School of Medicine, Inositol-1,4,5-trisphosphate (IP3) is a second messenger produced upon agonist Tokyo, Japan, 2Department of Drug Delivery Research, Kyoto University, binding to a G-protein coupled receptor (GPCR) and subsequently triggers SR- 2þ Kyoto, Japan. Ca release through openings of IP3 receptors (IP3Rs). In cardiac muscle, IP3R 2þ Background: Type 2 ryanodine receptor (RyR2) is the Ca release channel on type 2 (IP3R2) is the predominant isoform expressed both in ventricle and atrial sarcoplasmic reticulum and plays a pivotal role in cardiac E-C coupling. The tissue. Several studies have focused on the functional interaction between mutations in RyR2 have been implicated in various arrhythmogenic disorders ryanodine receptors (RyRs) and IP3Rs in atrial myocytes. However, it is still 2þ including catecholaminergic polymorphic ventricular tachycardia (CPVT), unclear how IP3-induced Ca release (IP3ICR) may contribute to idiopathic ventricular fibrillation (IVF) and long QT syndrome (LQTS). We excitation-contraction coupling in ventricle. Evidence suggests that IP3ICR þ have characterized their Ca2 release properties using a heterologous modulates the RyR function by affecting its local Ca2þ environment. Under HEK293 expression system and found that CPVT mutants are all gain-of- pathophysiological cellular remodeling conditions (e.g. atrial fibrillation and 2þ function (GOF) mutations whereas IVF and LQTS mutants include two groups, heart failure) a functional interplay of IP3R and RyR Ca events may be GOF and loss-of-function (LOF) types. Among them, the GOF mutations significantly pronounced. Our aim in this study was to examine this interaction 2þ 2þ exhibit reduced ER Ca level, depending on the Ca release activity. How- in a cardiac-specific IP3R2 overexpressing (TG) mouse model. Our experi- 2þ ever, ER Ca level of CPVT patients may not be so reduced because neither mental approach includes: characterization of the IP3 signaling pathway by structural abnormality nor contractile dysfunction is detected in them. This sug- various pharmacological interventions; electrophysiology under whole-cell gest that expression level of RyR2 in HEK cells may be very high and reduce configuration of the patch clamp technique in combination with rapid confocal þ ER Ca2 level. In this study we examined effects of expression level of RyR2 Ca2þ imaging and complemented with molecular biology approaches (RT- þ on Ca2 homeostasis in HEK293 cells. PCR, Western Blot, immunostaining). Western blot results show an increase Methods: Expression of WT and mutant RyR2s were induced by doxycycline. in IP3R2 protein expression in the TG model both in atria and ventricular tissue 2þ 2þ 2þ 2þ Cytoplasmic Ca ([Ca ]cyt) and ER Ca ([Ca ]ER) were determined with compared to its wild-type littermate (FVB). However, a similar distribution on 2þ 2þ fluo-4 and R-CEPIA1er, respectively. Ca induced Ca release (CICR) activ- the junctional SR was found, where IP3Rs co-localize with RyRs. Supporting ity of WT and mutant RyR2s was determined by [3H]ryanodine binding assay. the protein data, preliminary results show an increase in basal spark frequency Results: WT and mutant RyR2 protein similarly increased with time after in- (SpF) in TG atrial and ventricular myocytes compared to FVB. Upon IP3 stim- duction and reached steady state at ~24 h. WT and GOF mutants showed spon- ulation FVB atrial and ventricular cardiomyocytes presented an increase in 2þ 2þ taneous periodic increase in [Ca ]cyt (Ca oscillation) and corresponding SpF. Furthermore, a larger increment was seen in TG atrial myocytes. In order 2þ 2þ 2þ decrease in [Ca ]ER, and the oscillation frequency was higher in GOF mutants to examine the contribution of the SR-Ca leak mediated by IP3R2 a SR-Ca 2þ 2þ than in WT. The upper level of the [Ca ]ER (threshold [Ca ]ER) gradually leak protocol was established. However, even under control conditions a signif- decreased with time after induction and reached steady state at 24 h. There icant contribution of IP3ICR on the SR-Ca2þ leak was found. We conclude that 2þ 2þ was a very good correlation between threshold [Ca ]ER at 24 h and the overexpression of IP3R2 in the TG model affects Ca handling significantly CICR activity. On the other hand, LOF mutants showed no oscillations with by functional interplay with the RyR2. 2þ constant [Ca ]ER at any time after induction. At 2-4 h after induction, 2þ threshold [Ca ]ER was similar between WT, GOF and LOF mutants, whereas 2665-Pos Board B272 only GOF, but not WT or LOF mutants, showed Ca2þ oscillations. Dynamical Interactions of Early Afterdepolarizations with Stretch Acti- 2þ Discussion: Our results indicate that [Ca ]ER in HEK expression system is vated Channels determined by both CICR activity and expression level of RyR2, and that the Dexter K. Luu, Daisuke Sato, Yuanfang Xie. 2þ [Ca ]ER at ~24 h is a sensitive indicator of CICR activity of RyR2 mutants. University of California, Davis, Davis, CA, USA. Implication of LOF mutants in Ca2þ homeostasis will be also discussed based Cardiac arrhythmia is often triggered by premature ventricular contractions on the expression level. (PVCs), which have been linked to early and delayed afterdepolarizations (EADs / DADs). The cardiac action potential controls mechanical activity of 2663-Pos Board B270 the heart via excitation-contraction coupling. Conversely, mechanical contrac- Effective Suppression of Arrhythmogenic Ca Waves by Flecainide in tion also affects the action potential via stretch-activated channels (SACs). This Ventricular Myocytes from Casq2 -/- Mice Depends on CaMKII Activity is called mechano-electric feedback. However, the role of mechano-electric Dmytro Kryshtal, Nieves Gomez-Hurtado, Bjorn Knollmann. feedback in the formation of afterdepolarizations is not well-understood. In Department of Medicine, Vanderbilt University Medical Center, Nashville, this study, we investigate how activation of SACs promotes or suppress TN, USA. EADs using a physiologically detailed computational model of a rabbit ventric- We reported previously that flecainide effectively suppresses spontaneous Ca ular myocyte. The SAC current (ISAC) is modelled based on Galice et al (BJ waves in both membrane-permeabilized and freshly isolated intact ventricular 2016). We found that depending on the reversal potential of ISAC, the maximum myocytes from Casq2-/- mice. We also provided strong evidences that this conductance of ISAC, and the amount of stretch, ISAC can promote or suppress effect is due to the interaction between flecainide and RyR2 and is unrelated EADs. Mathematical analysis reveals the dynamical origin of these EADs. to flecainide’s block of Na channels. Moreover, this wave-suppressing effect These results may shed light on the role of SACs on cardiac arrhythmias. is dependent on the state of RyR2 (i.e. flecainide is much more potent in Casq2 -/- myocytes, where RyR2 activity is elevated comparing to WT). 2666-Pos Board B273 Here we demonstrate that the effect of flecainide on Ca waves is determined Modelling Calcium-Induced-Calcium-Release from Measurements of RyR by the activity of CaMKII. In membrane-permeabilized Casq2 -/- ventricular Gating myocytes, the IC50 for flecainide (20-25 min. incubation) was reduced form Derek R. Laver1, Cherrie H. Kong2, Mark B. Cannell2. 6 mMto1mM by the presence of 100 nM free CaM. Application of selective 1Biomedical Sciences, University of Newcastle, Callaghan, Australia, CaMKII blockers AIP2 (1 mM) or KN-93 (10 mM) abolished the suppressive 2Department of Physiology, Pharma cology and Neuroscience, University of effect of flecainide (10 mM) on the incidence, frequency and amplitude of spon- Bristol, Bristol, United Kingdom. taneous Ca waves both in the presence and absence of 100 nM free CaM. In Many cells depend on ‘calcium-induced calcium release’ (CICR), an inherently freshly-isolated intact cardiomyocytes, application of AIP2 (1 mM in patch regenerative process due to the Ca2þ-dependent gating and Ca2þ conduction of

BPJ 7904_7909 Wednesday, February 15, 2017 541a ryanodine receptors (RyRs) in the sarco-endoplasmic reticulum (SER). The sta- scale modeling approach that spans from single channel to whole-cell and spatial bility of Ca2þ release in cardiac muscle, in spite of inherent regeneration within simulations, we show that both CICR and SOICR gating modes can indeed acti- the CICR mechanism, arises from the local control of clusters of RyRs in the junc- vate RyR2 channels and modify Ca2þ spark dynamics in a manner consistent tions between the SER and surface membrane. Using a 3D spatial model of adja- with experimental observations. However, detailed comparison of Ca2þ wave cent pairs of release sites, we examine the factors determining ability of one site to generation and CRU-to-CRU Ca2þ wave propagation shows that CICR alone trigger Ca2þ release at an adjacent site, a process crucial to our understanding of is a sufficient, and necessary, mechanism to explain Ca2þ release in heart under propagating Ca2þ waves and cardiac arrhythmias. Importantly, the model incor- both physiological and pathological conditions. porated our measurements of RyR Ca2þ conductance and sensitivity to cyto- plasmic [Ca2þ], recorded in artificial lipid bilayers using near-physiological 2669-Pos Board B276 conditions (Kþ 100 mM, Ca2þ 100 nM-100 mM, Mg2þ 1 mM and ATP 2 mM). Mechanism of Dysfunction in a RyR2R420Q Mouse Model In the presence of 0.9 mM luminal Ca2þ, the RyR unitary Ca2þ current at Ofcatecholaminergic Polymorphic Ventricular Tachycardia 5 2þ Yue-Yi Wang1, Pietro Mesirca2, Elena Marque´s-Sule´3, 0 mV was 0.47 0.04 pA. RyR opening rate, k, had a hyperbolic [Ca ]-depen- 4 1 5 m Alexandra Zahradnikova Jr , Olivier Villejoubert , Pilar d’Ocon , dence with Kd=35 M, H = 2.7 and kmax = 900/s. We found that the triggering of 6 6 6 2 neighboring sites was extremely dependent on the distance (0.6 to 1.0 mm) be- Cristina Ruiz , Diana Domingo , Esther Zorio , Matteo E. Mangoni , nd 2þ Jean-Pierre Benitah1, Ana Maria Gomez1. tween RyR clusters and increased with the 2 power of SER luminal [Ca ] 1 (1-2 mM) but the number of RyRs in the triggering cluster had only minor effects. Faculte de Pharmacie, Inserm UMR-S1180, Chaˆtenay-Malabry, France, 2CNRS UMR-5203, Montpellier, France, 3Physiotherapy Department, 2667-Pos Board B274 University of Valencia, Valencia, Spain, 4Slovak Academy of Sciences, Modulation of Ca2D Signaling in HL-1 Cardiomyocytes by Arrhythmo- Institute of Molecular Physiology and Genetics, Bratislava, Slovakia, genic RyR2 Mutants 5Pharmacy School, University of Valencia, Valencia, Spain, 6Hospital Naoyuki Tetsuo, Nagomi Kurebayashi, Takashi Murayama, Takashi Sakurai. Universitario y Polite´cnico La Fe, Valencia, Spain. Juntendo University, Tokyo, Japan. Sinoatrial node (SAN) is the primary cardiac pacemaker. Ca2þ release via the 2þ Background and purpose: Type 2 ryanodine receptor (RyR2) is a Ca release ryanodine receptor (RyR2) plays a critical role in maintaining automaticity. channel on the sarcoplasmic reticulum and plays a pivotal role in excitation- RyR2 mutations lead to catecholaminergic polymorphic ventricular tachycardia contraction coupling in heart. RyR2 is the major target for arrhythmogenic dis- (CPVT), which is a lethal inherited disease characterized by exercise/stress- eases such as catecholaminergic polymorphic ventricular tachycardia (CPVT), induced syncope and/or sudden death. CPVT patients frequently bear SAN idiopathic ventricular fibrillation (IVF), long QT syndrome (LQTS), etc. We dysfunction. In order to find out its underling mechanisms, we created a KI have characterized various types of arrhythmogenic RyR2 mutations using mice model carrying a mutation in the N-terminal portion of the RyR2 found HEK cell expression system and foundthat some of IVF and LQTS mutants reveal in a CPVT family, RyR2 (R420Q). All KI mice showed sustained bidirectional loss-of-function phenotype whereas others are gain-of-function mutants. In this ventricular tachycardia and other arrhythmias after pharmacologic (epinephri- study, we aimed to compare the effects of loss-of-function and gain-of -function neþcaffeine) or emotional (hair dryer) stress, validating the model, and a RyR2 mutants on Ca2þ signaling in cardiac intracellular milieu of HL-1 cells, slower resting heart rhythm was denoted in KI female mice during day time, mouse cardiac cell line. Methods: Wild type (WT), 4 loss-of-function type and underlying alteration in SAN function. For further investigation, SAN tissues 2 gain-of-function type mutant RyR2s were expressed in HL-1 cells using bacu- were dissected from WT and KI mice and Ca2þ movements followed by 2þ 2þ lovirus system and intracellular Ca signals were monitored with Cal520. confocal microscopy. The cycle length of spontaneous [Ca ]i transients was 2þ Results and discussion: Under semiconfluent condition, most of HL-1 cells significantly longer in KI, and [Ca ]i transient amplitude was significantly did not show spontaneous beating. Expression of WT and gain-of-function shortened. Funny current (If) in isolated SAN cells are not altered but L-type 2þ 2þ 2þ mutants in such cells showed frequent Ca waves whereas loss-of-function mu- Ca current (ICa,L) was reduced in absence of intracellular Ca buffer. In tants exhibited no Ca2þ waves. On the other hand, cells forming a cluster under KI mice compared to WT littermates, Ca2þ sparks frequency between sponta- confluent condition showed spontaneous, highly rhythmic, and synchronized neous beats and the Ca2þ spark mass was augmented pointed out an increase in Ca2þ transients caused by automatic activity. Expression of gain-of-function mu- diastolic Ca2þ leak. In summary, our data show that KI mutation: 1) induces 2þ tants in the beating cells caused frequent Ca waves during spontaneous beating, CPVT phenotype in mice, 2) decreases the SAN rhythm without affecting If 2þ 2þ 2þ while loss-of-function mutants reduced amplitudes of Ca transients. Mecha- and, 3) has more [Ca ]i leak during diastole as Ca sparks. The latter may 2þ R420Q nism of perturbation of Ca signals in HL-1 cells will be discussed based on account for ICa inactivation. In conclusion, RyR2 N-terminal mutation 2þ the distribution of endogenous and exogenous RyR2s. causes [Ca ]i leak during diastole and leads to the SAN dysfunction. 2668-Pos Board B275 2670-Pos Board B277 How Does Calcium Overload Generate Calcium Waves in Heart? Ablation of the RyR2-Ser2030 Phosphorylation Site Limits Changes in George S.B. Williams1, Andrew P. Wescott1, Stephan E. Lehnart2, RyR2 Sensitivity during ß-Adrenergic Stimulation W.J. Lederer1. Duilio M. Potenza1, Radoslav Janicek1, Miguel Fernandez-Tenorio1, 1University of Maryland, Baltimore, Baltimore, MD, USA, 2University Hector H. Valdivia2, Ernst Niggli1. Medical Center Go¨ttingen, Go¨ttingen, Germany. 1Department of Physiology, Bern, Switzerland, 2Center for Arrhythmia Calcium-induced calcium (Ca2þ) release (CICR) is the fundamental signaling Research, Department of Internal Medicine, Ann Arbor, MI, USA. mechanism that enables the voltage-sensitive L-type Ca2þ channels (LCCs) in During physical exercise or stress, the sympathetic system stimulates cardiac 2þ the sarcolemma to activate the cellular [Ca ]i transient and thus contraction contractility via b-adrenergic receptor (AR) activation, resulting in protein ki- in the heart. In CICR the LCC Ca2þ influx activates nearby Ca2þ release channels nase A (PKA)-mediated phosphorylation of the cardiac ryanodine receptor, (ryanodine receptors, RyR2s) across a 15 nm ‘‘subspace’’ gap via CICR, just as RyR2. Hyperphosphorylation by PKA at the RyR2-Ser2808 site has been pro- the Ca2þ released from any open RyR2 can activate other RyR2s located within posed as a key mechanism responsible for cardiac dysfunction in heart failure the same RyR2 cluster. The junctional ensemble of LCCs and the opposing (HF). However, the sites of PKA phosphorylation in RyR2 and their phosphor- RyR2s constitute the cardiac ‘‘Ca2þ release unit’’ or CRU. When a CRU is acti- ylation status in HF are not well defined. Recently, a new PKA phosphorylation vated by any means it produces a Ca2þ spark. Excitation-contraction (EC) site has been identified and proposed as a mediator of the adrenergic response, coupling is the process by which CRUs are activated quasi-synchronously by Ser2030. We examined the contribution of RyR2-Ser2030 to the excitation the voltage-activated LCCs in a cell during the action potential (AP). Impor- contraction (EC)-coupling mechanism using experimental approaches on tantly, CICR also occurs independently when the probabilistic opening of cellular and subcellular levels and a transgenic mouse with ablated RyR2- RyR2s produce a Ca2þ spark in the absence of an LCC trigger. Ca2þ sparks S2030 phosphorylation site (RyR2-S2030A). EC-coupling gain was assessed and individual RyR2 openings constitute the endogenous Ca2þ leak pathway with the whole-cell patch-clamp technique and confocal Ca2þ imaging while from the sarcoplasmic reticulum (SR), the ‘‘cellular Ca2þ store’’. However, in the ß-ARs were stimulated with Isoprotenerol (Iso). At matched Ca2þ loading pathological conditions the Ca2þ leak can become unstable and produce cell- of the sarcoplasmic reticulum (SR), the EC-coupling gain in Iso was dimin- wide Ca2þ waves and life-threatening Ca2þ-dependent arrhythmias. During SR ished in mutant compared to WT cardiomyocytes. In addition, Ca2þ waves eli- Ca2þ overload, it has been suggested that a putatively novel phenomena dubbed cited by SR Ca2þ overloading showed no acceleration during Iso treatment, ‘‘Store Overload Induced Ca2þ Release’’ or ‘‘SOICR’’, that is distinct from unlike WT cells. Recent studies have suggested that refractoriness of Ca2þ CICR, may develop. SOICR’s distinctiveness hinges on the molecular ability spark triggering depends on the sensitivity of the ryanodine receptors. More- of SR lumenal Ca2þ to directly activate RyR2 openings without the need for over, it was demonstrated that adrenergic stimulation shortens RyRs refracto- elevated Ca2þ in the subspace to produce CICR. To investigate this hypothesis, riness. We measured Ca2þ spark restitution using the ‘‘low-dose ryanodine we extend our existing model of RyR2 gating to include a formulation for SOICR method’’ in which repetitive sparks originating from the same cluster of whereby SR Ca2þ can directly activate a RyR2 channel to open. Using a multi- RyRs are recorded. We found that mutant RyRs sensitivity was not enhanced

BPJ 7904_7909 542a Wednesday, February 15, 2017 by Iso application, contrary to WT cells. Together, our results suggest that abla- Voltage gated ion channels are integral membrane proteins responsible for tion of the RyR2-S2030 site may result in a blunted increase of RyR2 Ca2þ the propagation of electric signals in excitable cells such as cardiac myo- sensitivity upon ß-adrenergic stimulation, and that the site represents a link be- cytes. The voltage gated , Kv11.1, encoded by the human tween the adrenergic pathway and modulation of RyR2 channel activity. ether-a-go-go related gene (hERG), mediates the rapid repolarization phase of the cardiac action potential. Inherited mutations in this gene, as well as 2671-Pos Board B278 channel interactions with various organic molecules, including pharmaceuti- Molecular Cloning and Expression of cDNA Encoding the Ryanodine Re- cals, are associated with long QT syndrome (LQTS), a standard clinical in- Rattus Norvegicus ceptor Type 2 from Cerebral Artery Smooth Muscle dicator for increased risk of ventricular arrhythmias and sudden cardiac 1 1 2 Jianxi Liu , Guruprasad Kuntamallappanavar , Venkatasushma Kalava , death. In the absence of published experimentally resolved structures of Alex Dopico1. 1 hERG channel, we used ROSETTA structural modeling software and a Pharmacology, The University of Tennessee Hlth. Sci. Ctr., Memphis, TN, recent cryo-EM structure of a homologous EAG1 channel as a template to USA, 2Christian Brothers University, Memphis, TN, USA. 2þ generate a closed-state hERG channel model. Furthermore, open conducting Ryanodine receptors (RyRs) are a family of Ca release channels found in intra- and open inactivated models of hERG were modeled using ROSETTA and cellular organelles. RyR channels are ubiquitously expressed in many cell types the crystal structures of KvAP, Kv1.2, Kv1.2-Kv2.1 chimera and KcsA chan- and participate in a wide variety of physiological processes (e.g., neurotransmis- nels as templates, and incorporating structural constraints from mutagenesis sion, secretion, regulation of myogenic tone, cardiac contractility). We have 2þ studies. Model stability was assessed via microsecond-long all-atom molec- cloned and sequenced cDNA encoding the Ca release channel isoform 2 of ular dynamics (MD) simulations of the channels in an explicitly hydrated the ryanodine receptor (RyR2) from smooth muscle cells of Rattus norvegicus ce- lipid membrane environment. Additionally, hERG modulation by lipid mem- rebral arteries and determined RyR2 protein expression. The RyR2 cDNA is brane composition was probed by MD simulations of the channel models in 14,850 bp in length, resulting in a protein product of 4,950 amino acids with a lipid membranes with varied compositions. In particular, omega-3 polyunsat- theoretical pI/MW of 5.81/561,699.41 Da. We used total RNA from endothelium urated fatty acid, docosahexaenoic acid (DHA), is known to possess anti- free cerebral arteries and conducted reverse transcription. We divided the RyR2 arrhythmic properties, exhibiting time-, voltage- and use-dependent blockade cDNA in two equal parts because of its huge size; N-terminal part: 1-7,486 bp and of hERG, preferentially binding to the open state of the channel. Conversely, C-terminal part: 7,487-14,862 bp. We used gene specific primers to get both N some pharmaceuticals block hERG in a state-dependent manner, and exhibit and C-terminal parts of the cDNA sequence. Primers for the N-terminal part: for- pro-arrhythmogenic properties. These interactions were probed by MD sim- ward 5’ - CG ACG CGT ATG GCT GAT GCG GGC GAA G - 3’; reverse 5’ - GA ulations as well. This study will help elucidate such molecular mechanisms CGC GTC GAC CAG CCG TGT CTA GAG AG - 3’; for C-terminal part: for- of hERG modulation potentially leading to decreased risks of LQTS and ward 5’ - GGA CGC GTC GAC CAC TAA GTG CTA CAG AC - 3’; reverse deadly arrhythmias. 5’ - GAT AAG AAT GCG GCC GCT TAA TTT AAC TGG TCC - 3’. After clon- ing the cDNAs into the mammalian vector pIRESneo, we characterized the 2674-Pos Board B281 clones by restriction analysis and confirmed the resulting cDNA sequence by Effect of Membrane Composition on Ion Conduction in a Voltage-Gated automated analysis. Using immunohistochemistry, Western blotting and surface Potassium Channel biotinylation assays, we evaluated the cytosolic and membrane surface expres- Niklaus B. Johner, Simon Berneche. sion of RyR2 in HEK cells. Our results demonstrate that RyR2 protein effectively Biozentrum, Basel Universit€at, Basel, Switzerland. expresses in both plasma and internal membranes. Our study demonstrates pro- Potassium channels constitute a super-family of membrane proteins playing a tein expression from the newly cloned RyR2 from cerebral artery smooth muscle. key role in the function of neurons. Of particular importance are the voltage- Functional characterization of this RyR2 is underway. Support: HL104631, gated potassium channels (Kv), of which there are around 40 sub-types. These R37AA11560 (AMD). channels have been studied extensively for several decades and great ad- vances in the understanding of the mechanisms governing conduction and 2672-Pos Board B279 gating at the atomic scale have been made since the first crystal structure MCU and EMRE Binding is Mediated through Intermembrane Helix- of a potassium channel was solved almost 20 years ago. Nevertheless, as mo- Helix Interactions lecular dynamics (MD) simulations have suggested that the conformational Charles Phillips. states captured in the crystal structures of voltage-gated potassium channels Brandeis University, Waltham, MA, USA. sustained permeation rates much lower compared to experimental measure- Mitochondrial calcium uptake is critical for physiological processes such as ments, the details of the ion conduction mechanism and whether the crystal calcium signaling, energy production, and apoptosis, and is mediated by a cal- structures truly represent the conducting state are still debated. We are using cium activated calcium channel known as the mitochondrial calcium uniporter. MD simulations to study the impact of membrane composition and voltage on The uniporter is a protein complex, composed of the pore-forming MCU pro- the conformation of a Kv channel and its ion conductivity. Our results notably tein, and the important regulatory proteins EMRE, MICU1, and MICU2. It has suggest that membrane thickness affects the function of the channel. These been well established that EMRE, a small 10kDa single-pass transmembrane simulations illustrate how the microenvironment can impact the function of (TM) protein, is absolutely required for MCU activity. However, little is known ion channels. about how it interacts with MCU. Here, using a Tryptophan scanning assay, we identify key residues absolutely required for MCU-EMRE interaction and 2675-Pos Board B282 MCU activation by EMRE. Specifically, we show that EMRE contains a D GXXXG in the TM helix to pack against the TM1 of MCU. Residue swapping K Occupancy in the Cavity Determines the Ion Permeation Rate through the KV1.2 Channel along with co-immunoprecipitation experiments establish that EMRE and Takashi Sumikama, Shigetoshi Oiki. MCU directly bind each other without the need of a bridging protein. This work is an important first step toward understanding how EMRE regulates Faculty of Medical Sciences, University of Fukui, Eiheiji-cho, Yoshida-gun, MCU, and helps explain why animals have evolved a requirement for EMRE Fukui, Japan. to facilitate mitochondrial calcium uptake. The ion permeation mechanism through potassium channels has been examined extensively through experimental and simulation studies. Molecular dynamics (MD) simulations have demonstrated that rapidly permeating ions collide near Voltage-gated K Channels and Mechanisms of the selectivity filter (SF) (the ‘‘knock-on’’ mechanism), but this oversimplified Voltage Sensing and Gating IV view is insufficient to account for the experimentally observed single-channel current amplitudes. Here, we examined the MD-simulated ion trajectories 2673-Pos Board B280 through the Kv1.2 potassium channel by developing an event-oriented analysis. State-Dependent Structural Modeling and Atomistic Simulations of the In the potassium channels, a cavity stands between the intracellular bulk solu- hERG Potassium Channel tion and the SF. The analysis showed that two ions in the SF were immobilized Kevin R. DeMarco1, Phuong T. Nguyen1, Toby W. Allen2,3, when an ion was empty in the cavity. We found surprisingly that the queueing Vladimir Yarov-Yarovoy4, Colleen E. Clancy5, Igor Vorobyov5. ions in the SF became mobile and initiated outward motion when an ion entered 1Biophysics Graduate Group, University of California Davis, Davis, CA, the water-filled cavity. This cavity ion subsequently filled the space left in the USA, 2School of Applied Sciences, RMIT University, Melbourne, Australia, SF and the cavity is ion-empty again. After an expulsion of the outermost ion in 3Department of Chemistry, University of California Davis, Davis, CA, USA, the SF and a following short relaxation, the ions in the SF became immobilized. 4Department of Physiology and Membrane Biology, University of California Accordingly, outward ion movements were not continuous but exhibited alter- Davis, Davis, CA, USA, 5Department of Pharmacology, University of nating mobile and immobile (or queueing) phases, and the permeation process California Davis, Davis, CA, USA. can be described as a cyclic phase diagram having two phases. The period spent

BPJ 7904_7909 Wednesday, February 15, 2017 543a in the immobile (or queueing) phase was longest, indicating that the period de- 2678-Pos Board B285 termines the ion permeation rate or the single-channel current amplitude. The Quantum Calculations of Large Segments of a Voltage Sensing Domain of concentration dependency of the period was also investigated. We found that a Voltage Gated Channel the period having an ion in the cavity is independent of the concentration, Alisher M. Kariev, Michael E. Green. but the queueing period is inversely proportional to the concentration. Thus, Chemistry, City College of the City University of New York, New York, NY, the ion-bearing state in the cavity serves as a catalytic intermediate in the USA. Michaelis-Menten-type kinetics, accounting for the current-concentration Several large scale calculations of sections of the voltage sensing domain relationship. (VSD) of the Kv1.2 channel (pdb: 3Lut), including structure optimizations at HF/6-31G* level, were done with applied electric fields of both polarities, or 2676-Pos Board B283 without field. Atoms at the ends of the S1, S2, and S3 segments were frozen. On Resolution of the Selectivity/Conductivity Paradox for the Potassium With the S4 segment free to move, the backbone displacement of S4 relative Ion Channel to the S1,S2,S3 block remained below approximately 2 A˚ in calculations that Dmitry G. Luchinsky1,2, Will A.T. Gibby1, Igor Kh Kaufman1, included the side chains. Thus gating current would not come from the motion Dogan A. Timucin3, Peter V.E. McClintock1. of S4 perpendicular to the membrane surface. Optimizations with protons in 1Physics, Lancaster University, Lancaster, United Kingdom, 2SGT Inc., several possible positions are being carried out, and a possible response of pro- Greenbelt, MD, USA, 3Intelligent Systems Division, NASA Ames Research tons to field, in which they move through exchange of partners, is suggested by Center, Moffett Field, CA, USA. the energy of the systems in which the protons shift, e.g., from arginine to gluta- þ The ability of the potassium channel to conduct K at almost the rate of free mate. This leaves an unionized salt bridge with energy less than that of the þ diffusion, while discriminating strongly against the (smaller) Na ion, is of ionized salt bridge (see accompanying Kariev/Green abstract). Proton shifts enormous biological importance [1]. Yet its function remains at the center of are required: e.g., calculated open state, salt bridge R300 nitrogen to E226 ox- a ‘‘many-voiced debate’’ [2,3]. In this presentation, a first-principles explana- ygen distance, with Y266 very close, do not agree with the X-ray structure with tion is provided for the seemingly paradoxical coexistence of high conductivity the salt bridge ionized. Second, the E183-R297 salt bridge distance corresponds with high selectivity between monovalent ions within the channel. It is shown to the X-ray structure, ionized or not, but the neutral case is appreciably lower that the conductivity of the selectivity filter is described by the generalized Ein- in energy. Thus this salt bridge is likely to be neutral in the open state. Based on stein relation. A novel analytic approach to the analysis of the conductivity is related results, we suggest a hypothesis for a proton pathway that creates the proposed, based on the derivation of an effective grand canonical ensemble for gating current in Kv1.2. It also appears that the Hv1 channel deviates from ions within the filter. The conditions for barrier-less diffusion-limited conduc- Kv1.2 near R201/D108/R204(Hv1):R300/E226/R303(Kv1.2), leading to a hy- tion through the KcsA filter are introduced, and the relationships between sys- pothesis for a path for the protons in Hv1. Water molecules also are an essential tem parameters required to satisfy these conditions are derived. It is shown that part of the path in the upper and lower sections of the VSD. the Eisenman selectivity equation is one of these, and that it follows directly from the condition for barrier-less conduction. The proposed theory provides 2679-Pos Board B286 analytical insight into the ‘‘knock-on’’ [1] and Coulomb blockade [4] mecha- Quantum Calculations of Salt Bridges, their Ionization State in the Inte- nisms of Kþ conduction through the KcsA filter. It confirms and illuminates rior of Voltage Sensing Domains of Voltage Gated Channels, and Some an earlier argument [3] that the ‘‘snug-fit’’ model cannot describe the fast Consequences diffusion-limited conduction seen in experiments. Numerical examples are pro- Alisher M. Kariev, Michael E. Green. vided illustrating agreement of the theory with experimentally-measured I-V Chemistry, City College of the City Univ of NY, New York, NY, USA. curves. The results are not restricted to biological systems, but also carry impli- Quantum calculations on salt bridges with varying amounts of water show that cations for the design of artificial nanopores. ionization of the salt bridge requires 3 molecules of water to be certain, and a [1] Morais-Cabral, J. H. H., Zhou, Y. & MacKinnon, R., Energetic optimization minimum of 2 molecules to have reasonable probability, depending on other of ion conduction rate by the Kþ selectivity filter, Nature 414, 37-42 (2001). parts of the surroundings. Calculations relevant to VSDs show essentially the [2] Piasta, K. N., Theobald, D. L. & Miller, C., Potassium- selective block of same results. There is a hydrophobic region at the VSD center, where the barium permeation through single KcsA channels., J. Gen. Physiol. 138, 421- salt bridge appears unionized; i.e., the energy is lower when E183 of the 36 (2011). Kv1.2 potassium channel (3Lut numbering) is not ionized. However, there [3] Horn, R., Roux, B. & A˚ qvist, J., Permeation Redux: Thermodynamics and are complications in the calculations, as protons, hence charge, may transfer Kinetics of Ion Movement through Potassium Channels, Biophys. J. 106, 1859- to or from other neighboring residues (e.g. tyrosine) that do not belong to 1863 (2014). Kaufman, I. K., Luchinsky, D. G., Tindjong, R., McClintock, P. what is normally considered part of a salt bridge; these triangular arrangements V. E. & Eisenberg, R. S., Multi-ion conduction bands in a simple model of cal- may be ionized in ways that are not expected. Work is now in progress to deter- cium ion channels, Phys. Biol. 10, 026007 (2013). mine whether it is the expected arginine from the S4 transmembrane segment, which becomes neutral, or rather a neighboring tyrosine, which would become 2677-Pos Board B284 negatively charged, that provides the proton to neutralize the glutamate. A Weighted Ensemble Approach to In Silico Measure the I-V Relationship in possibly related topic: the standard gating models depend in part on R to C mu- D aK Ion Channel tations followed by reaction with MTS reagents, which are assumed to require Sara Capponi1, Joshua Adelman2, John Rosenberg2, Michael Grabe1. the cysteine to migrate to a water accessible region in order to ionize, the inte- 1Cardiovascular Research Institute, UCSF, San Francisco, CA, USA, rior of the VSD being assumed to be too hydrophobic to allow ionization. The 2Department of Biological Sciences, U Pitt, Pittsburgh, PA, USA. assumptions of ionized salt bridges and the impossibility of ionization of the Weighted Ensemble (WE) sampling is a rigorous multi-replica method for cysteine may be incompatible. Also the R to C mutation leaves a large cavity, full-atom simulations of equilibrium and not-equilibrium processes [1,2]. which may be filled by water or the MTS reactive headgroup, so it is not certain The strategy this method uses can be summarized in three steps: i) initiation that the cysteine would have to migrate to ionize. of independent simulations from a starting state of the system; ii) progress coordinate definition and replication of the trajectories advancing along it; 2680-Pos Board B287 iii) splitting/replicating or merging/culling simulations in undersampled or True Or False? ‘‘The Arginines and Lysines of the S4 Segment of a oversampled bins, in which the conformational space has been divided. Voltage-Sensitive Ion Channel Repel One Another Electrostatically’’ WE method has been demonstrated to accurately reproduce the results ob- H. Richard Leuchtag. tained by using full-atom molecular dynamics simulations, herein called Retired, Kerrville, TX, USA. brute-force dynamics [3]. The ionic current through a membrane ion channel The Channel Activation by Electrostatic Repulsion (CAbER) hypothesis1— model was calculated by means of WE simulations. The results show that that activation of a voltage-sensitive ion channel on depolarization is powered the I-V relationship accurately reproduce that obtained by using brute- by an increase in the electrostatic repulsions between the positively charged force dynamics. Moreover, in presence of low ionic current, the efficiency arginine and lysine residues of S4 segments—has been challenged: Criti- of the WE approach increases greatly. Here, we aim to calculate the I-V cisms2: The S4 positive charges are not necessarily real. Almost as many relationship of the Kþ TRAAK ion channel embedded in a POPC bilayer acid groups with putatively negative charges are on S2 and S3; they should by applying the WE method. Research supported by NIH grants R21 be salt bridged, making the combination neutral. The charges are not obviously NS091941-01. there; quantum calculations suggest that with no or only one water molecule in [1] G. A. Huber and S. Kim, Biophys J, 70, 97 (1996). a voltage sensor domain, salt bridges don’t ionize. When they do ionize, the [2] B. W. Zhang et al., J Chem Phys, 132, 052107 (2010). charges are less than one because of charge transfer. Rebuttal: The claim [3] J. L. Adelman and M. Grabe, JCTC, 11(4), 1907 (2015) that the positive charges are not real contradicts the accepted concept that

BPJ 7904_7909 544a Wednesday, February 15, 2017 the charges are real in their sensitivity to a depolarization. Positive S4 charges including the salt concentration, ion mobility in the pore. More surprisingly, greatly outnumber the nearby negatives—a Shaker Kv sensor contains 7 posi- with the increase of the electric field strength, ion concentration in the nanopore tives in S4 and 3 negatives in S2 and S33—so the majority cannot be neutral- sharply drops from the bulk concentration, while ion mobility in the nanopore ized and must repel. Claims that arg may not be ionized without water are increases. Ion mobility in the nanopore will decrease as the salt concentration debatable: ‘‘[The] unique capacity of Arg side chains to retain their charge in the nanopore is increased. These unordinary ion behaviors should be due to in dehydrated environments likely contributes toward/ functional roles of in- be a sum of three contributions: collision of ions, Wien effect, desolvation. ternal Arg residues/ in the interior of a protein.’’ 4 Coulomb’s law requires The results presented here will be helpful not only in understanding the behavior repulsion even if the charges are less than one; showing it wrong requires of ions and water transport but also in the design of novel nanofluidic devices. extraordinary proof. The predictions of the CAbER hypothesis should be sub- jected to critical experimental tests. 1. Leuchtag HR (2016) Biophys J 110(3) 2683-Pos Board B290 277a; http://doi.org/10.13140/RG.2.1.3360.2328; (2008) Voltage-Sensitive Ion Transport and Dehydration in Subnanoscale Pores Subin Sahu1, Massimiliano Di Ventra2, Michael Zwolak3. Ion Channels: Biophysics of Molecular Excitability, Springer. 2. Green ME, 1 pers. comm.; Kariev AM et al. (2014) Biophys J 106:548-555. 3. Papazian Department of Physics, Oregon State University, Corvallis, OR, USA, 2Department of Physics, University of California, San Diego, San Diego, CA, DM et al. (1991) Nature 349:305-310. 4. Harms MJ et al. (2011) PNAS 3 108(47):18954-18959. USA, Center for Nanoscale Science and Technology, National Institute of Standards and Technology, Corvallis, MD, USA. 2681-Pos Board B288 Ion channels play a critical role in regulating cell behavior and in electrical Findings from Condensed-State Physics on Branched-Chain Amino Acids signaling. In these settings, polar and charged functional groups – as well as Apply to Voltage-Sensitive Ion Channels protein response – give rise to ion selective transport, allowing the channels H. Richard Leuchtag. to perform specific tasks in the operation of cells. According to recent experi- Retired, Kerrville, TX, USA. ments, graphene nanopores can have both weak to strong selectivity, the origin Dramatic effects on channel function by mutations of branched to unbranched of which is unclear. Here, we establish that graphene displays an alternative, residues demonstrate the importance of branched-chain amino acids novel mechanism for selectivity: Dehydration – the most fundamental physical (BCAAs)—I, L and V—in voltage-sensitive ion channels (VSICs): L to A mu- process in ion transport – yields selective pores without the presence of charges tations in Shaker K channel S4 segments shift conductance-voltage curves.1 or structural changes of the pore. This fundamental mechanism – one that de- Substitution of unbranched for branched residues in fragments of Nav channels pends only on the geometry and hydration – is the starting point for selectivity alters their voltage response.2 Mutations of L to A in S4 segments of Nav1.4 for all channels and pores. Its likely detection in graphene pores resolves the channels alters steady-state activation curves and slows inactivation.3 conflicting selectivity results, as well as opens up a new paradigm for engineer- Voltage-sensor motion in Ci-VSP channels is tuned over two orders of magni- ing molecular/ionic selectivity in filtration and other applications. 4 tude or shifted 120 mV by I126 of S1. In a Kv channel, I237 controls the voltage 2684-Pos Board B291 dependence of charge movement, shifting the Q-V curve toward more positive 5 Molecular Determinants of Structural Coupling between C-Type Inactiva- voltages; I287 controls the energy barrier underlying activation. In 1986, Yosh- tion and Inner Gate Opening in KD Channels ino et al. investigated whether sidechains of amino acids engender ferroelectric Jing Li, Jared Ostmeyer, Eduardo Perozo, Benoit Roux. properties in molecules with aromatic rings and chiral centers. Three of them Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, did, dramatically: the BCAA sidechains. One compound with an isoleucine USA. sidechain, 4’-3M2CPOOB, exhibited a ferroelectric phase with extremely large þ ε 6, 7 C-type inactivation of K channels is a molecular process of great physiolog- spontaneous polarization and dielectric permittivity over 3000. Applying ical significance. It affects the firing patterns of neurons in the central nervous these condensed-state-physics findings to VSICs suggests that the high- ε system, and the repolarization of cardiac cells in the heart. The coupling be- electric-field resting channel is ferroelectric, with a high , so that the electro- tween selectivity filter and inner gate is a functional hallmark of Kþ channels. static repulsions between the positively charged arginine and lysine residues Identification the molecular determinants controlling the coupling could pro- of S4 are weak. Threshold depolarization, according to the Channel Activation vide a deeper understanding of the C-type inactivation. Our Umbrella by Electrostatic Repulsion (CAbER) hypothesis,8 converts the channel to a ε Sampling/Hamiltonian-Replica Exchange molecular dynamic simulations (an nonpolar phase with much lower , causing these repulsions to greatly increase, aggregate of 10 microsecond), for the first time, reveal relative propensity of which expands the S4 segments. CAbER proposes that this observed outward S4 the conductive filter versus the inactive filter, when the gate is in different motion drives the channel into a stochastically ion-conducting, active, confor- open states in KcsA. Meanwhile, our equilibrium MD simulations demonstrate mation. 1. Lopez GA et al. (1991) 7:327-336. 2. Helluin O et al. distinct dynamical behaviors of the selectivity filter with the inner gate in (2001) IEEE Trans Diel Elect Insul 8:637-643. 3. Bendahhou S et al. (2007) different partially and fully open state. In the fully open state, spontaneous tran- BBA 1768:1440-1447. 4. Lacroix JJ, Bezanilla F (2012) Biophys J 103: L23- sition of selectivity filter from conductive to constricted conformation was L25. 5. Lacroix JJ et al. (2014) PNAS 111(19):E1950-E1959. 6. Yoshino K et repeatedly observed. This series of simulations richly illustrate the molecular al. (1986) Japan J App Phys 25:L416-L418. 7. Yoshino K, Sakurai T (1991) process of c-type inactivation at atomic scale. Analysis and comparison of these In: Goodby JW et al., Ferroelectric Liquid Crystals, Gordon and Breach. 8. trajectories clearly show that two critical residues from the TM2 contacting the Leuchtag HR (2008) Voltage-Sensitive Ion Channels, Springer; (2016) Biophys bottom of selectivity filter are the structural determinants for the coupling be- J 110(3) 277a; http://doi.org/10.13140/RG.2.1.3360.2328. tween C-type inactivation and inner gate opening. Then mutation simulations reveal how these two residues work cooperatively to control the coupling of Ion Channel Regulatory Mechanisms II two gates in a symmetrical manner among four subunits of Kþ channel. Iden- tification of the coupling determinants also shed light on the exploration of the 2682-Pos Board B289 C-type inactivation structure for other Kþ channels. Transport of Water and Ions through a Graphene Nanopore in an Electric Field 2685-Pos Board B292 Anping Ji1,2, Zhongwu Li1, Haojie Yang1, Pinyao He1, Yunfei Chen1. Investigation of Ionic Coulomb Blockade in Sub-1 Nm Nanopores with 1School of Mechanical Engineering, Southeast University, Nanjing,jiangsu, Molecular Dynamics Simulations People’s Republic of China, China, 2School of Mechanical Engineering, Zhongwu Li, Kun Li, Pinyao He, Haojie Yang, Anping Ji, Yunfei Chen. Chongqing Three Gorges University, Chongqing, China. Jiangsu Key Laboratory for Design and Manufacture of Micro-Nano Graphene nanopore has been widely used in a nanofluidic device for sensing or Biomedical Instruments, School of Mechanical Engineering, Southeast analyzing biomolecules, as its single atom thickness provides high spatial reso- University, Nanjing, China. lution. The ionic conductance of nanopore is of primary importance for Ionic transport in nanopores could provide much useful information for many analyzing biomolecules transported through the pore. The continuum theory applications such as DNA sequencing and seawater desalination. Through all- holds that conductance comprise of three contributions: bulk, surface (for a atom molecular dynamics simulations, we found that the current-voltage char- charged nanopore) and access ones. However, when the pore diameter ap- acteristic of ionic transport shows a nonlinear behavior with voltage gap when þ þ þ þ proaches the size of the molecule, ion and water dynamics may deviate from metal ion types (K ,Na ,Ca2 ,Mg2 ) translocate through atomically thin sil- the bulk values and continuum analysis cannot be used to account for experi- icon nitride nanopores with small diameters (<1 nm), which is attributed to the mental data. Here we perform molecular dynamics simulations to systematically large energy barriers. The similar phenomenon has been reported in other’s investigate the relationship between conductance and the coupling transport of experiment recently, which was not explained clearly. We found that the en- water and ions through a graphene nanopore in electric fields. The results show ergy barriers of ionic transport also include the ionic coulomb blockade apart that the ionic conductance of nanopore strongly depend on the ionic conditions, from the dehydration effects. Ionic coulomb blockade is similar to the

BPJ 7904_7909 Wednesday, February 15, 2017 545a electronic coulomb blockade and resonant tunneling in quantum dots. It is a 2688-Pos Board B295 D fundamental electrostatic phenomenon based on charge discreteness theory. Structural and Functional Response of a Mechanosensitive K2P K Chan- We used the adaptive biasing method in the colvar module of NAMD to nel to Asymmetric Membrane Tension perform the free energy calculation. In the simulations, we found that the Viwan Jarerattanachat, Michael V. Clausen, Prafulla Aryal, coulomb blockade energy is the main contributor as the pore size ranges Elisabeth P. Carpenter, Mark S.P. Sansom, Stephen J. Tucker. from 0.6 to 1 nm, while for smaller pores the main contributor changes from University of Oxford, Oxford, United Kingdom. þ ionic coulomb blockade to ion dehydration effects. Moreover, by incorporating The mechanosensitive K2P K channels play important roles in a range of both the transport barriers together, we introduced an analytical model to calcu- diverse sensory processes, including touch, pain, and hearing. The ability of late the energy barriers and the current-voltage characteristic curve referring to these channels to sense changes in pressure within the membrane is central the electronic coulomb blockade model and Poisson-Boltzmann-Nernst-Planck to the process, but the structural and biophysical mechanisms underlying this model. The discrete conductance of ionic transport was well predicted by our remain unclear. Our previous studies have shown that the core transmembrane model. Therefore, we remark that our results could play an important role in domains of the TREK-2 K2P channel are intrinsically sensitive to changes in charged pores including biological ion channels and synthetic nanopores. We bilayer tension and that the channel switches conformation from the ‘Down’ also believe that the sub-1 nm pores can offer a new platform to explore novel state to the ‘Up’ state in response to increasing levels of membrane stretch. physics in the research areas of nanoscale fluidics and biology. However, the structure of the TREK-2 channel is highly asymmetric and increasing tension is expected to change the pressure profile equally in both 2686-Pos Board B293 the inner and outer leaflets of the bilayer. In this study we have examined Glutamate Receptor Ion Channel Activation Mechanism Revealed by the functional and structural effects of both negative and positive pressure on Cryo-EM Maps TREK-2 channels reconstituted into planar lipid bilayers, whilst their structural Xiongwu Wu, Bernard R. Brooks. responses were investigated using MD simulations predicted to alter tension NHLBI, National Institutes of Health, Bethesda, MD, USA. within only one leaflet of the bilayer. Our results demonstrate that TREK-2 ex- Ionotropic glutamate receptors (iGluRs) are cation channels that mediate signal hibits a highly asymmetric response to changes in membrane tension, and we transmission in the central nervous system by depolarizing the postdynaptic discuss the role that changes in the lateral pressure profile and local curvature membrane in response to L-glutamate release from the presynaptic neuron. of the membrane may play in this process. GluA2 is a subunit of AMPA, a major subtypes of iGluRs, which harbors a modular architecture composed of an amino-terminal domain (ATD), 2689-Pos Board B296 angonist-recognizing ligand-binding domain (LBD), a transmembrane domain Fluctuation-Driven Transport in Bacterial Channels under Acidic Stress (TMD) that forms the ion channel pore, and intracellular carboxyl-terminal do- Marı´a L. Lo´pez, Marı´a Queralt-Martı´n, Vicente M. Aguilella, mains. To understand the ion channel activation mechanism, many efforts have Antonio Alcaraz. been dedicated to obtain the structures of GluA2 in various functional states. Physics, University Jaume I, Castellon, Spain. While ATD, LBD structures in the close and open states have been obtained Fluctuation-driven ion transport can be obtained in bacterial channels with the through x-ray and cryo-EM, the TMD structure is only available in the close aid of different types of colored noise including biologically relevant Lorent- state because the open state TMD is difficult to capture with crystallography zian one. Using the electrochemical rectification of the channel current as and its cryo-EM maps are low in resolution. The Low resolution cryo-EM ratchet mechanism we observe transport of ions up their concentration gradient maps of GluA2 in the close, open, and desensitized states have been obtained, in conditions similar to that met in vivo, namely moderate pH gradients and which opens a window to investigate the structural mechanism of the ion chan- asymmetrically charged lipid membranes. We find that depending on the direc- nel activation. MapSGLD is a method to determine molecular structures from tion of the concentration gradient the channel can pump either cations or anions low resolution maps by utilizing structural information embedded in the force from the diluted side to the concentrated one. We discuss the possible relevance field. Through MapSGLD simulations with the low resolution maps of GluA2 of this phenomenon for the pH homeostasis of bacterial cells. in the close, open, and desensitized states, we determined the structures of GluA2 in the corresponding states. The structures show that LBD clamshell Ion Channels, Pharmacology, and Disease II is open in the close state and closed in the open and desensitized states. The TMD ion pore is closed in the close and desensitized states but open in the 2690-Pos Board B297 open state. Comparing these structures we obtained an atomic detailed under- Sodium Valproate Reverses Electrical Remodeling of Atrial Myocytes standing of the ion channel activation mechanism. Isolated from CREM-IbDC-X Transgenic Mice C. Florentina Pluteanu, Beatrix Scholz, Jan S. Schulte, Frank U. Muller.€ 2687-Pos Board B294 Inst. Pharmacology Toxicology, Westf€alische Wilhelms-Universit€at The C-Terminal Domain of Kv1.3 Interacts with KCNE4 to form Oligo- Munster,€ Munster,€ Germany. meric Channels Patients that suffer from paroxysmal and chronic atrial fibrillation (AF) Sara R. Roig1, Laura Sole´2, Albert Vallejo-Gracia1, Daniel Sastre1, show increased levels of CREM-IbDC-X, a truncated form of cAMP Antonio Serrano-Albarra´s1, Clara Serrano-Novillo1, response element modulator (CREM). In mice, cardiomyocyte directed Ramo´n Martı´nez-Ma´rmol3, Michael M. Tamkun2, Antonio Felipe1. over-expression of CREM-IbDC-X leads to atrial ectopy progressing to 1Biochemistry and Molecular Biomedicine, University of Barcelona, persistent AF. CREM represses gene transcription induced by cAMP Barcelona, Spain, 2Department of Biomedical Sciences, Colorado State response binding protein (CREB). Previously we showed that the treatment University, Fort Collins, CO, USA, 3Clem Jones Centre for Ageing Dementia of mice with anticonvulsant sodium valproate (VPA), which also inhibits Research, University of Queensland, Queensland, Australia. histone deacetylase (HDAC) classes I>II, prevented the atrial structural re- The voltage-gated potassium channel Kv1.3 plays crucial roles in the immune modeling. The aim of this study was to investigate whether chronic VPA system. KCNE4, present in leukocytes, physically associates with Kv1.3 acting treatment also reverses electrical remodeling of atrial myocytes (AM) in as a dominant negative subunit. KCNE4 inhibits Kþ currents and impairs mem- CREM-IbDC-X mice. brane surface targeting. Although canonical KCNE-Kv7 interactions have been AM were isolated from 12-weeks old CREM-IbDC-X mice (TG) and wild type under intense investigation, the molecular determinants implicated in this essen- (WT) littermates, both treated for 7-weeks with VPA (0.4mM in the drinking tial Kv1.3/KCNE4 interaction remain unknown. Structural signatures between water) or water (vehicle control). In TG mice, patch-clamp measurements Kv7 and Kv1 channels display enormous differences. Our results indicate that showed that VPA treatment shortens action potentials (AP) by reducing AP the carboxy terminal of Kv1.3 is an essential and sufficient interacting domain duration (APD) at 50, 70 and 90% repolarization (7.350.6 vs 1151.2ms, for KCNE4. However, similar to what described for the Kv7.1 association, this 13.851.1 vs 20.552.2ms and 34.153vs4754.1ms, n=24 VPA-TG vs. motif apparently is not implicated in the modulation of the Kv1.3 activity. We n=37 untreated TG AM, p<0.05). In line with this observation total outward þ also describe two independent and synergic mechanisms which potentiate the K current densities were decreased by 36% in TG vs WT mice (p<0.05 vs KCNE4-dependent intracellular retention of the Kv1.3/KCNE4 complex. First, WT) and were further increased by 38% only in VPA-TG mice (p<0.05 vs un- KCNE4 association masks the YMVIEE motif at the C-terminal domain of the treated TG). The inwardly rectifying IK1 currents were reduced by 35% in TG Kv1.3, which is crucial for the channel surface targeting; and second, we identify vs WT mice (p<0.05 vs WT) and increased by 47% in VPA-TG animals a basic endoplasmic reticulum retention signature in KCNE4. This element is (p<0.05 vs untreated TG). transferred to the channelosome that further limits cell surface expression and In conclusion, VPA treatment reduced APD by increasing outward Kþ currents triggers ER localization. Our results map specific molecular determinants which and may stabilize the resting membrane potential by increasing IK1 in atrial my- play a crucial role in the physiological function of Kv1.3 in leukocytes. Supported ocytes of CREM-IbDC-X mice. VPA has the potential to reverse atrial electri- by MINECO, Spain (BFU2014-54928-R, BFU2015-70067-REDC and FEDER). cal remodeling, possibly via class I HDAC inhibition.

BPJ 7904_7909 546a Wednesday, February 15, 2017

2691-Pos Board B298 in KCNJ6 associated with idiopathic ventricular tachycardia in a child. This Heart Failure is Associated with Activation of the Unfolded Protein is the first case reporting mutations in thisgene as a potential cardiac Response and Electrical Remodeling that is Partially Reversed by a arrhythmia susceptibility gene. PERK Inhibitor Man Liu, Guangbin Shi, Hong Liu, Samuel C. Dudley. 2693-Pos Board B300 Cardiology, Brown University and Rhode Island Hospital, Providence, RI, Restoring Defective Camp-Dependent Upregulation in Long-QT Syn- USA. drome Type-1 through Interventions that Promote IKs Channel Opening Introduction: Heart failure (HF) is associated with endoplasmic reticulum Cristina Moreno Vadillo, Roel L.H.M.G. Sp€atjens, Sandrine Seyen, stress and activation of the unfolded protein response (UPR). UPR inhibits pro- Gabriele Menini, Jordi Heijman, Paul Volders. tein transcription/translation. Ion channel downregulation is associated with Cardiology, Cardiovascular Research Institute Maastricht, Maastricht arrhythmic risk. We hypothesized that UPR could be contributing to electrical University, Netherlands. remodeling in HF. Patients with the KCNQ1 mutation A341V are at risk of sympathetically- Methods: Hypertensive HF was induced in C57BL/6 mice by unilateral ne- triggered ventricular tachyarrhythmias and sudden cardiac death. A341V de- phrectomy, deoxycorticosterone acetate pellet implantation, and salt water sub- creases basal IKs and disrupts cAMP-dependent upregulation due to impaired stitution. Sham operated mice were used as controls. After 6-7 weeks, isolated phosphorylation at S27, which can be rescued by the phosphomimetic substitu- cardiomyocytes were used for whole-cell patch clamp recording, mRNA and tion KCNQ1-S27D. We hypothesize that A341V imposes a different conforma- protein assay. GSK2606414 (GSK), a protein kinase R like ER kinase tional state of the IKs channel, which prevents full phosphorylation of KCNQ1- (PERK) inhibitor, was applied to myocytes at 4-30 nM for 20 h. S27 and therefore loss of upregulation. Here, we examine whether strategies Results: HF myocytes showed classical electrical remodeling with action po- promoting channel opening can rescue cAMP-dependent upregulation. tential duration prolongation (APD90: 203526 vs. 108516 ms of sham, Chinese hamster ovary cells were transfected with wild-type KCNQ1 (WT) or P<0.05) and DADs. Activation of the PERK and IRE1 branches of the UPR A341V-KCNQ1 in heterozygosis (WTþA341V-KCNQ1), together with in HF myocytes was verified by elevated protein levels of phospho-PERK, KCNE1 and Yotiao. In the absence and presence of cAMP/okadaic acid phospho-eIF2a, ATF4, phospho-IRE1/IRE1, and spliced XBP1/XBP1. PERK (OA), we examined IKs responses to (1) the KCNQ1 I346-K358 short peptide inhibition by GSK shortened the APD (APD90: 131512 ms, P<0.05 vs. (shown to unlock the closed state of IKs by binding to the endogenous S4-S5- HF). Different changes of channel mRNA, protein and current levels were linker of KCNQ1), (2) the double mutant A341V/L353K (L353K was previ- observed in HF myocytes and PERK inhibition showed different effect. Peak ously shown to promote constitutively open channels), and (3) the pharmaco- þ INa and three types of K currents (Ito,IK1, and IKslow) were decreased signif- logical channel activator ML277 (1 mmol/L). icantly in HF group, among which INa and IKslow were restored by PERK inhi- As previously shown, WT but not A341V IKs was upregulated by intrapipette 2þ þ bition with GSK. L-type Ca current (ICaL) and the sustained K current were cAMP/OA. The unlocking effect of the I346-K358 peptide and the constitutive not affected in HF. Corresponding to the current changes, we observed signif- conduction produced by the co-presence of L353K facilitated A341V IKs þ þ icant reductions at the mRNA and protein levels of Kv1.5 and channel protein enhancement by cAMP/OA ( 61% and 71%, respectively). Similar results level for Kv4.3 and Kir2.1, all of which were restored by GSK. Nav1.5 protein were obtained for the IKs activator ML277. Under baseline conditions, level was unaltered, although INa was reduced, suggesting a posttranslational ML277 increased both WT and A341V IKs by approximately 60%. The IKs effect of HF. This shows that the PERK branch of UPR has a canonical effect response to ML277 in cells stimulated with cAMP/OA was amplified more on Kv1.5 with concurrent reductions of mRNA, protein and current, but non- than twofold in WT, but strikingly also in A341V IKs. This larger ML277 effect canonical effects on the other channels with reduction of either protein or cur- is likely explained by an augmented cAMP-dependent regulation of A341V IKs. rent alone. Pharmacological and molecular interventions promoting IKs-channel opening Conclusions: HF is accompanied by activation of two of three branches of rescue from defective cAMP-dependent upregulation by the hot-spot mutation UPR and downregulation of a variety of ion channels and currents. The A341V. Our results suggest novel therapeutic strategies to restore the beta- PERK branch of UPR appears to mediate the Nav1.5 and Kv1.5 current reduc- adrenergic control of repolarization in long-QT1 syndrome. tions by noncanonical and canonical pathways, respectively. Inhibiting the UPR may be a novel antiarrhythmic strategy. 2694-Pos Board B301 KCa1.1 Channel Auxiliary Beta Subunit Composition in Glioblastoma 2692-Pos Board B299 Multiforme A De Novo Loss-of-Function Mutation in KCNJ6 Associated with Idio- Zoltan Denes Petho, Andras Balajthy, Almos Klekner, Laszlo Bognar, pathic Ventricular Tachycardia Zoltan Varga, Gyorgy Panyi. Franck Potet1, Ruth McGowan2, Karen McLeod3, Alfred L. Jr George1. Biophysics and Cell Biology, University of Debrecen, Debrecen, Hungary. 1Department of Pharmacology, Northwestern University, Chicago, IL, USA, Glioblastoma Multiforme (GBM) is the most aggressive glial cancer as well 2Department of Clinical Genetics, Laboratories Building, Queen Elizabeth as the most common primary malignant brain tumor. Even in the 21st cen- University Hospital, Glasgow, United Kingdom, 3Department of Cardiology, tury, regardless of aggressive treatment, the median survival remains less The Royal Hospital for Sick Children, Glasgow, United Kingdom. than two years following diagnosis. Therefore, it is crucial to study the We investigated the genetic basis for idiopathic ventricular tachycardia in an biology including the ion channel profile of GBM in order to develop new 18 month old Scottish girl. There was no family history of cardiac arrhythmia therapeutic and diagnostic options for the treatment of this disease. GBM tu- or sudden cardiac death and both parents were asymptomatic with normal mor cells and tumor model systems express KCa1.1 (BK, Slo1, MaxiK) and ECGs. Because of the young age of onset and lack of any identifiable cause predominantly the gBK transcript variant as their major Kþ channel. More- for ventricular arrhythmia, a genetic etiology was suspected. Exome over, it is known that auxiliary b and g subunits modulate the biophysical sequencing revealed a novel, de novo mutation in KCNJ6 (c.641G>A; properties of the KCa1.1 channel, and have restricted tissue expression. In p.Arg214Gln) and no other variants with plausible pathogenicity. KCNJ6 en- this study, our aim is to characterize the KCa1.1 b subunit composition in codes Kir3.2, also known as the G-protein activated inward rectifying potas- both tumor model systems and in freshly isolated tumor cells. We used sium channel 2 (GIRK2). GIRK2 is expressed in neurons but not in cardiac cultured tumor cell line U87-MG and freshly isolated tumor samples ob- myocytes. Previously, gain-of-function (enhanced Na permeability) mutations tained from the Neurosurgery Department of the University of Debrecen. in this gene have been associated with neurodevelopmental phenotypes in Following isolation, grade IV. GBM samples were homogenized, cultured, man and mice. Heterologous cells expressing homotetrameric GIRK2- then examined by rt-PCR, confocal microscopy for glial markers and R214Q channels with a receptor (needed for activity) exhibited whole-cell currents using patch-clamp technique. In our patch-clamp exper- þ essentially no Ba2 -sensitive current at 120 mV (0.1 5 0.2 pA/pF) iments of GBM cells we measured the activation and inactivation kinetics of whereas cells expressing homotetrameric wildtype (WT) GIRK2 exhibited the KCa1.1 channel as well as the pharmacological responses to various robust current density (-52.3 5 13.1 pA/pF). Acute application of ethanol KCa1.1 modulators (e.g. , paxilline). In accordance with cur- (GIRK2 positive allosteric modulator) or R-8-OH-DPAT (a serotonin agonist) rent literature, we observed predominantly KCa1.1 currents on GBM cells did not evoke current in cells expressing mutant GIRK2. Co-expression of showing consistent inhibition by paxilline. Moreover, we could deduce the GIRK2-R214Q with WT-GIRK2 demonstrated a dominant-negative effect presence of functional b subunits based on rt-PCR, current kinetics and phar- of the mutant. The mutation did not modify the reversal potential of the cur- macological response. In conclusion, our findings also support that KCa1.1 rent suggesting that ion selectivity was not affected. Because of the neuronal has an important role in GBM pathogenesis and the inhibition of this channel expression of KCNJ6, we propose that altered autonomic nervous system ac- specifically targeted via auxiliary subunits may have a potential therapeutic tivity may be responsible for ventricular tachycardia in this case. In summary, consequence in the future. we identified a novel, de novo, dominant-negative loss-of-function mutation Funding projects: OTKA K 119417 ; KTIA NAP 13B_2ABB813

BPJ 7904_7909 Wednesday, February 15, 2017 547a

2695-Pos Board B302 In inflammatory arthritis, the dysregulation of osteoclast activity by pro- The Uremic Toxin P-Cresol Reduces Cell Surface Expression of Human inflammatory cytokines such as TNF interferes with the homeostatic process Ether-A-Go Go-Related Gene (hERG) Channels via the Ubiquitin Ligase of bone remodeling and perpetuates inflammation through calcium- Nedd4-2 dependent and -independent mechanisms causing pathological bone loss. Ellen G. Avery, Shawn Lamothe, Shetuan Zhang, Jun Guo, Wentao Li, Bone resorption requires functional expression of the osteoclast-specific genes Tonghua Yang. MMP-9, TRAP and Ctsk during osteoclast differentiation. This process is Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, crucially dependent on NFATc1. The calcium-activated potassium channel Canada. KCa3.1 constitutes a known key player in calcium-induced NFATc-dependent The human ether-a-go-go–related gene (hERG) encodes the rapidly activating gene expression in leukocytes. Herein, we investigated the role of potassium delayed rectifier potassium channel (IKr), which is critical for cardiac repolar- channels in the calcium-dependent regulation of osteoclast differentiation in in- ization. A reduction in the hERG current can cause long QT syndrome, leading flammatory (RANKLþTNF) compared to non-inflammatory (RANKL only) to potentially fatal cardiac arrhythmias. In chronic kidney disease (CKD), the conditions using mouse bone marrow macrophages (BMMs) in vitro. Microar- risk of sudden death is increased beyond what is explicable by traditional ray analysis, immunostaining and qPCR revealed increased expression of risk factors for cardiovascular disease. The molecular mechanisms governing KCa3.1 during RANKL and RANKLþTNF-induced osteoclastogenesis. CKD-induced cardiac arrhythmia are unknown, but a significant portion of KCa3.1-/- cells and cells treated with the KCa3.1 inhibitor TRAM-34 compared CKD patients have prolonged QT interval. In CKD, a large number of com- to wild-type untreated controls exhibited an average of 40% less multinucleated pounds, which under normal conditions are excreted by kidney, are progres- TRAPþ cells in inflammatory and approximately 30% less in non-inflammatory sively accumulated. These compounds are known as uremic toxins, and are conditions, as well as significantly reduced osteoclast-specific gene expression. known to negatively affect the biological functions. Serum concentrations of Similar to TRAM-34, the calcineurin inhibitor cyclosporine A (CsA) or the the uremic toxin p-cresol increase up to 30-fold in CKD patients compared CaMK inhibitor KN-93 caused a reduction in multinucleated cells stimulated to normal population. Increased serum p-cresol level has been associated with RANKL of approximately 99% and 50%, respectively. However, the addi- with QT interval prolongation in patients with end stage renal failure, though tion of TNF partially rescued osteoclast formation resulting in an average of a causative link has not been established. Impairment of hERG function is a 77% (CsA) and 20% (KN-93) reduction of multinucleated cells. In calcium im- common cause of long QT syndrome, so we investigated the effects of p-cresol aging experiments we found TRAM-34 to decrease the amplitude of RANKL- on hERG channels using whole-cell voltage clamp and Western blot analysis. induced acute calcium transients by about 50%. Our data suggest that KCa3.1 We found that p-cresol selectively decreased the mature hERG protein plays an important role in the calcium regulation of osteoclast differentiation expression and the hERG current. However, p-cresol had no effect on the during inflammation and implicates it as a target for the treatment of bone Nedd4-2 interaction-deficient mutant hERG channels. These data suggest erosion in inflammatory arthritis. that Nedd4-2 may be involved in p-cresol mediated hERG reduction. In iso- 2698-Pos Board B305 lated rat neonatal ventricular myocytes, p-cresol decreased IKr and prolonged D action potential duration. These results suggest that p-cresol-mediated hERG Oxidation of K Channels in TBI reduction may contribute to the prolonged QT in CKD patients. Federico Sesti, Wei Yu, Randika Parakramaweera, Janet Alder, Smita Thakker-Varia, Shavonne Teng. 2696-Pos Board B303 Neuroscience and Cell Biology, Rutgers, Piscataway, NJ, USA. IPSC-Derived Neurons Harboring a Known Epilepsy Mutation Display The delayed rectifier potassium (Kþ) channel KCNB1 (Kv2.1), which conducts Known and Novel Electrophysiological Phenotypes a major somatodendritic current in cortex and hippocampus, is known to un- Coby Carlson1, Imran Quaraishi2, Yalan Zhang2, Michael McLachlan1, dergo oxidation in the brain but whether this can cause neurodegeneration Benjamin Meline1, Chris McMahon1, Tom Burke1, Eugenia Jones1, and cognitive impairment is not known. Here, we used transgenic mice Leonard Kaczmarek2, Kile P. Mangan1. harboring human KCNB1 wild type (Tg-WT) or non-oxidable C73A mutant 1Cellular Dynamics International, Madison, WI, USA, 2Yale University, New (Tg-C73A) in cortex and hippocampus, to determine whether oxidized Haven, CT, USA. KCNB1 channels affect brain function. Animals were subjected to moderate Epilepsy is a disturbance in the electrical activity of the brain manifested via traumatic brain injury (TBI), a condition characterized by extensive oxidative countless etiologies. 65 million individuals suffer from epilepsy and one- stress. Dasatinib, an FDA-approved inhibitor of Src tyrosine kinases was used third of these individuals live with uncontrollable seizures because no known to impinge on the pro-apoptotic signaling pathway activated by oxidized pharmacological treatment works for them. A portion of this population is ac- KCNB1 channels. Thus, typical lesions of brain injury, namely inflammation counted for by single-gene epilepsy disorders resulting from mutations within (astrocytosis), neurodegeneration and cell death were markedly reduced in sodium, potassium or inhibitory channels. For example, the Slack gene Tg-C73A and dasatinib-treated non-Tg animals. Accordingly, Tg-C73A mice (KCNT1) encodes a sodium-activated potassium channel that is very widely and non-Tg mice treated with dasatinib exhibited improved behavioral out- expressed in the brain. Mutations in this KCNT1 gene in humans presents comes in motor (rotarod) and cognitive (Morris Water Maze) assays compared with autosomal-dominant nocturnal frontal lobe epilepsy (ADNFLE), a disease to control. Moreover, the activity of Src kinases, along with oxidative stress, marked by brief, but violent, seizures during sleep and devastating effects on were significantly diminished in Tg-C73A . Together, these data demon- intellectual function. Advances in personalized medicine is crucial for these strate that oxidation of KCNB1 channels is a contributing mechanism to types of diseases. Central to this vision is induced pluripotent stem (iPS) cell cellular and behavioral deficits in vertebrates and suggest a new therapeutic technology, which provides a platform to expand our understanding of how approach to TBI. single-gene mutations result in disease states. This approach illustrates and le- verages the ‘‘disease-in-a-dish’’ iPSC-technology into phenotypic screening 2699-Pos Board B306 and drug development. We have engineered and generated human cortical neu- Protective Role of Olesoxime in Alpha-Synuclein-Induced Mitochondrial rons harboring the KCNT1 {P924L} single-gene mutations, as well as the Dysfunction isogenic wild-type control match. This ability provides unprecedented access Amandine M.F. Rovini. to in vitro models of all-types of neurological disorders. Here we present func- Molecular Transport, NIH / NICHD, Bethesda, MD, USA. tional data, via patch-clamp and multi-electrode array (MEA) electrophysiolog- Olesoxime, a cholesterol-like neuroprotective drug, promotes motor neuron sur- ical techniques, illustrating the known ‘gain-of-function’ ionotropic cellular- vival and neurite outgrowth in vitro. In vivo olesoxime rescues motor neurons level fingerprint, which has previously been linked to this mutation, along from axotomy-induced cell death and promotes nerve regeneration. It was re- with newly-discovered neural-network level hyper-active phenotypes. We ported that olesoxime plays a protective role against toxicity induced by wild- further show multiple examples that selective pharmacology can reverse these type alpha-synuclein (a-syn) in human neuronally differentiated cells. a-syn is observed phenotypes. Collectively, our results illustrate how human iPS cells the main component of Lewy bodies, a hallmark of Parkinson disease (PD) which can be model disease states and be leveraged in the personal medicine space. is usually diagnosed clinically from classical motor symptoms. Importantly, a-syn accumulation and mitochondrial dysfunction are believed to play pivotal 2697-Pos Board B304 roles in the pathogenesis of PD and others a-synucleinopathies. It is still an KCa3.1 as Master Regulator in Inflammatory Osteoclastogenesis open question regarding mechanisms by which a-syn exerts its neurotoxicity Eva M. Grossinger1, Mincheol Kang1, Laura Bouchareychas1, Ritu Sarin1, and especially how exactly it impacts mitochondria. It is presumed that some Dominik Haudenschild2, Laura Borodinsky3, Iannis E. Adamopoulos1. of olesoxime-induced effects occur via its direct interaction with mitochondrial 1Rheumatology, Allergy and Clinical Immunology, University of Califonia membranes. Here we address a tentative mechanism of olesoxime protection Davis, Davis, CA, USA, 2Department of Orthopedic Surgery, University of against neuronal cell death caused by a-syn -induced mitochondrial dysfunctions Califonia Davis, Sacramento, CA, USA, 3Department of Physiology and using different in vitro and cell-based methods. With channel reconstitution tech- Membrane Biology, University of Califonia Davis, Davis, CA, USA. nique and fluorescence correlation spectroscopy, we showed that a-syn binding to

BPJ 7904_7909 548a Wednesday, February 15, 2017 the membrane is lipid-dependent and precedes blockage and translocation of the pancreatic cancer cell lines. In this study, we show that TMEM16A/ANO1chan- protein through the mitochondrial outer membrane voltage-dependent anion nel promotes cell proliferation in pancreatic cancer cell lines and intracellular channel (VDAC). We now find that olesoxime reduces a-syn binding to mito- chloride through TMEM16A/ANO1 is important mechanism for cell prolifer- chondria isolated from neuroblastoma cells. Furthermore, our preliminary chan- ation. These results indicate that TMEM16A/ANO1 can be possible druggable nel reconstitution experiments showed that lipid mixtures containing up to 20% targets using ANO1/TMEM16A inhibitor. Further, TMEM16A/ANO1 might of olesoxime allow forming stable planar bilayers, and that the kinetics of a-syn/ play a role as prognostic factors for survival outcomes in pancreatic cancer. VDAC interaction in olesoxime-containing bilayers differ from those in cholesterol-containing bilayers. We anticipate that future work will clarify 2702-Pos Board B309 whether a-syn is sensitive to olesoxime due to its lipidic (cholesterol-like) nature The Role of BK Channel in Microglia Activation Xiaoying Yang1,2, Wenying Zhao1,2, Li Zhang1,2, Jianmin Cui1,3, through the changes in membrane integral properties or whether a direct interac- 1,2 1,2 tion of olesoxime with VDAC is at play. Xiaohui Sun , Xuechu Zhen . 1College of Pharmaceutical Sciences, Soochow University, Suzhou, China, 2700-Pos Board B307 2Jiangsu Key Laboratory of Translational Research and Therapy for Neuro- RIP3 Mediates Necroptotic Cell Death in Response to Acid Stimulation of Psycho-Diseases, Soochow University, Suzhou, China, 3Washington ASIC1a University in St. Louis, St. Louis, MO, USA. Yu Huang1, Qin Hu2, Jun-Long Huang2, Jaepyo Jeon1, Tian-Le Xu2, Microglial cells are the resident macrophages in the central nervous system, Michael Xi Zhu1. upon any detection of signs for brain lesions or nervous system dysfunction, 1Department of Integrative Biology and Pharmacology, UT Health Science they are activated and thus trigger inflammation. Large conductance Ca and Center at Houston, Houston, TX, USA, 2Department of Anatomy, Histology voltage gated K (BK) channel currents are specifically detected in activated mi- and Embryology, Shanghai Jiao Tong University School of Medicine, croglial cells of mice and patients who underwent surgery for pharmaco- Shanghai, China. resistant epilepsy. However, whether and how BK channel gets involved in Acidosis occurs commonly under pathological conditions when body homeo- microglia activation is unclear. We observed that BK KO mice develop motor stasis is disrupted due to inflammation, hypoxia, cancer and other illnesses. impairment and they are more sensitive to MPTP toxicity. The brain slides of However, the effects of acidosis on cell fates differ dramatically from one MPTP treated BK KO mice contain more microglia than that of WT mice. cell type to another. On one hand, acidosis is usually harmful to brain neurons, Furthermore, specific BK channel blocker paxilline significantly inhibits 100 which are particularly sensitive to tissue acidosis that occurs very often among ng/ml lipopolysaccharide (LPS) induced NO production, and iNOS and neurological disorders, including ischemic stroke. Because of this, acidotoxic- COX2 expression in immortalized mouse microglia cell BV2; indicating that ity represents one of the major causes of neurodegeneration and brain injury. BK channel activation is underlying LPS induced BV2 cells activation. The On the other hand, cancer cells thrive in acidity as the microenvironment of tu- expression level of BK channel in whole cell is not changed whereas the intra- mor is often acidic. Previously, we have shown that extracellular acid induces cellular distribution of the channel is altered after 12h LPS (100 ng/ml) appli- neuronal cell death through activation of acid-sensing ion channel isoform 1a cation. Moreover, the blockade of BK channel by paxilline prominently (ASIC1a) in a manner that involves recruitment and phosphorylation of recep- reduces 100 ng/ml LPS stimulated TNF-a release from BV2 cells within 4h. tor interacting protein 1 (RIP1), but independent of the ion channel function of These results suggested that the internalization and regulation on NF-kB acti- ASIC1a. Here, we demonstrate that receptor interacting protein 3 (RIP3) is also vation of BK channel might play important role in LPS induced inflammatory critically required for acid-induced cell death and the presence of ASIC1a and/ response of BV2 cells. or RIP3 determines the nature of cellular responses to acidosis. Whereas both ASIC1a and RIP3 are expressed in neurons, at least one of them tends to be ab- 2703-Pos Board B310 k Kv1.3 Inhibition Reduces Amyloid-Beta Induced Microglial Neurotoxicity sent in many cancer cell lines. In the absence of RIP3, acidosis activated NF- B 1 2 1 1 signaling to promote survival of the tumor through ASIC1a. Re-introduction of Heike Wulff , Jacopo Di Lucente , Hai M. Nguyen , Vikrant Singh , Lee-Way Jin2, Izumi Maezawa2. RIP3 then converted the cancer cells to undergo necrosis in response to 1 2 acidosis. Conversely, inhibiting RIP3 reduced both acid-induced death of Pharmacology, University of California, Davis, CA, USA, Pathology and mouse cortical neurons in vitro and brain infarction in mice elicited by middle Laboratory Medicine, University of California, Davis, Sacramento, CA, USA. cerebral artery occlusion in vivo. Our data also implicate the formation of ne- þ crosome downstream of ASIC1a/RIP1 in response to acidosis in the presence of The voltage-gated K channel Kv1.3 has long been known to play an important RIP3. Thus, ASIC1a works similarly as conventional death receptors that regu- role in microglia. Our group recently demonstrated that Kv1.3 expression in- late both cell survival and demise. Differently controlling RIP3 expression and creases following differentiation into M1-like proinflammatory microglia and function may be of therapeutic values in treating different diseases. that the Kv1.3 inhibitor PAP-1 improves pathology in both mouse and rat models of ischemic stroke. Based on the observation that Kv1.3 expression 2701-Pos Board B308 is elevated in microglia in human Alzheimer’s disease (AD) brains we hypoth- The Function and Mechanism of TMEM16A/ANO1 in Pancreatic Cancer esized that Kv1.3 might constitute an attractive novel target for reducing neuro- Yonjung Kim1, Hyung Soon Park2. inflammation in AD. To determine if our in vitro observation of enhanced 1Department of Pharmacology, Yonsei Univ. College of Medicine, Seoul, Kv1.3 expression in M1-like microglia can be generalized to adult microglia, Korea, Republic of, 2Department of Medical Oncology, Yonsei Univ. we injected LPS intracerebroventricular (ICV) into WT and Kv1.3-/- mice. College of Medicine, Severance Hospital, Seoul, Korea, Republic of. Acutely isolated WT CD11þ cells showed increased Kv1.3 current densities Pancreatic cancer is the eighth leading cause of cancer death and is difficult to and increased inflammatory cytokine production at 24 h. Hippocampal slices treat because clinical presentation is often late, and the disease is resistant to acutely obtained from LPS ICV-injected WT mice demonstrated a deficit of conventional chemotherapy. About 57% of pancreatic cancer patients are diag- LTP in Schaffer-commissural synapses. Kv1.3 knockout completely rectified nosed at distant metastasis status and five-year survival rate is only 2%. More this hLTP deficit. PAP-1 treatment after LPS-ICV had similar effects. We than 90% of pancreatic cancer has activating mutations in KRAS and recent next evaluated microglial Kv1.3 expression in the 5xFAD mouse model and exome sequencing studies have identified additional mutations in several kinds found that CD11þ cells from 4, 6, and 10 months old 5xFAD mice showed of genes such as AIRD1A, ARID1B, SMARCA1. However, most of mutational significantly increased Kv1.3 channel activity. In cultured microglia Kv1.3 in- genes are tumor suppressor genes and these are no effective druggable target in hibition with PAP-1 blocked AbO-induced microglia proliferation, p38MAPK pancreatic cancer. Hence, the study for new druggable target is unmet medical phosphorylation, NF-kB activation, and NO generation but not AbO phagocy- need. TMEM16 family genes consist of 10 genes that range from TMEM16A to tosis. In hippocampal slice cultures, which allow microglia and neurons to TMEM16K. Of these genes, TMEM16A, TMEM16B, and TMEM16F are known interact, PAP-1 inhibited AbO-induced microglia mediated neuronal killing. as ion channels. In addition TMEM16C, TMEM16D, TMEM16F, TMEM16G, We next evaluated PAP-1 in two mouse models of amyloid-induced neuropa- and TMEM16J are reported as phospholipid scramblase, but their exact physi- thology. In mice receiving bilateral intrahippocampal injection of AbO daily ological function is not yet known. Among TMEM16/ANO family, oral PAP-1 (50 mg/kg) for 8 days improved performance in the step-through TMEM16A/ANO1 has been shown to be a calcium-activated chloride channel passive avoidance test, while PAP-1 medicated diet administered for 5 months that is expressed in diverse tissues, including airway epithelia, smooth muscles, to APP/PS1 mice starting at 9 months of age improved their performance in and sensory neurons. Interestingly, over-expression of ANO1/TMEM16A has several tests evaluating memory deficits and reduced the cerebral Ab-amyloid been found in many tumor types including breast cancer, and head and neck load and soluble and insoluble Ab in brain homogenates as determined by cancer. However, the expression and function of TMEM16A in gastrointestinal ELISA. Taken together our results suggest that Kv1.3 should indeed be further cancer, such as pancreatic cancer, has not been revealed. To investigate the investigated as a potential pharmacological target for the treatment of Alz- function and mechanism of TMEM16A/ANO1 in pancreatic cancer, we heimer’s disease and other conditions accompanied by neuroinflammation. conduct MTT assay and immunoblotting using over expression system in Supported by NIH (NS098328, AG043788 and AG038910).

BPJ 7904_7909 Wednesday, February 15, 2017 549a

Other Channels II 2706-Pos Board B313 Tracking Pore Hydration within the Red-Activatable Channelrhodopsin 2704-Pos Board B311 ReaChR by Site-Directed Labeling with Infrared-Active Azido Probes Lysenin Gating Implies Strong Interaction between the Voltage-Gating Benjamin S. Krause1, Joel C.D. Kaufmann2, Jens Kuhne3, Sensor and the Bilayer Membrane: An Ionic Screening Study Johannes Vierock1, Thomas Huber4, Thomas P. Sakmar4, Klaus Gerwert3, 1 1 2 2 Franz J. Bartl2, Peter Hegemann1. Samuel R. Kosydar , Kaitlyn S. Ware , Sheenah Bryant , Nisha Shrestha , 1 2 2 Institute of Biology, Experimental Biophysics, Humboldt Universit€at zu Charles Hanna , Daniel Fologea . 2 1Department of Physics, Boise State University, Boise, ID, USA, Berlin, Berlin, Germany, Institute for Medical Physics and Biophysics, 2 Charite´ Berlin, Berlin, Germany, 3Department of Biophysics, Department of Physics/Biomolecular Sciences Graduate Program, Boise 4 State University, Boise, ID, USA. Ruhr-University Bochum, Bochum, Germany, Laboratory of Chemical Lysenin, a pore-forming toxin extracted from the earthworm E. foetida,in- Biology and Signal Transduction, Rockefeller University, New York, NY, serts large conductance channels into membranes containing sphingomyelin. USA. A salient feature of lysenin channels is the history-dependent gating in Ion channels like Channelrhodopsins (ChRs) constitute at least two distinct response to periodic transmembrane voltages for which the period greatly states, one is permeable (conducting) for charges and one is not (non-con- exceeds the relaxation time of the voltage-induced gating. Our work focuses ducting). Mostly, ion transport across a membrane requires a continuous wa- on understanding the molecular mechanisms by which lysenin channels gain ter channel throughout the entire protein, which has to build up upon history-dependent functionality. We hypothesized that lysenin channels, activation by a certain stimulus, e.g. ligand, substrate or for ChRs with light, upon interaction with electric fields, undergo conformational changes that and collapses succeeding cessation of the activating trigger. Within the last induce gating by pushing the voltage-domain sensor deep inside the bilayer years, researchers have intensively studied the mechanism how pore- membrane. Our experiments aim at testing this hypothesis by analyzing the formation is achieved in ChRs but still a profound understanding, especially effects of ionic screening induced by addition of mono and multivalent ions on the closing event, is lacking. In the current study, we introduced the arti- to the support electrolyte. Results demonstrate that ionic screening weakens ficial IR-active probe (p-azido phenyl alanine) in ChR at certain sites align- ing the ion pathway by amber stop codon suppression. By exploiting its the electrostatic interactions between the external electric field and the 1 voltage domain sensor, which manifests as a significant rightward shift of unique spectral properties (nas ~ 2100 cm ) and its sensitivity towards po- the channels’ open probability in response to ascending voltage ramps larity changes, we tracked hydration dynamics within the pore region and that elicit channel closure. The shift is both concentration and electrova- the inner gate in the red-activatable ChR (ReaChR) by infrared techniques. lence dependent, characteristic of ionic screening. However, once channels Our data imply that channel closure coincides a late dehydration event close, descending voltage ramps, which elicit channel reopening, show no happening within the interface of the central and the inner gate. Further- major effects of ionic strength on the open probability. Moreover, the reac- more, site-directed mutagenesis of inner gate residues suggest that this tivation open probability is invariant irrespective of ionic conditions. Conse- spatial constriction is essential for preventing intracellular water influx quently, we propose a model of hysteresis based on gating that is within the closed configuration of the ion channel. Thus, alteration of the accompanied by significant movement of the voltage domain sensor. While inner gate structure by mutations leads to water invasion inside the intracel- the voltage domain sensor is exposed to external aqueous solutions it is sub- lular part of the pore already in the dark state, establishing a prehydrated ject to screening, but gating pushes the domain deep within the membrane, closed state. from where the ions are excluded owing to the high energy cost. Therefore, gating induces major changes in the energy landscape, which may explain 2707-Pos Board B314 the molecular memory of lysenin channels and the invariance of the reacti- Mechanism of Water and Solute Cotransport by the Sodium Glucose vation pathway. Cotransporter SGLT1 Christine Siligan1, Andreas Horner1, Sergey Akimov2, Peter Pohl1. 1 2 2705-Pos Board B312 Institute of Biophysics, JKU Linz, Linz, Austria, A.N. Frumkin Institute of Subunits that form Trimeric DEG/ENaC Mechano-Electrical Transduc- Physical Chemistry and Electrochemistry, Russian Academy of Sciences, tion Channels in Touch Receptor Neurons Moscow, Russian Federation. Sylvia Fechner1, Frederic Loizeau2, Adam L. Nekimken2, Beth L. Pruitt2, The molecular mechanism of carrier mediated water and solute cotransport is Miriam B. Goodman1. unresolved. It has long been believed that water may be engulfed into protein- 1Molecular and Cellular Physiology, Stanford University School of aceous cavities and thus be moved along with the solute in a fixed stoichio- Medicine, Stanford, CA, USA, 2Mechanical Engineering, Stanford metric ratio from one side of the membrane to the other. Since solute and University, Stanford, CA, USA. solvent flux measurements through the potassium-chloride-cotransporter or In C. elegans, the behavioral response to touch and the underlying molecular the glucose sodium cotransporter (SGLT1) in epithelial monolayers and/or re- actors are thoroughly studied. However, it is still debated which and how constituted lipid bilayers did not confirm stoichiometric coupling, we tested many of several co-expressed pore-forming subunits assemble into a func- the hypothesis that water and glucose share part of their pathway through tional, multimeric mechano-electrical transduction (MeT) channel in vivo. SGLT1. To this end we reconstituted the purified SGLT1 into lipid vesicles Two members of the DEG/ENaC channel family, MEC-4 and MEC-10, are and exposed the proteoliposomes to osmotic gradients of different origin. important to convert touch into behavioral responses. MEC-4 is required to SGLT1 facilitates passive water flow. Its unitary water permeability pf was form native MeT channels, whereas MEC-10 plays a regulatory role. higher in the presence of than in the presence of glucose indicating Recently, a third homologous protein and potential subunit, DEGT-1, was a reflection coefficient s of glucose smaller than one. The decrease pf was identified; its role in touch sensation remains incompletely understood. Given due to occlusion of the water pathway by bound glucose molecules. Mutation that DEG/ENaC proteins are thought to assemble as trimers, the presence of a of the glucose binding pocket released the block. The glucose concentration at third, homologous protein opens new questions regarding the composition of which pf dropped to 50 % of its maximal value characterizes the glucose af- native MeT channels. A key outstanding question is which of these homolo- finity of SGLT1’s inwardly open conformation. It was two orders of magni- gous proteins co-assemble to form the channels responsible for touch sensa- tude larger than the affinity in the outwardly open conformation, i.e. the tion. With the gentle touch assay, we detected a more severe defect in previously reported concentration at which SGLT1 achieves half of its touch sensation if both MEC-10 and DEGT-1 were removed simultaneously maximum rate. Thus glucose transport requires conformational transitions than we did by removing them individually. This genetic enhancement sug- while water is channeled as long as the glucose binding site is unoccupied - gests that DEGT-1 is part of the native MeT channel complex. To investigate explaining as to why both substrates are transported at despairingly different this further, we are analyzing the contribution of DEGT-1 to native MeT rates. channels and to channel activity in Xenopus oocytes. Using in vivo patch clamp recordings of wild type and mutant touch receptor neurons lacking in- 2708-Pos Board B315 dividual subunits or expressing proteins with altered pore domains, we seek to Chimeric Reveal Complexities of Electrical Rectification delineate how DEGT-1 contributes to the mechanosensitivity, voltage- Jamal B. Williams, Martha Skerrett. dependence and adaptation of the native MeT current. In Xenopus oocytes, Biology, SUNY Buffalo State, Buffalo, NY, USA. we find that DEGT-1 is unable to form homomeric channels on its own, The innexins ShakingB Lethal (SBL) and ShakingB Neuralþ16 (SBN16) form but can assemble into functional channels in the presence of MEC-4. This heterotypic gap junctions in the Giant Fiber System of Drosophilia mela- finding suggests that, like MEC-10, DEGT-1 plays a regulatory role in chan- nogaster. The junctions favor arthodromic transmission of action potentials nel formation. and exhibit properties of rectification when expressed in vivo. Sequence

BPJ 7910_7914 550a Wednesday, February 15, 2017 analysis reveals that SBL and SBN16 are similar in size but differ significantly 2711-Pos Board B318 in sequence within the amino terminus (NT), first transmembrane domain Molecular Determinants of Photocurrent Kinetics of the Red Light (TM1) and first extracellular loop (EL1). It was previously shown that the Activable Channelrhodopsin Chrimson NT carries properties required for rectification in the sense that homotypic Johannes Vierock, Noam Nitzan, Peter Hegemann. ShakBL junctions can be induced to rectify by replacing the NT of SBL with Humboldt University of Berlin, Berlin, Germany. that of SBN16 in one of the cells. While this result provided a strong first Channelrhodopsin (ChR) are light gated ion channels which expressed in neu- impression about the importance of the NT in rectification, further experiments rons enable the temporal and spacial precise regulation of membrane voltage suggest complex interactions and structural features are required. This is high- and action potential firing by light [1]. Recently an extended screen of 61 po- lighted by the behavior of the reciprocal chimera (SBN16 with the NT of SBL) tential ChR genes for photocurrents in human kidney cells discovered a far-red which does not induce rectification when paired heterotypically with SBN16 activable ChR from the freshwater algae Chlamydomonas noctigama CnChR1 but displays voltage-dependent gating similar to SBL when paired homotypi- also named Chrimson [2]. Chrimson shows peak photocurrents at 590 nm more cally. We previously suggested that the NT but must be paired with an appro- than 50 nm further red shifted than previous ChRs and is therefor best suited for priate body in order to induce voltage-dependent rectification and are optogenetic experiments requiring deep tissue penetration or dual color activa- further investigating features of the innexin channel essential for rectification. tion with blue and red light. Photocurrent kinetics are key properties of ChRs We are also investigating the role of various innexin domains in transjunctional determining their potential optogenetic application. Whereas fast photocurrent (Vj)-dependent gating. Using chimeric constructs that focus on domains with kinetics enable the temporally precise triggering of high frequency action po- significant sequence variation between SBL and SBN16 (namely NT, TM1 tential trains, slow photocurrent kinetics increase the operational range of and E1) in combination with Xenopus oocyte expression and electrophysiology ChRs to lower light intensities and enable the prolonged manipulation of mem- we hope to provide insight into the structural determinants of electrical rectifi- brane voltage by single low intensity light pulses. In the well-characterized cation and Vj-dependent gating in innexin-based junctions. The attributes of us- CrChR2 from Chlamydomonas reinhardtii the residue D156 has been identified ing splice variants, which are unique to innexin-based gap junctions, will be as a key determinant of photocurrent kinetics possibly participating in the re- discussed. protonation of the retinal Schiff base [3]. In Chrimson this aspartate is not conserved. Interestingly photocurrent kinetics are still fast indicating different kinetic determinants in Chrimson than in CrChR2. By sight-directed mutagen- 2709-Pos Board B316 esis we investigated amino acid substitution in Chrimson compared to CrChR2 Figuring Out Fluoride Transport in the inner gate, central gate and in the retinal binding pocket and functionally Benjamin McIlwain, Randy Stockbridge. characterized kinetic properties of these mutants in whole cell patch clamp University of Michigan, Ann Arbor, MI, USA. measurements. Whereas specific substitutions in all three regions of the protein This newly discovered family of membrane proteins is able to select for F- were affecting channel kinetics, especially the retinal binding pocket proved over other anions on the order of 10,000- fold. It is a small, dimeric, dual- essential for fast photocurrent kinetics. topology protein with two large aqueous vestibules extending into the mem- References brane, bookended by F- conducting polar tracks. The X-ray crystal structure [1] F. Zhang and J. Vierock et al., Cell, 2011. shows four bound F- ions, in two parallel pores, but the route to the internal [2] N. C. Klapoetke et al., Nat. Methods, 2014. and external solutions is not apparent. Likewise, the role of the large aqueous [3] C. Bamann et al, Biochemistry, 2010. vestibules, and whether they make up part of that route, is unknown. Using a combination of mutagenesis, single-channel electrophysiology, and X-ray 2712-Pos Board B319 crystallography, we identify residues that define the full pathway of F- ions Searching for a Potential Voltage Sensor Intrinsic to Pannexin Channels through the channel. Kevin Michalski, Toshi Kawate. Cornell University, Ithaca, NY, USA. 2710-Pos Board B317 Pannexins are a family of large, oligomeric ion channels involved in a number Effect of Electroosmotic Flow on the Transport of a-Cyclodextrin through of broad physiological processes. As ATP release channels, pannexins have the Channel CymA important implications for purinergic signaling pathways, especially in context Jigneshkumar Dahyabhai Prajapati1, Satya Prathyusha Bhamidimarri2, of neurological signaling where ATP acts as a neurotransmitter, or in context of Mathias Winterhalter2, Ulrich Kleinekathoefer1. vascular signaling, where ATP regulates vasoconstriction. Pannexin-1 is the 1Department of Physics and Earth Sciences, Jacobs University Bremen, best characterized member of the pannexin family largely because it presents Bremen, Germany, 2Department of Life Sciences and Chemistry, Jacobs readily observable currents when expressed heterologously and studied using University Bremen, Bremen, Germany. patch clamp electrophysiology. Curiously, pannexin-1 channels can be acti- CymA, an outer of Klebsiella oxytoca, allows the passive vated using strong depolarizing voltage potentials (greater than þ20 mV), sug- diffusion of the bulky molecule a-cyclodextrin (a-CD, M.W. 972.8 Da) to the gesting the presence of a voltage sensing motif not found in other members of periplasm of the bacterium [1]. In single channel electrophysiology experi- the pannexin family. To localize a potential voltage sensor in pannexin-1, we ments, the flow of the uncharged a-CD was found asymmetric with respect borrowed domains from pannexin-3 - a member of the pannexin family not to the applied voltage and ionic salts used. The net water current associated activated by a voltage stimulus - and generated chimeric channels. Interest- with the ion movement, i.e., the so-called electroosmotic flow (EOF), is ingly, we find that the extracellular and intracellular domains of pannexin-1 induced by the ionic selectivity of the pore. This effects has been found to are dispensable for voltage sensing, suggesting an intrinsic voltage sensor be a major factor behind the modified interactions of the a-CD molecule within the transmembrane domains of pannexin-1. with the channel [2]. To get atomistic insight into the EOF on a-CD perme- ation, we have performed ~40 ms free energy calculations in presence of three 2713-Pos Board B320 different ionic conditions, i.e., in the absence of ions, in the presence of 1 M Characterization of Structural Changes via Lipid Regulation of KIR2.1 in KCl and of 1 M MgCl2, using well-tempered matadynamics simulations [3] a Lipid Environment applying an external field of 0 V, þ1Vand1 V. No major changes in the Joshua Brettmann, Sunjoo Lee, Shizhen Wang, Colin G. Nichols. free energy landscapes were observed in the absence of ions at both polarities Department of Cell Biology and Physiology, Washington University, of the voltage. This finding indicates the absence of an electrophoretic effect St Louis, MO, USA. on the neutral a-CD molecule and of an EOF mediated effect due to the Regulation of ion channel activity occurs through various mechanisms, via absence ions. However, using an electric field together 1 M KCl salt, we small molecule regulators, membrane potential, and specific interaction with observed significant free energy changes in the transport of the a-CD consis- lipid molecules. Inward rectifying potassium (Kir) channels conduction is tent with net EOF at both voltage polarities. Moreover, using 1 M MgCl2 salt, controlled both by small molecules, such as polyamines blocking ion conduc- we demonstrate an alteration of the pore selectivity from cationic to anionic. tion, and lipid molecules that stabilize a conductive state. Advancing molecular Thereby, the direction of the resulting EOF at a particular voltage polarity and understanding of this lipid regulation has been difficult as many structural tech- its effect on the a-CD permeation is inverted. These results highlight the role niques rely on detergent solubilized channels, which removes specific channel:- of the EOF in the transport of a-CD through the nanometer-sized CymA lipid interaction. FRET, both macroscopically and in single molecules, can help channel. surmount this issue, providing measurements of structural and dynamic [1] van den Berg B. et al., Proc. Natl. Acad. Sci. USA 2015, 112, E2991-E2999. changes induced by lipid regulation in a membrane environment. We have [2] Bhamidimarri, S. P. et al., Biophys. J. 2016, 110, 600-611. used macroscopic FRET to measure lipid specific structural changes in [3] Barducci, A. et al., Phys. Rev. Lett. 2008, 100, 020603. Kir2.1 in liposomes of defined composition. We have optimized purification

BPJ 7910_7914 Wednesday, February 15, 2017 551a and fluorophore labeling of the human Kir2.1 channel, which are reconstituted signaling molecules between cells. A single gap junction channel consists into liposomes of various lipid composition. We have measured PIP2- of two docked hemichannels composed of connexin proteins. While most con- dependent FRET changes at numerous sites, which indicate structural move- nexins form hemichannels that remain closed until docked, presumably to pre- ments of the intracellular domain of Kir2.1 upon PIP2 binding. This work spe- vent lysis, Cx50 and others can form functional hemichannels that open and cifically investigates how movements of the slide helix contribute to activation close in unopposed membranes. Gap junction and hemichannel function is of the channel. Further efforts will help to define the molecular details of bulk known to be sensitive to pH, with different showing different sen- anionic lipid and PIP2-dependent structural dynamics of Kir2.1 and help to un- sitivities. Mechanisms of pH-dependent closure of gap junctions can include derstand more generally how lipid-protein interactions regulate membrane pro- direct effects on the channel protein either through inter-domain interactions tein function. or protonation of residues in/near the channel pore, or indirect effects on modulating proteins or small compounds that subsequently interact with the 2714-Pos Board B321 channel. The cytoplasmic tail of connexins are the target of modulating pro- Electrophysiological Investigation of the Sodium Pump KR2 teins and can also participate in inter-domain interactions to affect channel Christiane Grimm, Arend Vogt, Peter Hegemann. properties, including pH sensitivity. Published studies have shown different Experimental Biophysics, Humboldt University, Berlin, Germany. and sometimes contradictory results regarding the effects of tail truncation KR2 is the first representative of a novel type of microbial rhodopsins and was on pH sensitivity, gating kinetics, and the ability to form functional hemichan- recently identified in the marine flavobacterium Krokinobacter eikastus. Like nels in non-junctional membranes. I will present studies focused on the other microbial rhodopsins, KR2 adopts a seven transmembrane helix fold voltage and pH sensitivity of a series of truncation sites in Cx50, thus and harbors a retinal cofactor covalently bound in helix seven making the pro- revealing the minimum tail length required to maintain functional hemichan- tein light sensitive. In the following years further pumps also belonging to this nels. One goal of these studies is to examine the remaining pH sensitivity of novel class of sodium-pumping microbial rhodopsins were identified. Neverthe- Cx50 channels after eliminating the confounding influences of inter-domain less, as the crystal structure of KR2 was solved twice in 2015, it remains the most and/or cellular protein interactions mediated through the cytoplasmic tail interesting representative of this new group. Inoue et al. (2013) claim that KR2 domain. in cell suspensions of the flavobacterium and E. coli pumps sodium out of the cell and converts into a proton pump in absence of sodium. Measurements on 2717-Pos Board B324 black lipid membranes and large unilamellar vesicles support a sodium pump Comparison of Second Messenger Permeabilities through Gap Junction activity of KR2. However, electrophysiological selectivity measurements in Channels Formed by CX43 and CX50 mammalian cells under defined intra- and extracellular ionic conditions as Virgis Valiunas, Laima Valiuniene, Peter R. Brink, Thomas W. White. well as membrane-voltage control are still lacking. We recently started a Physiology and Biophysics, Stony Brook University, Stony Brook, NY, USA. detailed electrophysiological analysis of KR2 and found that wild-type KR2 Connexins form intercellular channels, coordinating tissue activity via electri- is poorly membrane targeted in HEK293 and ND7/23 cells, which hampered cal and metabolic cell-cell coupling. A striking feature of connexins is their electrical characterization so far. Thus, we designed a variant of KR2, which diverse selectivity in intercellular passage of larger solutes, like second messen- shows significantly enhanced targeting of the pump to the cell membrane in gers. Previously, we quantified the permeability of cAMP for Cx43 channels, þ ND7/23 cells as seen by confocal microscopy. Analysis of the activity using determining the cAMP/K ratio to be 0.180, suggesting rapid cell to cell trans- whole-cell patch-clamp recordings showed that the new variant exhibits 6 times fer (J.Gen.Physiol,2008;131:293-305). 2þ higher photocurrents compared to wild-type KR2, which allowed electrophysi- Here, we compare the permeability of cAMP, IP3, and Ca through gap ological investigation of biophysical properties like light sensitivity or spectral junction channels of lens connexins: Cx43 and Cx50. Intercellular solute dependence of the activity. Ongoing electrophysiological experiments under transfer was investigated in HeLa cells expressing Cx43 or Cx50 and various extra- and intracellular ionic compositions reveal insights into the com- measuring cell to cell flux while simultaneously monitoring junctional plex ion selectivity and the ion transport mechanism of KR2. conductance via dual whole-cell/perforated patch clamp. For cAMP detec- tion, recipient cells were transfected with the cyclic nucleotide-modulated 2715-Pos Board B322 channel (SpIH), which acted as a reporter. cAMP was introduced via patch Voltage and Time Dependence of Amphotericin B Channel Activity in pipette into the cell not expressing SpIH within a pair. In the case of cAMP Lipidic Membranes diffusion to a neighboring SpIH transfected cell, SpIH- derived currents 2þ Karla S. Re´camier1, Iva´n Ortega-Blake1, Punit Parmananda2. increased over time. For IP3 and Ca detection, cells were loaded with 1 2þ 2þ Instituto de Ciencias Fı´sicas, Universidad Nacional Auto´noma de Me´xico, Fluo-8, the Ca binding dye. IP3 and/or Ca was delivered via patch Cuernavaca, Mexico, 2Department of Physics, Indian Institute of Technology pipette to one cell of a pair or to a monolayer while fluorescence intensity Bombay Powai, Mumbai, India. changes were recorded. 2þ Amphotericin B (AmB) is a polyene molecule that forms pores. It has been pro- Homotypic Cx43 channels were permeable to cAMP, IP3, and Ca over a wide posed that these pores consist of several monomers of the molecule in a barrel range of conductance. Conversely, homotypic Cx50 channels were impermeable 2þ configuration inserted in lipidic membranes. Although it is a drug used since the to IP3 and cAMP, while exhibiting high permeation to Ca . Negatively charged 1950’s, the molecular mechanisms by which it forms a pore are not well known. species (cAMP and IP3) showed a similar permeability order: Cx43>>Cx50. þ Earlier results showed that the time average conductance of Nystatin (another Positively charged Ca2 permeability was comparable: Cx43zCx50. polyene antibiotic) is function of the applied potential, greater potentials These results confirm that channels formed from certain connexins can discrim- inducing increased channel expression. In a recent work we found that the in- inate between solutes based on size and/or charge, suggesting that channel ternal dynamics on pore formation leads to the existence of an electric resonant selectivity is a key factor in cell signaling. Furthermore, second messenger 2þ frequency of voltage application. In this work, the tip-dip technique is used to permeability of Cx43 results in a rapid delivery of cAMP, IP3, and Ca be- characterize the voltage and time dependence of AmB channels in POPC and tween cells in sufficient quantities to trigger relevant cellular responses. Alter- 30% mol Cholesterol membranes. Therefore a square train of pulses with a fre- natively, reduced Cx50 permeability to cAMP and IP3 may play a role in quency of 0.5 Hz and jumps of 0-80, 0-100, 0-120 and 0-150 mV is applied to regulating cell division, differentiation, and homeostasis of the lens. the above membrane containing AmB. The results show similar behavior to the Supported by NIGMS 088181 (VV), NEI 013163 (TWW). Kþ macroscopic currents of Hudgkin and Huxley; a voltage and time depen- dence from the beginning of the pulse up to a stationary value. These experi- 2718-Pos Board B325 mental curves will be used to pursue the construction of a deterministic Investigation of Siderophore-Monobactam Antibiotic Derivatives: Their model that will help to understand the molecular processes involved in the Iron(III)-Complexes and Binding to Receptors expression of AmB channels. Mariano A. Scorciapino1,2, Giuliano Malloci3, Matteo Ceccarelli2,3, Funding: CONACyT 247031, INT/MEXICO/P-6/2012 DST (India) and Lucile Moynie4, James H. Naismith4, Eric Desarbre5, Malcolm Page6. DGAPA-PAPIIT-IG100416. 1Biomedical Sciences, Biochemistry Unit, University of Cagliari, Monserrato, Italy, 2Istituto Officina dei Materiali del Consiglio Nazionale 2716-Pos Board B323 delle Ricerche (IOM-CNR), Monserrato, Italy, 3Physics, University of Effects of Cytoplasmic Tail Truncations on Connexin 50 Hemichannel Cagliari, Monserrato, Italy, 4University of St. Andrews, St. Andrews, United Function Kingdom, 5Basilea Pharmaceutica, Basel, Switzerland, 6Jacobs University, Justine J. Jacobi, Derek L. Beahm. Bremen, Germany. Biology, State University of New York College at Buffalo, Buffalo, NY, USA. The ever increasing number of multiple drug resistant pathogens is one of the Gap junction channels form aqueous pores connecting the cystolic compart- most pressing global healthcare challenges. Novel antibiotic classes are ments of two cells, allowing for the direct transfer of ions, metabolites, and needed, but clear rules to drive optimization of existing drugs and de-novo

BPJ 7910_7914 552a Wednesday, February 15, 2017

design represent the actual innovation gap, especially against Gram-negative A/C g-aminobutyric acid receptor (GABAA/CR). Over the last years, impor- bacteria. These are particularly challenging due to their outer membrane, tant structural information emerged with the structure determination of the which works to modulate the entry of chemical species under strict control. b3 GABAR, the 5-HT3AR, the a1anda3GlyRandthea4b2nAChR. Among the possible strategies to overcome this barrier, siderophores suggest Despite this tremendous progress crystal structures of integral homopenta- an intriguing one, the Trojan horse strategy. Bacteria make use of endoge- meric nAChRs are still lacking. Previously, we identified a putative nAChR nous/exogenous chelating agents, the siderophores, to capture Fe3þ from in the genome of the extreme thermophile worm Alvinella pompejana, the environment. Complexes are then retrieved by specific outer membrane named Alpo4, which we can express and purify in sufficient amounts for receptors that work within multi-proteins machineries. By connecting/ structural studies. Following solubilization with a variety of detergents mimicking an antibiotic to a siderophore it is possible to trick bacterial de- we observed that Alpo4 is biochemically stable and assembles into pentam- fenses. Despite considerable efforts expended in both natural and synthetic ers as observed with electron microscopy. In this study, we further charac- conjugates, only few compounds have met with success. As crystal structures terize the functional properties of Alpo4 after reconstitution of purified of siderophore receptors are becoming available, increased understanding of protein in planar lipid bilayers and subsequent patch clamp electrophysi- siderophore-metal complex recognition will certainly help to develop new ology. Our recordings indicate that Alpo4 can be activated by the agonists siderophores and linkages with improved performance. BAL30072 is an or acetylcholine. Furthermore, currents can be modulated by allo- interesting monocyclic b-lactam antibiotic belonging to the sulfactams. It steric modulators such as ivermectin or PNU-120596 and subsequently was designed from tigemonam by introducing an iron chelating 1,3-dihy- blocked by a pore blocker such as memantine or a competitive antagonist droxy-4-pyridinone group. It shows remarkable activity against important such as a-bungarotoxin. This pharmacological profile suggests that Alpo4 Gram-negative pathogens, including multidrug-resistant Pseudomonas aeru- is a representative member of the nAChR subfamily. Ongoing experiments ginosa and Acinetobacter sp. isolates. In this work, BAL30072 and several aim to quantify the single channel properties of Alpo4. Collectively, the re- analogues have been compared in terms of metal-complex formation in sults from our functional and biochemical studies suggest that Alpo4 is a different environments, namely, dimethylsulfoxide and water at different suitable candidate for future structure determination of an integral homo- pH. NMR titrations have been performed with Ga3þ ions in order to retrieve pentameric nAChR. stoichiometry and relative stability of the complexes. Inter-proton distances were also determined in some instances and the corresponding molecular 2721-Pos Board B328 model formulated. The experimental data have been complemented by a Acetylcholine Receptor Gating: Click-Twist-Tilt-Rip-Pop combination of quantum and classical MD simulations to elucidate structural Shaweta Gupta, Srirupa Chakraborty, Ridhima Vij, Tapan K. Nayak, and dynamical properties of compounds. Molecular docking was then per- Anthony Auerbach. formed to investigate the binding to the putative BAL30072 receptor in P. Biophysics, University at Buffalo, Buffalo, NY, USA. aeruginosa, PiuA. We propose a testable model for AChR C4O gating that is consistent with the following. i) Mutational energy changes are mostly local. Mutant-cycle 2719-Pos Board B326 analyses show that most side chains separated by >15A˚ do not interact, TMEM16A Mediates the Fast Block to Polyspermy in Xenopus Laevis Eggs except for the aM2M3 linker that interacts significantly with residues at the Katherine L. Wozniak, Brianna L. Mayfield, Anne E. Carlson. binding sites. ii) Gating is a sequential process. Phi gives the free energy Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, character (structure) of an amino acid at the gating transition state on a scale USA. from 1 (O-like) to 0 (C-like). Phi values are distributed as 5 Gaussians and Ca2þ-activated Cl- channels (CaCCs) mediate some of the most important decrease domain-wise binding sites to gate, except for the aM2M3 linker physiologic processes, such as the regulation of neuronal and cardiac excit- that has the same phi as the binding sites. iii) There is a transition state ability, electrolyte balances in secretory epithelia, and olfactory transduction. ensemble (TSE). The number and relative heights of energy barriers in the However, it wasn’t until 2008 that transmembrane proteins with unknown TSE can be computed from phi values. There are 4 short-lived intermediate function 16 (TMEM16a) was identified as a bona fide CaCC. One of the states, and opening is uphill. iv) It takes time to cross the TSE. We assume cell types that TMEM16a was originally identified in was Xenopus laevis oo- a~5ms shut component in single-channel recordings (‘flip/primed’) reflects cytes. Although TMEM16a is present in these oocytes, we do not know if it sojourns in the TSE. Simulations suggest that the lifetime of each TSE inter- is maintained during oogenesis and functional in the fertilization-competent mediate is ~1 ms. v) C and O are stable. The opening/closing rate constants of egg. During fertilization in X. laevis,aCa2þ-activated Cl- current is evoked WT adult AChRs are (ms1) ~50/2 with 2 bound ACh and ~106/10 without in a pathway known as the fast polyspermy block, which inhibits sperm from any agonists. vi) Structures. There are locally-closed GLIC structures. In entering an already fertilized egg. Here, we sought to uncover the molecular opening the extracellular domain (ECD) rotates/compacts, membrane helices identity of the Cl- channel mediating this polyspermy block. We tested move and the gate unpacks. whether the rate of the fertilization-evoked depolarization was altered in In our model, opening starts with coupled fluctuations at the aM2M3 linker and eggs inseminated in the presence of TMEM16a-targeting small molecule in- the transmitter-binding sites (click), followed by rotation of the ECD (twist), hibitors including: MONNA, Ani9, CaCCinh-A01, and T16ainh-A01. We rearrangement of the helices (tilt), dilation of the gate (rip) to form a bubble reasoned that the rate of depolarization is proportional to the number of that collapses to initiate ion flow (pop). Closing is the reverse and starts with channels opened and thus looked for changes in the depolarization rate. bubble formation (the main structural correlate of ‘flip’). The most We found that the depolarization of the fast block was significantly slowed energetically-unfavorable step in opening is twisting. or completely prevented in eggs inseminated in the presence of these TMEM16a inhibitors. To verify that the TMEM16a-targeted inhibitors block 2722-Pos Board B329 2þ - the X. laevis version of the channel, we recorded the Ca activated Cl cur- A Fluorescent Agonist of the Muscle Nicotinic Acetylcholine Receptor 2þ rent evoked by uncaging IP3 to evoke Ca release from the endoplasmic re- Abhilasha Ladha1, Vera Martos2, Andrew Plested3, Jana Kusch1. ticulum, in X. laevis oocytes using two electrode voltage clamp. We found 1Institute of Physiology II, University Hospital Jena, Jena, Germany, that these inhibitors indeed were effective on the X. laevis TMEM16a chan- 2Medicinal Chemistry, Leibniz Institute of Molecular Pharmacology, nels. Taken together, these results suggest that TMEM16a is conserved Berlin, Germany, 3Molecular Neuroscience and Biophysics, Leibniz Institute through the oogenesis process and that TMEM16a mediates the fast block of Molecular Pharmacology, Berlin, Germany. to polyspermy in X. laevis. The muscle-type nicotinic acetylcholine receptor (nAChR) is expressed at the neuromuscular junction, where it mediates the fast synaptic response Ligand-gated Channels II upon ACh release from a motoneuron. It is one of the most studied ion channels and the gating mechanism has been described intensively. How- 2720-Pos Board B327 ever, the reciprocal relationship between activation state and agonist binding Functional Characterization of a Nicotinic Acetylcholine Receptor from an is still elusive. Herein, we adapted a challenging approach, the so-called Extremophile Worm confocal patch-clamp fluorometry (cPCF) (Biskup et al., Nature 2007, Eveline Wijckmans, Florian Delbart, Chris Ulens. 446:440-3) to study state-dependent agonist binding and unbinding behavior Laboratory of Structural Neurobiology, KU Leuven, Leuven, Belgium. in nAChRs. We introduce a novel fluorescently tagged ACh derivative, Cys-loop receptors (CLRs) or pentameric ligand-gated ion channels fACh, which is an excellent congener of the native neurotransmitter agonist (pLGICs) are a class of integral membrane proteins involved in fast excit- for at adult muscle-type nAChRs. For whole-cell recordings, HEK cells atory or inhibitory synaptic neurotransmission. Known representatives of transiently transfected with muscle nAChRs were lifted and positioned the family are the nicotinic acetylcholine receptor (nAChR), the in front of a double-barreled application pipette. Fast solution exchange serotonin-3 receptor (5-HT3R), the glycine receptor (GlyR) and the subtype was realized by a piezo-driven device. fACh was found to be similarly

BPJ 7910_7914 Wednesday, February 15, 2017 553a

efficacious as ACh (Imax,fACh/Imax,ACh=0.9750.01) and, surprisingly, 2725-Pos Board B332 slightly more potent (fACh: EC50=1.1mM; ACh: EC50=2.8 mM). The Thermoanalytical Characterization of Pentameric Elic Based on Detergent desensitization kinetics was similar for both compounds. For confocal fluo- Size rescence imaging, fACh was excited with a 543 nm HeNe laser line. To Ty E. Whisenant1,2, Benjamin W. Elberson1,2, Doris M. Cortes1,2, define the cell position for confocal imaging and for background signal Luis G. Cuello1,2. subtraction, background was stained with a reference dye Dy647 (1mM), 1Molecular Biophysics, Texas Tech University HSC, Lubbock, TX, USA, excited with a 633 nm HeNe laser line. Exposure of transfected cells to 2Center for Membrane Protein Research, Lubbock, TX, USA. fACh, led to fluorescence signals that were absent in non-transfected con- Differential Scanning Calorimetry has long been used for thermal stability trol cells. Agonist binding and unbinding followed a bi-exponential time studies of proteins. Erwinia Chrysanthemi Ligand-Gated Ion Channel (ELIC) course. Interestingly, the second phase of fluorescence increase during is a prokaryotic analogue of the nicotinic Acetylcholine Receptor (nAChR), application of saturating fACh was similar to the time course of desensitiza- a eukaryotic pentameric Ligand-Gated Ion Channel (pLGIC) responsible for tion, which might reflect the widely accepted concept of the desensitized action potential propagation through nerve cells. Elucidation of the melting state having a higher affinity for ACh than the resting state (Barrantes, temperatures (Tm) of the extracellular domain (ECD) and the transmembrane Biochem Biophys Res Commun. 1976, 72:479-88). In summary, we demon- domain (TMD) were achieved using ‘‘Intermediate’’ (C10, C11 and C12) and strate the feasibility of exploiting fluorescent agonists to shed light on the ‘‘Long-Chained’’ maltoside detergents (C13 and C14). Studies using the ‘‘In- activation mechanisms of the nAChR and other members of the Cys-loop termediate’’ maltosides suggest increased stability by over 8C to the hydro- superfamily. phobic TMD without altering the hydrophilic ECD’s stability. There is an obvious correlation between increased length of the detergent and increased 2723-Pos Board B330 stability of the TMD. However, ‘‘Long-Chained’’ maltosides appear to provide The Intracellular Domain of Pentameric Ligand-Gated Ion Channels and such a large increase in thermal stability of the TMD (>15C) that it is its Effect on Receptor Conductance in GLIC Chimeras conveyed to the ECD, as well, resulting in an increase of over 4C. Thus, by Antonia Stuebler, Pablo Artigas, Michaela Jansen. manipulating the detergent length we were able to separate and identify the Texas Tech University Health Science Center, Lubbock, TX, USA. Tm’s of the TMD and ECD. As well, we have provided novel stability data The pentameric ligand-gated ion channel (pLGIC) superfamily includes the for improving crystal structures and other detergent-based experimentation. nicotinic acetylcholine (nACh), 5-HT3, -aminobutyric acid type A (GA- AHA- 11SDG5440003, NIH 1RO1GM097159-01A1 and Welch Foundation BAA), and glycine receptor. These channels are made up of five homologous BI-1757. subunits surrounding a central ion channel pore and can be divided into three pentameric domains. The extracellular domain (ECD) and transmem- 2726-Pos Board B333 brane domain (TMD) of the channels show high levels of homology and Biochemical and Thermodynamic Characterization of the Erwinia have been well studied and structures have been published. In contrast, Chrysanthemi Ligand-Gated Ion Channel the intracellular domain (ICD) shows sequence and length divergence in Benjamin W. Elberson1,2, Ty E. Whisenant1,2, D. Marien Cortes1,2, the different receptors. Mutations in the ICD have been shown to alter re- Luis G. Cuello1,2. ceptor conductance. Gloeobacter violaceus LGIC (GLIC) is a prokaryotic 1Molecular Biophysics, Texas Tech University HSC, Lubbock, TX, USA, pLGIC without a native ICD, which can be expressed and studied in Xeno- 2Center for Membrane Protein Research, Lubbock, TX, USA. pus oocytes. To elucidate the mechanisms by which the ICD affects the Pentameric ligand-gated ion channels (pLGICs) are responsible for producing channel pathway and conductance we have created chimeras of GLIC either an inhibitory or excitatory downstream response - depending on the per- ECD and TMD with ICDs of both anion- and cation-conducting pLGICs meant ion - when a nerve stimulus reaches a synaptic cleft. Prominent exam- (GABAr1, Glya1, nAChRa7, 5-HT3A). Functional expression of chimeras ples include the GABA receptor, which is responsible for inhibitory was first confirmed by applying low pH (pH 5) pulses to oocytes under neurotransmission and is the target of some types of anti-epileptic medica- two-electrode voltage clamp. We have begun to study the single channel tions, and the nicotinic acetylcholine receptor, which is responsible for prop- conductance and gating kinetics of chimeric channels and confirmed that agation of nerve impulses resulting in muscle contraction. As these eukaryotic the GLIC wildtype has a single-channel conductance of 10pS. For the channels can be difficult to express, purify, and study, much of the work is GLIC-5-HT3A-ICD chimera, we expect to see a decrease in conductance done using prokaryotic analogues. Erwinia chrysanthemi Ligand-gated Ion as we observed an increase in a construct of 5-HT3A upon removing the Channel (ELIC) is one such prokaryotic channel which we have found to ICD. Amino acid engineering in both the chimeras and eukaryotic parent be amenable to biochemical and structural characterization. The first portion channels will then be used to further understanding of how the ICDs of this study - and the topic of this presentation - consists of a novel purifica- contribute to ion conducting properties. tion scheme such that the quality, yield, and cost of ELIC are substantially improved. We are able to produce 16x more ELIC at 202x less cost per milli- 2724-Pos Board B331 gram when compared to typical protocols. These improvements are validated Barbiturates Bind in the GLIC Ion Channel Pore and Cause Inhibition by by Differential Scanning Calorimetry (DSC) and Fast Protein Liquid Chroma- Stabilizing a Shut State tography (FPLC). Our successes have enabled us to begin work determining Zeineb Fourati1, Reinis R. Ruza1, Duncan Laverty2, Emmanuelle Drege3, the structural dynamics of ELIC primarily using Substituted Cysteine Scan- Sandrine Delarue-Cochin3, Delphine Joseph3, Patrice Koehl4, Trevor Smart2, ning Mutagenesis (SCAM) and Continuous-Wave Electron Paramagnetic Marc Delarue1. Resonance spectroscopy (CW-EPRs). 1Structural Biology and Biological Chemistry, Institut Pasteur, Paris, France, AHA- 11SDG5440003, NIH 1RO1GM097159-01A1 and Welch Foundation 2Neuroscience, University College London, London, United Kingdom, BI-1757. 3Faculte de Pharmacie, Universite Paris-Sud, Chatenay Malabry, France, 4Computer Science, UC Davis, Davis, CA, USA. 2727-Pos Board B334 Barbiturates induce anesthesia by modulating the activity of anionic and Deciphering Anesthetic Action of Noble Gases through their Modulation of cationic pentameric ligand-gated ion channels (pLGICs). Despite more than Membrane Protein and Lipid Bilayer Properties a century of use in clinical practice, the prototypic binding site for this class Samuel Murail1,2, Pluton Pullumbi2, Marc Baaden1. of drugs within pLGICs is yet to be described. In this study, we present the 1Laboratoire de Biochimie The´orique, UPR 9080, IBPC, Paris, France, first X-ray structures of barbiturates bound to GLIC - a cationic prokaryotic 2Centre de Recherche Paris-Saclay, Air liquide, Jouy-en-Josas, France. pLGIC sensitive to general anaesthetics and, as shown here, to barbiturates, The molecular mechanism of action underlying general anesthesia remains a at clinically-relevant concentrations. Several derivatives of barbiturates con- matter of controversy. One proposed mode of action involves binding of anes- taining anomalous scatterers were synthesized and helped to unambiguously thetic molecules to membrane proteins such as ligand gated ion channels, identify a unique barbiturate binding site within the central ion channel thereby modulating their function. An alternative mode of action discussed pore at the level of the 6’ residue, in a closed conformation. Barbiturates in the literature is through binding to the lipid bilayer. We chose to explore do not bind the open state. In addition, docking calculations around the both modes of action at the molecular level by studying two types of targets: observed binding site for all three states of the receptor, including a model pure lipid bilayer systems such as POPC, and a lipid bilayer with the pentame- of the desensitized state, showed that barbiturates preferentially stabilize the ric ligand-gated ion channel GLIC inserted into it. Sauguet et al. recently solved resting state. The identification of this pore binding site sheds light on the the structure of GLIC in complex with the general anesthetic Xenon [1], mechanism of barbiturates inhibition of cationic pLGICs and allows the ra- describing a multitude of binding sites. tionalisation of several structural and functional features previously observed Here, we studied the effect of a series of three Noble gases: Argon, Krypton and for barbiturates. Xenon. We have performed molecular dynamics (MD) simulations to gain key

BPJ 7910_7914 554a Wednesday, February 15, 2017 insights on the Noble gases binding location and affinity. Different gas:lipid ra- the accelerated channel dehydration observed in the ketamine-bound system. tios were used to characterize their effect on the physical properties of the The study provides a structural and dynamical basis for the inhibitory modula- membrane, as well as their affinity for the core of the lipid bilayer. tion of ketamine on pLGICs. Research supported by NIH R01GM066358. Microsecond-long flooding simulations, combined with free energy calcula- tions, were run to characterize access to binding sites and quantify binding af- 2730-Pos Board B337 finities of Nobles Gases on the GLIC channel. This work extends and Structural and Functional Evidence for Multi-Site Allostery Mediated by generalizes our previous study on bromoform action on GLIC, that revealed General Anesthetics in a Model Ligand-Gated Ion Channel 1 1 2 a complex network of interconnected binding sites, possibly all contributing Stephanie A. Heusser , Rebecca J. Howard , Zeineb Fourati , 2 1 in concert to the anesthetic effect [2]. We go beyond previous work by consid- Marc Delarue , Erik Lindahl . 1Department of Biochemistry and Biophysics, Stockholm University, ering the measurable effect on lipid bilayer properties induced by the Noble 2 gases therefore providing a better description of key pathways for anesthetic Stockholm, Sweden, Department of Structural Biology & Chemistry, Institut action. Pasteur, Paris, France. [1] Sauguet et al. Plos One. 2016. doi: 10.1371/ journal.pone.0149795 General anesthetics act as either positive or negative allosteric modulators of [2] Laurent, Murail et al. Structure. 2016. doi: 10.1016/j.str.2016.02.014 several pentameric ligand-gated ion channels, including physiologically impor- tant receptors for g-aminobutyric acid and acetylcholine. Although functional 2728-Pos Board B335 studies have implicated conserved sites of modulation in this channel family, Transmembrane Structural Determinants of Alcohol Binding and the limited scope and resolution of structural data for human Cys-loop receptors Modulation in a Model Ligand-Gated Ion Channel have hampered mechanistic studies of anesthetic action. We previously showed Rebecca J. Howard1,2, Stephanie A. Heusser1,O¨ zge Yoluk3, Oliver Snow1,3, the prokaryotic homolog GLIC to be a useful model system that recapitulates Go¨ran Klement1,3, Alex R. Mola2, Travers MD Ruel2, Erik Lindahl1,3. functional modulation of human ion channels, and enables structure determina- 1Biochemistry & Biophysics, Stockholm University, Stockholm, Sweden, tion both in apparent open and nonconducting states. Specifically, anesthetic in- 2Chemistry, Skidmore College, Saratoga Springs, NY, USA, 3Theoretical hibition of GLIC can be removed, reversed, or rendered bimodal by site- Physics, KTH Royal Institute of Technology, Stockholm, Sweden. directed mutations in the transmembrane domain (TMD). In this work, we pro- Pentameric ligand-gated ion channels are heavily implicated in neurological ef- vide crystallographic and electrophysiological evidence for a multi-site mech- fects of alcohol, yet a structural understanding of this process remains limited anism of bimodal modulation by anesthetizing agents, including the common by a lack of high-resolution data for pharmacologically relevant receptors. The surgical medication propofol. With the pore in an apparent nonconducting prokaryotic ligand-gated ion channel GLIC is a potentially valuable model sys- state, propofol bound at the intracellular (lower) end of the channel pore, tem whose structure has been determined in multiple conformations and bound similar to other inhibitors. Consistent with this result, single hydrophobic sub- to various ligands. In particular, modification of a key transmembrane position stitutions at the lower-pore site enhanced functional inhibition. Conversely, in in GLIC rendered it potently sensitive to potentiation, and enabled co- the apparent open state, anesthetics bound to one or more contiguous sites in the crystallization with ethanol as well as other anesthetic agents. To elucidate extracellular-facing (upper) end of the TMD, particularly for variants in which alcohol interactions with channel structure and function, we substituted a vari- anesthetic inhibition was reduced or reversed. In one novel variant exhibiting ety of amino acids in the ethanol site and channel pore, expressed the mutated anesthetic potentiation, propofol binding in the upper-TMD converted the channels in frog oocytes, and measured their gating and modulation properties channel from an apparent nonconducting- to open-pore conformation under by two-electrode voltage-clamp electrophysiology. We compared the resulting otherwise identical conditions, providing direct evidence for potentiation via changes in agonist and modulator sensitivity with standard amino acid proper- specific sites in the upper TMD. Based on these findings, we propose a struc- ties and molecular modeling of the predicted binding site and gating transitions. tural model for allostery in which anesthetic binding to spatially distinct We identified structural determinants of binding that may reflect distinct prop- TMD cavities differentially stabilizes opposing functional states of pentameric erties of alcohols relative to classic drugs, and could provide insight into other ligand-gated ion channels, providing valuable insights into channel modulation low-affinity modulators such as anesthetics and lipids. We further found that and drug development. modifying the ethanol pocket removed or even reversed effects of mutations 2731-Pos Board B338 in the ion channel pore, implicating tight coupling between the allosteric and Determinants of 5-Ht3A Intracellular Domain Oligomerization and RIC-3 active sites. Our ongoing work aims to integrate functional data with computa- Interaction tional models of receptor binding and channel gating. Elham Pirayesh, Akash Pandhare, Michaela Jansen. Cell Physiology and Molecular Biophysics, Center for Membrane Protein 2729-Pos Board B336 Research, Texas Tech University Health Sciences Center, Lubbock, TX, Ketamine Inhibition of Pentameric Ligand-Gated Ion Channels - Insights USA. from Molecular Dynamics Simulations The serotonin type 3A (5-HT3A) receptor is a homopentameric cation-selective Bogdan F. Ion, Marta M. Wells, Yan Xu, Pei Tang. member of the pentameric ligand-gated ion channel (pLGIC) superfamily. Anesthesiology, University of Pittsburgh, Pittsburgh, PA, USA. Members of this superfamily assemble from five subunits, each of which con- The anesthetic ketamine is well known as a non-competitive antagonist of N- sists of three domains, extracellular (ECD), transmembrane (TMD), and intra- methyl-D-aspartate receptors (NMDARs). However, not all the clinical effects cellular domain (ICD). Recently, it was demonstrated that 5-HT3A-ICD fused of ketamine can be fully explained by its inhibition of NMDARs. An alternative to maltose binding protein (MBP) forms stable pentamers in solution (Pan- mode of ketamine action is through inhibition of pentameric ligand-gated ion dhare, Grozdanov, Jansen, Sci. Rep. 2016). Also it has been shown that channels (pLGICs), such as nicotinic acetylcholine receptors. Ketamine in- MBP-5-HT3A-ICD directly interacts with the chaperone protein resistance to hibits the bacterial pLGIC from Gloeobacter violaceus (GLIC) and the inhibitors of choline esterase (RIC-3). To elucidate the nature of the oligomer- ketamine-GLIC co-crystallized structure was determined previously. Ketamine ization of the 5-HT3A-ICD and its interaction with the chaperone protein RIC-3 was bound to an intersubunit cavity that partially overlaps with the homologous we developed different MBP-fused constructs of this domain by deletion of antagonist-binding site. Here, we performed molecular dynamics simulations large portions of its amino acid sequence. We have expressed two mutants of GLIC crystal structures in the absence and presence of ketamine. Three par- (MBP-5-HT3A-ICD-A and MBP-5-HT3A-ICD-B) in Escherichia coli and puri- allel simulations were run for each GLIC system. Ketamine was parameterized fied them to homogeneity. Additionally, we have also purified RIC-3 to be uti- using the Force Field Toolkit based on protocols from CGenFF. Simulations lized in protein-protein interaction experiments. The oligomeric states of both show that asymmetric binding (only one or two ketamine molecules) is deletion constructs is probed using size exclusion chromatography together preferred to the symmetric binding of five ketamine molecules observed in with multi-angle light scattering. Additionally, using RIC-3 affinity pull the crystal structure. Asymmetric ketamine binding introduces a series of struc- down the interaction of MBP-5HT3A-ICD constructs and RIC-3 is investigated. tural changes starting from the binding site to the pore lining TM2 helices. The Further studies are directed toward deciphering the molecular determinants for presence of ketamine stabilized intersubunit salt bridges between loop C and ICD oligomerization as well as RIC-3 protein-protein interaction. the adjacent complementary subunit but destabilized salt bridges and critical hydrophobic interactions at the interface between the extracellular and trans- 2732-Pos Board B339 membrane domains (b1-b2$$$pre-TM1, b6-b7$$$TM2-M3). Interestingly, Characterizing the Intrinsic Assembly Behavior of the 5-HT3A Receptor these interactions in the subunits where ketamine was not bound were weak- Intracellular Domain ened to a greater extent than in the subunits asymmetrically bound to ketamine. Akash Pandhare, Michaela Jansen. Ketamine binding also introduced a significant increase in the population of Cell Physiology and Molecular Biophysics, Center for Membrane Protein TM2 lateral tilting angles shifting towards a closed channel conformation Research, Texas Tech University Health Sciences Center, Lubbock, TX, when compared to apo GLIC. These conformational changes contributed to USA.

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The domain topology of eukaryotic pentameric ligand-gated ion channels modes in the accessible b-/aþ and b-/gþ sites; of these, the b-/aþ site was (pLGICs) from the Cys-loop receptors superfamily exclusively contains three shown via AFEP to have an affinity stronger than the sevoflurane EC50, domains: the most diverse and at the same time least studied intracellular while the second-ranked site, b-/gþ was similar to sevoflurane EC50. These domain (ICD), the extracellular domain (ECD) and the transmembrane domain results are consistent with a requirement for binding of two molecules for (TMD). The recently published report from our laboratory has clearly estab- the general anesthetic to have an effect. If so, the higher potency of propofol lished that the engineered chimera comprised of the ICD of the 5-HT3A receptor relative to sevoflurane would originate from its high affinity for its second- downstream of a maltose binding protein (MBP) assembles into stable pentam- ranked site a-/bþ; sevoflurane did not spontaneously bind to either a-/ ers in solution (Pandhare A. et al., Sci. Rep. 6, 23921(2016)). Notably, the or- bþsite. dered assembly behavior assigns a novel role for the ICD in receptor oligomerization along with the other two domains, and further enriches its 2735-Pos Board B342 functional repertoire. Therefore, in order to fully characterize ICD oligomeri- Investigating Structural Transitions of GABA-A Receptors using zation, current studies are directed at identifying the molecular determinants Accelerated Molecular Dynamics of pentameric organization by employing site-directed mutagenesis as well Ole J. Andersen, Philip C. Biggin. as heterologous expression in E. coli and protein purification followed by Department of Biochemistry, University of Oxford, Oxford, United size exclusion chromatography in line with multi-angle light scattering Kingdom. (SEC-MALS). Additionally, we are studying the intrinsic assembly kinetics GABA (g-aminobutyric acid) is the main inhibitory neurotransmitter in the of pentamer formation to determine self-association affinities by utilizing mammalian central nervous system, and its actions are crucial for maintain- isothermal titration calorimetry. ing proper brain function. It binds to the GABA-A ion channel receptor that 2733-Pos Board B340 belongs to the cys-loop receptor family, controlling the flow of chloride ions into nerve cells. While several structures have been published of various cys- Development of a Pentameric, Non-Chimeric 5-HT3A Receptor Intracel- lular Domain Construct loop receptor states, including the resting, open and desensitized states, the underlying mechanisms controlling the functional transitions between these Mina Ahmadi, Akash Pandhare, Michaela Jansen. states are still poorly understood. Molecular dynamics (MD) simulations Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX, are often able to provide valuable information on protein dynamics, but USA. the time scale of the state transitions are beyond what is currently accessible 5HT-3 (serotonin) receptors are members of the pentameric ligand-gated ion to conventional MD simulations. To resolve this issue we have used an channels (pLGIC) superfamily. Each channel consists of five homologous enhanced sampling method, accelerated MD, to study these transitions using a recent crystal structure of the GABA-A b3-homopentamer. Simulations of subunits with three domains, namely extracellular domain (ECD), transmem- the desensitized-to-resting state transition correlate well with prevailing brane domain (TMD), and intracellular domain (ICD). So far, the ICD has not been well-studied due to the assumption of largely disordered structure. views in the literature, including closing of the activation gate at the 9’ To study the ICD in more detail, we have developed a soluble form of the 5- Leu, straightening of the channel-lining M2 helices, and an inward move- HT -ICD fused to the C-terminus of a modified maltose binding protein ment of the M2-M3 linker consistent with a recent electron microscopy 3A resting-state model of the closely related glycine receptor. GABA-A recep- (MBP). Expression and purification of this MBP-5-HT3A-ICD-chimera re- sults in a stable pentamer in solution, without evidence for other oligomeric tors are highly important drug targets due to the widespread actions of GABA in the central nervous system, and an improved understanding of assemblies (Pandhare, Grozdanov, Jansen, Sci. Rep. 2016). This indicates the functional state transitions would be highly valuable for developing that the ICD itself may contribute to pentamerization of pLGICs as a whole, a function that has previously been attributed to the two other domains. In drugs against a range of diseases such as amnesia, anxiety, insomnia, and epileptic seizures. this study, we aimed at generating a 5-HT3A-ICD construct without the large MBP. To this end we engineered a cleavage site between the MBP and the 5- HT3A-ICD. Further optimization is geared towards modifying the construct 2736-Pos Board B343 itself and identifying isolation, purification and cleavage conditions that Structural Elements Governing GABA-A Receptor Channel Activation will yield stable pentameric 5-HT3A-ICD without a large helper protein and Drug Modulation such as MBP. Such a construct would provide the basis for structural studies Tzu-Wei Tsao1, Connor Ford2, Chong Lor2, Robert Pearce2, of the ICD and it would also be useful to study conformational transitions of Cynthia Czajkowski1. the ICD caused by posttranslational modifications or interactions with cyto- 1Department of Neuroscience, University of Wisconsin-Madison, Madison, solic proteins. WI, USA, 2Department of Anesthesiology, University of Wisconsin- Madison, Madison, WI, USA. 2734-Pos Board B341 Inhibition in the brain depends on rapid opening and closing of GABA-A re- Relative Affinities of General Anesthetics for Pseudo-Symmetric ceptor (GABAR) channels. Therapeutic drugs such as benzodiazepines Intersubunit Binding Sites of Heteromeric GABA(A) Receptors (BZDs), barbiturates and general anesthetics modulate GABAR currents by Sruthi Murlidaran1, Reza Salari1, Jerome He´nin2, Grace Brannigan1. binding to distinct allosteric sites that are far from the agonist binding sites. 1Computation and Integrative Biology, Rutgers University, Camden, NJ, Structural mechanisms underlying how GABA binding promotes channel USA, 2Laboratoire de Biochimie The´orique, CNRS, Paris, France. gating and how drug binding modulates channel function are still unclear. GABA(A), a pentameric ligand gated ion channel is critical for regulating In a recent elements-based kinetic model of GABAR gating (JGP 144:27- neuronal excitability. These inhibitory receptors, gated by -amino butyric 39, 2014), intermediate transduction elements mediate the coupling between acid (GABA), can be potentiated and also directly activated by intravenous the two GABA binding sites to the channel activation gate and desensitization and inhalational anesthetics. Although this receptor is a widely-studied gates. Moreover, the BZD binding site also interacts with one of the transduc- target for general anesthetics, the mechanism of receptor modulation re- tion elements to promote BZD modulation of GABA-mediated currents. mains unclear. These receptors are predominantly found in 2a:2b:1g stoi- Whether specific regions of the GABAR are associated with these intermedi- chiometry, with four unique inter-subunit interfaces. Here we use ate transduction elements is not known. We hypothesize that a region in the a thermodynamically rigorous free energy perturbation (AFEP) techniques subunit that physically links the BZD and GABA binding sites (ab4-b5 and Molecular Dynamics simulations to rank the different intersubunit sites linker, K105-M111) is an important transduction element. Previously, when by affinity. AFEP calculations predicted selective propofol binding to inter- glycine residues were inserted into this linker, BZD modulation of GABA- facial sites, with higher affinities for a-/bþ and b-/aþ interfaces than g-con- mediated currents was significantly decreased (PNAS 287: 6714- taining interfaces; the strongest affinity (b-/aþ), was about ten times 6724,2012). Here, we expressed wild-type abg GABARs and mutant GA- stronger than propofol EC50, while the second-ranked affinity (a-/bþ) BARs with 4-glycine insertions in the ab4-b5 linker (aGly4) in human em- was equivalent to EC50. The simulations revealed the key interactions lead- bryonic kidney 293 cells and measured GABAR currents using outside-out ing to propofol selective binding within GABAA receptor subunit interfaces, patch clamp electrophysiology and ultra-rapid ligand exchange. Compared with stable hydrogen bonds observed between propofol and a-/bþ or b-/aþ to wild-type GABARs, aGly4 currents were slow to activate and fast desen- cavity residues. Propofol competed with water molecules for hydrogen sitization was eliminated indicating that this linker region is a key structural bonding in the more hydrophilic b-/gþ interfacial cavity, resulting in a element that influences GABAR kinetics. Kinetic models will be used to lower affinity despite an increased number of hydrogen bonding partners. explain aGly4 effects on GABAR macroscopic current responses and to Weaker affinities were measured for sevoflurane, consistent with its greater identify the role the ab4-b5 linker plays in GABA activation and BZD EC50. ‘Flooding’ molecular dynamics simulations identified stable binding potentiation.

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2737-Pos Board B344 that are ubiquitously expressed in the heart (e.g. cMyBP-C or cTnT) could Binding Modes of Full and Partial Agonists in the Orthosteric Binding Site also affect atrial function. Here we investigate whether HCM-associated atrial of the Glycine Receptor myopathy is a consequence of mutation-driven sarcomere dysfunction or re- Marc A. Damgen€ 1, Timo Greiner2, Remigijus Lape2, Lucia G. Sivilotti2, sults from atrial remodeling due to the diastolic dysfunction and increased Philip C. Biggin1. LV filling pressures. In one HCM patient carrying the Lys814del cMyB-C mu- 1Structural Bioinformatics and Computational Biochemistry, Department of tation, changes in sarcomere function (increased myofilament Ca2þ sensitivity Biochemistry, University of Oxford, Oxford, United Kingdom, 2Department and increased cross bridges detachment rate under isometric conditions) were of Neuroscience, Physiology and Pharmacology, University College London, similar in atrial and ventricular myofibrils compared to donor preparations. London, United Kingdom. However, isometric twitch mechanics and kinetics of intact trabeculae from The glycine receptor is a well-characterized member of the Cys-loop superfam- the right atrium of 4 cMyB-C-mutant patients were unaffected as compared ily of receptor proteins. Upon the binding of agonist the transmembrane pore to trabeculae from non-HCM patients (N=8), or mutation negative HCM pa- opens to allow the passage of chloride ions into the cell. Previous research tients (N=3), or HCM patients carrying mutations in beta-myosin (N=2). We has identified agonists that bind with comparable affinity to the receptor’s or- extended the study to HCM mouse models carrying mutations in cTnT. In the thosteric binding site, but differ in their ability to open the ion channel pore. E163R mouse, atrial and ventricular sarcomere kinetics and energetics were This phenomenon of partial agonism is still poorly understood at atomistic res- similarly altered compared to WT mice. Isometric ATPase, both at rest and olution. Knowledge of how different agonists modulate the receptor’s response at maximal Ca2þ-activation and the energy cost of tension generation were is not only of fundamental importance, but also has direct implications for drug increased in both atrial and ventricular preparations of E163R vs WT. How- design. Previously, on the basis of homology models, we suggested that water- ever, isometric twitch kinetics were prolonged in intact ventricular trabeculae mediated interactions may play a significant role in determining the binding of E163R mice vs WT while they were unaffected in atrial trabeculae. In the modes of agonists. We have extended this approach using the recent cryo- R92Q mouse model, that is associated with a much more severe degree of LV EM and crystal structures. Our molecular dynamics simulations support our diastolic dysfunction and left atrial dilatation compared to the E163R, left original hypotheses with respect to the role of water in the binding pocket. In atrial trabeculae showed prolonged twitch contractions, increased sponta- addition, via the use of Markov State Modelling, we are able to provide a neous activity and a number of E-C coupling alterations that resemble those detailed picture of the complex nature of ligand-protein interactions for the observed in ventricular preparations. HCM-mutations in cMyBP-C and glycine receptor that may have implications for the interpretation of efficacy cTnT induce similar alterations in both atrial and ventricular sarcomeres. of different agonists. However, likely due to the different working conditions of the two chambers, sarcomere dysfunction can significantly alter the mechanics and kinetics of 2738-Pos Board B345 the intact myocardium only in the ventricles. Atrial muscle dysfunction in Structure-Based Discovery of Novel Glycinergic Modulators HCM is induced by remodeling processes that depend on the increased filling Marta M. Wells, Nathan Reinert, Pei Tang, Yan Xu. pressures. University of Pittsburgh, Pittsburgh, PA, USA. Marijuana’s analgesic effects can be attributed to the allosteric modulation of 2740-Pos Board B347 glycine receptors (GlyRs) by D9- (THC). GlyRs are Myosin Activator Omecamtiv Mecarbil Differentially Impacts the pentameric ligand-gated chloride channels that control inhibitory neurotrans- Contractile Properties of Skinned Myocardium from Failing and Donor mission in the brainstem and spinal cord. The glycinergic mechanism of Human Hearts -induced analgesia is independent of the other psychoactive effects Ranganath Mamidi1, Kenneth S. Gresham2, Jiayang Li1, Julian E. Stelzer1. of THC. Compounds specifically targeting the well-defined THC-binding 1Case Western Reserve University, Cleveland, OH, USA, 2Temple site in GlyRs are likely to provide pain relief with fewer unwanted side University, Philadelphia, PA, USA. effects. Here, we screened over 2 million drug-like molecules from the Omecamtiv mecarbil (OM) is a compound that selectively targets the cardiac ZINC database on an ensemble of a3GlyR structures obtained from molecu- sarcomere and enhances myosin crossbridge (XB) activation and force gener- lar dynamics simulations of the closed-state a3GlyR crystal structure (PDB ation. Because of these properties OM has been proposed as a treatment for sys- ID: 5CFB) and a homology model derived from the open-state a1GlyR tolic heart failure in humans, however, there are no studies that have directly NMR structure (PDB ID: 2M6I). Computational dockings were specifically examined the functional effects of OM, at XB level, in remodeled human targeted to the known THC-binding site in the a3GlyR transmembrane failing myocardium. Thus, we tested whether OM differentially impacts con- domain. Each screened compound was ranked based on its total predicted tractile properties in failing and donor human myocardium. Isometric force, 2þ binding affinity across the ensemble of a3GlyR structures. Top ranked com- myofilament Ca -sensitivity (pCa50), rates of XB detachment (krel), recruit- pounds were selected for functional measurements in Xenopus laevis oocytes ment (kdf), and magnitude of XB recruitment (Pdf) were measured both before expressing human a3GlyR. Several lead candidates have been identified as and after OM incubation in chemically-skinned myocardial preparations iso- strong modulators of a3GlyR, exhibiting positive and/or negative allosteric lated from failing and donor hearts. Because the failing myocardium exhibited effects at micromolar concentrations. Further molecular dynamics simula- an enhanced pCa50, dynamic XB parameters were measured at pCa’s 6.6 and tions of a3GlyR in the presence of modulators revealed that in the closed- 6.3, respectively for failing and donor groups in order to achieve equivalent state a3GlyR, potentiating compounds showed an increased probability of levels of activation (~22% and ~35% of maximal force, respectively for pre- contacting with residue S296 compared to inhibitors occupying the same and post-OM incubations). OM incubation did not alter the pCa50 but signifi- THC-binding site. S296 on the third transmembrane helix has been previ- cantly enhanced the sub-maximal force production at forces lower than 50%, ously shown to be critical for THC potentiation of GlyRs. In contrast, inhib- an affect that was less pronounced as Ca2þ levels increased. Furthermore, itors showed a higher probability of close contact with residue S241 in the OM differentially impacted the contractile properties of failing and donor first transmembrane helix. Interestingly, the adjacent residue I240 has been myocardium. Specifically, OM-induced force increases were more pronounced previously reported as an inhibitory site. This study suggests that different in the failing myocardium (~79% increase vs. ~28% in donor myocardium). interaction residue partners, even within the same binding site, may lead to Additionally, the OM-induced slowing of krel and kdf, and increase in Pdf distinctly different allosteric modulations in a3GlyR. Research supported were also more pronounced in the failing myocardium. Our results suggest by NIH grants. that OM is effective in increasing force generation in myocardium isolated from failing hearts by increasing XB on-time and enhancing cooperative XB Cardiac Muscle Mechanics and Structure II recruitment.

2739-Pos Board B346 2741-Pos Board B348 Atrial Remodeling in Hypertrophic Cardiomyopathy Biochemical and Mechanical Properties of the S532P and R712L Cecilia Ferrantini1, Jose` Manuel Pioner2, Francesca Gentile1, Cardiomyopathy Myosin Mutants Raffaele Coppini1, Cristina Morelli1, Nicoletta Piroddi1, Beatrice Scellini1, Ailian Xiong1, Yingying Liu1, Bipasha Barua2, Betty Belknap1, Elisabetta Cerbai1, Jil Tardiff2, Chiara Tesi1, Iacopo Olivotto1, Howard White1, Donald Winklemann2, Eva Forgacs1. Corrado Poggesi1. 1Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA, 1Physiology, University of Florence, Florence, Italy, 2Physiology, University USA, 2Pathology, Robert Wood Johnson Medical School, Rutgers of Arizona, Tucson, AZ, USA. University, Piscataway, NJ, USA. Changes in myofilament function related to HCM-associated mutations Human cardiomyopathiesincludes hypertrophic cardiomyopathy (HCM) and contribute to the diastolic dysfunction observed in the in vivo patient heart dilated cardiomyopathy (DCM), which affect 1 in every 500 and 1 in every and in intact ventricular preparations from patient samples. HCM mutations 250 people respectivelyin the U.S. The highest percentage of disease-causing

BPJ 7910_7914 Wednesday, February 15, 2017 557a mutations occur in MYH7, encoding the b-cardiac myosin heavy chain gene. reduced binding affinity for actin in vitro. We then used echocardiography We used an adenoviral mediated mammalian expression system to express and pressure-volume recordings to assess cardiac function under sedentary human b-cardiac heavy-meromyosin (cHMM). The S532P DCM mutation and stress conditions. is associated with a severe phenotype. The S532 residue is in the actin bind- Both systolic function (e.g. stroke volume, ejection fraction and maximal rate ing region in a Helix-Loop-Helix motif involved in weak hydrophobic bind- of pressure development (þdP/dt)) and diastolic function (e.g. –dP/dt, isovolu- ing to actin. The R712L HCM mutation disrupts a salt bridge linking the metric relaxation time, E/A) were normal in E330K-Tg mice under sedentary relay helix to the converter domain. We have investigated the effects of conditions. Acute b-adrenergic stimulation with isoprenaline also led to normal both S532P and R712L mutations on the cardiac myosin ATPase cycle, mo- increases in both contractility and relaxation in E330K-Tg mice. However, tor activities and their interaction with Omecamtiv Mecarbil (OM), a small chronic cardiac stress by increasing preload to the heart by aortocaval fistula molecule allosteric effector of cardiac myosin. All of those effects have was fatal to all E330K-Tg mice within 10 days after surgery. been compared with wild type (WT) cHMM. The S532P mutation has a Our results demonstrate that reduced interaction between the M-domain of 50% reduced steady-state ATPase rate. The phosphate release rate is 3 cMyBP-C and actin did not impair cardiac function in E330K-Tg mice under fold slower for the S532P mutant than for wild type, resulting in a signifi- sedentary conditions, but that this interaction may be essential for the heart cantly reduced rate of transition from the weak to strong actin binding state to cope with chronic volume overload. and a significantly lower power output in the mutant. The most obvious ef- This work is supported by NIH HL-080367 and AHA 15POST25700403. fect of R712L is a shift of ATP hydrolysis equilibrium towards ATP over products ADP-Pi. Other key kinetic steps are not affected. Both mutations 2744-Pos Board B351 slow the unloaded shortening velocity measured by in vitro motility Structural Dynamics of Human Cardiac Myosin-Binding Protein C compared with WT cHMM. OM restores the motility of the R712L construct Revealed by Time-Resolved FRET but further slows the motility of S532P. Understanding the biochemical and Thomas A. Bunch, Victoria C. Lepak, Brett A. Colson. mechanical changes resulting from cardiac myosin mutations will aid in the Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA. development of therapeutic approaches to mitigate the dysfunction leading to Cardiac myosin-binding protein C (cMyBP-C) tunes myocardial contraction cardiomyopathies. through interactions with the thick and thin filaments in a phosphorylation- dependent manner. However, cMyBP-C’s structural arrangements and mecha- 2742-Pos Board B349 nism of action in cardiac muscle remains highly elusive. We have performed Peptides Designed to Destabilize the Myosin Coiled Coil Enhance time-resolved FRET to measure the structural dynamics of human cMyBP-C Myofibril Shortening While Peptides that Stabilize the Coiled Coil Inhibit in solution and determine effects of protein kinase A (PKA) phosphorylation Myofibril Shortening treatment with high spatial resolution. PKA treatment leads to a more bent Rohit Singh, Motamed Qedan, Negar Aboonasrshiraz Dua’a Quedan, and compact conformation detected in N terminal domains C0 through C2 Daniel Wang, Douglas D. Root. (C0-C2), and could thereby mediate cMyBP-C sarcomeric interactions to Biological Sciences, University of North Texas, Denton, TX, USA. enhance contractility. Molecular dynamics (MD) simulations further suggest Myosin subfragment-2 (S2) is believed to influence the accessibility of the that a hinge-like rotation within the M domain upon phosphorylation facilitates myosin head’s binding to actin in muscle. Measurements indicate that the sta- C0-C2 bending and exposes a putative binding site. Here, we engineered pairs bility of the S2 coiled coil can modulate myofibril shortening and that modula- of cysteine residues within the M domain and/or in neighboring domains C0, tion may be related to the degree of myosin head association on the thick C1, and C2 for thiol-reactive probe labeling. We then used a fluorescence filament. Anti-S2 peptides designed using conformation searching, dynamics lifetime-based FRET assay to map C0-C2 structure and conformational dy- simulations, interaction energies, and simulated force spectroscopy were chem- namics in the absence and presence of PKA phosphorylation. Our spectroscopic ically synthesized for microscopic and spectroscopic analyses. One set of studies suggest unique structural differences between human and mouse iso- computationally modeled peptides was designed to stabilize the myosin S2 forms, particularly among the immunoglobluin-like domains. Consistent with by wrapping around the S2 coiled coil. A second set of computationally MD simulations, we observed an equilibrium of closed and open structural modeled peptides was designed to destabilize the myosin S2 by competing states of the M domain. Upon treatment with PKA, the M domain structural for the intrinsic interactions of the coiled coil strands with each other. Nanomo- states shifted in equilibrium to favor the open structural state, in agreement lar concentrations of peptides that stabilized the coiled coil inhibited myofibril with MD simulations. Our survey of the dynamic N terminal cMyBP-C struc- shortening, while nanomolar concentrations of peptides that destabilized the tural landscape provides insight into the molecular basis for the regulation of coiled coil enhanced myofibril shortening. The targeted region of myosin S2 cardiac muscle function. This insight into the structural changes in C0-C2 is appeared to be more mobile than light meromyosin using fluorescence anisot- a foundation for identification of biologically-active compounds and small mol- ropy measurements. Stoichiometries of the predicted interactions of the anti-S2 ecules that perturb structure in ways that mimic the regulatory phosphorylation peptides with the myosin coiled coil were verified by spectroscopic analysis us- states. Ultimately, this furthers our goal to develop novel heart failure therapies ing hypochromism to measure alpha-helical content and FRET to gauge coiled to fine-tune contraction. coil associations. ELISA further confirms the high affinity interactions of the anti-S2 peptide with both skeletal and cardiac myosins with a greater affinity 2745-Pos Board B352 for the human cardiac myosin sequence than for rabbit skeletal myosin. The Increased Phosphorylation of Cardiac Myosin Binding Protein C and ability to modulate force production in muscle by this mechanism has potential Increased Expression of Troponin I in Normal Human Ageing applications in familial hypertrophic cardiomyopathies, dilated cardiomyopa- Lisa Nguyen, James McNamara, Cris Dos Remedios, Amy Li, Sean Lal. thies, and chronic congestive heart failure, among others. The observation Anatomy and Histology, University of Sydney, Sydney, Australia. that force production is never completely extinguished even by the stabilizing Cardiac myosin binding protein C (cMyBPC) is a large myofibrillar protein that peptide, even at high concentrations, suggests that safety from overdoses could regulates muscle contraction. Phosphorylation of cMyBPC is required to main- be a major advantage of this mechanism. tain normal cardiac function as decreased levels are seen in human heart failure. Troponin I (TropI) stabilizes the actin tropomyosin complex, inhibiting myosin 2743-Pos Board B350 binding to actin in the relaxed state. We are the first to examine human Reduced Binding of the M-Domain of Cardiac Myosin Binding Protein C cMyBPC phosphorylation and TropI expression in normal donor hearts be- to Actin Impairs the Ability of the Heart to Respond to Chronic Stress tween the ages of 4 and 64 years. These hearts were procured but not used Sabine J. van Dijk, Joshua Strom, Samantha P. Harris. for heart transplantation (not post-mortem). Pro-Q Diamond staining of Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA. 1D gels revealed increasing phosphorylation levels of cMyBPC with age Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein that reg- (P value = 0.008) in the absence of any significant change in protein expression. ulates cross bridge cycling to control timing and strength of cardiac contraction. Further Western Blot analysis of other major myofibrillar proteins such as However, the molecular mechanisms by which cMyBP-C influences cross myosin light chain and TropT (including its isoforms) showed unaltered phos- bridge cycling are incompletely understood. The M-domain of cMyBP-C can phorylation and expression levels with age. However, TropI protein expression interact with actin and myosin, and phosphorylation of cMyBP-C in response significantly increased with age and this was confirmed at the transcript level, to b-adrenergic stimulation during cardiac stress diminishes its affinity for both. with a 2-fold increased between young and old hearts. We propose that these In this study we determined the functional relevance of cMyBP-C binding to enhanced levels of cMyBPC phosphorylation and TropI expression favour a actin on cardiac function. Binding of cMyBP-C to actin can activate the thin system that breaks cross-bridge formation, reduces contractile force and in- filament and we hypothesize that dissociation of cMyBP-C from actin is essen- hibits relaxation; features that would be consistent with reductions in left ven- tial for cardiac relaxation. To test our hypothesis, we created a transgenic tricular compliance with ageing, in addition to having important implications mouse model with a mutation in the M-domain of cMyBP-C (E330K) that for diastolic heart failure.

BPJ 7910_7914 558a Wednesday, February 15, 2017

2746-Pos Board B353 mechanisms of cardioskeletal myopathy associated with MYL2. Supported by Pseudo-Phosphorylation Mediated Rescue of R58Q-Linked Familial NIH-HL123255 (DSC). Hypertrophic Cardiomyopathy Phenotype Sunil Yadav, Katarzyna Kazmierczak, Jingsheng Liang, 2748-Pos Board B355 Danuta Szczesna-Cordary. Distinct Lattice Structure Altreations in DCM and HCM Mouse Models Molecular and Cellular Pharmacology, University of Miami, Miami, FL, Associated with Mutations in Myosin Regulatory Light Chain USA. Chen-Ching Yuan1, Jingsheng Liang1, Katarzyna Kazmierczak1, Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant Zhiqun Zhou1, Rosemeire Kanashiro-Takeuchi1, Thomas C. Irving2, Danuta Szczesna-Cordary1. disease characterized by ventricular hypertrophy, myofibrillar disarray and 1 2 often caused by mutations in genes encoding for major sarcomeric proteins. University of Miami, Miami, FL, USA, Illinois Institute of Technology, This study is focused on Arginine to Glutamine substitution (R58Q) in the Chicago, IL, USA. ventricular myosin regulatory light chain (RLC) resulting in a malignant hy- In this study we used small angle X-ray diffraction to analyze changes in the pertrophic phenotype. We previously showed a mutation-induced decrease interfilament lattice spacing (IFS) and equatorial reflections’ intensity ratio in the endogenous level of cardiac RLC phosphorylation in R58Q mice, re- (I1,1/I1,0) due to mutations in myosin RLC shown to cause hypertrophic sulting in compromised communication between the functional regions of (HCM) or dilated (DCM) cardiomyopathy. In mice, D166V was found to result in systolic and diastolic dysfunction, reduced contractile force and abnormally the RLC and affecting the interaction of myosin and actin and cardiac mus- 2þ cle contraction. In this report we explored the therapeutic potential of high Ca -sensitivity, highlighting the severity of the HCM phenotype. The pseudo-phosphorylation, in which Aspartic Acid is replaced for phosphory- D94A mutation resulted in DCM manifested by increased LV-chamber diam- eter and reduced ejection fraction with no changes in maximal force production latable Serine-15 (S15D) in the R58Q background. In vitro actin-activated 2þ myosin ATPase activity was assessed using S15D-R58Q and R58Q vs. but with D94A-induced decrease in the Ca -sensitivity of force. In this report WT reconstituted porcine cardiac myosin. R58Q largely decreased maximal we collected the X-ray data on skinned papillary muscles from D166V-HCM and D94A-DCM vs. WT mice at serial pCa (8, 7, 6, 5.4, 5.2, and 4) and at ATPase activity, Vmax (~30% lower) compared to WT. S15D-R58Q rescued the maximal ATPase activity to the WT level. The Michaelis–Menten con- SL=2.1mm. For D166V-HCM vs. WT-fibers, IFS was significantly increased in pCa8, 6 and 5.4 but significantly decreased at pCa4. For D94A-DCM and stant (in mM) for R58Q (Km=4.0450.45) was significantly increased for WT, no significant changes in IFS were observed within or between the compared to WT (Km=1.6850.19), suggesting that a higher concentration of actin is needed to activate cross-bridges. S15D-R58Q-RLC showed a groups for each pCa. There was a clear correlation between I1,1/I1,0 readings vs. increasing [Ca2þ] for all three groups of fibers. A significant increase in small but significant increase in Km (2.5650.32), compared with WT. Steady-state acto-myosin binding was studied using fluorescently labelled I1,1/I1,0 for all pCa solutions (except pCa4) was observed in D166V-HCM vs. WT fibers. These data correlated with D166V-induced changes in force/ actin to quantify the dissociation constants (Kd) and stoichiometry of bind- 2þ ing (n) between myosin and actin in the absence of nucleotide. Binding of Ca -sensitivity observed in fiber mechanics study. For D94A-DCM, a near WT-RLC to actin was strong (K value =5.5 nM, n=0.89). A large decrease significant decrease in I1,1/I1,0 at pCa 5.2 vs. WT was observed d 5 5 in binding (~67-fold increase in K ) was observed for R58Q-RLC. K of (WT:0.43 0.08 vs. D94A: 0.35 0.09 P=0.072). Fitting I1,1/I1,0 –pCa plots d d 5 5 5 S15D-R58Q-RLC was only slightly increased (~3-fold) compared with yielded pCa50=5.2 0.03 (WT), 4.98 0.05 (D94A) and 5.43 0.10 WT indicating a rescue in binding affinity by pseudo-phosphorylated RLC (D166V), showing a clear correlation in pCa50-values between X-ray and fiber mutant. Our findings on S15D-R58Q support the idea that myosin phosphor- mechanics experiments. Our collective data indicate that RLC mutations lead ylation may have important translational applications for the treatment of to HCM or DCM through structural rearrangements of myosin cross-bridge RLC induced hypertrophic cardiomyopathy. Supported by NIH-HL123255 mass with D166V bringing it closer to actin, and D94A moving it farther (DSC). away from the thin-filaments. Supported by NIH-HL123255 (DSC).

2747-Pos Board B354 2749-Pos Board B356 Molecular Mechanisms Involved in Cardioskeletal Dysfunction Caused by Abnormal Cardiac Cross-Bridge Kinetics in a Troponin T ILE79ASN Mutations in Myosin RLC Linked to Hypertrophic Cardiomyopathy Transgenic Mouse Model Danuta Szczesna-Cordary1, Chen-Ching Yuan1, Katarzyna Kazmierczak1, David Gonzalez-Martinez1, Karissa D. Jones1, Maicon Landim-Vieira1, Zhiqun Zhou1, Jingsheng Liang1, Sunil Yadav1, Thomas C. Irving2, Kwangjun Lee2, Yeojung Koh2, Bjorn C. Knollmann3, P. Bryant Chase4, Jenice X. Cheah3, Aldrin V. Gomes3. Hyun S. Hwang2, Jose R. Pinto1. 1Molecular and Cellular Pharmacology, University of Miami School of 1Biomedical Sciences, Florida State University College of Medicine, Medicine, Miami, FL, USA, 2Biological, Chemical and Physical Sciences, Tallahassee, FL, USA, 2Nutrition, Food and Exercise Sciences, Florida State Illinois Institute of Technology, Chicago, IL, USA, 3Neurobiology, University, Tallahassee, FL, USA, 3Medicine and Pharmacology, Vanderbilt Physiology, and Behavior, University of California, Davis, CA, USA. University School of Medicine, Tallahassee, FL, USA, 4Biological Science, The MYL2 gene concurrently expresses the human myosin regulatory light Florida State University, Tallahassee, FL, USA. chain (RLC) in the ventricles of the heart and in the slow-twitch skeletal mus- The missense mutation Ile79Asn in human cardiac troponin T (HcTnT-I79N) cle. Mutations in MYL2 are known to cause hypertrophic cardiomyopathy has been associated with phenotypic outcomes of familial hypertrophic car- (HCM), but in some cases they also lead to cardioskeletal myopathy in humans. diomyopathy, arrhythmias, and sudden cardiac death. Previous characteriza- Using transgenic animal models of HCM, we studied the mutation-induced tion of a transgenic mouse model bearing the HcTnT-I79N mutation, which morphological, structural and functional changes and the signaling pathways expressed 52% HcTnT-I79N of total troponin T, demonstrated increased þ that trigger pathological remodeling of the HCM heart and slow-twitch skeletal Ca2 sensitivity of isometric force. However, little is known about the muscle. Electron microscopy data showed severe myofilament disarray, a hall- changes in cross-bridge kinetics, if any, that result from the mutation. This mark of HCM, in the hearts and in the soleus muscle of RLC-mouse models of study investigates isometric force, sinusoidal stiffness and rate of tension HCM. Likewise, functional measurements of contractile force in skinned papil- redevelopment (kTR) of skinned papillary muscle fiber bundles at two sarco- lary and soleus muscle fibers showed similar direction of changes in HCM mere lengths (1.9 mm and 2.1 mm); cardiac muscle was isolated from the mice. Mutation-induced structural abnormalities in papillary and soleus mus- left ventricle of non-transgenic wild-type (WT) mice or transgenic mice cles from HCM-R58Q mice were confirmed by high resolution X-ray diffrac- bearing the HcTnT-I79N mutation. Sarcomere length (SL) was set at pCa8 us- tion showing similar changes in d1,0 (interfilament lattice spacing) and in ing HeNe laser diffraction after the attachment points of each skinned fiber equatorial reflections’ intensity I1,1/I1,0 ratio in both papillary and soleus bundle were fixed with glutaraldehyde to minimize end compliance. Sinusoi- muscles. Quantitative proteomic analysis revealed that out of >1560 TMT- dal stiffness and kTR were obtained after recording steady-state isometric ten- labeled proteins identified with high confidence, 372 were different in the sion by oscillating the fiber bundle 0.2% of its relaxed length or by applying a hearts of R58Q vs. WT mice, while in the soleus skeletal muscle out of ramped shortening followed by a quick re-stretch to original fiber bundle >1100 TMT-proteins, 255 were different in R58Q vs. WT. The two major bio- length, respectively. As previously reported, the HcTnT-I79N mutation re- þ logical processes affected in both types of muscles in R58Q were the metabolic sulted in increased Ca2 -sensitivity of isometric force although no difference and cellular processes, with a greater percentage of metabolic processes in maximum force per cross-sectional area was observed. Length-dependent þ affected in cardiac muscle. Common metabolic proteins decreased in heart/so- activation at the longer SL resulted in increased Ca2 -sensitivity of isometric leus of R58Q vs. WT mice included the energy production proteins, while the force and sinusoidal stiffness, and decreased kTR for both WT and HcTnT- cellular processes included the calcium handling proteins. Our comprehensive I79N. However, HcTnT-I79N exhibited faster kTR and greater sinusoidal stiff- þ data clearly indicated adverse heart/soleus muscle remodeling in HCM mice ness at sub-maximum pCa (-log[Ca2 ]) solutions compared to WT regardless making them an ideal model to study the molecular, energetic and cellular of SL. These data indicate that HcTnT-I79N mutation, and SL, markedly

BPJ 7910_7914 Wednesday, February 15, 2017 559a influence not only the dynamics of individual regulatory units, but also the ki- with the cTnC-A8V, sensitizing the thin filament with Bepridil resulted in an netics of cross-bridge cycling. increased pCa50 and kTR. These data suggest an important role of the thin fila- ment controlling not only the number of cross-bridges during Ca2þ-activation, 2750-Pos Board B357 but may also influence cross-bridge kinetics. A Novel DCM-Associated Mutation in the N-Helix of Cardiac Exhibits Impaired Contractile Kinetics and Reduced Ca2D-Sensitivity 2752-Pos Board B359 In Vitro The Actc A295S Hypertrophic Cardiomyopathy Mutation Promotes Thin Jamie Johnston1, David Gonzalez-Martinez1, Einat Birk2, Nili Zucker2, Filament Disinhibition and Enhances Contractile Activity Maicon Landim-Vieira1, P. Bryant Chase3, Yael Wilnai4, Jose R. Pinto1. Meera C. Viswanathan1, William Schmidt1, Marek Orzechowski2, 1Biomedical Sciences, Florida State University College of Medicine, William Lehman2, Anthony Cammarato1. Tallahassee, FL, USA, 2Cardiology, Schneider Children’s Medical Center, 1Johns Hopkins University, Baltimore, MD, USA, 2Boston University School Tel Aviv University, Petah Tikva, Israel, 3Biological Science, Florida State of Medicine, Boston, MA, USA. University, Tallahassee, FL, USA, 4Genetics Unit, Schneider Children’s Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease of the Medical Center, Petah Tikva, Israel. heart. HCM mutations in sarcomeric genes often engender hypercontractile Point mutations in genes encoding the subunits of the cardiac troponin com- myocardium that displays increased Ca2þ-sensitivity of force, enhanced plex are known to result in various forms of cardiomyopathy. We report a clin- cross-bridge cycling, and incomplete relaxation, which precede abnormal ical case of a 1-year old female who presented in the neonatal period with wall thickening. The alpha-cardiac actin (ACTC) Ala295Ser substitution severe dilated cardiomyopathy (DCM) and hypotonia. Whole-exome causes a highly penetrant disease with diverse phenotypes. To help resolve sequencing detected a previously unreported heterozygous variant of uncer- the mechanistic basis of the disorder, we generated several Drosophila models tain clinical significance in exon 1 of the TNNC1 gene, c.12C>G (p.Ile4Met). that limit genetic diversity and pathological complexity. We created lines with This variant was not detected in the proband’s parents, suggesting that this inducible wildtype UAS-Act57BWTand mutant UAS-Act57BA295Scardiac actin variant has likely arisen de novo. To begin understanding the pathogenicity, transgenes to examine the effects of A295S actin on the fly heart. High- we investigated the functional effects of this mutation in vitro by measuring speed video microscopy and motion analysis of beating hearts revealed Ca2þ-sensitivity of isometric force development and kinetics of isometric ten- A295S actin expression significantly reduced diastolic volumes and prolonged sion redevelopment (ktr). Bundles of porcine ventricular papillary muscle systolic intervals, consistent with elevated contractile properties at rest and dur- were dissected, detergent-skinned, mounted between a force transducer and ing contraction. In situ treatment of Act57BA295S hearts with blebbistatin, under a motor, and the sarcomere length was set to 2.1 mm using HeNe laser diffrac- low intracellular Ca2þ conditions, caused significantly larger increases in my- tion. After native TnC extraction with CDTA solution, fibers were reconsti- ocyte length vs. Act57BWTcontrols. These data suggest A295S actin induces tuted with either recombinant WT TnC or TnC-I4M, and immersed in excessive thin filament disinhibition, myosin binding, and resting tension. solutions ranging from low to high [Ca2þ] (pCa8-4) to measure isometric We also expressed indirect flight muscle (IFM)-targeted Act88FWT or Act88- force production at two temperatures. The [Ca2þ] required to reach 50% FA295S transgenes in an IFM actin-null background. Confocal microscopy A295S A295S maximum-force (pCa50)at21C for the mutant was 5.44, compared to 5.54 resolved hypercontracted Act88F fibers. Reconstituted Act88F thin for WT. At 30 C, pCa50 for TnC-I4M was 5.74, compared to 5.87 for WT. filaments, using bovine cardiac troponin-tropomyosin, displayed significantly 2þ At 21 C, TnC-I4M showed a significant decrease in ktr only at maximal enhanced Ca -sensitivity of activation vs. control filaments in regulated Ca2þ-activation compared to WT, while at 30C, the TnC-I4M showed a sig- in vitro motility experiments. Finally, in silico modelling revealed the A295S 2þ nificant decrease in ktr at submaximum and maximum Ca levels. Altogether, mutation influences the orientation of nearby actin residues that mediate critical these results suggest that Ca2þ-sensitivity of force development and cross- electrostatic interactions with tropomyosin. Such mutation-induced propagated bridge kinetics are perturbed as a consequence of this thin-filament mutation effects likely lower the energy required to relieve tropomyosin-mediated inhi- with a more pronounced effect near physiological temperature, and likely bition and promote force production. Overall, enhanced contractile activity contribute to the pathogenesis of DCM. from the molecular through the cellular level is consistent with the early hyper- dynamic contractile properties frequently associated with HCM and could 2751-Pos Board B358 trigger ACTC A295S-associated pathology. This work was supported by Thin Filament-Mediated Modulation of Mouse Cardiac Cross-Bridge R37HL36153 and 1R01HL124091. Kinetics by Ca2D-Sensitizing Mutation CTNC-A8V or Bepridil David Gonzalez-Martinez1, Maicon Landim-Vieira1, Jamie Johnston1, 2753-Pos Board B360 Weikang Ma2, Brittany Griffin1, Olga Antipova2,3, Omar Awan1, Cardiac-Specific Expression of VCP/TER94 RNAi or Disease Alleles P. Bryant Chase4, Thomas Irving2, Jose R. Pinto1. Disrupts Drosophila Heart Structure and Impairs Function 1Biomedical Sciences, Florida State University College of Medicine, Anna C. Blice-Baum1, Meera C. Viswanathan2, Tzu-Kang Sang3, Tallahassee, FL, USA, 2Biological Sciences, Illinois Institute of Technology, Anthony Cammarato2. Chicago, IL, USA, 3X-ray Science Division, Advanced Photon Source, 1Department of Biological Sciences, Sam Houston State University, Argonne National Laboratory, Chicago, IL, USA, 4Biological Science, Huntsville, TX, USA, 2Department of Medicine, Division of Cardiology, Florida State University, Tallahassee, FL, USA. Johns Hopkins University, Baltimore, MD, USA, 3Institute of Biotechnology Recent genetic and functional data with the Ala8Val missense mutation in car- and Department of Life Science, National Tsing Hua University, Hsinchu diac troponin C (cTnC-A8V) suggest its pathogenicity for the development of City, Taiwan. hypertrophic cardiomyopathy (HCM) in humans and mice. Previous functional Valosin-containing protein (VCP) is a highly conserved, homohexameric studies performed using cTnC-A8V knock-in mice revealed increased Ca2þ- ATPase that helps maintain protein homeostasis in all cells and serves special- sensitivity of contraction and impaired kinetics of cardiac muscle relaxation ized functions in distinct cell types. In skeletal muscle, it is critical for myofi- of skinned and intact cardiomyocytes. This study aims to further understand brillogenesis and in the accelerated degradation of muscle proteins during the molecular mechanisms that underlie the development of HCM caused by atrophy. However, little is known about the role(s) of VCP in the heart. Its the cTnC-A8V mutation by measuring the kinetics of tension redevelopment functional diversity is determined by differential binding of distinct cofac- (kTR) and sinusoidal stiffness at two distinct sarcomere lengths (SL). Papillary tors/adapters, which is likely disrupted during disease. VCP mutations cause muscles were isolated from the left ventricle of cTnC-A8V knock-in multisystem proteinopathy (MSP), a pleiotropic degenerative disorder that in- homozygote and WT mice. SL of skinned preparations was set to 1.9mmor volves inclusion body myopathy. MSP patients display progressive muscle 2.1mm using HeNe laser diffraction after the ends were chemically fixed weakness. They also exhibit cardiomyopathy and die from cardiac and respira- with glutaraldehyde to minimize end-compliance. SL dependent changes in tory failure, which are consistent with critical myocardial roles for the enzyme. mechanical properties were similar in WT and cTnC-A8V preparations: Nonetheless, efficient models to interrogate VCP in cardiac muscle remain un- increased pCa50, with increased stiffness and decreased kTR at any given acti- derdeveloped and poorly studied. We have investigated the significance of VCP vation level. However, regardless of SL, cTnC-A8V preparations exhibited an and mutant VCP in the Drosophila heart. Cardiac-restricted RNAi-mediated 2þ increased pCa50, kTR and sinusoidal stiffness at submaximum Ca levels rela- knockdown of TER94, the Drosophila VCP homolog, severely perturbed tive to WT. Maximum sinusoidal stiffness increased 2x in WT preparations myofibrillar organization, heart function, and lifespan in developing and adult stretched from SL 1.9mm to 2.1mm, while for the cTnC-A8V preparations the flies, suggesting that TER94 is required for heart tube formation, maintenance, SL-dependent increase in maximum sinusoidal stiffness was blunted. To deter- structure, and function. Conditional RNAi expression, post-development, mine whether the difference in cross-bridge kinetics at SL 2.1mm between induced extensive cardiac defects soon after activation. Furthermore, expres- cTnC-A8V and WT were due to the increased Ca2þ-sensitivity of cTnC, WT sion of MSP disease-causing alleles engendered cardiomyopathy in adults preparations were treated with 300mM Bepridil, a Ca2þ-sensitizer. As seen and structural defects in embryonic hearts. Drosophila may therefore serve

BPJ 7910_7914 560a Wednesday, February 15, 2017 as a valuable model for examining role(s) of VCP in cardiogenesis and for iden- ing domains which induce fragmentation. ADF/cofilin has also been proposed tifying novel heart-specific VCP interactions, which when disrupted via muta- to accelerate the depolymerisation at the pointed end. However these different tion, contribute to or elicit cardiac pathology. reactions have not been fully characterised and a mechanism that fully explain ADF/cofilin-induced actin disassembly is yet to be proposed. Through single- Actin Structure, Dynamics, and Associated molecule experiments, we aim to take a fresh look at the various effects of ADF/cofilin on actin filaments. Proteins We use a microfluidic setup developed in the group in which actin filaments are anchored to the surface by one end and are aligned by the fluid flow, parallel to 2754-Pos Board B361 the surface. We are able to simultaneously observe tens of actin filaments, the Comparing the Effects and Regulations of Cofilin on Actin Barbed binding of ADF/cofilin and the effect on actin depolymerisation. We have con- Ends Generation in Lamellipodia vs. Invadopodia using Mathematical ducted this study using all three human isoforms of ADF/cofilin, and alpha- Modeling skeletal as well as cytoplasmic actin. Nessy Tania1, John Condeelis2. We are able to observe the formation of ADF/cofilin domains on actin filaments 1Mathematics & Statistics, Smith College, Northampton, MA, USA, and for the first time directly measure their assembly and disassembly rate con- 2Department of Anatomy and Structural Biology and Gruss Lipper stants. In addition we quantify ADF/cofilin ability to fragment filaments. We Biophotonics Center, Albert Einstein College of Medicine of Yeshiva then measure the variation of filament depolymerisation rates at the barbed University, Bronx, NY, USA. and pointed end. We found ADF/cofilin to simultaneously bind the side and Cofilin is an important regulator of actin polymerization and cell motility. It the end of filaments with unexpected effects. In particular ADF/cofilin acceler- severs old actin filaments, generates new barbed ends, and thus enhances actin ates bare barbed end depolymerisation, elucidating controversial results on polymerization from capped F-actin. We build mathematical models and global actin disassembly. We further investigate the cooperation with other analyze the dynamics of cofilin activation and subsequent actin barbed end gen- actin binding proteins such as profilin and capping protein in disassembling fil- eration based on data from the lamellipodia and invadopodia of mammary car- aments and preventing actin regrowth. cinoma cells. At first glance, it appears that cofilin plays similar roles during the Taken together, our experiments reveal that targeted severing, accelerated de- early dynamics/formation of these two distinct motility structures. EGF stimu- polymerisation and cooperation with other actin binding protein synergise to lation induces cofilin activation followed by a peak of actin barbed ends on the meticulously disassemble the actin cytosketon. timescale of 1 min.We had previously characterized the regulation of cofilin within the lamellipodia and showed that ~60% of cofilin must be sequestered 2757-Pos Board B364 via PIP2 binding to obtain dynamics consistent with experimental observation. Structure of the Anti-Cancer Compound TR100 Target-Sites on Tpm3.1 Furthermore, the lamellipodia data showed that a ~50% drop in PIP2 via hydro- Tropomyosin lysis results in a ~12-fold increase in actin barbed ends, indicating a large de- Michael Rynkiewicz1, Justine Stehn2,3, Peter Gunning3, William Lehman1. gree of signal amplifications induced by nonlinear effects of cofilin 1Dept. Physiology & Biophysics, Boston University School of Medicine, cooperativity. However, closer inspections of invadopodia data (first 3 mins. Boston, MA, USA, 2Novogen Pty. Ltd., Hornsby, Australia, 3School of after stimulation) shows that a lesser degree of signal amplifications is Medical Sciences, University of New South Wales, Sydney, Australia. observed: a ~50% drop between cortactin-cofilin interaction translates to a There are upwards of 40 tropomyosin isoforms that are variously expressed in ~6-fold increase in actin barbed ends, which is then followed by a drop below mammalian cells. Binding head-to-tail, tropomyosin coiled-coils form cables the steady-state level. Thus, a simple extension of our previous mathematical along actin-filaments that influence filament-stability and remodeling. Posi- model fails to capture the observed data. These differences lead us to look tioned as filament-gatekeepers, tropomyosins also regulate access to cyto- further into differences between lamellipodia and invadopodia. We use math- plasmic actin-binding proteins, thus defining cytoskeletal organization, while ematical modeling to further quantify the effects of pH-modulation on controlling myosin-binding and hence cell motility and muscle contraction. cofilin-cortactin binding, the availability of actin monomers and existing F- Amino acid sequences of the head-to-tail overlapping-domains responsible actin at steady state, and the role of Arp2/3, in shaping the actin dynamics dur- for tropomyosin polymerization are isoform specific. Therefore, compounds ing the initial stage of invadopodia formation. that bind to these domains will likely be selective for one or another tropomy- 2755-Pos Board B362 osin isoform. TR100 (Stehn et al. 2013; Bonello et al., 2016) was designed to The Role of Intermolecular Interactions in the Polymerization of Actin by target the non-muscle tropomyosin isoform Tpm3.1 (Tm5NM1/2) overlapping- Formins domain, and modify actin-dynamics without compromising actin-tropomyosin Fikret Aydin1, Naomi Courtemanche2,3, Thomas D. Pollard3, binding. It is a tumor cell antagonist without affecting cardiac cell contractility. Gregory A. Voth1. Here, we ran extensive Molecular Dynamics simulations on unliganded 1University of Chicago, Chicago, IL, USA, 2University of Minnesota, Tpm3.1 overlapping-domain homology structures (Greenfield et al., 2009; Minneapolis, MN, USA, 3Yale University, New Haven, CT, USA. PDB-2K8X), which converged on a conformation previously determined for Formins are large multidomain proteins that nucleate and modulate the elon- other Tpm models (Li et al., 2014). We then docked TR100 into the refined gation of actin filaments. The FH2 domains (~400 amino acids) remain asso- overlapping-domain, which fitted snuggly into a corresponding binding pocket ciated with the growing barbed end and slow elongation by 5 to 95% without obvious clashes, and ran further MD on the energy minimized com- depending on the particular formin. We use all atom and coarse-grained mo- plexes. Resulting average structures indicate that the binding specificity of lecular dynamics simulations to investigate the interactions of three formins the compound appears to be determined sterically and dictated by the cumula- FH2 domains (fission yeast Cdc12, budding yeast Bni1p, mouse mDia1) tive effect of several small changes induced in the interior of the overlapping- and the barbed end of the actin filament to understand these formins poly- domain. TR100 changes the bending angle of the overlapping-domain without merize actin at different rates. The simulations demonstrated that dissimilar- marked effect on the domain flexibility. However, acting as a molecular wedge, ities in the structures and intermolecular interactions of three formin-actin TR100 alters the radial dimensions of the Tpm3.1 overlapping-domain complexes, which can affect their elongation rates. In addition, the interac- modestly. Such steric-effects may interfere with cooperative, azimuthal move- tions between FH2 domains and actin filament affect the configuration of ment of tropomyosin on actin-filaments and hence with critical actin-binding the barbed end of the filament, and we identified critical regions of formins protein interactions on actin. These perturbations may influence cytoskeletal that play role in regulating the barbed-end configuration. Our coarse- stress-fiber formation and/or architecture as well as motor-protein traffic in ma- grained simulations predicted these formins have different degrees of steric lignant cells. Supported by NIH grant R37-HL036153 and a contract from Nov- interference to the addition of actin monomer to the barbed end, and it was ogen Pty. Ltd. found to be inversely proportional to their rates of polymerization. This work can help to resolve the underlying molecular mechanisms used by 2758-Pos Board B365 different formins to assemble actin networks. Modulating Formin Processivity with Mechanical and Biochemical Factors 2756-Pos Board B363 Guillaume Romet-Lemonne. Disassembling the Cytoskeleton: Effects of ADF/Cofilin on Actin Dynamics Institut Jacques Monod, CNRS, Paris, France. Hugo Wioland, Sarah Myram, Antoine Je´gou, Guillaume Romet-Lemonne. In cells, actin filament assembly is regulated by actin regulatory proteins and Institut Jacques Monod, CNRS, Paris Diderot, Paris, France. mechanical context. Formin proteins are able to track a growing filament One essential function of a well-built cell is its ability to disassemble its con- barbed end and dramatically enhance its elongation from profilin-actin. They stituents. The ADF/cofilin protein family is certainly a central player in the can do so while being anchored to a surface, thereby allowing the transmission actin cytoskeleton disassembly: it binds cooperatively to actin filaments, form- of mechanical tension while the filament keeps on elongating. We have recently

BPJ 7910_7914 Wednesday, February 15, 2017 561a shown that tension applied to the filament accelerates its elongation and that, in 2761-Pos Board B368 the absence of actin monomers, formins were able to track depolymerizing fil- C0 Domain of Cardiac Myosin Binding Protein-C Modulates Interaction aments in spite of an opposing tension of a few picoNewtons (Jegou et al., Nat. of the Neighboring C1 Domain with Tropomyosin Through the Allosteric Commun. 2013). Here, with similar single filament experiments using micro- Interaction with F-Actin fluidics, we investigate the impact of tension and protein concentration on Jamie Eisner1, Cristina Risi1, Betty Virok1, Julio Kovacs2, another key feature of formins, processivity. We show that tension drastically Samantha P. Harris3, Willy R. Wriggers2, Howard D. White1, enhances the detachment of the formin from the filament barbed end, both in Vitold E. Galkin1. the elongation and the depolymerization regime. Moreover, we find that tension 1Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA, and Capping Protein act in synergy to rapidly detach the filament from the for- USA, 2Mechanical and Aerospace Engineering, Old Dominion University, min. We also characterize the impact of actin and profilin concentrations on Norfolk, VA, USA, 3Cellular and Molecular Medicine, University of formin processivity. Arizona, Tucson, AZ, USA. Mutations in cardiac myosin binding protein C (cMyBP-C) are the most com- 2759-Pos Board B366 mon cause of hypertrophic cardiomyopathy affecting millions of people world- A Model System for the Reconstitution of the Cellular Actin Cortex wide. Recent studies established that the N-terminal domains (NTDs) of Or Gill1, Anne Bernheim-Groswasser1,2. cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the 1Department of Chemical Engineering, Ben-Gurion University of the thin filament (TF). However, the molecular mechanism(s) by which NTDs Negev, Beer-Sheva, Israel, 2Ilse Kats Institute for Nanoscale Science and modulate interaction of myosin with the TF remains unknown. Recently, we Technology, Ben-Gurion University of the Negev, Beer-Sheva, Israel. demonstrated that despite being structural homologs, C0 and C1 NTDs exhibit The cell cortex is a dense actin network, few hundreds of nanometers thick, different patterns of binding on the surface of F-actin. Moreover, we showed which localizes just below the plasma membrane of animal cells. Actin nucle- that C1 but not C0 can activate the TF by means of shifting the tropomyosin ators link the cortical network to the membrane surface and it undergoes dy- (Tm) cable from the ‘‘closed’’ to the ‘‘open’’ structural state via direct interac- namic remodeling which allows cells to rapidly transform, move, and exert tion with Tm. Here we used cryo electron microscopy and image analysis to forces in response to internal and extracellular signals. The structural and me- reveal how C0 and C1 Ig-domains connected by the Proline/Alanine-linker chanical properties of the cell cortex depend on its protein composition. Several communicate with each other when interacting with the TF in tandem. Our re- actin bundling and cross-linking proteins localize to the cortex as well as sults show that C0 binds to the front of actin subdomain-1 in two distinct myosin II motors that provide the cortex with its contractile ability. Myosin mo- modes, and that in these two modes C0 has different effect on the interaction tors were shown to affect cortex thickness, structural organization, and mechan- of C1 domain with both actin filament and Tm cable. In one mode C0 enhances ical properties. the activating effect of C1 domain so that the Tm cable is shifted towards the A difficulty inherent in studies in vivo is that cells perform many processes to subdomain-4 of actin to an even larger extent than found in the ‘‘open’’ state of control their mechanical properties and it can thus be difficult to draw conclu- Tm. In the second mode C0 reverses the activation effect of C1 by means of sions about the principles of cytoskeletal organization from these experiments. disrupting the interaction of C1 with Tm. Importantly, we demonstrate that Reconstituted systems, on the other hand, allow for full control of the constit- the two domains communicate with each other allosterically through the actin uents and as such to obtain a direct relation between function and composition. filament. Our data suggest that the internal dynamics of the actin molecule is This work investigates the self-organization of actomyosin gels grown on flat essential for the regulation of the actomyosin interaction in cardiac muscle. supported membranes with the aim to reconstitute an actin cortex under We propose a mechanism by which cMyBP-C may modulate actomyosin inter- well-defined and controlled conditions. Specifically, we varied the concentra- actions in cardiac muscle. tion of myosin motors in wide concentration range to explore the effect of contractility on the cortical actin gel dynamics and structural organization. 2762-Pos Board B369 We show that the addition of myosin motors affects both the thickness of the Using Photonic Force Microscopy to Investigate the Mechanics of cortical actin layer as well as the density of the gel across the actin layer. Spe- Filopodia cifically we find that the thickness decreased with the increase in myosin con- Rebecca Michiels, Alexander Rohrbach. centration. The contractile stresses generated by the motors seem to affect University of Freiburg, Freiburg, Germany. density profile of actin, from exponential to a more step-like function. Filopodia are thin, needle-like protrusions of the cell surface which many cells use to probe their environment. The filopodial backbone consists of tight bun- 2760-Pos Board B367 dles of filamentous actin that protrude from the actin cortex and are connected The Mechanosensitivity of Actin Bundles with the cell membrane via linker molecules. Retraction towards the cell body Emiko Suzuki. is thought to be driven by molecular motors. In macrophage cells, filopodia Institut Jacques Monod, Paris, France. serve a special function. Macrophages are immune cells which take up and Cells’ ability to sense their environment is essential for many cellular pro- digest cell debris and bacteria in the body, a process which is called phagocy- cesses including cell division, migration and morphogenesis. The actin cyto- tosis. They use filopodia to bind the particles and pull them towards the cell skeleton, which has been shown to be mechanosensitive, is organized into body where they can be internalized. The underlying physical principles that different architectures that carry out various functions within the cell. Filo- allow macrophages to tune their actions during phagocytosis are only little un- podia, which are finger-like structures consisting of actin filaments bundled derstood. The binding and retraction of the particles is a process which requires in parallel, emerge at the cell front and orient the cell in response to its me- the cell to both sense mechanical stimuli as well as apply controlled mechanical chanical environment. These actin filaments are elongated at their barbed forces to the environment. Macrophages’ success in phagocytosis is crucially ends by formins and Ena/VASP and cross-linked by the bundling protein fas- dependent on filopodia function. We use a Photonic Force Microscope in which cin. These two machineries are thought to collaborate to design a unique type we combine DIC microscopy and fluorescence microscopy with optical twee- of actin network that governs filopodium dynamics, yet the exact mechanism zers and interferometric particle tracking. Polystyrene beads are held in an op- by which these two key proteins interact, and how mechanosensing is tical trap to enable controlled placement in the vicinity of the cells. The motion achieved in filopodia, are not well understood. Core questions such as: of the bead in the trap can be tracked in 3D with nanometer precision at a how actin filaments self-assemble in a bundle; how forces are transmitted microsecond timescale using back focal plane interferometry. By changing along filopodia; how fascin and formin synergize to control the growth of the optical forces spatially or temporally, we investigate how the cell reacts actin filament in filopodia remain to be addressed. To tackle these questions, to external forces and which mechanisms determine success or failure when at- we use a microfluidics-based approach to reconstitute, in vitro, a minimal tempting to bind and digest particles. We present novel experiments which system to recapitulate the mechanosensitivy of actin bundles : from single reveal binding, pulling and unbinding events of macrophage filopodia on a mo- filaments to bundles of several filaments, using the actin binding proteins for- lecular scale. min and fascin. On single filaments, the activity of formins and fascins in a large range of biochemical conditions will be probed: first separately, then 2763-Pos Board B370 when present together on the same filament. We will scale up to bundles Actin Based Pulling Forces in Endocytosis of several filaments mimicking bundles in filopodia. Again, microfluidics Fowad Motahari, Anders Carlsson. will be used to apply physiologically relevant pulling and pushing forces Physics, Washington University, St. Louis, MO, USA. to actin bundles. This bottom-up approach will allow us to understand actin Endocytosis in yeast requires actin polymerization to overcome the large bundles mechanosensitivity and bring us closer to obtaining a comprehensive turgor pressure opposing invagination. While the generation of pushing forces description of force generation and transmission in formin and fascin gener- by actin polymerization is understood, it is not clear how actin polymerization ated bundles. generates the required pulling forces. Previous work using a finite element

BPJ 7910_7914 562a Wednesday, February 15, 2017 approach has suggested that spatial variation of the polymerization rate could 60.351.5 and 106.452.2 pN. Using mutations that destroy actin-binding lead to pulling forces1. We extend this work by performing calculations using sites of Tmod2, we assigned specific unbinding forces to the individual discrete actin filaments with all subunits explicitly treated. We simulate a actin-binding sites. Based on these data, the lower peak force corresponds growing array of up to 200 actin filaments in a hexagonal network. Each fila- to the N-terminal actin-binding site and the higher peak force corresponds ment interacts with the membrane via an interaction potential that has both to the actin-binding site within the C-terminal domain. We also quantified attractive and repulsive components. The inner filaments of the array are the unbinding forces between Lmod2 and G-actin. Lmod2 showed a trimodal bound more strongly to the membrane and thus grow more slowly. Elasticity unbinding force distribution with peak values of 56.052.9, 85.652.9 and of the actin network is modeled by linear springs connecting the filaments to 114.3521.9 pN. For the peak assignment, we used Lmod2 fragments. each other. We calculate the spatial distribution of the filament-generated Lmod21-514 showed a bimodal distribution with peak values of 47.351.1 forces. The time-averaged force of the outer filaments pushes on the mem- and 78.451.5, and Lmod21-201 showed a unimodal distribution with a peak brane, while the time-averaged force of the inner filaments pulls on the mem- value 58.251.4. Our results confirm the existence of the N-terminal actin- brane. We calculate the total force of the pulling filaments as a function of binding site in Lmod2 and explain why Lmod2 is a stronger nucleator than several model parameters, including the potential depths, the free filament Tmod2. Altogether, our data demonstrate how the differences between the on-rates, the numbers of fast- and slow-growing filaments, and the network number and the strength of actin-binding sites of Tmod or Lmod translate rigidity. to their functional abilities. [1] A. E. Carlsson and P. V. Bayly, Biophys. J. 106:1596-1606(2014). 2766-Pos Board B373 2764-Pos Board B371 The Effects of Nucleotide and Polymerization on the Structure and Active Cargo Positioning from Actin-Polarity Sensing by Small Myosin Dynamics of Actin Assemblies Lauren Jepsen, David Sept. Mathieu Richard1, Hajer Ennomani2, Carles Blanch-Mercader1, University of Michigan, Ann Arbor, MI, USA. Enrique de la Cruz3, Jean-Franc¸ois Joanny1, Frank Julicher€ 4, Actin’s affinity for polymerization as well as its interaction with actin binding Laurent Blanchoin2, Pascal Martin1. proteins is strongly dependent on the nucleotide state. To examine the effects of 1Laboratoire Physico-Chimie Curie, Institut Curie - CNRS - UPMC - PSL both nucleotide and polymerization on structure and dynamics, we performed Research University, Paris, France, 2iRTSV, CEA, Grenoble, France, multi-microsecond molecular dynamics simulations on both ADP and ATP 3Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, muscle G-actin, as well as the two most recent F-actin models. We find that USA, 4Biological Physics, Max-Planck-Institute for the Physics of Complex the ADP and ATP G-actin monomers show large deviations from their crystal Systems, Dresden, Germany. structures. The F-actin models show a more modest shift from their starting Molecular motors navigate the cytoskeleton to position vesicles and organ- points, and surprisingly the conformational spaces of all simulations have sig- elles at specific locations in the cell. Cytoskeletal filaments assemble into nificant overlap, suggesting that the G- and F-actin structures are more similar parallel, antiparallel or disordered networks, providing a complex environ- than previously thought. We find nucleotide-dependent changes in several re- ment that constrains active transport properties. Using surface micro- gions of the protein that have been previously implicated, but also now in patterns of nucleation-promoting factors to control the geometry of actin the C-terminus and portions of subdomain 2. The F-actin simulations also polymerization, we studied in vitro the interplay between the actin-network reveal both structural and dynamic differences between the barbed and pointed architecture and cargo transport by small myosin assemblies. With two par- ends that provide insight into differences in phosphate release and polymeriza- allel nucleation lines, we produced an antiparallel network of overlapping fil- tion rates at the two ends of the filament. aments. We found that 200-nm beads coated with processive myosin-5 2767-Pos Board B374 motors displayed directed movements towards the midline of the pattern, Spectroscopic Probes of Actin and Actin-Binding Domains of Dystrophin where the net polarity of the actin network was null, and accumulated there. and b-III-Spectrin Reveals Disease-Related Changes in Structural The bead distribution was dictated by the spatial profiles of bead velocity and Dynamics diffusion coefficient, indicating that a diffusion-drift process was at work. Michael E. Fealey, Adam W. Avery, Thomas S. Hays, David D. Thomas. Interestingly, beads coated with skeletal heavy mero-myosin-2 motors University of Minnesota, Minneapolis, MN, USA. showed a similar behavior. However, although velocity gradients were We have used site-directed spectroscopy to examine disease-related struc- sharper with myosin 2, the much larger bead diffusion observed with this tural transitions in the N-terminal actin binding domains (ABDs) of the non-processive motor resulted in less precise positioning. Strikingly, bead proteins dystrophin and b-III-spectrin. Both ABDs contain tandem calpo- positioning did not depend on the spacing between the nucleation lines. nin homology (CH) domains that are implicated in disease, dystrophin in Our observations are well described by a three-state model of bead transport, muscular dystrophy and spectrin in spinocerebellar ataxia type 5. By placing in which active beads locally sense the net polarity of the filament network spin and fluorescent labels within each CH domain and measuring the by frequently detaching from and reattaching to the filaments. A stochastic microsecond- and nanosecond-resolved dipolar electron-electron resonance sequence of processive runs and diffusive searches results in a biased random (DEER) and fluorescence resonance energy transfer (FRET), we found that walk with an effective drift velocity and diffusion coefficient. Positioning re- dystrophin’s CH domains transition from a compact closed conformation lies on spatial gradients of the net actin polarity, as well as on the run length to a more open extended conformation upon complex formation with actin. of the cargo in the attached state. Altogether, our results on a minimal acto- In complementary studies, time-resolved phosphorescence anisotropy of myosin system demonstrate the key role played by the actin-network archi- actin reveals cooperative restriction of actin dynamics upon complex forma- tecture on motor transport. tion. Mutations in dystrophin that cause muscular dystrophy affect both measurements, suggesting that cooperative structural dynamics is intimately 2765-Pos Board B372 involved in disease progression. Molecular dynamics simulations indicate An AFM Investigation of the Nanoscale Forces that Govern the that structural transitions in dystrophin are regulated in part by hydrophobic Interactions between Actin and Proteins of Tropomodulin Family interactions between CH domains. A disease-causing mutation within b-III- Baran Arslan, Mert Colpan, Kevin Gray, Nehal Abu-Lail, Alla Kostyukova. spectrin’s ABD, which results in a 1000-fold increase in actin binding affin- School of Chemical Engineering & Bioengineering, Washington State ity, perturbs the natural hydrophobic contacts between its CH domains, and University, Pullman, WA, USA. spectroscopic probes provide crucial insight into the molecular mechanism. Tropomodulin family of proteins includes several isoforms of tropomodulins We propose that dystrophin and b-spectrin proteins share a common (Tmod) and leiomodins (Lmod), which are differentially expressed in various mechanism of actin regulation, based on coupling between structural dy- tissues. They bind to the pointed end of an actin filament and regulate dy- namics of actin and ABD. Funding to DDT (NIH grant AR63007). Funding namics at that end. By binding to G-actin they sequester actin or nucleate to TSH (NIH grant RO1GM44757). actin polymerization. Of Tmod isoforms, Tmod2 is the best nucleator and Tmod3 is the best in sequestering. Although these properties are isoform- 2768-Pos Board B375 dependent, knowledge on how they vary in their strength of interactions to Tropomyosin Isoforms Show Unexpected Differential Effects on Actin actin is missing. In a step to explore this gap in the knowledge, atomic force Polymerization microscopy (AFM) was used to quantify the interactions between G-actin and Robin Maytum1, Khadar Dudekula2. proteins of the Tmod family. Unbinding forces between G-actin and Tmod1, 1Life Sciences, University of Bedfordshire, Luton, United Kingdom, Tmod2, Tmod3, or Tmod4 were quantified. Our results indicated that Tmod1, 2University of Edinburgh, Edinburgh, United Kingdom. Tmod3 and Tmod4 had unimodal unbinding force distributions while Tmod2 Tropomyosin is a rod-like coiled-coil protein that forms a continuous filament had a bimodal distribution. The two peak values observed for Tmod2 were that is weakly associated, but firmly-attached to the surface of the actin

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filaments in all eukaryotic cells. Simple eukaryotes such as yeasts have only (PTMs) on the C-terminal tails (CTTs). The intrinsically disordered CTTs one or two different tropomyosin isoforms which are known to be essential are involved in mediating binding interactions with tubulin and affecting larger and perform roles in regulating the actin cytoskeleton. However higher eukary- scale microtubule dynamics. Using the organism Tetrahymena thermophila,we otes have larger numbers of tropomyosins, the number of which appear linked are able to purify tubulin with varying degrees of post-translational modifica- to organismal complexity. Mammals have 4 genes producing over 40 different tions. We have developed a labeling scheme to isotopically label endogenous isoforms by alternative splicing. tubulin for NMR. Our purification scheme yields predominantly unmodified In higher organisms tropomyosin is best known and characterized in the and poly-glycylated tubulin, as determined by mass spectrometry and NMR. regulation of striated muscle contraction. The role of tropomyosin outside We see evidence of two populations of the CTTs, suggesting interaction with of muscle is less well understood. It is generally thought to have a regulatory the tubulin surface and discrimination between different states of tubulin. Com- role in controlling interactions of actin-binding proteins and in providing parison to isolated CTT peptides shows that the CTTs attached to the dimer are additional stability to actin-filaments. In the latter case has been considered sensitive to the tubulin surface as well as the PTMs present. This suggests the that tropomyosin binds to actin-filaments some time after their formation, importance of studying the CTTs in the context of the dimer rather than as iso- both making them mechanically stiffer and protecting them from breakdown. lated peptides to understand fully the mechanism by which they aid in tubulin We have produced a range of recombinant tropomyosins from all four and microtubule regulation. mammalian genes and characterized their actin-binding affinities in a cose- dimentation assay. We have then used them to systematically study the ef- 2771-Pos Board B378 fects of different isoforms of tropomyosin on actin polymerization for the An Optogenetic Approach to Control Microtubule Acetylation in Living first time. We have monitored actin polymerization by the well- Cells characterised change in fluorescence of a pyrene-label attached to actin. Neha Kaul, Hui Wang, Onur Dagliyan, Klaus M. Hahn. Actin polymerisation is monitored by measuring the significant fluorescence University of North Carolina, Chapel Hill, NC, USA. enhancement on polymerization. The association and conformation of proteins at the perimeter of moving Our results characterize the actin-affinities of some of the TPM3 and TPM4 iso- cells is affected by post-translational modifications of microtubules. Acety- forms for the first time, These are in the same general range as mammalian iso- lation of specific microtubules regulates cargo selection and trafficking to forms previously characterized by our group and others. specific regions of the cell edge. Microtubule acetylation correlates with We demonstrate differential effects of the different isoforms on actin- metastasis, indicating that microtubule post-translational modification is polymerisation for the first time. The data unexpectedly show the most signif- important in cancer cell biology. To study the spatio-temporal control of icant effects of the different isoforms appears to be in the early initiation / elon- microtubule acetylation in live cells, we designed an analog of alpha tubulin gation stages of polymerizations. This is unexpected as tropomyosin is only acetyl transferase1 (aTAT1) that can be controlled with light. Unlike other considered to have significant affinity for actin filaments through itself forming tubulin modification enzymes, aTAT1 is highly specific for tubulin in its a polymer along the surface of an actin filament. Different isoforms appear polymerized form. aTAT1 binds and transfers an acetyl moiety from acetyl capable of both enhancing and inhibiting the early stages of polymerization, coA to lysine 40 on alpha tubulin. A fragment of aTAT1 was fused to the with examples of the shorter 6-actin spanning TPM1 gene isoforms showing photo-responsive LOV2 domain from Avena Sativa phototropin and to a a significant reduction in the lag-phase of early polymerization. These differen- peptide that we had engineered to bind selectively to the dark state of tial effects on different isoforms provides a new role for tropomyosin in not LOV2 (Wang et al. Nature Methods 13 755-758, 2016). Only in the dark, only stabilizing filaments, but also in helping catalyze their formation. the LOV2 and Zdk bound to each other, occluding the microtubule binding interface. Photoactivatable aTAT1 (PA aTAT) could be fully activated in Microtubules, Structure, Dynamics & Associated less than a second, and the half-life for return to the off state could be adjusted between 1.7 and 496 seconds by mutating residues around the Proteins LOV2 flavin. Cell assays showed extensive acetylation of microtubules upon irradiation of cells expressing PA aTAT1. Use of the analog in living 2769-Pos Board B376 cells to elucidate the role of microtubule acetylation in directed motility will Detailed Structure of Single, Unaveraged Microtubules Revealed by be described. Electron Microscopy Tomography Andrea Fera1, Thomas S. Reese2, Dan L. Sackett1. 2772-Pos Board B379 1NICHD, NIH, Bethesda, MD, USA, 2NINDS, NIH, Bethesda, MD, USA. Revised Model, with Experimental Verification, for Motor Densities in Tubulin carboxyterminal tail peptides are short (10-20 amino acids) acidic, Gliding Assays unstructured peptides that vary significantly in sequence between tubulin iso- Anh T.N. Hoang, Augustus J. Lowry, Douglas S. Martin. types and are exposed on the outer surface of microtubules. These peptides Physics Department, Lawrence University, Appleton, WI, USA. have, so far, eluded direct visualization. Electron microscopy tomography is Gliding assays are used to investigate cytoskeletal polymers and motor proteins used here to analyze stabilized microtubules embedded in a tungsten-based in vitro, including measurements of cytoskeletal polymer stiffness and the negative stain previously found to resist sustained electron beam irradiation. cooperative effects of motors. A theoretical model (Duke et al., 1995) devel- Sample stability is compatible with high electron doses, permitting collection oped to extract the biophysical quantities of interest from gliding assays has of 142 images at various tilt angles with high signal-to-noise ratio. The result- been used extensively, yet the model remains incompletely validated. In gliding ing tomogram is reconstructed without the need of averaging multiple sam- assay experiments using kinesin and microtubules, we found inconsistencies in ples. After further optimizing collection of data and signal elaboration, model-dependent measurements of average kinesin spacing along microtu- images reveal the three dimensional structure of subunits as well as previously bules, a key parameter for motor cooperativity and bending stiffness interpre- unresolved small stalks on the outer surface of the microtubule. Inspection of tations. We traced these inconsistencies to an implicit approximation, an virtual sections demonstrates that the stalks are ~ 2.5 nm long and ~ 1 nm wide infinite processivity for kinesin. We report an updated theoretical model for (at half length). Stalks protrude intermittently every ~ 4 nm along the micro- gliding assays incorporating the finite processivity of kinesin, and report exper- tubule from the outer face of each monomer. These protrusions are well imental verification of the active motor density. Deviations between densities resolved from background in virtual sections perpendicular to the long axis inferred from the original model and updated model range from under 25% of microtubule. These protrusions are likely the carboxyterminal peptides of for short microtubules (0.5 micrometers long) to greater than four-fold for tubulin. This hypothesis is supported by comparison with results obtained in long microtubules (more than 2 micrometers long). We use these revised motor the same way from microtubules whose C-terminals have been cleaved enzy- densities to measure the length-dependence of microtubule bending stiffness matically. In summary, it is possible to resolve single peptides by amplitude (persistence length). contrast in a conventional electron microscope without the need of averaging data from multiple microtubules. 2773-Pos Board B380 Protofilament Bundles Created by Mechanical Splitting of Microtubules 2770-Pos Board B377 by Surface-Tethered Kinesin-1 The Role of the C-Terminal Tails of Tubulin in Microtubule Dynamics Virginia VanDelinder1, Peter Adams1,2, George D. Bachand1. Kathryn P. Wall1, Maria Pagratis1, Geoffrey Armstrong1, 1Center for Integrated Nanotechnology, Sandia National Laboratories, Jeremy Balsbaugh1, Chad Pearson2, Loren E. Hough1. Albuquerque, NM, USA, 2Molecular and Nanoscale Physics Group, 1University of Colorado - Boulder, Boulder, CO, USA, 2University of University of Leeds, Leeds, United Kingdom. Colorado - Anschutz Medical Campus, Denver, CO, USA. Microtubules are hollow cytoskeletal filaments composed of about 13 protofila- Tubulin is the heterodimeric building block of microtubules. Regulation of ments, which are made of a,b-tubulin dimers connected end to end. Motor tubulin in the cell occurs primarily through post-translational modifications proteins, such as kinesin-1, walk along microtubules in accordance with the

BPJ 7910_7914 564a Wednesday, February 15, 2017 polarity prescribed by the anisotropy of the microtubule’s tubulin dimer build- chronic traumatic encephalopathy), remains not well-understood due, in part, ing blocks. Surface-tethered kinesin can transport microtubules across surfaces, to its intrinsically disordered nature; Tau does not form a secondary structure and have been proposed as molecular shuttles for capture and transport of an- in solution. Here, we expand on our recent SAXS and electron microscopy alytes in microdevices. In device applications, microtubules, which are highly measurements that show Tau mediates microtubule bundle architectures dynamic in vivo, are stabilized with the anti-cancer drug . Even so, mimicking fascicles found in the axon initial segment (P. J. Chung et al. molecular wear and breakage into shorter microtubules result from the mechan- Nat. Commun. 2016, 7, 12278) to demonstrate this Tau-mediated interaction ical forces exerted on the microtubules during transport. We observe that between microtubules is contigent on the intrinsic disorder of Tau, allowing microtubule can also be split laterally into two protofilament bundles. These Tau to bind to microtubules and assume properties of an extended, grafted poly- protofilament bundles continue to be transported across the surface by the teth- electrolyte with biologically-encoded weak attractions. The extended state is ered kinesin, although they follow trajectories with a much high curvature than consistent with prior work (P.J. Chung, M.C. Choi et al. PNAS 2015, MTs and can curl up into submicron diameter rings. We characterized these 112, E6416) confirming a mushroom to polyelectrolyte brush state transition bundles using fluorescence microscopy, AFM, and SEM. We also investigated for Tau in the high (but physiologically-relevant) coverage regime using the effect of the kinesin surface density and the kinesin-surface linker on the the SAXS-osmotic pressure technique. Implications of this novel interaction rate of protofilament bundle formation. include facilitating transport of axon-specific cargoes and a possible, Sandia National Laboratories is a multi-mission laboratory managed and oper- physiological role for post-translational modifications (phosphorylation, acety- ated by Sandia Corporation, a wholly owned subsidiary of Lockheed Martin lation) that have been traditionally associated with Tau dysfunction and Corporation, for the U.S. Department of Energy’s National Nuclear Security neurodegeneration. Administration under contract DE-AC04-94AL85000. 2776-Pos Board B383 2774-Pos Board B381 Remodeling of Microtubule-Protein Tau Bundles in the Presence of Reversible Morphological Control of Tubulin-Encapsulating Giant Biologically Relevant Cations Liposomes by Hydrostatic Pressure Chaeyeon Song1, Phillip Kohl1, Peter Chung1,2, Bretton Fletcher1, Masayoshi Nishiyama1, Masahito Hayashi2, Kingo Takiguchi2, Herbert P. Miller1, Youli Li1, S.C. Feinstein1, Leslie Wilson1, Yoshie Harada3. Cyrus R. Safinya1. 1Department of Chemistry, Graduate School of Science, Kyoto University, 1UCSB, Santa Barbara, CA, USA, 2James Franck Institute, University of Kyoto, Japan, 2Nagoya University, Nagoya, Japan, 3Osaka University, Chicago, Chicago, IL, USA. Osaka, Japan. Microtubule-associated protein (MAP) Tau, an intrinsically disordered protein, Liposomes encapsulating cytoskeletons have drawn much recent attention to binds to and regulates microtubule dynamics in the axonal compartment in develop an artificial cell-like chemical-machinery; however, as far as we mature neurons. Using synchrotron small-angle X-ray scattering (SAXS) and know, there has been no report showing isothermally reversible morpholog- electron microscopy we recently reported on the discovery that under dissipa- ical changes of liposomes containing cytoskeletons because the sets of various tive out-of-equilibrium conditions (i.e. with added GTP at 37C), Tau mediates regulatory factors, that is, their interacting proteins, are required to control the microtubule bundling architectures which mimic microtubule fascicles in the state of every reaction system of cytoskeletons. Here we focused on hydro- axon initial segment, resulting from aggregate sub-kBT Tau-mediated attrac- static pressure to control the polymerization state of microtubules (MTs) tions (NATURE COMMUNICATIONS 2016, 7:12278). Here we will report within cell-sized giant liposomes (diameters ~10 mm). MT is the cytoskeleton on ongoing time-dependent SAXS studies of the response of the reconstituted formed by the polymerization of tubulin, and cytoskeletal systems consisting MT-Tau bundling architectures to the addition of biologically relevant cations of MTs are very dynamic and play many important roles in living cells, under dissipative out-of-equilibrium conditions. such as the morphogenesis of nerve cells and formation of the spindle appa- ratus during mitosis. Using real-time imaging with a high-pressure micro- 2777-Pos Board B384 scope [1, 2], we examined the effects of hydrostatic pressure on the Physical Interactions between Disease-Linked Tau Mutants and morphology of tubulin-encapsulating giant liposomes. At ambient pressure Microtubules (0.1 MPa), many liposomes formed protrusions due to tubulin polymerization Megan T. Valentine1, Benjamin J. Lopez1, Dezhi Yu2, Jamianne C. Wilcox1. within them. When high pressure (60 MPa) was applied, the protrusions 1Mechanical Engineering, University of California, Santa Barbara, Santa shrank within several tens of seconds [3]. This process was repeatedly induc- Barbara, CA, USA, 2Materials, University of California, Santa Barbara, Santa ible (around three times), and after the pressure was released, the protrusions Barbara, CA, USA. regenerated within several minutes. These deformation rates of the liposomes We investigate the physical interactions of tau proteins harboring disease- are close to the velocities of migrating or shape-changing living cells rather linked point mutations (R406W, P301L or DN296) with microtubules. Using than the shortening and elongation rates of the single MTs, which have single-molecule fluorescence microscopy, we measure the binding lifetimes been previously measured. These results demonstrate that the elongation and diffusion coefficients of fluorescently-labeled wildtype and mutant forms and shortening of protrusions of giant liposomes is repeatedly controllable of tau. We find similar values for mean diffusion coefficient and binding life- by regulating the polymerization state of MTs within them by applying and time in all cases when using relatively high ionic strength solutions (80 mM releasing hydrostatic pressure. PIPES). Under each condition we measure a broad distribution of diffusion [1] Nishiyama M. and Y. Sowa. 2012. Biophys J. 102:1872-1880. doi: 10.1016/ coefficients, but do not observe a significant fraction of immobile proteins. j.bpj.2012.03.033. Simple Monte Carlo simulations are used to explore the effects of binding life- [2] Nishiyama M. 2015. Subcell Biochem. 72:593-611. doi: 10.1007/978-94- time on the accurate determination of diffusion coefficients via single- 017-9918-8_27. molecule tracking, and to assess our ability to identify real heterogeneity in [3] Hayashi M., M. Nishiyama, / K. Takiguchi. 2016. Langmuir. 32:3794- short diffusive trajectories. Finally, we explore the effects of disease-linked 3802. doi: 10.1021/acs.langmuir.6b00799. tau mutants on microtubule stiffness using spectral analysis of movies of ther- mally fluctuating microtubules assembled by or coated with wildtype or 2775-Pos Board B382 mutant forms of tau. We find that the mutant tau proteins significantly alter Cell-Free Reconstituions of Tau and Tubulin Mediate Complex microtubule mechanics in a manner that depends on the presence of taxol, a Microtubule Architectures Found in the Axon small molecule inhibitor of microtubule dynamics. These studies provide Peter J. Chung1, Chaeyeon Song1, Joanna Deek2, Herbert P. Miller3, new insight into the physical interactions of disease-linked tau mutants and Youli Li4, Myung Chul Choi5, Leslie Wilson3, Stuart C. Feinstein3, microtubules, and suggest possible avenues for therapeutic intervention in Cyrus R. Safinya1. tauopathy diseases. 1Physics, Materials, and Molecular, Cell, and Developmental Biology, University of California, Santa Barbara, Santa Barbara, CA, USA, 2778-Pos Board B385 2Chemistry and Biochemistry, University of California, Santa Barbara, Santa Multi-Scale Modeling of the Nanomechanics of Microtubule Filaments Barbara, CA, USA, 3Neuroscience Research Institute and Molecular, Nan Jiang1, Jennifer Ross2, Ruxandra Dima1. Cellular, and Developmental Biology, University of California, Santa 1Chemistry, University of Cincinnati, Cincinnati, OH, USA, 2Physics, Barbara, Santa Barbara, CA, USA, 4Materials Research Laboratory, University of Massachusetts, Amherst, MA, USA. University of California, Santa Barbara, Santa Barbara, CA, USA, 5Bio and Microtubules are prime examples of large-size biomolecular systems that Brain Engineering, KAIST, Daejon, Korea, Republic of. assemble, disassemble, and self-repair by controlled inputs. Despite the known The physiological function of Tau, a microtubule-associated protein unequivo- role of mechanics in both the function of microtubules for example as motors cally linked to neurodegenerative disorders (e.g. Alzheimer’s, FTDP-17, driving the poleward chromosome motion during mitosis and in the remodeling

BPJ 7910_7914 Wednesday, February 15, 2017 565a of the microtubule lattice during depolymerization and severing by molecular feedback-controlled laser trap to measure the work done by curling protofi- motors, an understanding of the link between the structure of filaments and their laments. Using this ‘wave’ assay, we show that curling protofilaments carry dynamical properties is still in its infancy. Establishing such a link requires a a significant amount of the energy of GTP hydrolysis, in the form of combined experimental and computational approach. We will discuss the bending strain, which allows them to drive movement with an efficiency development of our approach to investigate the mechanics of filaments with similar to conventional motor proteins. To further understand whether relevance for chromosome motion and microtubule severing. Because of the microtubule disassembly speed depends on protofilament bending strain, modular architecture and strong inter- and intra-molecular coupling that influ- we used ‘hyperstable’ tubulin mutants in the wave assay. Mutating threo- ence the properties of microtubule filaments, we developed a multi-scale meth- nine 238 in b-tubulin to valine (T238V) resulted in microtubules that disas- odology, using coarse-graining and atomic details, to map out mechanical sembled7-foldmoreslowlythanwild-type.Sincefastdisassemblyis properties of microtubules on experimental timescales through large-scale mo- thought to be driven by protofilament strain, we expected that these mutant lecular simulations. We will present our findings, combining results from sim- microtubules might carry less strain than wild-type microtubules. Surpris- ulations with results from in vitro experiments, of the microscopic origin of ingly, however, the conformational wave energy for T238V microtubules physico-chemical properties of microtubules and their protofilaments and the was indistinguishable from wild-type. Thus, even though they disassemble molecular mechanisms of their response to controlled mechanical inputs. Our at very different rates, wild-type and T238V microtubules must store similar results focus on the thermodynamics of the lattice and the large bending rigidity amounts of curvature-derived strain. A simple physical model shows for a protofilament, which support the idea that the disassembling microtubule how these two properties can be uncoupled. Our work establishes that the is capable of generating a large mechanical force to move chromosomes during conformational wave mechanism can make a major contribution to kineto- cell division. Furthermore, we will present our findings regarding the role of chore motility and it provides a new, direct way to examine tubulin lattice defects and constraints on the crushing and recovery of the microtubule mechanochemistry. structure, which provide quantitative information relevant for severing mecha- nisms and support the proposed role of vacancies as binding sites for severing 2781-Pos Board B388 enzymes. Microtubule Sorting by Persistence Length and Surface Charge Density of Microtubules 2779-Pos Board B386 Naoto Isozaki1, Hirofumi Shintaku1, Hidetoshi Kotera1, Building Up and Force Probing the Microtubule Cytoskeleton from Taviare L. Hawkins2, Jennifer L. Ross3, Ryuji Yokokawa1. Scratch 1Micro Engineering, Kyoto University, Kyoto-shi, Japan, 2Physics, Matthias D. Koch1,2, Natalie Schneider3, Peter Nick3, Alexander Rohrbach2. University of Wisconsin — La Crosse, La Crosse, WI, USA, 3Physics, 1Princeton University, Princeton, NJ, USA, 2Imtek, University of Freiburg, University of Massachusetts — Amherst, Amherst, MA, USA. Freiburg, Germany, 3Botanical Institute, Karlsruhe Institute of Technology, We have been focusing on a kinesin-driven microtubule as a tool for direct Karlsruhe, Germany. transportation of target molecules in microfluidics. However, random direc- The eukaryotic cytoskeleton is a complex and dynamic network that regulates tion of gliding microtubules makes it difficult to utilize microtubules as an in important cellular functions and is driven by a large variety of forces or me- vitro molecular transporter. Here, we controlled microtubule gliding direc- chanical stimuli. Due to their mechanical rigidity, microtubules are able to tions by designing both persistence length (Lp) and surface charge density transport such stimuli allowing integrating distant regions of a cell instanta- of microtubules. Gliding directions under a given electric field corresponded neously. This is relevant for the response to pressure, gravity, or osmotic to their properties, and the difference in gliding directions led to the first changes and during mechanotransduction, a critical process during many severe demonstration of autonomous microtubule sorting in a microfluidic device. diseases such as deafness or cancer. We measured Lp of microtubules polymerized under various buffer condi- So far, only equilibrium mechanical properties of single microtubules have tions—nucleotide (GTP or GMPCPP), tubulin concentration, and tau protein. been characterized. Since intra- and extracellular forces occur on a brought Tubulin concentration controlled microtubule growth rate; faster growing in range of time scales, we fill this void by using an in vitro bottom-up approach higher tubulin concentration. Microtubules were exposed to thermal fluctua- to determine the frequency response of single microtubules and small networks tion, and their Lp were investigated by equating thermal energy and bending thereof that mimic the basic cytoskeletal structure. We combine a label-free energy of microtubules. As a result, tau binding showed no effect on Lp of darkfield imaging technique with multiple time-shared optical tweezers to flex- either GTP- or GMPCPP-polymerized microtubules. However, GMPCPP ibly construct and force probe such networks with a well-defined, user-selected and slow-growing produced microtubules with larger Lp than GTP and geometry over a broad frequency range. fast-growing. In addition, we controlled surface charge density of microtu- We report on a length dependent stiffening of individual microtubules above a bules by labeling them with DNA molecules. The negative charge of DNA physiologically relevant transition frequency between 1-30Hz due to the exci- increased the surface charge density. Then, we evaluated trajectory curvature tation of higher order bending modes. This increased transport efficiency for of microtubules under a given electric field. Larger Lp and smaller surface high frequencies displays a mechanical high-pass filter with a tunable cutoff charge density produced small trajectory curvature, which enabled to make frequency, e.g., allowing the cell to react to fluctuations at distant sites. Further- two kinds of microtubules with extremely different trajectory curvature, more, we identify and relate different mechanical responses of different slow-growing GMPCPP-polymerized and fast-growing GTP-polymerized network geometries to different functions inside the cell. Triangular networks, microtubules. Finally, we designed a microfluidic device to guide each for example, are sensitive to tiny imbalances and display a high stiffness even microtubule toward different destinations according to their curvatures. It for low frequencies, resembling a load bearing scaffold protecting the nucleus. sorted two kinds of microtubules with the sorting efficiency of 85%. There- The mechanistic comparison between basic network geometries, the known fore, we developed a method to design microtubule properties to tune the cytoskeletal topologies and the general function of different cell lines will sub- controllability of microtubule gliding directions and achieved a high- stantially strengthen our understanding of the function and structure of the efficient microtubule sorting in a microfluidic device. cytoskeleton. 2782-Pos Board B389 2780-Pos Board B387 Identification and Mechanism of a Novel Microtubule Nucleation Factor Direct Measurement of the Force-Generating Capacity of Protofilaments Akanksha Thawani1, Rachel S. Kadzik2, Sabine Petry2. Curling Outward from Disassembling Microtubule Tips 1Department of Chemical and Biological Engineering, Princeton University, Jonathan W. Driver1, Elisabeth A. Geyer2, Megan E. Bailey1, Luke M. Rice2, Princeton, NJ, USA, 2Department of Molecular Biology, Princeton Charles L. Asbury1. University, Princeton, NJ, USA. 1Physiology and Biophysics, University of Washington, Seattle, WA, USA, Microtubule (MT) cytoskeleton forms diverse structures such as the mitotic 2Biophysics and Biochemistry, UT Southwestern Medical Center, Dallas, spindle and cellular appendages. These defined MT architectures are created TX, USA. by spatiotemporally regulating MT dynamics, transport and most importantly, Microtubules can generate substantial force as they disassemble, which is nucleation. Since the identification of g-tubulin (g-TB) in 1989, it was thought important for the movement of chromosomes during mitosis. One explana- to be the sole MT nucleating protein in the cell. However, purified g-TB com- tion for this microtubule-driven motion is the conformational wave model, plexes display very low nucleation capacity and upon knockdown of g-TB, the which predicts that protofilaments pull on a kinetochore as they curl back cell can still form MTs. Therefore, it has long been speculated that nucleation during disassembly. However, it is unknown how much force a curling pro- factors other than g-TB may exist. However, what protein factors are critical tofilament produces and whether it is enough to drive chromosome move- for nucleating MTs and how this process occurs in the cell has remained a ment. We have developed an assay using recombinant yeast tubulin and a mystery.

BPJ 7910_7914 566a Wednesday, February 15, 2017

We discovered a novel protein that nucleates MTs. By using an isolated mitotic 2785-Pos Board B392 spindle assembly pathway, branching MT nucleation, we resolved individual Exploring Microtubule Breaking and Recovering Mechanisms using MTs and directly observed every MT nucleation event. In the absence of this Large Scale Molecular Simulations protein, no MTs emerge at all, thus resembling the phenotype of g-TB deple- Nan Jiang, Ruxandra Dima. tion. The recombinant protein rescues the phenotype in a concentration- University of Cincinnati, Cincinnati, OH, USA. dependent manner, indicating that the protein has bonafide nucleation activity. Microtubules (MTs), polymerized from dimer units, are the main cytoskel- Next, we identified the domains required for the nucleation activity of this pro- etal filaments providing structural support for cells. The reorganization of tein by generating truncation constructs. In addition, we defined its role in rela- MTs is often initiated by removing dimers through mechanical destruction tion to g-TB and other proteins involved in MT nucleation. In summary, we by motor enzymes, such as katanins and spastins. Those enzymes convert identified a novel and critical MT nucleation factor and elucidated its mecha- chemical energy from ATP hydrolysis into mechanical work. Previous nism, which provides insight into how MT nucleation occurs to assemble experimental work suggests that lattice defects act as active spots for those MT structures in the cell. enzymes working on the MT lattice. In our research, we investigate the me- chanical behavior leading to the crushing and recovering of MTs using mo- 2783-Pos Board B390 lecular dynamic simulations. Our in-silico experiments provide molecular Measurement of the Persistence Length of Cytoskeletal Filaments using details of the MT breaking pathways as well as the distribution of mechan- Curvature Distributions ical forces needed to break the lattice. Our results strongly support the pro- Pattipong Wisanpitayakorn1, Keith J. Mickolajczyk2, posal that defects represented by lattice vacancies are important in MT William O. Hancock2, Luis Vidali3, Erkan Tuzel€ 1. 1 crushing, as the force needed to break a filament with vacancies is much Department of Physics, Worcester Polytechnic Institute, Worcester, MA, lower than the one required to break an intact one. Importantly, we found USA, 2Department of Biomedical Engineering, Pennsylvania State 3 that our results, including the critical force, the breaking pathways and the University, University Park, PA, USA, Department of Biology and respective kinking angle distributions, match well their experimental coun- Biotechnology, Worcester Polytechnic Institute, Worcester, MA, USA. terparts. We also studied the recovery process of MTs with or without va- Cytoskeletal filaments such as microtubules and actin filaments play an cancies and in the presence and absence of constrains. Our results indicate important roles in mechanical signal transduction allowing cells to respond that constraints prevent MTs from fully recovering, particularly for a lattice to their environment. Measuring the mechanical properties of cytoskeletal with high degree of vacancies. Taken together with the pushing studies, our structures is crucial for gaining insight into intracellular mechanical stresses lattice recovery data strongly supports the view that vacancies in the filament and their role in regulating cellular processes. One of the ways to charac- can weaken the MT stability and serve as targets for MT cutting (severing) terize such bio-filaments is by measuring their persistence length, the enzymes. average length over which filaments stay straight. There are several ap- proaches in the literature to measure filament deformations, including Four- 2786-Pos Board B393 ier and tangent correlation analysis of images obtained using fluorescence Microtubule Mechanics in the Presence of Trimethylamine N-Oxide microscopy. Here, we show how curvature distributions can be used as an Adrienne M. Hester1, Corina C. Valencia2, Tegan L. Marianchuk3, alternative tool to quantify bio-filament deformations, and investigate how Dan L. Sackett4, Taviare L. Hawkins1. the apparent stiffness of filaments depends on the resolution and noise of 1Physics, University of Wisconsin- La Crosse, La Crosse, WI, USA, the imaging system. We present analytical calculations of the scaling curva- 2Biology, University of Wisconsin- La Crosse, La Crosse, WI, USA, ture distributions as a function of filament discretization, and test our predic- 3Physics, Arizona State University, Tempe, AZ, USA, 44Eunice Kennedy tions by comparing Monte Carlo simulations to results from existing Shriver National Institute of Child Health and Human Development, techniques. We also apply our approach to microtubules and actin filaments National Institute of Health, Bethesda, MD, USA. obtained from in vitro gliding assay experiments with high densities of non- Microtubules, the largest filament in the cytoskeleton, contribute to cellular functional motors. Finally, we provide an ImageJ Plugin for users to measure structure, transport, and division. The natural osmolyte Trimethylamine N-ox- the persistence length of bio-filaments from in vivo or in vitro fluorescence ide (TMAO) is known to stimulate tubulin dimer polymerization and coun- microscopy images. teract microtubule destabilization. Microtubule mechanics can be altered 2784-Pos Board B391 based on TMAO concentration. Here we present the results of microtubule me- How to Break a Ring: Exploring the Mechanisms of SAS-6 chanics, measured using the freely fluctuating method. Fluorescently labeled Oligomerisation microtubules confined to a two dimensional thin chamber (<3mm) were iso- Julia M.C. Busch1, Minos T. Matsoukas2, Philip C. Biggin1, lated, imaged, and analyzed using Fourier Mode analysis and Bootstrapping Ioannis Vakonakis1. statistics. 1University of Oxford, Oxford, United Kingdom, 2Cloudpharm P.C., Athens, Greece. 2787-Pos Board B394 Centrioles are cylindrical organelles that comprise the microtubule organising Quantifying Bisphenol a Absorbed in a Model Organism and Studying the centres in eukaryotic cells. As such they play important roles for diverse Effects this Endocrine Disrupting Compound Has on Microtubule cellular functions including locomotion, signalling and division. As basal Formation bodies centrioles anchor flagella and cilia to the cell membrane, while as cen- Pamela M. St John1, Abigail Fagan1, Spencer S. Mass2. trosomes they are involved in coordinating the spindle apparatus during chro- 1Chemistry, SUNY New Paltz, New Paltz, NY, USA, 2Biology, SUNY New mosome segregation. [Bornens 2012]. Paltz, New Paltz, NY, USA. Centriole numbers are strictly controlled with their duplication being a highly The endocrine disrupting compound bisphenol A (BPA) was detected using regulated and conserved process. [Azimzadeh and Marshall 2010] Deeper HPLC with fluorescence following its extraction from planaria that were insight into the molecular mechanisms at play in the centriole duplication pro- exposed to the substance for a period of days. The HPLC fluorescence tech- cess would have implications on our understanding of fundamental cell pro- nique can detect concentrations of BPA that fall well below 1 x 10 12 M in con- cesses and a number of disease including cancer, microcephaly and even centration and therefore it can be used to quantify the amount of BPA absorbed protozoan infections like malaria. [Chavali et al 2014, Marques et al. 2015]. by this model organism. Examination of the cytoskeleton of planaria exposed to SAS-6 is the structural core of the forming centriole. It oligomerises into rings BPA showed a disruption in microtubule formation that is currently being char- or spirals that ensure the nine-fold symmetry of the organelle. Oligomerisation acterized in vitro using atomic force microscopy. Microtubules formed in solu- of SAS-6 is crucial for successful centriole duplication. [Winey and O’Toole tion in the presence and absence of BPA have been deposited onto mica 2015, Hirono 2014] SAS-6 contains two dimerisation domains facilitating surfaces derivatized with self-assembled monolayers made from aminopropyl- oligomerisation; a long C-terminal coiled-coil and a globular N-terminal di- triethoxysilane vapor to promote adsorption of the protein. The prepared sur- merisation domain. We have explored the molecular interactions and mecha- faces have been imaged in fluid using AFM to better understand how BPA nisms contributing to SAS-6 N-terminal dimerization by combining X-ray may affect microtubule formation. AFM images of microtubules showed struc- crystallography, biophysical assays, virtual screening and molecular dynamics tures with diameters of approximately 20 nm and variable lengths. Roughness simulations. Our aim is to disrupt SAS-6 oligomerisation using small molecule measurements of the microtubule surface along with force spectroscopy used to modulators as an option for new approaches in cell cycle research and the determine the extent of adhesion between AFM tip and protein surface will be potential for drug design. presented.

BPJ 7910_7914 Wednesday, February 15, 2017 567a

Bacterial Mechanics, Cytoskeleton & Motility fimbriae which have adhesive molecules called FimH at its distal end. In vivo studies of the fimbriae attachment are difficult to perform due to the small size 2788-Pos Board B395 of these structures. In order to gain insight of how FimH catch bounds and Magneto-Aerotaxis: Bacterial Motility in Magnetic Fields fimbriae dynamics are coupled in the context of adhesion and fluid flow, we Stefan Klumpp1,2, Christopher Lefevre3, Livnat Landau1, Agnese Codutti1, developed a computer simulation based on the Lattice-Boltzmann method Mathieu Bennet1, Damien Faivre1. (LBM) couple to a dynamic simulation of the fimbriae. 1Max Planck Institute of Colloids and Interfaces, Potsdam, Germany, 2Georg The LBM offers the advantage, over more traditional numerical solution of the August University, Go¨ttingen, Germany, 3CEA/CNRS/Aix-Marseille Navier-Stokes equations, in that is highly parallelizable and that complex Universite, Saint Paul lez Durance, France. boundary conditions can be easily programmed. A three dimensional realistic Magnetotatic bacteria align along magnetic field lines with the help of a linear model of the bacteria with adhesive structures was developed and the interac- chain of magnetic organelles called magnetosomes. Magnetosomes contain mag- tion between flow, bacteria and fimbriae was analyzed. netic nanoparticles, (iron oxide or iron sulfide) and are aligned along a cytoskel- 2791-Pos Board B398 etal structure, the magnetosome filament, forming a cellular compass needle. The Atomistic Simulations of OmpA: A Step Towards the Virtual Bacterial physiological role of the magnetotactic apparatus is to guide aquatic bacteria to Envelope their preferred habitat in the oxic-anoxic transition zone. The corresponding tac- Firdaus Samsudin1, Maite L. Ortiz-Suarez1, Thomas J. Piggot1,2, tic behavior is thus called magneto-aerotaxis. We use a combined experimental Peter J. Bond3,4, Syma Khalid1. and theoretical approach to study magneto-aerotactic motility. We have charac- 1School of Chemistry, University of Southampton, Southampton, United terized magneto-aerotaxis under controlled magnetic fields and oxygen concen- Kingdom, 2CBR Division, Defence Science and Technology Laboratory, tration gradients. Using 12 different strains of cultured magnetotatic bacteria, we Wiltshire, United Kingdom, 3Bioinformatics Institute, A*STAR, Singapore, observed six different magneto-aerotactic behaviors that can be described as a Singapore, 4Department of Biological Sciences, National University of combination of three distinct mechanisms, in which the magnetic field either pro- Singapore, Singapore, Singapore. vides an axis or a direction of motion. Combined with mathematical modeling, Gram-negative bacteria have an extremely complex cellular envelope encom- our results suggest that sensing of the oxygen gradient (but not the oxygen con- passing two separate layers of membranes of distinct lipid compositions, which centration) can be replaced by following the direction given by the magnetic field. are segregated by a periplasmic space containing a network of peptidoglycan Use of the magnetic field as a proxy for the oxygen gradient appears to be the (PGN) cell wall. The outer membrane is adhered to the PGN scaffold via inte- dominant magneto-aerotactic behavior in nature. In addition, we implemented gral outer membrane proteins like the ubiquitous OmpA. Despite numerous an individual-based mathematical model (within a rather general framework of biophysical studies, the molecular details of OmpA interactions with PGN multi-state active Brownian particles) to address how the bacteria cope with sit- remain sparse. Here we report the atomistic simulations of full-length OmpA uations with a nonzero angle between the magnetic field and the oxygen gradient. monomer and dimer models embedded in a realistic representation of the outer membrane, comprising full-rough (Ra) lipopolysaccharide, phospholipids and 2789-Pos Board B396 cardiolipin, with and without PGN strands attached to the C-terminal domain. Cell Population and Electrophysiology Approaches to Osmotic Fitness of Pseudomonas Aeruginosa The models were stable in both monomeric and dimeric states with a large buried surface area found in the latter suggesting a robust dimerisation. The Ugur Cetiner1,2, Ian Rowe3, Anthony Schams2, Christina Mayhew2, flexible linker connecting the N- and C-terminal domains presents a possible Deanna Rubin2, Andriy Anishkin2, Sergei Sukharev1,2. mechanism by which OmpA provides a dynamic mechanical support to the 1Institute for Physical Science and Technology, University of Maryland, cell wall. Comparative analysis of OmpA C-terminal domains from different College Park, MD, USA, 2Department of Biology, University of Maryland, bacteria revealed a universally conserved PGN binding mode, while dimerisa- College Park, MD, USA, 3Department of Chemistry and Biochemistry, tion of OmpA may be crucial to prevent local distortions in the PGN network. University of Maryland, College Park, MD, USA. Our work provides a step forward towards understanding the structure-function Pseudomonas aeruginosa (PA) is an opportunistic pathogen with exceptional relationship of the major components of bacterial cell envelope to facilitate adaptability to a range of environments. Upon a sudden dilution of external me- future developments of antibiotics. dium, PA, as other bacteria evades mechanical rupture by engaging tension- activated channels releasing osmolytes. In this study we compare fast osmotic 2792-Pos Board B399 permeability responses in wild-type PA and E. coli (EC) strains in stopped- High Throughput Identification of Bacterial Cell Stiffness Regulators flow experiments and provide further electrophysiological description. Osmotic Rishi R. Trivedi, George Auer, Doug B. Weibel. dilution experiments showed that PA tolerates a broader range of shocks than Biochemistry, University of Wisconsin-Madison, Madison, WI, USA. EC. Under the assumption that the cell has a chance to survive the shock when Bacterial contamination impacts a wide range of industries with serious eco- osmolyte release is fast enough to curb osmotic swelling, we recorded the kinetics nomic consequences. To combat contamination, many antimicrobials target of light scattering responses to osmotic up- and down-shifts. While exhibiting a the bacterial cell envelope to disrupt the mechanical integrity of the cell lower water permeability, PA showed faster osmolyte release which correlated and promote lysis. Despite the central importance of cell mechanical proper- with better survival. To characterize the PA tension-activated channels, we gener- ties, many of the proteins that contribute to cellular integrity are unidentified ated giant spheroplasts from this microorganism and recorded current responses due to technological challenges in measuring the material properties of bac- in excised native patches. Unlike EC which relies on two types of channels, teria. In this talk, I will describe a high-throughput optical method for quan- EcMscS and EcMscL, to generate a distinctive two-wave pressure ramp response, tifying cell stiffness to comprehensively screen a genome-wide collection of PA exhibits a more gradual response dominated by MscL-type channels present 6,000 Pseudomonas aeruginosa mutants. We identified novel genes with in higher density. Genome analysis, cloning and expression revealed that PA pos- diverse physiological processes spanning cell-wall synthesis, energy produc- sesses one MscL-type (PaMscL) and two MscS-type proteins (PaMscS-1 and 2). tion, and DNA replication and contribute to cell stiffness when deleted. Our Both PaMscS channels exhibited an earlier activation by pressure as compared to methodology also enables the rapid evaluation of how chemical perturba- EcMscS. PaMscS-2 showed a smaller conductance, higher anionic preference, tions or genetic disruptions, from single amino-acid changes to whole-gene strong inactivation and slow recovery. While PaMscS-1 and PaMscL rescued deletions, alter the mechanical properties of cells. In total, our work demon- the unprotected MJF641 E. coli strain from osmotic damage, PaMscS-2 did strates that bacterial cell stiffness is a property of both the cell wall and not. Homology models and functional analysis of mutants have identified in broader cell physiology and lays the groundwork for future systematic PaMscSs the same ‘hinge’ elements responsible for inactivation as in EcMscS. studies of mechano-regulation. We discuss distinctive parameters of PA cells, the capacity of its osmolyte release system, and a path toward quantitative models of the osmotic rescue mechanism. 2793-Pos Board B400 Probing the Mechanosensitivity of the Bacterial Flagellar Motor by Load 2790-Pos Board B397 Manipulation Application of Lattice-Boltzman to the Study of the Adhesion Dynamics of Ashley Nord, Gachon Emilie, Barducci Alessandro, Pedaci Francesco. E. Coli under Flow Centre de Biochimie Structurale, Montpellier, France. Juan Diego Arango-Montoya1, Andre´s Gonza´lez-Mancera2, The bacterial flagellar motor (BFM) powers the swimming and swarming of Manu Forero-Shelton1. many motile bacteria. The torque of this rotary motor is provided by stators, 1Physics, Universidad de los Andes, Bogota´, Colombia, 2Mechanical ion motive force powered protein complexes anchored at the periphery of the Engineering, Universidad de los Andes, Bogota´, Colombia. rotor. It has recently been discovered that the stators are mechanosensitive, During E. Coli infection of the urinary tract, bacteria are able to withstand sig- with the number of engaged stators dependent upon the external load acting nificant forces from fluid flow. Bacteria are anchored to the surface via Type 1 on the motor, though the mechanism of BFM mechanosensitivity has yet to

BPJ 7915_7919 568a Wednesday, February 15, 2017 be revealed. In order to better understand the mechanosensitive properties of were always right-handed regardless of the rotation direction of the motor, the stators, we combine high-resolution motor torque measurements in and rotation rate was estimated to be 22.5 and 22.2 Hz in CW and CCW rota- E. coli with a step detection algorithm with which we can estimate stator tion direction, respectively. Using these structural and kinetic parameters, we stoichiometry in single-motor traces. We detect stator dynamics at both computationally reproduced the swimming and precession motion with a hy- steady-state and after a rapid and controlled change in external load, via a drodynamic model. The torque was estimated to be 50 pN nm, and the effi- magnetic field. ciency of the motor is calculated to be 6-10% assuming unitary steps we In this way, we directly probe the mechanosensitivity of the stators, we observe observed in the motor is caused by single turnovers of chemical reaction of and characterize single stator association and dissociation events triggered by ATP hydrolysis. the change in external load, and we quantify for the first time the dependence (1) Kinosita, Y., Uchida, N., Nakane, D. & Nishizaka, T. Direct observation of between stator number and external load. We incorporate these results into an rotation and steps of the archaellum in the swimming halophilic archaeon Hal- adsorption model of stator kinetics, providing the first step into understanding obacterium salinarum. Nature Microbiology 1, 16148. the mechanism of mechanosensitivity of the BFM. 2797-Pos Board B404 2794-Pos Board B401 Quantifying Biophysical Differences between Planktonic and Biofilm Micro Manipulating a Single Bacterial Cell: Studying the Strength of Bacteria in Response to Antibiotic Application Bacterial Cell Adhesion Catherine Volle1, Megan Nunez2, Temiloluwa Olaoluwa1, Ryo Kawamura. Kanesha Overton1. Physics\MBI, National University of Singapore, Singapore, Singapore. 1Cottey College, Nevada, MO, USA, 2Wellesley College, Wellesley, MA, Bacteria switch their lifestyle from motile individuals to immobile colonies USA. of clustered cells called biofilm on the surface when the environmental con- While many think of a bacterium as a solitary organism, most bacteria likely ditions such as pH, temperature and nutrients etc. are not favorable. In bio- spend at least some time living in complex communities called biofilms. In film, bacteria express extra cellular polymeric substance (EPS) such as fact, bacteria can exist in two separate forms: a motile planktonic bacterium proteins, DNA and polysaccharides to protect themselves. Once formed, bio- or a stationary biofilm member. These biofilms might be made of a single spe- film is stable and it it difficult to eliminate bacteria in biofilm. However at cies or multiple species, and provide a distinct organization that makes the the initial stage of biofilm development, bacterial cells have to attach the sur- biofilm resistant to removal by chemical or physical means. This becomes face before expressing EPS to protect themselves. We have developed a mi- a problem when biofilms are causing infections by growing on indwelling cro manipulation technique to manipulate single cells to study the initial medical devices or fouling the bottoms of boats. However, much of what surface attachment. In this presentation I will introduce a single-cell manip- we know about the antibacterial activity of a chemical compound comes ulation technique and the quantification of cell-surface and cell-cell adhesion from studying their effect on planktonic cells, and evidence suggest that strength. chemical antibiotics will have a different level of effectiveness and cause different biophysical changes on cells in a biofilm. The research we present 2795-Pos Board B402 here is a first step in determining antibiotic effectiveness on matched samples A Cardiolipin-Deficient Mutant of Rhodobacter Sphaeroides has an of planktonic and biofilm cells, and quantifying the biophysical effects of Altered Cell Shape and is Impaired in Biofilm Formation antibiotic application. Ti-Yu Lin, Douglas B. Weibel. UW-Madison, Madison, WI, USA. Membrane Pumps Cell shape has been suggested to play an important role in regulating bacterial attachment to surfaces and the formation of communities associated with sur- 2798-Pos Board B405 faces. We found that a cardiolipin synthase deletion mutant of the rod-shaped Protonation Dependent Water Permeation of Ion Binding Pocket of NaD bacterium Rhodobacter sphaeroides—in which synthesis of the anionic, highly Bound NaD,KD-ATPase curved phospholipid cardiolipin (CL) is reduced by 90%—produces Minwoo Han1, Wojciech Kopec2, Ilia A. Solov’yov3, Himanshu Khandelia1. ellipsoidal-shaped cells that are impaired in forming biofilms. Reducing the 1MEMPHYSCenter for Biomembrane Physics, University of Southern concentration of CL did not cause significant defects in R. sphaeroides cell Denmark, Odense, Denmark, 2Computational Biomolecular Dynamics growth, swimming motility, lipopolysaccharide and exopolysaccharide produc- Group, Max Planck Institute for Biophysical Chemistry, Go¨ttingen, tion, surface adhesion protein expression, and membrane permeability. Germany, 3Department of Physics, Chemistry and Pharmacy, University of Complementation of the CL-deficient mutant by ectopically expressing CL Southern Denmark, Odense, Denmark. synthase restored cells to their rod shape and increased biofilm formation. The six key acidic amino acid residues in the ion binding pocket of the Naþ, Treating R. sphaeroidescells with a low concentration (10 mg/mL) of the small Kþ -ATPase (NKA) proposed to drive ion binding, release and possibly molecule MreB inhibitor, S-(3,4-dichlorobenzyl) isothiourea (A22), produced determine Naþ or Kþ selectivity by dynamically altering their protonation ellipsoid-shaped cells that had no obvious growth defect, yet reduced R. states during the ion transport cycle. We investigate the effect of each pro- sphaeroides biofilm formation. This study demonstrates that CL plays a role tonation state using molecular dynamics (MD) and density functional theory in R. sphaeroides cell shape determination, biofilm formation, and the ability (DFT) simulations and determine the probable protonation schemes of the of this bacterium to adapt to its environment. Naþ bound conformation of NKA. MD simulations of all possible proton- ation schemes show that the bound Naþ ionsaremoststablewhenthree 2796-Pos Board B403 or four protons reside in the binding sites, and that Glu954 near site III is Cross-Kymography Reveals the Structural and Kinetic Parameters of always protonated. Glutamic acid residues in the three binding sites act as Archaellum water gates, and their deprotonation triggers water permeation to the binding þ Yoshiaki Kinosita, Takayuki Nishizaka. sites. From DFT calculations of Na binding energies, we conclude that þ Physics, Gakushuin Univ., Tokyo, Japan. three protons in the binding site are needed to effectively bind Na from wa- Motility is critically important for all unicellular microorganisms. The flagel- ter and four are needed to release them in the next step. We speculate that the lum is the representative motility machinery of bacteria and archaea, but its water permeation of site III provokes the protonation of Asp926 which will þ fluctuation and morphological dynamics have hindered the precise determina- induce Na release, and Glu327, Glu954 and Glu779 are all likely to be pro- þ tion of flagella structure and the motor properties. To overcome problems, here tonated in the Na bound occluded conformation. Our data provides key in- þ we developed an advanced analysis method that we named ‘cross-kymography’ sights into the role of protons in the Na binding and release mechanism of under total internal reflection fluorescence microscope. The cell body was NKA. attached to the glass, and labelled flagella were illuminated by evanescent wave. From the same image sequence, two kymographs, one parallel to the 2799-Pos Board B406 flagella and the other perpendicular to it, were extracted. Notably, the combi- Exchange of Sodium Or Potassium Ions against Protons at Cytoplasmic nation of directions of the propagation of spots in two kymographs uniquely in- Side of Na,K-ATPase dicates both structural helicity and rotation direction at any one time (1). The Vsevolod Tashkin. application to Salmonella enterica serovar Typhimurium demonstrated the Frumkin Institute of Physical Chemistry and Electrochemistry, Moscow, left-handed helix structure as a normal mode. We observed a rotary filament Russian Federation. of archaea, the archaellum, as surface appendage that resembles bacterial fla- The Na,K-ATPase performs active transmembrane transport of sodium and gellum but is homologous to bacterial type IV pilus; little is known about the potassium ions. In addition, exchange of sodium or potassium ions against mechanism by which archaella produce motility. Notably, archaella bundle protons has been found under appropriate conditions. To study this exchange

BPJ 7915_7919 Wednesday, February 15, 2017 569a on the cytoplasmic side of the protein, the admittance method has been K, while site-III exclusively binds Naþ. We mutated Y780 in site-III of the Xen- applied to an experimental setup consisting of membrane fragments with opus a1-subunit with the ouabain resistance-conferring mutation Q120R/ Na,K-ATPase adsorbed to a bilayer lipid membrane (BLM) or a solid sup- N131D (RD). Previous studies presented conflicting reports of how Y780F af- þ ported membrane. The small changes of the membrane admittance (capaci- fects Na o affinity. We used two-electrode voltage clamp to evaluate the effect þ tance and conductance) initiated by the fast steps of the pH or Na of Y780F on extracellular ion-binding reactions. The center (V1/2) of the Boltz- concentration were measured in the absence of ATP. pH jumps were per- mann distribution describing the charge-voltage curve for the transient charge formed by release of protons from a photosensitive compound (caged Hþ) movement that reports the voltage-dependent binding and unbinding of extra- þ þ þ triggered by a UV light flash, the Na concentration jumps were carried cellular Na in the absence of K o, was shifted to more negative voltages: out by fast solution exchange. The changes of the membrane capacitance V1/2 = 46.9 5 2.5 mV (n=10) in RD to V1/2 123 5 9 mV (n=6) in Y780F, þ þ triggered by a pH jump depended on the initial pH and the concentration indicating a ~6-fold reduction in affinity for Na o. The K o affinity was reduced þ of sodium or potassium ions. The effects of these ions are explained by a ~2-fold without Na o, but increased in its presence, particularly at negative þ theoretical model assuming competition of protons with sodium or potassium voltages. Apparent affinity for Na i activation of pump currents was measured þ þ ions in the binding sites on the cytoplasmic side of the Na,K-ATPase. The in inside-out patches, exposed to 5 mM K o in NMG o pipette solution, without þ þ approximation of the experimental data by theoretical curves yields the Na o. The K0.5 for Na i activation in the presence of 4 mM ATP at 0 mV was dissociation constants and the cooperativity coefficients of the binding sites 3.3 5 0.1 mM (n=3) for RD and 18 5 1 mM (n=2) for Y780F. Our results indi- for sodium ions and potassium ions. In the presence of magnesium ions the cate a strong influence of the hydroxyl group on Naþ binding affinity. To study apparent dissociation constants of sodium were increased. The effects of K the importance of Y780’s hydroxyl moiety and potential for a cation-p interac- ions on the pH driven capacitance changes were attenuated in presence of tion during Naþ binding, we are attempting incorporation of unnatural tyrosine 100 mM of ATP and no magnesium. This observation indicates that derivatives with fluorination around its phenol ring to gradually ablate tyro- the Na,K-ATPase is driven into a conformation with low affinity for the sine’s ability to form this interaction. After introducing an amber (TAG) potassium ions presumably because of trapping the enzyme in state stop codon at position 780, we have been able to rescue normal RD function with ATP bound in the low-affinity site. Supported by RFBR project (~1/5th of current with RD cRNA) by coinjecting tyrosine tRNA with the 16-04-01162. Y780TAG-cRNA.

2800-Pos Board B407 2802-Pos Board B409 A Na/K Pump with Altered Stoichiometry Contributes to Brine Shrimp Ion Binding and Charge Movements in the Function of the Na/K-Pump Adaptation to High Salinity Huan Rui1, Jorge Sanchez-Rodriguez2, Miguel Holmgren3, Dylan J. Meyer1, Jessica Eastman1, Huan Rui2, Kevin Stanley3, Francisco Bezanilla1, Benoıˆt Roux1. Benoit Roux2, Craig Gatto3, Pablo Artigas1. 1Department of Biochemistry and Molecular Biology, The University of 1Cell Physiology and Molecular Biophysics, Texas Tech University Health Chicago, Chicago, IL, USA, 2Departamento de Fı´sica, Universidad de Sciences Center, Lubbock, TX, USA, 2Department of Biochemistry and Guadalajara, Guadalajara, Mexico, 3Molecular Neurophysiology Section, Molecular Biology, The University of Chicago, Chicago, IL, USA, 3School National Institute of Neurological Disorders and Stroke, National Institutes of of Biological Sciences, Illinois State University, Normal, IL, USA. Health, Bethesda, MD, USA. Brine shrimp are osmoregulating crustaceans capable of adapting to satu- The Naþ/Kþ-pump resides in the plasma membrane and maintains the phys- rating salt environments, allowing them to thrive in isolated salt lake envi- iological Kþ and Naþ concentration gradient across the cell membrane. It ronments away from potential predators. Under hypersaline conditions, operates via an ‘alternating-access’ mechanism, making iterative transitions þ þ brine shrimp upregulate expression of a Na ,K -ATPase a-subunit between inward-facing (E1) and outward-facing (E2) conformations. The þ þ (a2(KK)) containing two asparagine-to-lysine substitutions in the highly- outward-facing, phosphorylated P-E2 is able to bind both Na and K . conserved ion-binding region. We utilized electrophysiology and radioactive The binding of each ion produces a distinctive, experimentally detectable ion uptake to evaluate the effect of equivalent substitutions (N333K and charge movement, i.e., the gating charge. This gating charge reflects the frac- þ þ N785K) in the Na ,K -ATPase a1-subunit from Xenopus, heterologously tion of the transmembrane potential traversed by the charged species in the expressed in Xenopus oocytes, to understand the molecular mechanism by system upon ion binding and can be computed from molecular dynamics which a2(KK) contributes to the brine shrimp’s hypersalinity adaptation. simulations. Previously simulations overestimated the gating charge upon Two-electrode voltage clamp measurements of ouabain-sensitive, extracel- extracellular Naþ binding. This is likely caused by a counter movement of þ þ lular Na (Na o)-dependent charge movement yielded steady-state charge- protons, which was not accounted for in the simulations. To test such a pos- voltage plots. A shift in the center (V1/2) of the Boltzmann distribution sibility and to resolve the binding site protonation configuration upon the E1 describing such curves to positive or negative voltages reflects an increase to E2 transition, we performed extensive molecular dynamics simulations of or decrease, respectively, in affinity (2-fold change per 25 mV shift) of all possible binding site protonation states. The results present evidence sup- þ mutated pumps. The V1/2 was (in mV) 47 5 3 for wild type (WT), porting the proposal that there is a counter movement of proton upon Na 116 5 3 for N333K, 115 5 3 for N785K, and 84 5 3 for N333K/ binding to P-E . We also constructed a working kinetic model of the þ þ 2 N785K. Without Na o, the apparent affinity for K o-induced outward pump cycle, which reveals that there is an additional state of P-E with þ þ 2 Na ,K -ATPase current was similar for WT, N333K, and N333K/N785K, one proton bound, lending supports again to the counter charge movement but reduced 20-fold by N785K. Excised inside-out patches were used to proposal. The findings from simulations and kinetic modeling offer a plau- þ þ measure the Na i-dependence of uncoupled inward H current, revealing sible explanation as to why pH affects the extracellular ion binding and þ reduced apparent Na i affinities of ~3-fold for N333K, ~4-fold for N785K, help illustrate what happens structurally during the structural transition be- and ~10-fold for N333K/N785K. Molecular dynamics simulations of tween E1 and E2. N333K/N785K showed decreased ion-binding site stability that was reversed after removing one ion. This suggested a reduced transport stoichiometry for þ 2803-Pos Board B410 N333K/N785K, which was subsequently measured using 86Rb uptake under Tuning Voltage Dependent Transitions during the Extracellular NaD TEVC. WT pumps imported 2.1 50.1 (n=40) Rbþ/charge extruded D D þ þ Binding/Release of the Na /K -ATPase by External Protons (3Na :2K stoichiometry), while N333K/N785K imported 1.0 50.1 ´ 1,2 2 2 þ þ þ Jorge E. Sanchez-Rodrı´guez , Huan Rui , Benoıˆt Roux , (n=13) Rb / charge extruded (2Na :1K stoichiometry). These modified Miguel Holmgren3, Francisco Bezanilla2. transport characteristics of a2(KK) play an essential role in the brine 1Departamento de Fı´sica, Universidad de Guadalajara, Guadalajara, Jal., shrimp’s ability to adapt to hypersaline environments. Mexico, 2Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA, 3Porter Neuroscience Research 2801-Pos Board B408 Center, NINDS, National Institutes of Health, Bethesda, MD, USA. Functional Effects of Substitutions Modifying the Hydroxyl or Phenol Ring Fueled from the energy of one molecule of ATP’s hydrolysis, the Naþ/Kþ- of Tyrosine 780 at Ion Binding Site-III of the Na, K-Atpase ATPase exports 3Naþ and then imports 2Kþ across the cell membrane play- þ þ Kerri Spontarelli1, Dylan J. Meyer1, Daniel T. Infield2, ing an essential role in maintaining the Na and K ionic gradients across Christopher A. Ahern2, Pablo Artigas1. the cell membrane. Using the cut-open oocyte voltage clamp technique we 1Cell Physiology and Molecular Biophysics, Texas Tech University Health measured transient currents associated with external Naþ binding/occlusion Sciences Center, Lubbock, TX, USA, 2University of Iowa, Iowa City, IA, steps of the squid Naþ/Kþ-ATPase expressed in Xenopus oocytes. From USA. these transient currents, three temporally well-defined components can be þ þ The Na -K ATPase uses three ion-binding sites to export Na and import K isolated: fast (Qf), medium (Qm) and slow(Qs). Presumably, each one corre- against their electrochemical gradients. Site-I and site-II bind either Naþ or sponds to a separate Naþ binding/occlusion transition. Here we study the

BPJ 7915_7919 570a Wednesday, February 15, 2017

effects of external pH on the kinetics and voltage-dependence of Qm and Qs, action energy suggested PLB can form a stable complex with either the canon- which can be studied within the voltage range accessible. Preliminary results ical site (856.1 kJ/mol) or the proposed secondary site (509.8 kJ/mol). show that an increase of 1.5 pH units shifted the steady-state charge distri- Notably, docking of PLB to the M3 helix on the opposite side of SERCA bution of both components to the right by more than 15 mV. These results shows a much less favorable interaction energy (108 kJ/mol). Additional an- are inconsistent with protons contributing directly to the charge movement alyses to be described include umbrella sampling simulations to determine the steps, but rather they are influencing the protonation state of key residues binding free energy of PLB for several putative binding sites on the SERCA along the external Naþ access pathway and/or in the binding sites. To illus- transmembrane domain. Overall, the data are consistent with a 2-site model trate in detail the protonation change in response to the altering pH and trans- for the PLB-SERCA regulatory complex, in which PLB interacts primarily membrane voltage, we performed molecular dynamics simulations of the with a high affinity conventional site and translocates to a nearby lower affin- outward facing Naþ/Kþ-ATPase with different ion/proton configurations in ity secondary site when SERCA binds calcium. Significantly, this 2-site model the binding sites and computed their displacement charge. This is followed reconciles our observations that FRET is maintained in high calcium with by Bayesian analysis to determine their population distribution under various other groups’ observations that site-specific crosslinking is lost with calcium external conditions. The results offer a mechanistic explanation as to binding. how external pH affects external Naþ binding and also highlight the impor- þ þ tance of protons in the function of the Na /K -ATPase. Supported by 2806-Pos Board B413 PROSNI2016-UdeG, U54GM087519 and GM030376. Targeting the Serca-Plb Complex using FRET in Living Cells Daniel Stroik1, Ang Li1, Tory Schaaf1, Simon Gruber2, Razvan Cornea1, 2804-Pos Board B411 Roger Hajjar2, David Thomas1. Structural and Functional Differences between Sarcoplasmic Reticulum 1Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Calcium Pump (SERCA) and Plasma Membrane Calcium Pump (PMCA) Minneapolis, MN, USA, 2Biophysics and Systems Pharmacology, Mount Reaction Cycle Intermediates Sinai Hospital, New York City, NY, USA. Nicola´s Andres Saffioti1, Marilina de Sautu1, Irene C. Mangialavori1, We are using time-resolved FRET (TR-FRET) in an intact cellular envi- Mariela Ferreira Gomes1, Rolando C. Rossi1, Joshua R. Berlin2, ronment to measure the function, binding, and structural dynamics of the Juan Pablo F. Rossi1. sarco(endo)plasmic reticulum Ca-ATPase (SERCA) in complex with its 1Dept. Quı´mica Biologica, IQUIFIB, Capital Federal, Buenos Aires, cardiac regulator phospholamban (PLB). A central factor in heart failure is þ Argentina, 2Department of Pharmacology and Physiology, New Jersey an increase in basal cytosolic Ca2 resulting in decreased cardiac contrac- Medical School, Rutgers University, Newark, NJ, USA. tility; thus fine-tuning the dynamics of the SERCA-PLB complex to augment þ SERCA and PMCA belong to the P-ATPases family. SERCA’s structure and Ca2 transport is a promising therapeutic approach. Our laboratory has function have been widely characterized whereas PMCA has a relatively developed an inducible system expressing SERCA and PLB fused to fluo- high sequence identity with SERCA but its structure is less known. Although rescent proteins in HEK293 cells and demonstrated a wide dynamic range both proteins pump Ca2þ out of the cytoplasm of cells, their kinetic properties with respect to biosensor expression and FRET response. Both proteins are very different. To understand this on the basis of differences in their struc- co-localize in the endoplasmic reticulum. Using TR-FRET, the binding ture, we employed fluoride complexes with beryllium, aluminium or magne- and structural states of the SERCA-PLB complex have independently sium, which stabilize different analogues of the phosphorylated intermediates resolved. FRET between GFP-SERCA and RFP-PLB in live cells shows in P-ATPases, although they have never been tested on PMCA. To study the hyperbolic dependence on protein concentration, with a maximum efficiency protein structure we employed the hydrophobic photoactivatable probe 3-(tri- of 17.2 þ/- 1.6%. This observed FRET is described by a two-component fluoromethyl)-3-(m-iodophenyl)diazirine (TID) which labels the transmem- model with a Gaussian distribution corresponding to a probe separation brane domains of these proteins and the fluorescent probe 2’,3’-O-(2,4,6- distance of 54.4 þ/- 2.5A and 90.8 þ/- 8.5A. This is consistent with models Trinitrophenyl)adenosine-5’-triphosphate (TNP-ATP) which binds to the in which PLB undergoes a structural transition between a relaxed (R) and - nucleotide binding-domain of both pumps. Results show that BeF3 and tense (T) state while bound to the calcium pump. Furthermore, increases - 2þ AlF4 inhibit PMCA activity at micromolar concentrations, whereas millimolar in Ca in permeabilized cells results in only modest changes in SERCA- concentrations of Mg2þ and F- are required to achieve that effect. TID labeling PLB FRET suggesting that the binding interface is remodeled by SERCA in different conformations of SERCA correlates well with the protein surface structural changes. Key residues in the transmembrane and hinge regions exposed to the bilayer calculated from crystallographic models, with less label- of PLB have been investigated for their contribution to affinity for SERCA, ing in the presence of calcium. Irrespectively of the presence of metal-fluoride leading to a better understanding of the mechanism of this allosteric regula- complexes, TID labeling of PMCA only decreases when the pump is incubated tion. As a proof-of-concept, PLB variants were shown to efficiently compete with calcium and calmodulin, indicating a lower exposure of the transmem- with wild-type PLB for SERCA binding. A recently discovered transmem- brane region. When incubated with metal-fluoride complexes, TNP-ATP in- brane peptide named DWORF (Nelson et al., 2016) activates SERCA by creases its quantum yield when it is bound to SERCA but decreases by a displacing the endogenous calcium pump inhibitors (e.g., PLB). We have half when it is bound to PMCA. Our results indicate that calmodulin binding developed a TR-FRET assay to characterize the binding and conformational to PMCA allows conformational changes in the transmembrane region similar dynamics of SERCA and its regulators - including DWORF - to charac- to those observed in SERCA in presence of calcium, but the nucleotide biding terize these novel interactions. These biosensors can readily be applied for domain behaves very differently when these proteins are in a phosphorylated- the screening of small molecules for potential heart failure therapy. This like state. work was supported by NIH grants (GM27906, HL129814, AR07612, and With grants from ANPCYT, CONICET and UBACYT. DA037622).

2805-Pos Board B412 2807-Pos Board B414 Molecular Dynamics Simulations Suggest Multiple Binding Sites for X-Ray Crystallography, Fluorescence, and Molecular Simulations Studies Phospholamban on SERCA on Regulators of SERCA Nikolai Smolin, Vidhya Sivakumaran, Seth L. Robia. Bengt Svensson1, Joseph M. Autry1, Chengguo Xing2, Razvan L. Cornea1, Department of Cell and Molecular Physiology, Loyola University Chicago, John K. Lee1, David D. Thomas1. Maywood, IL, USA. 1Biochemistry, Molecular Biology and Biophysics, Univ. of Minnesota, The transport activity of the cardiac calcium pump (SERCA2a) is inhibited by Minneapolis, MN, USA, 2Medicinal Chemistry, Univ. of Florida, its regulatory partner phospholamban (PLB). PLB has been shown to bind in a Gainesville, FL, USA. cleft composed of SERCA transmembrane helices M2,4,6, resulting in a X-ray crystallography was used to solve the structure of the sarco/endo- reduction in the pump’s apparent calcium affinity. A comparison with related plasmic reticulum Ca-ATPase (SERCA) in complex with a novel small- ion transporters led us to hypothesize that PLB might also bind to SERCA he- molecule inhibitor (CXL017) that targets multidrug-resistant leukemia. The lix M9, alternating between this position and the canonical site during SERCA structure, solved at 3.0A˚ resolution, reveals CXL017 bound at a new site structural transitions between calcium-bound (E1) and calcium-free (E2) con- that is distinct from those of other known inhibitors of SERCA. This new formations. This model is consistent with our previous fluorescence resonance site is near the ATP binding site, and suggests blockage of the phosphoryla- energy transfer (FRET) measurements. In the present study we observed pho- tion site as the main mechanism of inhibition. This inhibitor binding site ex- toactivatable crosslinking of PLB to all major enzymatic SERCA substates, plains the synergistic relationship previously uncovered between CXL017 including calcium-bound E1 states. The energetics of the PLB-SERCA inter- and other SERCA inhibitors. Identifying a novel inhibitor binding site pro- action was explored with molecular dynamics simulations of several different vides insights for structure-guided improvements of this new class of poten- configurations of the PLB-SERCA regulatory complex. An analysis of inter- tial anti-cancer agents. Molecular modeling and simulation, in combination

BPJ 7915_7919 Wednesday, February 15, 2017 571a with fluorescence spectroscopy, was used to generate new structural and well established that binding and subsequent hydrolysis of ATP drives substrate mechanistic models that better describe the conformational landscape and transport. However, details of the mechanism remain enigmatic. Two ATP mol- regulation of SERCA by phospholamban (PLB) and sarcolipin (SLN). Fluo- ecules bind at the two nucleotide binding sites (NBS) at the interface of the two rescence spectroscopy, including fluorescence resonance energy transfer nucleotide binding domains (NBD). ABCB1 has two canonical NBS, while (FRET) between fluorescent-fusion-protein constructs of SERCA, provided ABCB11 (49% sequence identity to ABCB1) has only one canonical NBS. structural dynamics information about the cytosolic headpiece of SERCA We capitalize on the similarity of ABCB1 and ABCB11, which are both active and also on its interaction with regulatory peptides. Simulations were per- transporters, to determine how the chemical energy stored in ATP is converted formed to sample conformations of fusion protein constructs, CFP- into conformational motion. These motions of the NBDs lead to directional sub- SERCA, YFP-PLB and YFP-SLN. The simulations were based on crystal strate transport through the membrane. We find that canonical and degenerate structures of SERCA/PLB and SERCA/SLN complexes. FRET parameters, NBS have different conformational preferences. It is this difference that enables 2 such as the inter-probe distance, RDA, and the orientation factor k were transporters with a degenerate NBS to remain active, while transporters with calculated from simulations and used to generate predictions of fluorescence two canonical NBS would be blocked. The canonical NBS can fully occlude lifetimes and FRET efficiency distributions that were compared with exper- ATP, which does not seem to occur at degenerate NBS. We discovered that imental data. Based on these results, we propose a model of a super- both, binding as well as hydrolysis of ATP have a large impact on the free energy inhibitory complex of SERCA, in which SERCA first binds SLN and then profile of NBD dimerization, thereby leading to changes in the geometry of binds PLB. Spectroscopic studies were performed at the Biophysical Tech- NBD dimer, thereby energizing the transport cycle. nology Center and computational work at the Minnesota Supercomputing Institute. This work was funded by NIH grants to DDT (GM27906 and 2810-Pos Board B417 AR07612). Measuring Transport Kinetics of Light Driven Chloride Pump, Halorhodopsin 2808-Pos Board B415 Hasin M. Feroz. - The HLA3 Protein of C. Reinhardtii Enhances HCO3 Transport Activity Chemical Engineering, Penn State University, University Park, PA, USA. of Mammalian Cells Applications in optogenetics and biophotonic devices have led to an increase in Michael A. McCloskey1, Deqiang Duanmu2, Nicholas Benge1, interest in light-driven ion pumps and channels. However, quantitative infor- Martin H. Spalding1. mation about the transport kinetics of these membrane proteins cannot be 1GDCB, Iowa State University, Ames, IA, USA, 2State Key Laboratory of directly obtained due to challenges in measuring transport and determining pro- Agricultural Microbiology, Huazhong Agricultural University, Wuhan, tein concentration and/or orientation of proteins in reconstituted liposomes China. (1, 2). We have developed a liposome-based assay to directly quantify light- Many aquatic photoautotrophic microorganisms expend metabolic energy to driven ion transport of the Cl- pump, halorohodopsin, pHR from Natronomonas - concentrate CO2 and HCO3 for efficient carbon fixation by RubisCO, under pharaonis. First, we used stopped flow fluorimetry to quantify the net rate of conditions where CO2 concentration is well below atmospheric levels (~10 light-driven ion concentration change per proteoliposome. Next, we used mM). The CO2-concentrating mechanism (CCM) is incompletely resolved, time-correlated single-photon counting (TCSPC) to evaluate the number of but in cyanobacteria and algae, low CO2 induces a suite of CCM proteins fluorescently-tagged proteins per proteoliposome. Combining the results crucial for survival. In Chlamydomonas reinhardtii these include the plasma from the two techniques and accounting for directional orientation using selec- membrane protein HLA3, a member of the ATP-binding cassette (ABC) super- tive inactivation of outwardly facing pHR proteins using mercury chloride, we family of active transporters. HLA3 is most similar to the cystic fibrosis trans- find that under saturating conditions of illumination, pHR pumps 350(590) membrane conductance regulator (CFTR) and the sulfonylurea receptor— ions/protein/s. This result is consistent with novel consecutive single- - neither an active transporter. Knockdown of HLA3 inhibits HCO3 uptake by turnover photocycle experiments conducted at variable actinic repetition rates Chlamydomonas in low CO2 media, and previous work suggests it may func- with purified protein that suggest a maximum turnover of 390(520) ions/pro- - tion as a HCO3 transporter. Here we tested whether HLA3 can transport tein/s. These experiments suggest that under continuous illumination the rate - - HCO3 in heterologous systems lacking other CCM components. We expressed limiting step in the pHR photocycle is the Cl uptake step (t1/2 = 1-2 ms) rather HLA3 in three mammalian cell lines and followed bicarbonate uptake in pH than the final relaxation of P6 to the ground state (t1/2 = ~ 20 ms)). This fluores- - jump assays using the reporters BCECF or pHRed. At 1.25 - 25 mM HCO3 , cent liposome-based transport assay can be extended to other light-driven ion HLA3 increased rates of pH recovery from an acid load, 5.4-fold at the lowest transporters as a standard in vitro technique for biophysical characterization - 3 HCO3 (7.0 vs. 1.3 x10 DpH/s)—without affecting buffer capacity. It also of reconstituted opsins. - enhanced rates of cytosolic alkalization upon step increase in [HCO3 ]o. References HLA3 had similar Mr expressed in Xenopus and Chlamydomonas, greater 1. Seki A, et al. (2007) Heterologous Expression of Pharaonis Halorhodopsin than in mammalian cells—perhaps reflecting inter-species glycosylation differ- in Xenopus laevis Oocytes and Electrophysiological Characterization of Its ences, or established effects of temperature on glycoprotein maturation. Muta- Light-Driven Cl- Pump Activity. Biophysical journal 92(7):2559-2569. tion G544D in the canonical ABC signature sequence of HLA3 abrogated 2. Bogomolni R, Taylor M, & Stoeckenius W (1984) Reconstitution of purified - enhancement of HCO3 uptake without altering HLA3 expression. The corre- halorhodopsin. Proceedings of the National Academy of Sciences 81(17):5408- sponding CFTR mutation G551D inhibits ATPase activity100-fold, preventing 5411. ATP-dependent gating of CFTR channels. These findings are consistent with 3. Chizhov I & Engelhard M (2001) Temperature and halide dependence of the the notion that HLA3 devoid of other CCM components can directly mediate photocycle of halorhodopsin from Natronobacterium pharaonis. Biophysical - ATP-dependent transport of bicarbonate, even at HCO3 concentrations above journal 81(3):1600-1612. those inducing the CCM. 2811-Pos Board B418 2809-Pos Board B416 Function of Bacterioruberin in Archaerhodopsin 4, from Expression to Nucleotides Control the Conformation of the Motor Domain of ABC Characterization Transporters Chao Sun1, Yujiao Gao1, Xiaoyan Ding1,2, Xiaoyan Ding1, Juan Wang1, Daniel Szo¨llo¨si1, Yaprak Doenmez-Cakil1, Imran Sohail2, Gabor Szaloki3, Juan Wang1, Haolin Cui1, Haolin Cui1, Yanan Yang1, Xin Zhao1. Katalina K. Goda3, Gergely Szakacs4, Peter Chiba2, Thomas Stockner1. 1Department of Physics, East China Normal University, Shanghai, China, 1Pharmacology, Medical University of Vienna, Vienna, Austria, 2Institute of 2Department of Biochemistry and Molecular Biology, Penn State College of Medical Chemistry, Medical University of Vienna, Vienna, Austria, Medicine, Hershey, PA, USA. 3Department of Biophysics and Cell Biology, University of Debrecen, Archaerhodopsin 4 (AR4), a new member of the microbial rhodopsin family Debrecen, Hungary, 4Institute for Cancer Research, Medical University of proteins in the claret membrane of Halobacterium species XZ515, functions Vienna, Vienna, Austria. as a proton pump similar to bacteriorhodopsin (BR) but with an opposite tem- The human genome codes for 42-44 ABC membrane transporters. The multi- poral order of proton uptake and release at neutral pH. Furthermore, it contains drug resistance transporter P-glycoprotein (ABCB1) is expressed in barrier tis- a second chromophore bacterioruberin. In order to elucidate mechanism of this sues. It transports an extraordinarily diverse range of structurally unrelated unusual proton translocation mechanism and the function of bacterioruberin drugs, xenobiotics compounds and endogenous substrates. In contrast, the bile further, it is essential to establish a suitable system in which the recombinant salt transporter BSEP (ABCB11) shows high sequence homology to ABCB1, AR4 with the two chromophores can be expressed. In this work, we report a but its expression is limited to the canalicular membrane of epatocytes. It has reliable method to express the recombinant AR4 in Halobacterium salinarum a narrow substrate specificity, recognizing and transporting essentially only MPK409. Experimental results show that the recombinant AR4 not only retains bile salts. Several models of the transport cycle have been proposed and it is the light-driven proton pump characteristics and photocycle kinetics, but also

BPJ 7915_7919 572a Wednesday, February 15, 2017 contains the two chromophore, similar to that in the native claret membrane. nals to large number of binding sites. We demonstrate that this ’’time-scale The function of bacterioruberin in AR4 is investigated through light-induced crisis’’ can be resolved by actively regulating residence times through molec- kinetic measurements, two-dimensional solid-state NMR experiments, dy- ular stripping. We illustrate these ideas by studying the stochastic dynamics of namic light scattering, FT-IR spectroscopy, and ATP formation rate measure- the genetic network of the central eukaryotic master regulator NF-kB which ment. This new expression system provides a functional platform for further broadcasts its signals to many downstream genes that regulate immune study of structure-function relationships of AR4, and other microbial rhodopsin response, apoptosis etc. family proteins in which there exist the two functional chromophores. Reference: Cao, Z.; et al. Biochim. Biophys. Acta 2015, 1847, 390. 2815-Pos Board B422 Multigenerational Study of Spaceflight-Responsive Gene Networks Genetic Regulatory Systems Irem Celen1,2, Jung Doh2, Aroshan Jayasinghe2, Michael Moore2, Andrew Moore3, Chandran Sabanayagam2. 2812-Pos Board B419 1Center for Bioinformatics and Computational Biology, University of A Biophysical Model of Supercoiling Dependent Transcription Predicts a Delaware, Newark, DE, USA, 2Delaware Biotechnology Institute, Newark, Structural Aspect to Gene Regulation DE, USA, 3Biological Sciences, University of Delaware, Newark, DE, USA. Christopher H. Bohrer, Elijah Roberts. As the interest in human missions to Mars has elevated drastically, under- Biophysics, Johns Hopkins, Baltimore, MD, USA. standing the genome-wide responses to the space environment has become Transcription in Escherichia coli generates positive supercoiling in the DNA, an urgent task. To address this, we investigated the transcriptomic responses which is relieved by the enzymatic activity of gyrase. Recently published for spaceflight-like microgravity conditions on C. elegans. Multigenerational experimental evidence suggests that transcription initiation and elongation effects of simulated spaceflight conditions were examined by exposing the are inhibited by the buildup of positive supercoiling. It has therefore been animals to the altered conditions and observing the continuing effects for proposed that intermittent binding of gyrase plays a role in transcriptional two to four generations after return to the ground control environment. Bio- bursting. Considering that transcription is one of the most fundamental informatics analyses were conducted to identify the spaceflight-specific gene cellular processes, it is desirable to be able to account for the buildup and networks. Our results depicted that approximately 88% of the gene networks release of positive supercoiling in models of transcription. Here we present are significantly enriched in previously found protein-protein interactions a detailed biophysical model of gene expression that incorporates the effects (P<0.01). Moreover, the networks predict 19% - 98% more interactions of supercoiling due to transcription. By directly linking the amount of pos- than the previously reported. A high correlation was found between the func- itive supercoiling to the rate of transcription, the model predicts that highly tion of the gene networks and the observed phenotype. For example, our transcribed genes’ mRNA distributions should substantially deviate from fluorescence microscopy results demonstrated that the animals experience Poisson distributions, with enhanced density at low mRNA copy numbers. a change in muscle mass under microgravity. The microgravity-related Additionally, the model predicts a high degree of correlation between gene networks also showed an enrichment for tissue development and mus- expression levels of genes inside the same supercoiling domain. Our model cle fiber development Gene Ontology terms. Interestingly, up to 78% of the makes specific predictions that differ from previous models of gene expres- differentially expressed genes (compared to control) maintained these sion. Genes in the same supercoiling domain influence the expression level expression patterns for approximately three generations. In particular, of neighboring genes. Such structurally dependent regulation predicts corre- Longevity Regulating Pathway genes were differentially expressed and the lations between genes in the same supercoiling domain. The topology of the Sphingolipid Metabolism Pathway was downregulated under microgravity. chromosome, therefore, creates a higher level of gene regulation, which has Given that inhibition of this pathway increases lifespan, our data suggests broad implications for understanding the evolution and organization of bac- an extended lifespan under microgravity. Surprisingly, these pathways main- terial genomes. tained the differential expression patterns for two more subsequent genera- 2813-Pos Board B420 tions on the ground conditions. We further investigated the epigenome and Interactions among Individual Phages Result in Different Cell-Fate regulatory non-coding RNA elements and identified the controlling switches Developments of these networks. Lanying Zeng. Biochemistry and Biophysics, Texas A&M University, College Station, TX, 2816-Pos Board B423 Escherichia Coli USA. Pressure Induced Sos Response in Involves Mrr Restric- Living cells make fate-determining decisions based on signals from their envi- tion Endonuclease 1 2 2 ronment. Understanding the decision making process is essential for unveiling Anaı¨s Bourges , Oscar E. Torres M. , Anirban Ghosh , 2 3 3 4 the mysteries of life and for improving human health. As decision-making is Wubishet M. Tadesse , Gilles Labesse , Nathalie Declerck , Abram Aertsen , Catherine A. Royer1. complex and ubiquitous, we use a simple paradigm for cell-fate decision-mak- 1 2 ing to deconstruct its fundamentals, the bacterium E. coli and its virus, phage Rensselaer Polytechnic Institute, Troy, NY, USA, Microbial and Molecular Systems, Laboratory of Food Microbiology, Leuven, Belgium, 3Centre de lambda. Upon infection by phage lambda, E. coli undergoes one of two alter- 4 nate pathways - lytic (violent) or lysogenic (dormant). Despite its extensive Biochimie Structural, Montpellier, France, Microbial and Molecular studies over the past fifty years, new insights into the decision-making mecha- Systems, Laboratory of Food Microbiology, Leuven, France. nism have been revealed with higher resolution techniques and mathematical Subjecting the mesophilic Escherichia coli bacterium to a pressure of ~100 modeling. Here, with our newly developed live-cell 4-color system combined MPa (1kbar) elicits a bona fide DNA damage (SOS) response, despite the with stochastic modeling at the single-virus/single-virus-DNA level, we find fact that pressure itself cannot compromise DNA covalent integrity. While that individual phages vote and interact within the cell: they cooperate during E. coli is not naturally subjected to high hydrostatic pressure (HP) in its envi- lysognization resulting in mutual propagation, compete among each other dur- ronment, food products are treated by pascalization to inactivate food-borne ing lysis to maximize their own prorogation, and confusion or coexistence be- pathogens. Screens for resistance to pressure shock revealed the constitutive tween the two pathways occasionally occurs to optimize its survival. We also presence in several strains of E. coli of an inactive, endogenous restriction find that the fluctuating/stochastic phage DNA ejection time and limited endonuclease type IV, Mrr (Methylated adenine Recognition and Restriction), cellular resources for phage DNA replication enable the interplay among that cleaves methylated DNA. Since the enzyme is present and non-toxic in the different developmental paths. absence of pressure treatment, activation must be pressure dependent. We investigated the mechanism of pressure dependent activation of Mrr in 2814-Pos Board B421 E. coli MG1655strains expressing a chromosomal GFPmut2-Mrr fusion as Stochastic Dynamics of Genetic Broadcasting Networks well as Mrr catalytic mutants identified by screens. We used highly quantitative Davit Potoyan. fluorescence fluctuation microscopy that provides a spatial map of the absolute Rice University, Houston, TX, USA. values for the concentration (n= number of molecules/PSF) and stoichiometry The complex genetic programs of eukaryotic cells are often regulated by key (molecular brightness, e, counts/s/molecule) of fluorescently labeled com- transcription factors occupying or clearing out of a large number of genomic plexes. We also examined the behavior of a phage methyltransferase HhaII, locations. Orchestrating the residence times of these factors is therefore impor- whose expression also leads to Mrr activation. Our results suggest that tant for the well organized functioning of a large network. The classic models pressure-mediated triggering of Mrr activity and the concomitant generation of genetic switches sidestep this timing issue by assuming the binding of tran- of DNA damage involve the conversion of inactive Mrr tetramers into active scription factors to be governed entirely by thermodynamic protein-DNA affin- Mrr dimers by pressure. This could occur either via a direct effect of pressure ities. We show that relying on passive thermodynamics and random release on the Mrr tetramer-dimer equilibrium, a common effect of pressure on oligo- times can lead to a ’’time-scale crisis’’ of master genes that broadcast their sig- mers, or via a pressure effect on structural features of the chromosome leading

BPJ 7915_7919 Wednesday, February 15, 2017 573a to more efficient recruitment of active dimeric Mrr. Homology modeling of the Chromosomal organization plays a crucial role both in the regulation of gene tetrameric Mrr catalytic domain supports the proposed mechanism of action expression and in cellular reproduction. Prior studies of chromosomal confor- drawn from the in vivo results of the mutants. mations in bacteria have shown evidence for a variety of architectures in different organisms, from a global twisted-ellipse structure in Caulobacter 2817-Pos Board B424 crescentus to highly self-interacting ‘‘macrodomains’’ in Escherichia coli. Accurate Non-Equilibrium Velocity and Flux Fields of Stochastic Reaction At finer grain levels of detail, evidence has accumulated in E. coli for smaller Networks topologically isolated domains that act to organize the chromosome, and Anna Terebus1, Chun Liu2, Jie Liang1. 1 2 several studies have suggested crucial links between genomic location, genome University of Illinois at Chicago, Chicago, IL, USA, The Penn State organization, and gene expression levels. University, University Park, PA, USA. Achieving a complete understanding of the organization of the E. coli chromo- Biochemical reaction networks are generally in non-equilibrium and often sto- some, and its impact on cellular regulatory logic, requires us to link the bio- chastic because of the different time scales of reactions as well as low copy physical interactions underpinning chromosome architecture, to the numbers of molecules of participating species. The discrete Chemical Master architecture itself, and to fully map the interplay between chromosome confor- Equation (dCME) provides a fundamental framework for studying time- mation, protein-DNA interactions, and transcription. evolving and steady state probability landscapes. A widely used approach to We have applied a combination of global mapping of protein-DNA interactions, study stochastic non-equilibrium probability flux is to approximate dCME genome-wide chromosomal conformation capture, and high-throughput mapping with Fokker-Plank equation based on Gaussian processes. Previous analysis of reporter expression at different genomic sites to obtain a multi-level picture of of stochastic thermodynamics for dCME is based on analysis of single reaction the interactions shaping both chromosome architecture and gene expression. We trajectories in terms of probability flux, probability velocity, entropy produc- find that extended protein occupancy domains (EPODs), previously identified us- tion and other thermodynamics characteristics. Here we describe a general ing the IPOD method for profiling protein-DNA interactions, play a crucial role in approach of analysis of the global flow map of probability mass in the direction organizing the chromosome, and account for the majority of information in chro- of each of the species. Our method is based on exact ACME solution of dCME mosome conformation capture maps in E. coli. We show that EPODs themselves [1,2]. It takes into full account the discreetness of both states and jump reac- arise due to the combined action of multiple nucleoid proteins, and that the loca- tions, and provides an exact quantification of the vector fields along the bound- tions of EPODs are highly robust across conditions even as the composition of the aries of the state space dictated by the reaction network. It allows computation protein component of the nucleoid changes dramatically. Finally, we find that of probability velocity and flux at every state. We show details of non- EPODs have a strongly repressive impact on genes contained in them, indicating equilibrium steady state probability fluxes and velocity fields for the oscillatory that they participate in gene regulation at both local and global levels. Our findings Schnakenberg model and the toggle switch model. We also point out key dif- provide a comprehensive framework for understanding the interplay of protein- ferences of exactly computed probability flux and that from approximation of DNA interactions, genome conformation, and gene expression in E. coli,and Fokker-Planck, including the nonexistence of solenoidal curls found in mag- additionally reveal a huge variety of fine-grain detail in the regulatory network netic field. Our results identify important cellular states as well as sources of this organism that will scaffold our ability to understand and modify it. and sinks of probability mass. [1] Cao, Youfang et al. Bulletin of mathematical biology 78.4(2016):617-661. 2820-Pos Board B427 [2] Cao, Youfang et al SIAM Multiscale Modeling and Simulation Whole-Cell Simulations of mRNA Production in Eukaryotic Cell Models 14.2(2016):923-963. Zhaleh Ghaemi, Zaida Luthey-Schulten. 2818-Pos Board B425 Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA. Clinorotation to Simulate Microgravity: Defining a Model Gravitome in To construct a comprehensive model of a eukaryotic cell, we considered in de- C. elegans tails, processes that are essential for life. An important component of transcrip- Chandran R. Sabanayagam1, Irem Celen2, Jung H. Doh1, tion process is RNA splicing accomplished by the spliceosome. Spliceosome is Michael T. Moore1. a complicated and highly dynamic machine that removes introns from precur- 1Delaware Biotechnology Institute, University of Delaware, Newark, DE, sor mRNA (pre-mRNA) in eukaryotic cells. It consists of five protein–RNA USA, 2Center for Bioinformatics and Computational Biology, University of complexes in the form of small nuclear ribonucleoproteins (snRNP). Delaware, Newark, DE, USA. We study the sequential formation of the spliceosome, the removal of an intron We introduce the term, gravitome, to include the set of genes that are respon- and the production of an mRNA in a spatially resolved model of a Saccharo- sive to changes in the gravitational vector. Understanding how the gravitome myces cerevisiae cell. functions will be important as humans begin a new era of long term space We start with the assembly of snRNP from its components, which are small nu- exploration and exploitation. To this end, we have developed a novel micro- clear RNA (snRNA), Smith (Sm) proteins and other proteins which are specific scope clinostat to visualize microorganisms and cell cultures growing in real- to each snRNP particle. The assembly of snRNP particle is a highly compart- time during simulated microgravity. Microspheres were used to calibrate the mentalized process with multiple steps consisting of the transport of snRNAs clinostat rotation and verify orbital trajectories. We take advantage of the through nuclear pore complexes (NPCs) to the cytoplasm, their association unique ability of the nematode, C. elegans, to live and reproduce in liquid cul- with Sm proteins, the transport of the snRNA-Sm complex to the nucleus tures, and use our clinostat to essentially cause these animals to live their lives through NPCs, and finally association with specific proteins and formation of in free-fall, nullifying the gravitational force. In addition to microscopy data, the full snRNP particle. the clinorotated animals were harvested and their transcriptomes and histone- We have derived a series of kinetic equations that describe the assembly pro- marked epigenomes were determined using next-generation sequencing. cess of snRNP. Preliminary results from this kinetic model in our S. cerevisiae- Here, we present the first genetic confirmation that ground-based clinostats geometry show that we can successfully generate the first and most abundant can capture elements of the space environment by comparing our results component of spliceosome, namely U1snRNP and observe the noisiness of with previous studies of C. elegans on the International Space Station (ISS). the U1snRNP formation at physiological condition. The simulated time scale In particular, we also find significant changes in genes belonging to the insulin, of snRNP particle formation compares well with experimental results, serving longevity, metabolic and muscle-related pathways in simulated microgravity, to validate our model. and also have identified large alterations in the dorsal-ventral pathway. Because These simulations lay the foundation for studying eukaryotic systems with next-generation sequencing provides more information than the previously spatial resolution and pathogen-related processes such as alternative splicing used gene arrays onboard the ISS, we also detect the expression of hundreds in detailed models. of novel long non-coding RNAs as a result of simulated microgravity. These new findings will help elucidate the intricate genetic regulatory networks that Electron Microscopy are responsible for adaptation of higher organisms in microgravity. 2821-Pos Board B428 2819-Pos Board B426 Cryo-EM of the Bacteriophage Tail Tube at Better than 3.5 A˚ Resolution Effects of Large Scale Protein Occupancy on E. Coli Genome Structure Weili Zheng1, Nicholas M I Taylor2, Petr G. Leiman3, Edward H. Egelman1. and Gene Expression 1Biochemistry and Molecular Genetics, University of Virginia, Peter L. Freddolino1, Thomas J. Goss1, Grace M. Kroner1, Scott Scholz2, Charlottesville, VA, USA, 2E´cole Polytechnique Fe´de´rale de Lausanne Xiaoxia Lin3. (EPFL), Lausanne, Switzerland, 3Biochemistry & Molecular Biology, 1Biological Chemistry, University of Michigan, Ann Arbor, MI, USA, University of Texas Medical Branch at Galveston, Galveston, TX, USA. 2Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI, The first electron microscopic three-dimensional reconstruction was from the USA, 3Chemical Engineering, University of Michigan, Ann Arbor, MI, USA. bacteriophage T4 tail at ~ 35 A˚ resolution using negative stain with a

BPJ 7915_7919 574a Wednesday, February 15, 2017

Fourier-Bessel approach. Progress in EM has been great since then, particu- 2824-Pos Board B431 larly over the past three to four years with the advent of direct electron de- Cryotomography of Pleomorphic Viruses tectors. Using the Iterative Helical Real Space Reconstruction method Amar D. Parvate1, Jason Lanman1, Colleen Jonsson2. implemented in SPIDER, we have been able to use images of the T4 base- 1Biological Sciences, Purdue University, West Lafayette, IN, USA, plate assembly to generate reconstructions of the tail tube at better than 2Department of Microbiology, University of Tennessee, Knoxville, 3.5 A˚ resolution, a 1,000-fold improvement in information content over Knoxville, TN, USA. what was possible in 1968. Surprisingly, we can show that a reasonable 61st Annual Meeting of Biophysical Society reconstruction is possible with a dose of only ~ 1.5 electrons/A˚ 2, using a Hantaviruses belong to the Bunyaviridae family and constitute a group of hu- higher dose set of frames for alignment. Considering that SPIDER was man pathogens causing hemorrhagic fevers with 15-40% mortality. Currently largely written more than 20 years ago, for some specimens at least legacy no vaccine is available for these infections, and treatment only relieves symp- software may not be a limiting factor. To test this, a comparison will be toms. The Gn-Gc glycoprotein complex on the virus surface is involved in made with Relion 2.0, a new version of Relion which allows for helical binding to host cell receptors and fusion with host membrane to release the viral reconstruction. genome. It has been postulated that Bunyavirus glycoproteins may belong to class II fusion proteins. Our objective is to determine whether the arrangement 2822-Pos Board B429 of the fusion proteins is similar to that of other class II fusion proteins such as those of Flaviviruses. Using cryo-electron tomography and sub-volume aver- Strain Between Leading and Trailing Heads of a Stepping Kinesin Dimer ˚ Visualized in 3D by Cryo-EM aging we propose to solve the structure of Hantavirus spike to 15-20 A resolu- Daifei Liu1, Xueqi Liu1, Zhiguo Shang2, Charles V. Sindelar1. tion. Hantaviruses are classified as BSL3 agents. We have optimized a method 1Yale University, New Haven, CT, USA, 2UT Southwestern Medical Center, to purify and inactivate Orthobunyavirus (a BSL2 virus) using 1% glutaralde- Dallas, TX, USA. hyde for cryoET imaging in a BSL1 containment facility. This method was The structural basis of walking by dimeric molecules of kinesin along micro- replicated in a BSL3 facility to prepare and inactivate Andes virus (a Hanta- tubules has remained unclear, partly because available structural methods virus) samples for cryo-ET. The Andes virus particles were classified as round, have been unable to capture microtubule-bound intermediates of this pro- elongated and irregular based on tomographic data. Further observations re- cess. We developed a novel method, FINDKIN, that allowed us to solve vealed the arrangement of glycoprotein spikes was clearly visible on the surface sub-nanometer resolution cryo-EM maps in which the two heads of a kinesin of both glutaraldehyde fixed viruses, possibly with a 4-fold symmetry in Andes, dimer are attached at sequential sites along a single protofilament of the as in case of Hantaan and Tula virus. microtubule, in a stepping configuration. The resulting structures indicate that the upper half of the nucleotide cleft shifts downward in the trailing 2825-Pos Board B432 head and upward in the leading head. Consequently, closure of the nucleo- Endophilin-Dynamin Complex Assembly - A General Mechanism of tide cleft in the trailing head supports the binding of an ATP analog, while Membrane Remodeling Control opening of the cleft in the leading head is accompanied by loss of nucleotide Anna C. Sundborger1, Veer Bhatt1, Robert Ashley1, Jenny E. Hinshaw2. density. These results supply a detailed explanation for how tension between 1The Hormel Institute, Austin, MN, USA, 2LCMB, National Institutes of the two heads of dimeric kinesin keeps the enzymatic cycles of the heads out Diabetes & Digestive & Kidney Diseases, Bethesda, MD, USA. of phase in order to stimulate directional motility. Moreover, the novel cryo- Endophilin belongs to a group of proteins containing membrane binding and EM image-processing method presented here paves the way for future struc- bending BAR domains. Neuronal-specific endophilin A1 mediates membrane tural studies of a variety of challenging systems that bind to microtubules bending in association with dynamin 1-catalyzed membrane fission. In a previ- and other biological filaments. ous study, we show that endophilin A1 co-localizes with dynamin 1 on necks of clathrin-coated pits in nerve terminals and assembles into a complex with dy- 2823-Pos Board B430 namin 1 on tubulated liposomes in vitro. Using cryo-EM and iterative helical Comparison of 3-D Cell and Tissue Imaging Techniques Based on real space reconstruction (IHRSR) we have generated a preliminary 3D density Scanning Electron Probes map of the endophilin A1–dynamin 1 complex. Our data suggests that endophi- Emma L. McBride1, Amith Rao1, Guofeng Zhang1, Irina D. Pokrovskaya2, lin A1 may regulate dynamin 1-catalyzed membrane fission by preventing Maria A. Aronova1, Brian Storrie2, Richard D. Leapman1. inter-molecular interactions within the dynamin scaffold. Such interactions 1NIBIB, National Institutes of Health, Bethesda, MD, USA, 2Department of promote dynamin stimulated GTPase activity and trigger fission. Thus, endo- Physiology and Biophysics, University of Arkansas for Medical Sciences, philin A1 may function as a negative regulator of dynamin 1-catalyzed plasma Little Rock, AR, USA. membrane fission by means of controlling dynamin scaffold organization. The use of scanned electron probes, rather than the wide-beam illumination This notion is further supported by in vitro observations. Endophilin B1 is a tu- of standard transmission electron microscopy, allows the structural biologist mor suppressor involved in regulation of mitochondrial dynamics and to image 3-D cellular and tissue ultrastructure by taking full advantage of the Bax-mediated apoptosis. Loss of Endophilin B1 was recently shown to inhibit physical interactions between the incoming electrons and the specimen. For dynamin 2-catalyzed fragmentation of Atg9 vesicles from Golgi during example, in serial block face scanning electron microscopy (SBF-SEM), a autophagosome formation. This suggests that endophilin B1 may act as a nega- low-energy (~1 keV electron probe) produces a backscattered electron signal tive regulator of dynamin 2 via formation of an endophilin B1-dynamin 2 com- originating from a thin approximately 25-nm layer below the face of a plex, in a manner similar to endophilin A1. This further implies a general heavy-atom stained, resin-embedded block. However, due to the low average mechanism for membrane remodeling control, by coordinating membrane atomic number of the atoms in the block, the scattering is in the forward di- bending and fission through endophilin-dynamin complex assembly. In this rection providing a lateral resolution of around 5 nm. By cutting successive study, we show that endophilin B1 and dynamin 2 indeed interact in vitro. layers from the block face, large (> million cubic micrometers) 3-D volumes We aim to solve the structure of the endophilin-dynamin complexes at atomic of a biological sample can be generated. Bright-field electron tomography in resolutions using a cryo-EM. Determining the organization of these protein the scanning transmission electron microscope (STEM) is performed with a complexes will shed light on potentially general mechanisms that control intra- high-energy (~300 keV) electron probe, which is focused to a 2-nm diameter cellular membrane remodeling during essential cellular processes, such as probe using a low convergence angle, which provides a depth of field of ~2 apoptosis, endocytosis and autophagy, and identify novel targets for drug micrometers in a thick section of a stained, embedded specimen. Unlike in discovery. conventional transmission electron microscopy, there are no post-specimen lenses in STEM, so that chromatic aberration does not affect image quality 2826-Pos Board B433 due to multiple inelastic scattering; however, it is necessary to limit the con- Sub-Surface Serial Block Face SEM of Biological Structures at Near centration of the heavy-atom stain to avoid attenuation of the probe by Isotropic Spatial Resolution elastic scattering. We have applied both SBF-SEM and STEM tomography Qianping He1, David C. Joy2,3, Guofeng Zhang1, Richard D. Leapman1. extensively to determine cellular ultrastructure. In the present study we 1National Institute of Health, Bethesda, MD, USA, 2Department of Materials compare performance and relative advantages of the two techniques in terms Science and Engineering, University of Tennessee, Knoxville, TN, USA, of spatial resolution, specimen size, and speed of acquisition. For example, 3Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, we present data from human blood platelets and secretory cells in pancreatic Oak Ridge, TN, USA. islets of Langerhans. This work was supported by the Intramural Research Serial block face scanning electron microscopy (SBF-SEM) provides nano- Program of the National Institutes of Biomedical Imaging and Bioengi- scale 3D ultrastructure of entire cells and tissue volumes. In SBF-SEM, an ul- neering, NIH. tramicrotome built into the SEM specimen stage successively removes thin

BPJ 7915_7919 Wednesday, February 15, 2017 575a sections from a plastic-embedded, heavy metal-stained specimen. After each 2829-Pos Board B436 cut, the freshly exposed block face is imaged at a low incident electron energy Accelerated Cryo-EM Structure Determination with Parallelisation using using a backscattered electron detector to provide 3D ultrastructure with a res- GPUs in Relion-2 olution of approximately 5 nm in the plane of the block face and around 25 Dari Kimanius, Bjo¨rn Forsberg, Erik Lindahl. nm in the perpendicular z-direction, as limited by the slice thickness. We Department of Biochemistry & Biophysics, Stockholm University, have explored the feasibility of improving the z-resolution in SBF-SEM by Stockholm, Sweden. recording images at multiple primary beam energies, thus sampling different Single-particle electron cryo-microscopy (cryo-EM) structure determination is depths below the block surface. transforming structural biology by enabling near-atomic resolution for wide A linear relationship was found between the depth of test structures, generated classes of structures. However, the most powerful Bayesian approaches for im- by Monte Carlo simulations, and the ratio of backscattered image intensities re- age reconstruction are very costly from a compuational point-of-view. This has corded at primary beam energies between 1.0 keV and 1.4 keV. This enabled us turned the computational step into a new bottleneck that is limiting both to reconstruct the 3D model within a 25-nm surface layer at a z-resolution of throughput and new method development, in particular as an increasing number around 12.5 nm based on a physical model. We have combined this method of facilities have access to direct-electron detectors. Here, we present a new im- with the serial block face cutting process. Automatic focusing of images re- plementation of the Bayesian regular likelihood optimization used in the RE- corded at different primary beam energies and tracking movement of the LION program that has been reformulated to use parallelization on graphics beam were achieved within each cut. A Zeiss Sigma-VP SEM equipped with processors (GPUs) to address the most computationally intensive steps in the a Gatan 3View SBF system was used to acquire data. A better z-resolution cryo-EM structure determination workflow. Both image classification and (~ 5nm) is possible in theory although the feasibility of achieving this resolu- high-resolution refinement have been accelerated up to 50-fold, and tion is highly dependent on conditions such as detector sensitivity and sample template-based particule selection running on a single workstation now pro- stability under radiation damage. vides performance equivalent to 1000 CPU cores at a tiny fraction of the cost. Memory requirements of the reconstruction algorithms have been reduced 2827-Pos Board B434 to make it possible to use widely available low-cost consumer hardware, and Towards a Multicomponent Cryo-EM Density Flexible Fitting Tool we show that the use of single precision arithmetic does not adversely affect Jose´ Ramo´n Lope´z-Blanco1, David Ritchie2, Pablo Chacon1. results. This routinely enables high-resolution cryo-EM structure determination 1Biological Chemical Physics, Institute of Physical Chemistry CSIC, Madrid, in a matter of days on a single workstation even for the largest datasets, and in Spain, 2Biological Chemical Physics, INRIA Nancy, Villers-le`s-Nancy, many cases the processing is completed in mere hours. This new processing France. approach removes the need for advanced computational infrastructure, and it Building a high resolution model from a cryo-EM density map of large pro- will make it possible to use many more classes in reconstruction - which in tein complex often involves puzzle multiple 3D atomic structures fitting turn will improve detection of alternative conformations and handle much together. To achieve this complex model-building task, two major computa- larger datasets than previously possible. tional challenges must be addressed. Firstly, each individual protein compo- 2830-Pos Board B437 nent, depending of the resolution, can fit almost anywhere in the density Visualizing the Myosin VI-F-Actin Interface during Force Generation map. Secondly, due to conformational flexibility, the available atomic confor- Pinar S. Gurel1, Laura Y. Kim2, Tosan Omabegho3, Paul V. Ruijgrok3, mation frequently differs from its (unknown) conformation in the cryo-EM Zev Bryant4, Gregory M. Alushin1. sample. To overcome these bottlenecks, we merge gEMfitter, a GPU- 1Rockefeller University, New York, NY, USA, 2Cell Biology and Physiology accelerated and sensitive 3D Fourier rigid-body fitting tool, with iMOD, an Center, National Heart, Lung, and Blood Institute, Bethesda, MD, USA, efficient tool to explore protein conformational flexibility using normal 3Department of Bioengineering, Stanford University, Stanford, CA, USA, mode in internal coordinates. In this way, feasible initial conformations pro- 4Departments of Bioengineering and Structural Biology, Stanford University, duced by iMOD are handled by gEMfitter for generating a repertoire of Stanford, CA, USA. feasible conformations for each component structure. We will present first re- The large superfamily of myosin motor proteins is responsible for movement sults for this combined approach on a selection of experimental and simulated and force generation at multiple scales of biology, ranging from muscle test cases. contraction to intracellular transport. While the mechanisms of myosin motor function have been the subject of extensive biophysical, biochemical, and 2828-Pos Board B435 structural characterization, high-resolution insight into the myosin-F-actin All-Atom Ensemble Refinement to Cryo-EM Densities with a Bayesian interface has been limited. Using cryo-electron microscopy (cryo-EM) and Measure of Goodness-Of-Fit an adapted protocol for Iterative Helical Real-Space Reconstruction (IHRSR) Christian Blau, Erik Lindahl. which implements refinement and reconstruction of independent-half datasets Theoretical and Computational Biophysics, Stockholm University, Solna, to minimize noise bias in resolution estimation and alignment, we present re- Sweden. constructions of myosin VI in nucleotide-free (rigor, 4.5 A˚ average resolution) Beneath any density map resolved by cryo electron microscopy (cryo-EM) lies and MgADP (strong-bound ADP, 5.5 A˚ average resolution) states bound to F- an ensemble of all-atom structures. However, established refinement methods actin, as well as corresponding atomistic models. Comparison to a recent high- are designed with a single best-fitting structures in mind. Further, established resolution structure of the myosin IIC-F-actin interface demonstrates that while refinement protocols require intricate sampling schemes and/or additional con- the regions of contact are similar, the specific residues involved and the chem- straints on, e.g., secondary structure due to the ruggedness of their refinement ical nature of the interactions differ between the two myosins, highlighting the potentials. need to characterize diverse myosins in complex with F-actin to gain insight Here, we introduce a goodness-of-fit measure of all-atom model to density that into motor-specific properties. Comparing the strong-bound ADP and rigor reflects the specific physics of cryo-EM in contrast to general measures like structures, we observe rearrangements in both the myosin motor domain and cross-correlation or density sum at atom positions. Its smoothness allows for actin relative to structures of these components in isolation, suggesting a recip- all-atom refinement against cryo-EM densities without any further constraints rocal relationship between actin and myosin conformation during force gener- on, e.g., secondary structure, and capturing large conformational transitions be- ation. Additionally, we observe formation of a single, unanticipated contact in tween initial model and target density. Bayesian cryo-EM ensemble refinement the transition from the strong-bound ADP to rigor state, which supporting func- was tested for refinement of closed conformation adenylate-kinase (AKE) tional experiments demonstrate makes a critical contribution to motility. In against a target density created from an ensemble of structures in the open addition to providing mechanistic insights specific to myosin VI, the recon- conformation. Our ensemble is closer to the underlying ensemble as measured struction and modeling approaches we have developed are applicable to visu- by Kullback-Leibler divergence after dimensionality reduction by 1.1 bits alizing diverse actomyosin complexes in multiple states at near-atomic compared to a cross-correlation based potential, and 1.5 bits compared to resolution. inverted-density potential. Bayesian refinement on novel cryo-EM data yielded excellent agreement in integrated Fourier shell correlation curves (>0.5 for a 6 2831-Pos Board B438 A˚ density map). Obliterating further constraints, our refinement protocol en- Optimization of a Cryo-EM Structural Model Representation ables secondary structure prediction while providing an ensemble of high- Wah Chiu. quality structures. Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Overall, our method yields reliable refinement results for challenging TX, USA. cases using a Bayesian potential and structure ensembles to describe cryo- Electron cryo-microscopy (cryoEM) has been used to resolve atomic resolu- EM densities. tion biological structures. However, the resulting density maps and models

BPJ 7915_7919 576a Wednesday, February 15, 2017 still lack assessment of accuracy and reproducibility. The densities of some 2834-Pos Board B441 residues particularly, the negative charged residues have been found to be Cryo-EM Structure of KV1.2 Channels in Liposomes consistently absent or have low values in near-atomic resolution cryoEM Hideki Shigematsu1, Youshan Yang2, Yangyang Yan2, Yi Chen2, maps. We thus introduce a procedure to derive an atomic model that faithfully Fred J. Sigworth2. replicates the experimental map and then use this map to ensure data accuracy 1RIKEN Center for Life Science Technologies, Yokohama, Japan, 2CM and reproducibility. We have applied this protocol to describe the 3.3 A˚ struc- Physiology, Yale University, New Haven, CT, USA. ture of the mature P22 bacteriophage capsid, a large and complex macromo- We present a sub-nanometer cryo-EM structure the Kv1.2 channel complex in a lecular assembly. This structure identifies previously unknown molecular lipid bilayer, obtained from protein reconstituted into liposomes. Using this interactions between capsid subunits, crucial to maintaining particle stability. system we expect that the voltage-gated channels can be subjected to physio- Acknowledgement: This research has been supported by NIH grants logical membrane potentials and ion gradients. Our present map was obtained (R01GM079429, P01GM063210, T15LM007093) and R Welch Foundation at zero membrane potential for comparison with X-ray structures, but we report (Q1242) progress in establishing and verifying the presence of membrane potentials in the small (~40 nm) liposomes used for cryo-EM imaging. 2832-Pos Board B439 2835-Pos Board B442 Efficient Cryo-Em: Measuring the Effect of Particle Orientation Distribu- Characterization of Cryo-EM Motion Correction Algorithms for tion on Resolution Movie-Mode Imaging Katerina Naydenova, Christopher J. Russo. James M. Bell, Isaac Forrester, Joanita Jakana, Steven J. Ludtke. MRC Laboratory of Molecular Biology, Cambridge, United Kingdom. Biochemistry, Baylor College of Medicine, Houston, TX, USA. Single-particle electron cryomicroscopy (cryo-EM) uses multiple 2D pro- The recent emergence of routine near-atomic resolution structures in CryoEM jection images of a bio-molecular complex to compute a 3D reconstruction can largely be attributed to the development of direct electron detectors as re- of its structure. This requires a sufficient number of views to populate the placements for older fiber-optic coupled scintillator-CCD technology. In addi- 3D Fourier space representation to a particular resolution. Often the sample tion to dramatically improved modulation transfer characteristics, these new preparation process results in a specimen where the orientation of the mol- detectors are also capable of acquiring data at frame rates sufficiently high ecules is not random, and this can limit resolution. These preferred orien- for individual electron event counting, which can then be averaged to produce tations are problematic because they lead to information gaps in Fourier movies at typically 10-40 frames/s. These movies can then be corrected for space and they indicate the presence of strong interactions between the mol- specimen motion to further improve the high-resolution signal present in the ecules and surfaces present in the support. Here we develop a simple math- final averaged image. While conceptually simple, the algorithms used to ematical model to quantify the effect of the particle orientation distribution perform this motion correction vary widely. At present there are six algorithms on 3D reconstruction in cryo-EM. We define a statistical quantity, the effi- in widespread use; however, because each alignment routine uses different ciency (E), which measures the quality of an orientation distribution in criteria to guide, smooth, and otherwise bias frame translations toward the terms of its ability to provide a spherically uniform coverage of Fourier optimal alignment, results vary, sometimes significantly. In this study we space, and thus uniform resolution in all directions. The quantity is analyt- compare the alignment quality of each algorithm on a set of movies through ically derived from the optical point spread function imposed by the distri- use of gold fiducials, which are excluded from the alignments through use of bution. We also provide an algorithm for predicting tilt angles for data a novel gold erasing technique, to approximate the ground truth. Because frame collection which will improve efficiency and resolution. We demonstrate alignment ultimately determines the obtainable resolution in single particle an- this improvement experimentally; while tilting the specimen can help in alyses, assessing the strengths and weaknesses of each algorithm may have a some instances it cannot overcome the problems of reduced image quality critical impact on the selection of a specific algorithm, and ultimately on quality at tilt and strong molecule-surface interactions. These can cause degrada- of the final reconstructions. Through understanding the reasons behind the dis- tion of the specimen that cannot be recovered in the imaging and recon- agreements among packages, we may achieve insights to better design the next struction process. We expect the metric described here will allow rapid generation of alignment software. evaluation of specimen support materials and experimental conditions dur- ing sample preparation. This will enable one to minimise potentially detri- 2836-Pos Board B443 mental surface interactions while optimising the orientation distribution for 3D-Visualization of the Precise Location of Symbiotic Organelle Crosstalk structure determination with minimal data. Surprisingly, the efficiency is as Throughout Mitosis in the Primitive Unicellular Eukaryotic Cell, C. Merolae important as the amount of data in determining the resolution of a cryo-EM 1,2 1,3 1,2 structure. Takako Ichinose , Keisuke Ohta , Atsuko H. Iwane . 1Riken, Suita, Japan, 2Osaka Univ., Suita, Japan, 3Kurume Univ., Kurume, Japan. 2833-Pos Board B440 In recent years, symbiosis research has been progressing rapidly with metage- Development of Thin-Ice Tem Grids to Control the Ice Thickness for Cryo- nomics analysis using Next-generation DNA sequencing, gnotobiotic technology Electron Microscopy and metabolome analysis technology. In the most primitive known form of sym- Liguo Wang, Lige Tonggu. biosis, referred to as ‘‘primary endosymbiosis’’, the subcellular organelles were University of Washington, Seattle, WA, USA. bacteria taken up by a heterotrophic eukaryote, specifically plastids and mito- Cryo-electron microscopy (cryo-EM) is a powerful technique to determine chondria originated from ancestral Cyanobacteria and Alphaproteobacteria structures of biological macromolecules and complexes embedded in a respectively. We are studying how the different organisms are integrated and thin layer of vitreous ice. More than 400 protein structures have been deter- began to live as one. We selected C. merolae, a primitive unicellular red algae, mined to be better than 5-A˚ resolutions in the EM databank. In cryo-EM, one as an excellent symbiosis model organism and focus on the mitosis process using limiting factor in image contrast is the ice thickness. Experiments have dualbeam EM and confocal LM. C. merolae is a small (2-5 micron) eukaryotic shown that by extensive efforts to optimize the vitrification process, the cell and is extremely simple, containing only an few double-membraned and contrast of recorded cryo-EM images increased dramatically. Currently, single-membraned organelles and lacking a cell wall, and has survived from the vitrification process is still a trial and error method, and there is very ancient times. During cell division, the parent cell divides and distributes each poor control of the ice thickness. In this study, a new type of TEM grid, organelle equally into daughter cells. We identified each mitotic phase using spe- termed as Thin-ice TEM grid, will be developed using nanofabrication pro- cific fluorescent tags as well as by the dynamic shape change of individual cells cesses. The thickness of the spacer layer on a Thin-ice TEM grid determines obtained from optical time-lapse observation. By using FIB-SEM and 3D-recon- the thickness of the liquid layer, thus the thickness of the vitreous ice. This struction techniques we successfully observed the shape changes of the individual makes the vitrification process a well-controlled step, and guarantees the ice main organelles in greater detail which revealed specific strong interactions and thickness to be the desired value. In addition, the sample volume is suggests organelle crosstalk specific to mitotic phase is essential to the symbiotic decreased by a factor of 100-10,000. The Thin-ice TEM grid not only elim- relationship. Furthermore, using UHVEM tomography of the plastid, we inates the intensive labor and cryo-EM involvement, but also results in maxi- observed the light harvesting antennae complex onto the thylakoid membrane mized contrast (i.e. signal-to-noise-ratio, SNR) of recorded cryo-EM images in detail. The outer division ring and inner division ring were found to be con- and higher resolution of the determined biological structure. This will in- structed of proteins which are present in modern eukaryotes and bacteria respec- crease the efficiency and success rate of cryo-EM in structural biology, speed tively. Thus a fundamental element of C. merolae symbiotic organelle crosstalk up the understanding of biological functions at molecular level, and poten- in mitosis is revealed. In revealing the precise symbiotic relationship between tially facilitate the development of new therapeutic agents to treat diseases eukaryote and bacteria we can develop the parameters which will also apply to in broad areas. larger ecological systems consisting of greater biological diversity.

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2837-Pos Board B444 that the truncated Sec13/31 edges can adopt. This will hopefully shed light on Focused Ion Beam-Sem as a Tool for Versatile Quantitative Imaging of the structural basis of the increased flexibility of cages, and provide a way for us Cellular Structures to circumvent it. Edward T. Eng, Ashleigh M. Raczkowski, William J. Rice, Kelsey D. Jordan, Alex J. Noble, Anchi Cheng, Bridget O. Carragher, 2840-Pos Board B447 Clinton S. Potter. Cryo-Electron Tomography and Sub-Volume Averaging Reveal the Asym- Simons Electron Microscopy Center, New York Structural Biology Center, metric Structure of the Leptospira Flagellla New York, NY, USA. Kimberley Gibson, Charles Sindelar. Modern 3D electron microscopy methods like focused ion beam scanning Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, electron microscopy (FIB-SEM) cover a large variety of applications at USA. different resolution scales. An advantage of this technique is that internal tis- Leptospirosis is an illness caused by bacteria of the genus Leptospira spp., sue architecture as well as intracellular structures can be directly quantified which can infect a wide range of mammalian host species. The disease infects and measured. We demonstrate these applications using examples from several million people globally. Infections can develop into liver disease, gen- research projects at the Simons Electron Microscopy Center where we are eral organ failure, and meningitis, leading to death. Belonging to the Spirochete applying 3D microscopic techniques to a wide variety of research problems. class of bacteria, Leptospira spp. possess a periplasmic flagella that lies be- First, using FIB-SEM we are able to visualize the internal structure of neu- tween the inner and outer bacterial membranes. Rotation of the flagella within rons in a neurodegenerative mouse model. Second, combining correlative the periplasmic space propels the bacterium in a corkscrew-like motion, allow- light microscopy and FIB-SEM we resolve the subcellular structures formed ing the pathogen to drill into tissue. Unlike the Salmonella flagella, which is during HIV-1 dissemination through cell-cell contacts. Lastly, we demon- composed of only one repeating flagellin monomer that forms a symmetrical strate our progress in developing a pipeline combining FIB-SEM with helical filament, Leptospira flagella are composed of several flagellin FlaA electron tomography to visualize internal structures of the cell at high and FlaB proteins, and additional novel surface-expressed flagellar-coiling pro- resolution. teins (FcpA and FcpB). FcpA is required to establish the curvature and motility of the flagella. Gaining further insights into this unique flagellar structure in 2838-Pos Board B445 Leptospira requires utilizing high resolution cryo-electron microscopy (cryo- Cryo-EM Tomographic Analysis of a Universal Influenza Virus Vaccine EM) techniques. Cryo-electron tomography (Cryo-ET), combined with a novel Candidate computational method for sub-volume averaging of filaments is applied to Erin Tran1, Kira Podolsky1, Alberto Bartesaghi1, Oleg Kuybeda1, flagella purified from the wildtype Leptospira biflexa serovar Patoc. Sub- Giovanna Grandinetti1, Teddy John Wohlbold2, Gene Tan2, volume averaging of purified Leptospira flagella has revealed partially helical, Raffael Nachbagauer2, Peter Palese2, Florian Krammer2, highly unique and asymmetric flagella structure. Future research will investi- Sriram Subramaniam1. gate the role of FcpA and FcpB in flagella curvature, periplasmic rotation, 1National Institutes of Health, Bethesda, MD, USA, 2Icahn School of and bacterial motility. Medicine at Mount Sinai, New York City, NY, USA. Influenza viruses cause hundreds of millions of infections every year, and vac- 2841-Pos Board B448 cines currently in use need to be reformulated annually due to the rapid muta- Feasibility of Cryo-EM for Structural Characterization of Small Mem- tion potential of these viruses. A universal influenza vaccine would alleviate the brane Proteins need for the yearly vaccine. Chimeric hemagglutinin (cHA) proteins are Daniel Asarnow, Yuan Gao, Eugene Palovcak, Yifan Cheng. currently in development as potential universal influenza virus vaccines based University of California, San Francisco, San Francisco, CA, USA. on the idea that when the is sequentially exposed to HA pro- Single-particle cryo-EM has recently emerged as a tool for characterization of teins that express the same stalk, but different head domains, the resulting anti- protein structure at atomic resolution, with applications both to soluble and body response will be directed toward the conserved stalk domain instead of the membrane proteins. Cryo-EM relies on computational alignment of single- rapidly mutating head domain. This vaccination strategy increases the body’s particle images, permitting calculation of interpretable two-dimensional aver- production of broadly neutralizing antibodies. Due to the conformational nature ages and three-dimensional reconstructions. Prior to data collection, cryo-EM of stalk epitopes, it is necessary to ensure that these epitopes remain correctly samples are trapped in vitreous ice by plunge freezing, protecting biological folded in the chimeric proteins, yet no prior structural information about cHA macromolecules from radiation damage and high-vacuum, yet maintaining hy- proteins exists. We used cryo-electron tomography to determine structures of a dration and native structure. Soluble proteins are thus surrounded by water, virus-bound cHA, cH5/1, comprising an H1 stalk and an H5 head domain. which has a small electron scattering cross-section relative to the protein itself. Compared to native H1 or H5 HA, the cHA shows a more open head domain However, membrane proteins in detergent or lipid nanoparticle systems (nano- with clear separation between HA monomers. The cHA structure also shows discs, saposin), are embedded in contrastive and predominantly unstructured a dramatic rotation between the stalk and head domains compared to the native material. The impact of this contrastive, unstructured mass on image alignment structures. Viruses expressing cH5/1 HA also had a significantly higher density and three-dimensional reconstruction is unknown. Indeed, traditional cryopro- of HA on the viral surface than those expressing native HA. Despite these dra- tectants (glycerol) are not used in cryo-EM because their protein-like scattering matic differences, cH5/1 retains its antibody-binding function with both head is deleterious to particle contrast. Nevertheless, a growing number of mem- and stalk-targeting antibodies, suggesting a surprising plasticity in HA protein brane proteins have been characterized using cryo-EM. To date all such pro- structure that may benefit future vaccine design. teins bear large, soluble domains (often dwarfing the membrane bound component, e.g. nanodisc-anchored ribosomes), which are hypothesized to þ 2839-Pos Board B446 drive image alignment. Recent structures of TRPV1 (a tetrameric Ca2 chan- Cryo-EM Structure Determination of Truncated SEC13/31 Proteins in nel) reconstituted in lipid nanodiscs show that current cryo-EM pipelines can COPII Vertices deliver high resolutions even when the ratio of structured to unstructured Mohammadreza Paraan, Scott Stagg. mass falls as low as 2:1. Here, we undertake to ascertain the limits to this ratio Institute of Molecular Biophysics, Tallahassee, FL, USA. (if any), as well as the feasibility of reconstructing structures lacking any sol- Recently, localized reconstruction algorithms have been used to determine the uble extension. structure of full length Sec13/31 COPII vertices to a resolution of 12 Ang- Starting from micrographs of TRPV1 in lipid nanodiscs, we use signal sub- stroms. Inherent flexibility of COPII cages, which is vital to their biological traction to simulate data for progressively smaller ratios of structured to un- function, has hindered high-resolution reconstructions by introducing confor- structured mass, eventually eliminating all contributions from the soluble mational heterogeneity into datasets. Localized reconstruction deals with the domains. Our results, including a 3.4 Angstrom structure of a hypothetical conformational heterogeneity of whole COPII cage particles by extracting membrane protein with no soluble domain, suggest that high-resolution align- the more homogenous vertices and classifying them separately. Although ment and reconstruction is feasible for membrane proteins with no soluble this method has successfully increased resolution, achieving density maps extension, including when the ratio of structured to unstructured mass is with a high enough resolution to demonstrate secondary structures requires less than 1:1. With continued technological development, it may be possible multiple approaches. We have hypothesized that the C-terminal unstructured to adopt cryoprotectants that greatly enhance the formation of vitreous ice domain of Sec13/31 could degrade the resolution of single particle reconstruc- even during slow freezing. Finally, this work also serves as a tutorial for pre- tions of the COPII cage by causing misalignment of projections. However, cise signal subtraction using open-source software, including creation of a truncating those domains increases the flexibility of the cages, but leaves the suitable subtraction map, simulation of particle image contributions, recenter- COPII proteins’ propensity to assemble into cages unchanged. Using localized ing of subtracted particles, and per-particle normalization based on Fourier reconstruction of the COPII cage vertices, we will show the range of structures correlations.

BPJ 7915_7919 578a Wednesday, February 15, 2017

2842-Pos Board B449 ever, the spatial separation of fluorescent molecules in a typical cryo-CLEM Cryo-EM Studies of Cullin-Ring Ubiquitin E3 Ligase (CRL)2 Regulation sample is sufficient for single-molecule or single-particle biophysics measure- by the COP9 Signalosome ments, with a similar geometry to TIRF measurements. This suggests the pos- Sarah V. Faull1, Fabienne Beuron1, Hugo Yebenes2, Andy Lau3, sibility of combining the structural information from electron microscopy with Argyris Politis4, Edward P. Morris1. information on function or dynamics of the particles being studied, using 1Structural Biology, Institute of Cancer Research, London, United Kingdom, the fluorescent signal to bridge cryo- and room-temperature measurements. 2Consejo Superior de Investigaciones Cientı´ficas, Centro de Investigaciones Previously, the Briggs group reported a cryo-CLEM system with 50 nm preci- Biolo´gicas (CIB), Madrid, Spain, 3Chemistry, University College London, sion given a high copy number of fluorescent proteins [2]. We have adapted this London, United Kingdom, 4Chemistry, King’s College London, London, system for biophysical characterization of particles labelled with synthetic United Kingdom. dyes, using laser illumination and additional analysis to access the data Cullin-RING E3 ubiquitin ligases (CRLs) are a key component of the ubiquiti- within the fluorescent signal. Here, we present a characterization of the cryo- nation cascade which targets proteins for degradation though the 26S protea- fluorescence microscope with an application to membrane fusion. A common some. This is a central mechanism in protein turnover and therefore assay involves dequenching of a concentrated lipid dye upon fusion with an un- influences many cellular processes. As such, abnormal regulation of the labelled vesicle. We show that auto-quenching is still possible under cryo- ubiquitin-proteasome system can lead to cancer. There are eight members of CLEM conditions, providing a method to determine the extent of lipid mixing the cullin family, with each member specifically degrading different protein in a single particle undergoing membrane fusion. In this way, the fluorescent targets (1). We have focused our studies on the CRL2 system, which uses signal can be utilized to provide functional information missing in the electron the VHL receptor protein (along with a cullin-2 scaffold, Rbx1 RING protein microscopy. and substrate adaptors Elongin B and C) to degrade hypoxia-inducible fac- tor-a (HIF-a). Due to the role of VHL in regulating cellular oxygen responses, 2845-Pos Board B452 inherited mutations in VHL pre-dispose carriers to the development of retinal, Secretory Glands Imaged in Aqueous Solution by Atmospheric Scanning spinal and cerebellar haemangioblastomata, clear cell renal carcinoma, and Electron Microscopy Toshiko Yamazawa1, Naotoshi Nakamura2, Mari Sato3, Chikara Sato3. phaeochromocytoma (2). Regulation of the CRL2 complex by the COP9 sig- 1 nalosome (CSN) via the removal of ubiquitin-like Nedd8 is therefore of thera- Department of Physiolgy, The Jikei University School of Medicine, Tokyo, Japan, 2Department of Statistical Genetics, Graduate School of Medicine, peutic interest. To address a lack of structural information on the CRL2 3 complex we have used cryo-electron microscopy to study the regulation Kyoto University, Kyoto, Japan, Biomedical Research Institute, National CRL2 by the CSN. We have used a catalytically-impeded CSN mutant Institute of Advanced Industrial Science and Technology, Tsukuba, Japan. (CSN5H138A (3)) in order to capture the molecular interactions between There are three pairs of major salivary glands in mammals, the parotid glans, CRL2 and CSN. We have also used mass spectrometry to provide insights submandibular glands and sublingual glands. These glands secrete serous, mu- into the structure and dynamics of CRL2 and the effects of neddylation. cous or mixed saliva via the proper main excretory ducts connecting the glan- 1) Zhao Y and Sun Y. Curr Pharm Des. 2013; 19(18): 3215-3225. dular bodies with the oral cavity. The hallmark characteristic of Sjo¨gren’s 2) Pugh CW and Ratcliffe PJ. Seminars in Cancer Biology 2003, 13(1): 83-89 syndrome is diminished secretory production from the primary exocrine gland 3)Mosadeghi R et al., Elife. 2016 Mar 31;5. pii: e12102. and the lacrimal or salivary glands resulting in symptoms of dry eye and mouth, by poorly understood mechanisms. Gland-related diseases have been studied by 2843-Pos Board B450 optical microscopy, and at higher resolution by transmission electron micro- Three-Dimensional Image Analysis of Melanocyte in Human Skin scopy of Epon embedded samples, which necessitates hydrophobic sample pre- Ji Young Mun1,2, Il-Hwan Kim3, Hyo Sun Choi2. treatment. Here, we report the direct observation of tissue in aqueous solution 1Eulji University, Seongnam, Korea, Republic of, 2Department of Senior by atmospheric scanning electron microscopy (ASEM). Salivary glands and Healthcare, Eulji University, BK21 Plus Program, Daejeon, Korea, Republic lacrimal glands were fixed, sectioned into slabs, stained with phosphotungstic of, 3Department of Dermatology, Korea University College of Medicine, acid (PTA), and inspected in radical scavenger D-glucose solution from below Ansan, Korea, Republic of. by an inverted scanning electron microscopy, guided by optical microscopy There is a global trend of increase in demand for 3 dimensional electron micro- from above to target the tissue substructures. A 2 to 3-mm specimen thickness scopy (3DEM) based on molecular resolution. The investigation of cellular was visualized by the SEM. In salivary and lacrimal glands, secretory vesicles structural change and related functional study are necessary for analyzing bio- and other organelles were clearly imaged at high resolution, and the former logical processes. In this study, currently available 3D reconstruction techniques could be classified according to the degree of PTA staining. The results suggest of electron microscopes (electron tomography, serial section analysis) were used the use of in-solution ASEM for histology and to study vesicle secretion sys- for study of hyper-pigment disease in human skin. Tomogram method applied to tems. Further, the high-throughput of ASEM makes it a potential tool for the medical devices such as MRI (Magnetic Resonance Imaging) and CT (Comput- diagnosis of exocrine -related diseases, such as Sjo¨gren’s syndrome. erized Tomography) uses images taken from various angles, and the tomogram 2846-Pos Board B453 linked with electron microscopy was named electron tomogram (ET). The 3D Implementing a Single Particle Pipeline for High Resolution Cryo-EM 5 structure was obtained from back projection using 61 images in 60 degrees. Ashleigh M. Raczkowski. Scanning electron microscopy (SEM) with 3-view system (diamond knife Simons Electron Microscopy Center, New York Structural Biology Center, installed SEM) or FIB (Focused Ion Beam installed SEM) was used for 3D New York, NY, USA. reconstruction using serial section (Briggman & Denk, 2006). The image seg- Recent advances in imaging technology and data processing have resulted in a mentation from ET and serial sections showed the detail of melanocytes (and ‘‘resolution revolution’’ in electron microcopy. There are now many structures keratinocytes) in pigmentary disorder such as seborrheic keratosis and melasma. determined by single particle cryo-EM which extend to near-atomic resolution. Briggman KL & Denk W (2006) Towards neural circuit reconstruction with Here we present the sub-3A˚ resolution single particle workflow of the T20S pro- volume electron microscopy techniques. Curr Opin Neurobiol, 16, 562-70. teasome to highlight the our efforts to establish a standardized automated pipe- This research was supported by Basic Science Research Program through the line. Our data collection and processing workflow for single particle analysis National Research Foundation of Korea (NRF) funded by the Ministry of Sci- starts from exploratory negative stain studies and proceeds to high resolution ence, ICT & Future Planning (2015R1C1A1A02037153). single particle reconstruction. The microscope was controlled by Leginon soft- ware and early stage processing of images were performed using the Appion 2844-Pos Board B451 workflow concurrent with data collection. This standardization lets us compare Incorporating Single-Particle Biophysics into Correlated Cryo- and optimize different experimental parameters on the fly, allowing each recon- Fluorescence and Cryo-Electron Microscopy struction shown here to be determined from images collected in a single day. The Lauren Ann Metskas, Giulia Paci, Edward A. Lemke, John A.G. Briggs. goal with this pipeline is for researchers from non-structural biology fields to ac- Structural and Computational Biology Unit, European Molecular Biology cess and make use of these new techniques and methodologies in their research. Laboratory, Heidelberg, Germany. In recent years, rapid advances in cryo-electron microscopy and tomography 2847-Pos Board B454 have been coupled with advances in correlated light and electron microscopy Why One Atomic Model? a Rationale for Atomic Ensembles (CLEM), where a fluorescent image of the sample is used to locate regions Mark A. Herzik Jr., Gabriel C. Lander. of interest prior to data collection. In particular, cryo-fluorescence microscopy Department of Integrative Structural and Computational Biology, The has been brought into single-molecule fluorescence regimes despite known lim- Scripps Research Institute, La Jolla, CA, USA. itations for objective lenses under cryogenic conditions [1]. Nearly all progress Our interpretation of high resolution EM structures is heavily influenced by to date has focused on particle localization or high-resolution imaging. How- crystallographic legacy, resulting in the now common practice of presenting

BPJ 7915_7919 Wednesday, February 15, 2017 579a a single atomic model to represent a structure. This is unfounded, given that the City University, Yokohama, Japan, 4Frontier Research Institute for Materials analysis of crystallographic and EM data are very disparate. A number of crys- Science, Nagoya Institute of Technology, Nagoya, Japan, 5Graduate School tallographic packages are being adapted to deal with EM data, which has re- of Frontier Science, The University of Tokyo, Kashiwa, Japan. sulted in the EM community adopting the practice of ‘‘one structure/one X-ray fluorescence holography (XFH) is a 3-dimensional visualization method atomic model’’, which can be severely misleading due to the fact that we often of atomic structures around specific atoms in single crystals (Tegze, M., Faigel, have variable resolutions within a map. In fact, even the highest resolution G. (1996) Nature 380, 49). In XFH, X-ray fluorescence atoms (usually metal maps have regions of poorly resolved density for certain residues and, for atoms) are excited by the external X-rays. The fluorescence intensity is spatially this reason, we should deposit multiple atomic models to the PDB, similar to modulated to form the holograms, because the fluorescent waves are partially what is done for NMR structures. Showing multiple solutions for an atomic scattered by neighboring atoms and interfere with unscattered reference waves. model would instantly provide naive users with a read-out of well-ordered Compared to standard X-ray diffraction methods, a remarkable feature of XFH is vs. less-resolved regions of an atomic map when they download a model that it provides a means of extracting both intensity and phase information, al- from the PDB. We present a methodology that combines Rosetta & Phenix lowing ‘‘model-free’’ structural analysis using Fourier-like transform of the to explore conformational landscape offered by a density, to generate multiple observed hologram (Barton, J. J. (1991) Phys. Rev. Lett. 67, 3106). Here we atomic solutions, as well as provide a per-residue convergence criterion. apply XFH to protein crystals with the recently developed measurement system (Sato-Tomita, A. et al. (2016) Rev. Sci. Inst. 87, 063707). The model protein Diffraction & Scattering Techniques used in this study is hemoglobin (Hb), an (ab)2 tetrameric protein with an iron-containing heme group per subunit. Since the Hb crystal has orientationally 2848-Pos Board B455 different multiple molecules in the unit cell, we used a pseudo symmetric- A New Technique for Molecular Dynamics Observation using Laboratory operation on each heme group to extract structural information of a particular X-Ray Source molecule of interest. Using such an analysis, we obtained the atomic images Keigo Ikezaki1, Ken Matsubara1, Yufuku Matsushita1, Chang Jaewon1, of the heme environments in the individual subunit of Hb. The results indicate Hiroshi Sekiguchi2, Yuji C. Sasaki1. that the degree of planarity of the a heme is higher than that of the b heme, consis- 1 2 The University of Tokyo, Kashiwa, Chiba, Japan, Japan Synchrotron tent with the result of high-resolution X-ray crystallography. In the conference, Radiation Research Institute (JASRI), Sayo, Hyogo, Japan. we will also present the results of a monomeric hemoprotein, myoglobin, with a Our laboratory has developed a single molecule observation method using X- similar folding as a Hb subunit, serving as a simpler model protein. ray which named Diffracted X-Ray Tracking (DXT). DXT monitors the infor- mation of three dimensional motions of single protein molecules by tracking 2851-Pos Board B458 diffracted spots from individual gold nanocrystals which labeled on each of New X-Ray Single Molecular Methodology using a Normal Monochro- the protein molecules. The accuracy of DXT reaches microsecond order of matic X-Ray time resolution and picometer order of spatial resolution. So for, DXT has suc- Yuji C. Sasaki1, K. Ikezaki2, N Ohta3, H. Sekiguchi3. ceeded to discover dynamics of many physiological molecules such as major 1The University of Tokyo, Kashiwa-city, Japan, 2The University of Tokyo, histocompatibility complex; MHC, Acetylcholine binding protein; AChBP, Kashiwa, Chiba 277-8561, Japan, 3SPring-8/JASRI, Sayo-cho, Japan. alpha-synuclein and so on. However, DXT has a weak point against a promo- The technique for high-speed and high-accuracy single molecular observations tion of general use because DXT needs the large facility of synchrotron orbital using X-rays, we called Diffracted X-ray Tracking (DXT) have been observed radiation source to detect diffraction spots from single gold nanocrystals. dynamical internal rotational motions in the individual single functional protein Therefore, DXT using a small light source is very effective. When using a lab- molecules in the cell membrane. In this single-molecule detection system, DXT oratory X-ray source, it is very clear that the signal to background ratio is better can monitor the rotating motions of an individual nanocrystal labeled to a spe- for the monochromatic X-ray diffraction than Laue diffraction using white x- cific site in individual protein molecules by using the time-resolved Laue rays. Furthermore, the monochromatic X-ray reduces the damage of measuring diffraction technique. This methodology was achieved the first observation of target. Recently, we developed a new technique to observe the molecular dy- x-ray single molecular dynamics in 2000. However, DXT have two weak points. namics using laboratory X-ray source; Rigaku FR-D (Cu anode, 50kV, One is to utilize the white x-rays (pink beam), and another one is to fabricate the 60mA) and a small detector; PILATUS-100K. In our technique, observing perfectly gold nanocrystals. The original concept in DXT method is to track the and tracking single diffraction spots like DXT are no longer necessary but diffracted spots from the labelled gold nanocrystal with individual single pro- we could estimate the molecular dynamics from intensity or variance of the teins. New DXT utilize a monochromatic x-ray and colloidal golds. Why diffraction ring form multiple gold nanocrystals. Now, we try to distinguish new DXT can track the diffractions using a monochromatic x-ray. Because molecular dynamics of AChBP between with or without toxin. In the meeting, movement information on diffraction spots is included in a lot of diffracted im- we will report the details of our analytical technique. ages. For example, very low-speed diffraction spot can be detected tightly, but a 2849-Pos Board B456 high-speed one can’t be detected. Additionally, when observing movements of Serial Synchrotron Cryallography with a Fixed Target more than one diffraction spot, the dynamic feature also appears in characteristic Henrike M. Mueller-Werkmeister1, Eike C. Schulz1, Darren Sherrell2, distribution of intensity fluctuation. Now, we demonstrated a new DXT tech- Danny Axford2, Friedjof Tellkamp1, Robin L. Owen2, R.J. Dwayne Miller1. nique to observe the molecular dynamics using a digital detector(PILATUS- 1Max-Planck-Institute for Structure and Dynamics of Matter, Hamburg, 100K) in SPring-8 BL40B2. The camera distance is about 300mm. But, we de- Germany, 2Diamond Light Source, Didcot, United Kingdom. tected the diffraction pattern from colloidal gold (diameter size=200nm) with in- Serial crystallography has been driven mainly from sample requirements tegrated time of 1-2s. The difference between the temperature changes of imposed by X-ray free electron lasers, however there is huge potential for Brownian movements of colloidal gold and the specific behaviors of a protein new applications and experiments as well at regular (microfocus) synchrotron could be detected. We made the total x-ray irradiation amount consequently beamlines normally used for rotation based structure determination from single decrease, so new DXT became possible to monitor longer movements. DXT protein crystals. Our approach uses a combination of fixed target arrays for would be used by this technological development more widely. sample delivery of micron sized protein crystals and a fast, accurate translation system and thus allows high throughput serial data collection at high hit-rates 2852-Pos Board B459 and with low sample consumption. Data obtained in this approach yield high Probing Manganese Ion Distributions Around Nucleic Acids using Small resolution structures and are collected at at room temperature with a low radi- Angle X-Ray Scattering ation dose per individual crystal, highly desirable for many proteins, where Suzette A. Pabit, Joshua M. Tokuda, George D. Calvey, Andrea M. Katz, structures can normally suffer from radiation damage. Yujie Chen, Lois Pollack. Further the possibility to extend the approach to time-resolved crystallography AEP, Cornell University, Ithaca, NY, USA. for the study of protein dynamics on the millisecond timescale is shown. Manganese ions are divalent cations that have profound biological roles in cellular processes, e.g. they are involved in folding of some RNAs and in 2850-Pos Board B457 some DNA conformational changes. We use two experimental Small-Angle X-Ray Fluorescence Holography for Proteins: Application to Hemoglobin X-ray Scattering (SAXS) based approaches to probe the distribution of Manga- and Myoglobin nese ions around nucleic acids. SAXS is a solution-based technique that is sen- Ayana Sato-Tomita1, Naohisa Happo2, Sam-Yong Park3, Koichi Hayashi4, sitive to the ion cloud and hydration layer around nucleic acids. By absolutely Yuji C. Sasaki5, Naoya Shibayama1. calibrating SAXS data, we can accurately characterize the ion cloud distribution 1School of Medicine, Jichi Medical University, Shimotsuke, Japan, and the water layer around DNA. In a recent publication (Meisburger et al., Bio- 2Graduate School of Information Sciences, Hiroshima City University, physical Journal, 2015), we used heavy atom isomorphous replacement to vary Hiroshima, Japan, 3Graduate School of Medical Life Science, Yokohama the SAXS scattering strength of the ion atmosphere to probe monovalent ion

BPJ 7915_7919 580a Wednesday, February 15, 2017 distributions around DNA. Here, we apply the isomorphous replacement tech- WAXS is a powerful method to study fine structural changes in short RNAs. nique to measure divalent ions. We compare the distribution of manganese We compare WAXS data with single molecule FRET data to see how the duplex with other divalent ions. We also measure Manganese ion distributions using structure of double-stranded RNA responds to different salt atmospheres. Anamolous Small Angle X-ray Scattering measurements (ASAXS) which required that we reconfigured our ASAXS instrumentation for low energy 2856-Pos Board B463 x-ray scattering measurements near the absorption edge of the Manganese In Situ Structural Changes of Biological Macromolecules with BioSAXS atom (6.55 keV). We show anomalous scattering of Manganese ions around Gerd Langenbucher1, Andreas Keilbach2, Heike Ehmann2. DNA of different sequences and an RNA riboswitch. These results provide 1Anton Paar USA, Inc., Ashland, VA, USA, 2Anton Paar GmbH, Graz, insight into the roles of divalent Manganese ions in nucleic acid studies. Austria. Biological small angle X-ray scattering (BioSAXS) is a nowadays well- 2853-Pos Board B460 established technique to study low resolution structural changes within biolog- Optimizing the Preparation, Analysis and Formulation of Liposomes for ical macromolecules in their native environment. In addition, SAXS is a power- Drug Delivery Applications ful tool for structural characterization and the quantitative analysis of flexible Diana Nash1, Betty Petrillo2, Dominic Senkl3. 1 2 3 system as a highly complementary method to the high resolution methods of Anton Paar USA, Ashland, VA, USA, Anton Paar, Graz, Austria, Chemie- X-ray crystallography and NMR. In general, BioSAXS allows studying three Ingenieurschule Graz, Graz, Austria. dimensional low resolution structures in different chemical environments under Liposomes, and other lipid based carriers, are useful vehicles for drug delivery defined conditions (e.g., temperature, pH, dielectric properties, etc.) using not applications because they are relatively simple to make, are biocompatible and only ab initio methods but also rigid body modeling. Furthermore, the shape their size, chemical functionality and stability can be modulated to optimize the (form factor) and interaction potential (structure factor) of the active quaternary drug delivery process. Additionally, targeting moieties can be added to the sur- protein structure can be assessed using the pair distance distribution function face of liposomes that enable specific targeting of tumor or other tissues and (PDDF) with the use of the generalized indirect Fourier transformation help to maximize drug delivery to the targeted area and to reduce non- (GIFT). Especially in situ measurements of biological macromolecules have specific delivery and toxicity. gained a lot of attention in the last decades due to the related structural changes However, liposome size, stability and surface chemistry are all susceptible to and correlated bioactivity (i.e., native and denatured state). changes in the surrounding environment including changes in pH, salt concen- As model substance, Lysozyme was analyzed in terms of its quaternary struc- tration, temperature and storage time. Thus it is critically important to optimize tural changes upon in situ heat treatment and changes in the dielectric constant liposome preparation, analysis techniques and storage conditions to ensure that of the chemical environment. The validation of the form and structure factor the final liposome formulation will be effective in its desired application. components of the analyzed system allows determining the influence of the Here we discuss liposome preparation, analysis and formulation using light scat- changed environmental conditions on the protein structure. The changed chem- tering techniques to assess liposome size and zeta potential, under a wide range ical environment causes an elongation of the previously globular protein struc- of environmental conditions, including variations in temperature, salt concen- ture. In aqueous environment, Lysozyme is stabilized via electrostatic tration, lipid/liposome concentration. It is observed that variations in these pa- stabilization. In ethanolic solution, the stabilization mechanism is changed, re- rameters significantly affect both liposome size and zeta potential outcomes. sulting in a change of structure (e.g., elongation). 2854-Pos Board B461 Bayesian Refinement of Accelerated Molecular Dynamics Simulations for 2857-Pos Board B464 In Vivo Interpreting SAXS Experiments Nonlinear Light Scattering Probe of Drug-Induced Activation of Samuel Bowerman, Amy Rice, Jeff Wereszczynski. Bacterial Mechanosensitive Channels 1 2 1 Department of Physics and Center for the Molecular Study of Condensed Soft Mohammad Sharifian Gh , Charles D. Cox , Michael J. Wilhelm , 1 Matter, Illinois Institute of Technology, Chicago, IL, USA. Hai-Lung Dai . 1Department of Chemistry, Temple University, Philadelphia, PA, USA, Small angle X-ray scattering (SAXS) is an experimental method that is useful 2 for capturing the full ensemble of flexible biomolecules, but it typically results Molecular Cardiology and Biophysics, Victor Chang Cardiac Research in low-dimensional data that is difficult to interpret without additional structural Institute, Sydney, Australia. knowledge. In principle, this information may be provided by molecular dy- The lack of homologues of the mechanosensitive (MS) channel of large namics (MD) simulation, but conventional MD trajectories rarely represent conductance (MscL) in the human genome makes it a novel antimicrobial the complete ensemble. Accelerated MD (aMD) was developed to overcome target. Specifically, MscL activators have been proposed as potential antibac- these sampling inadequacies by introducing a bias to the underlying energy terial species as they perturb cellular membrane potential and/or provide an landscape, which distorts the observed ensemble as a result. Here, we present alternative route of entry for antimicrobials into the cytosol. An important ques- a method for fitting aMD simulations with experimental SAXS data to accu- tion regarding the mechanism of drug-induced activation in MscL is: Does the rately model the relative populations of representative solution states. Scat- activated channel also allow other large species (i.e., secondary-molecules tering states are first identified from MD trajectories, and then their such as antibiotics) to pass through? To investigate this question, we have populations are re-weighted against empirical data through a Bayesian Monte developed a label-free optical technique, second-harmonic light scattering Carlo approach. Resistance to ensemble over-fitting is achieved by iteratively (SHS), for real-time analysis of channel activity in a living organism. Using considering increasing subsets of scattering states and by reducing experimental the principle of competitive transport [1], we apply time-resolved SHS to quan- data to the Shannon sampling limit. We apply this technique to several ubiquitin titatively characterize the transport rates of the second-harmonic active cation, trimers and find that aMD models converge upon agreement with the observed malachite green (MG) across the cytoplasmic membrane (CM) of living E. coli SAXS profiles up to an order of magnitude faster than conventional simulations. in the presence and absence of an MscL activator. MG is known to slowly diffuse across the hydrophobic core of the CM [2]. Subsequently, if MG trans- 2855-Pos Board B462 port across an activated MscL is permitted, the measured transport rate should Using WAXS to Study RNA Conformations increase substantially. As proof-of-concept, we used two molecules recently Yen-Lin Chen, Suzette A. Pabit, Andrea M. Katz, Lois Pollack. suggested to activate MscL, streptomycin (Strp) and dihydrostreptomycin Applied & Engineering Physics, Cornell University, Ithaca, NY, USA. (dStrp) [3]. Our results reveal that application of either activator results in a sig- Wide-angle x-ray scattering (WAXS) can reveal finer details of molecular struc- nificant increase in the MG transport rate, indicating that both Strp and dStrp tures than its counterpart, small-angle x-ray scattering (SAXS). WAXS also re- are inducing a more efficient route for MG transport across the CM. These re- ports small scale changes in the structural features induced by surrounding ions sults support the notion that Strp and dStrp are indeed MS channel activators. and ligands. WAXS is easily implemented by decreasing the sample-to-detector This study also shows that the SHS technique is a useful experimental paradigm distance in a SAXS experiment, increasing the scattering angles and momentum for in vivo study of channel activators, which is of particular interest in antimi- transfer q reached. Though the experimental setup is straightforward, the crobial drug discovery. computational tools required to fully understand WAXS spectra are not yet fully References developed. Currently, data interpretation focuses on variations in intensity [1]. Sharifian Gh. et al. ‘‘Label-Free Optical Method for Quantifying Molecular within the high-q regime. Our recent work compared WAXS profiles of 25 Transport Across Cellular Membranes In Vitro’’, J. Phys. Chem. Lett. (2016), base-pair (bp) double-stranded DNA and RNA in NaCl with and without added 7:3406. trivalent cobalt hexamine ions (CoHex). These profiles were compared with all- [2]. Wilhelm et al. ‘‘Gram’s Stain Does Not Cross the Bacterial Cytoplasmic atom molecular dynamics (MD) simulations (The Journal of Chemical Physics Membrane’’, ACS Chem. Biol. (2015), 10:1711. 2016, 144, 205102). The MD simulations of RNA conformational changes under [3]. Wray et al. ‘‘Dihydrostreptomycin Directly Binds to, Modulates, and different salt conditions were captured by WAXS. Here, we further show that Passes through the MscL Channel Pore’’, PLoS Biol. (2016), 14:e1002473.

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Optical Microscopy and Super Resolution domain with sub-mm lateral resolution. A sinusoidally modulated laser beam is used for excitation of the chromophores, thereby generating fluorescence Imaging: Novel Approaches and Analysis III and photoacoustic signals. For modulation, frequencies in the low MHz regime are used. The fluorescence light is collected by an objective and detected with 2858-Pos Board B465 an avalanche photodiode, whereas the photoacoustic signal is measured with an Non-Invasive Label-Free Imaging of Oxidative Processes in Human Skin ultrasonic transducer. Both signals, acoustical and optical signals simulta- Michaela Poplova1,2, Eduard P.A. Van Wijk3,4, Michal Cifra1. neously, are recorded using a lock-in detection technique. Recording of two 1 Bioelectrodynamics, Institute of Photonics and Electronics, The Czech signals enables the access to complementary information, i.e. the different 2 Academy of Sciences, Prague, Czech Republic, Faculty of Electrical chemical composition of the structures. In case the laser beam is absorbed by Engineering, Czech Technical University in Prague, Prague, Czech Republic, the sample, photoacoustic signals are preferably generated if non-radiative 3 Sino-Dutch Centre for Preventive and Personalized Medicine, Leiden, recombination dominates, whereas luminescence occurs preferably for radia- 4 Netherlands, Division of Analytical Biosciences, LACDR, Leiden tive recombination. University, Leiden, Netherlands. In this contribution we present images of polymer structures doped with dyes Oxidative processes present across all types of organisms, including humans, and fabricated with multiphoton lithography, labeled cells, and blood smears cause chemical formation of electron excited species with subsequent endoge- to demonstrate the importance of multimodality. Important information could nous ultra-weak photon emission. Thus, imaging of this endogenous chemilu- be missed, if these structures are inspected by photoacoustic and fluorescence minescence using ultra-sensitive devices potentially enables label-free microscopy only. monitoring of oxidative stress in optically accessible areas of human body, such as human skin. However, no quantified imaging of oxidative processes 2861-Pos Board B468 in human skin has been performed until now using endogenous chemilumines- Recovering Cellular Shape and Size in the Bright Field Optical Microscope cence under controlled extent of oxidative stress conditions. Furthermore, the Braulio Gutierrez-Medina, Carmen Noemi, Hernandez Candia, mechanisms and dynamics of endogenous chemiluminescence is not fully ex- Manuel de Jesu´s, Sa´nchez Miranda. plored.Here we demonstrate that different degrees of oxidative processes on IPICYT, San Luis Potosi, Mexico. skin can be spatially resolved through non-invasive label-free endogenous Bright field (BF) is one of the most widely used observation modes in optical chemiluminescence imaging in a quantitative manner. Additionally, to obtain microscopy, effective to image amplitude objects but inadequate to observe insight into the underlying mechanisms, we developed and employed a minimal phase objects—optically thin materials that only introduce phase changes in chemical model of skin based on a mixture of lipid (linoleic acid) / melanin / the illuminating wavefront plus limited scattering—such as cells. Because of water to show that it reproduces essential features of the response of a real this limitation, BF is rarely used in microbiological studies that aim to observe skin to oxidative stress. Our results contribute to novel non-invasive label- and quantify cell shape and size in unstained samples. Instead, staining or alter- free methods for quantitative monitoring of oxidative processes and oxidative native microscopy techniques (phase contrast, dark field, differential interfer- stress. ence contrast, among others) are used. Here, we present a methodology to We acknowledge support by the Czech Science Foundation, project no. P102/ obtain well-defined, three-dimensional images of unstained cells based exclu- 15-17102S. Authors participate in COST Action BM1309 and bilateral ex- sively in BF optics plus conventional digital image processing techniques. First, change project between Czech and Slovak Academies of Sciences, no. SAV- we determine the phase point spread function (PSF) of the BF microscope 15-22. through experiment or by using a standard scattering model. Second, we use 2859-Pos Board B466 the PSF to restore BF images of unstained cells through deconvolution. Using Of Absolute Concentrations of NADH in Cells using the Phasor Flim these steps we process images of Escherichia coli cells in BF to recover well- Method defined shapes and boundaries. To validate our technique, we compare the Enrico Gratton, Ning Ma, Michelle A. Digman, Leonel Malacrida. cellular boundaries obtained through BF deconvolution with those obtained Biomedical Engineering, University of California, Irvine, Irvine, CA, USA. by fluorescence imaging of stained cells, showing excellent agreement. In addi- One of the properties of fluorescence lifetime is its independence on concentra- tion, we demonstrate that BF deconvolution works well following living cells tion. While this is considered to be an advantage, when analyzing images of cell and that three-dimensional information can be restored. Compared to alterna- and tissues we often need to also measure the absolute concentration of a fluo- tive methods designed to observe unstained cells, our procedure does not rophore. The concentration value is commonly obtained by comparison of the require additional optical components and is readily applicable in any BF intensity of the fluorophore in the tissue and in a solution of known concentra- microscope. tion. The underlying hypothesis is that the quantum yield of the fluorophore is the same in the sample and in the solution used for calibration. In cells and tis- 2862-Pos Board B469 sue this assumption is not always valid especially for a fluorophore like NADH Coherent Anti Stokes Raman Scattering Microscopy for Visualizing that changes the quantum yield by a large factor when free in solution with Diffusion of Water in Skin 1 1 2 respect to when bound to enzymes. We propose a graphical method using Ste´phanie Yuki Kolbeck Hotta , Bjarne Thorsted , Jens Ahm Sørensen , Jonathan R. Brewer1. the phasor representation of the fluorescence decay to derive the absolute con- 1 2 centration of NADH in cells. The method requires the measurement of a solu- BMB, University of Southern Denmark, Odense, Denmark, Odense tion of NADH at a known concentration. The phasor representation of the Universitets Hospital, Odense, Denmark. fluorescence decay accounts for the differences in quantum yield of the free Diffusion and transport of water in cells and tissue is a vital part of all life. and bound form of NADH, pixel by pixel of an image. The concentration of However directly visualizing water in cell and tissue is not possible using NADH in every pixel in a cell is obtained after adding to each pixel in the pha- traditional microscopy techniques. In the current study Coherent anti Stokes sor plot a given amount of unmodulated light which causes a shift of the phasor Raman scattering (CARS) microscopy was used to investigate the diffusion towards the origin by an amount that depends on the intensity at the pixel and of D2O through human skin. CARS microscopy is a real-time label-free the fluorescence lifetime at the pixel. The absolute concentration of NADH is non-invasive imaging technique that provides spatial resolution and acquires obtained by comparison of the shift obtained at each pixel of an image with the contrast based on intrinsic molecular vibrations. Human skin and its barrier shift of the calibrated solution. Work supported in part by NIH grants NIH P41- function are of major interest in multiple fields. Increased knowledge GM103540 and NIH P50-GM076516. regarding skin structure and the dynamics of molecules in skin is crucial for the understanding of healthy and diseased skin conditions and the devel- 2860-Pos Board B467 opment of treatments. Transdermal penetration and diffusion measurements Multimodal Optical Resolution Photoacoustic and Fluorescence Micro- on different substances have traditionally been performed using methods scopy in the Frequency Domain that either cannot provide spatial resolution or only to a limited extend Bianca Buchegger1, Gregor Langer2, Jaroslaw Jacak1,3, Thomas A. Klar1, through invasive approaches. Using CARS it was found that the diffusion Thomas Berer2. characteristic of D2O in skin was depth dependent regarding the outer layer 1Institute of Applied Physics, Johannes Kepler University, Linz, Austria, of the skin called the stratum corneum. It was found that the stratum corneum 2Research Center for Non Destructive Testing (RECENDT), Linz, Austria, could be subdivided into three distinct regions based on the diffusion charac- 3Upper Austria University of Applied Sciences, Linz, Austria. teristics. In the outer most layer of stratum corneum the diffusion occurred Optical-resolution photoacoustic microscopy (OR-PAM) is a microscopy tech- too fast to be resolved. The diffusion coefficient for the middle part of the nique where acoustic waves are generated due to absorption of a pulsed or stratum corneum was determined to be 0.01 5 10.10 3 mm2 s 1, while the intensity modulated focused laser beam. In this work, we demonstrate simulta- diffusion coefficient for the inner stratum corneum inferior was 0.02 5 neous fluorescence and photoacoustic scanning microscopy in the frequency 3.6.10 3 mm2 s 1.

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2863-Pos Board B470 and backward steps, respectively, consistent with the length of the product, Holographic Tracking of Archaea and Bacteria Over Millimeter Length chitobiose (1 nm). The ratio of forward to backward steps was 18, corre- Scales sponding to the energy difference of 2.9 kBT. Interestingly, 2.4 nm forward Katie L. Thornton, Laurence G. Wilson. steps were also observed, and chitotetraose without one acetyl group was de- Physics, University of York, York, United Kingdom. tected with MALDI-TOF Mass as a by-product. These results indicate that Swimming microorganisms usually move in three dimensions, but camera SmChiA skips one chitobiose unit if deacetylated, because SmChiA cannot sensors are two-dimensional. This can restrict the scope of experiments using hydrolyze deacetylated chitin. Furthermore, distribution of pause was fitted standard techniques, which typically rely on tracking objects close to a two- by single exponential decay with time constant of 22 ms, indicating single dimensional image plane. This approach is the same used in the macroscopic rate-limiting step. domain, where ‘machine vision’ approaches to object recognition and tracking have been very successful. The work in our lab takes a different approach by 2866-Pos Board B473 using aspects of classical optics and signal processing to design new image Micro-Spectroscopy of Bio-Assemblies at the Single Cell Level processing algorithms, to ‘mine’ more information out of digital images. Jeslin Kera, Debopam Chakrabarti, Alfons Schulte. This has two advantages: (i) We can extract three dimensional imaging data University of Central Florida, Orlando, FL, USA. from two-dimensional images; (ii) by moving away from traditional ‘ma- Confocal absorption microscopy has the benefits of requiring no labels and low chine-vision’ ideas, we can redesign imaging systems that are cheaper and light intensity for excitation while providing a signal from the contrast gener- more flexible. I will present a few examples from recent work in holographic ated by the attenuation of propagating light due to absorption. This enables tracking of microorganisms that use image-processing algorithms to obtain spatially resolved measurements of single live cells and bio-molecules in three-dimensional data on swimming trajectory and cell shape. In particular, nano-liter solutions. We present experiments on model systems over the spec- the ability to follow hundreds or thousands of individual swimming bacteria tral range from the near-infrared to the ultraviolet. The spectral identification of in volumes of up to the scale of cubic millimetres allows us to address ques- biomolecules with characteristic absorption bands in the ultraviolet at spatial tions on the statistics and variability of cell swimming trajectories. We have resolution in the micron range will be discussed. demonstrated this technique to characterise the swimming behaviour of several motile, co-habiting halophilic archaea, as well as standard laboratory 2867-Pos Board B474 strains of bacteria such as E. coli. High Precision Indirect Optical Manipulation of Live Cells with Function- alised Microtools 2864-Pos Board B471 Gaszton Vizsnyiczai1, Badri Aekbote2, Andra´s Bu´za´s3, Istva´n Grexa3, Differential Interference Contrast Microscopes with Switchable Shear Pal Ormos3,Lo´ra´nd Kelemen3. Direction and Quadrilateral Shear 1Dipartimento di Fisica, Universita` di Roma ‘‘La Sapienza’’, Rome, Italy, Michael Shribak, Elena Iourieva. 2Center for Soft and Living Matter, Ulsan National Institute of Science and Marine Biological Laboratory, Woods Hole, MA, USA. Technology, Ulsan, Korea, Republic of, 3Institute of Biophysics, Biological A conventional differential interference contrast (DIC) microscope is based on Research Centre, Szeged, Hungary. the interference of two beams, which optic axes are sheared by a sub-resolution Mechanical manipulation of live cells is crucial to numerous biological ex- distance. When the beams are traveling through the specimen the interference periments. Optical trapping and manipulation is a popular and widely used creates image contrast, which depends on the gradient of optical path difference approach, however, direct optical trapping suffers from serious limitations: (OPD) encountered along the shear. The contrast disappears if the OPD The intensive laser illumination damages the cells, in addition, since trapping gradient is perpendicular to the shear direction. The image contrast also de- occurs due to interaction of light with high index of refraction regions in the pends on the bias OPD introduced by microscope and specimen’s absorption cells, the trapping is not defined sufficiently well for high precision manip- and/or scattering. The DIC image is not quantitative. ulation. To overcome these problems microbeads have already been used for The orientation-independent (OI-) DIC microscope overcomes these limita- trapping cells indirectly thereby reducing the irradiation damage and tions. The OI-DIC rapidly switches the shear direction by 90 and controls increasing trapping efficiency with their high refractive index contrast. The the bias. The microscope captures two complementary sets of DIC images drawbacks can be completely eliminated by the use of tailor made microtools with orthogonal shear directions. The images are processed to compute designed specifically for this task, so that indirect trapping and manipulation OPD map, which displays the specimen’s morphology and can serve as a is applied. The trapping light interacts only with the tools, and the shape of landmark for fluorescent staining. The OPD map can be used to measure the tool can be optimized for accurate and efficient manipulation around all the sold content (DNA, protein) of the cells and to reconstruct the refractive spatial coordinates as well as for optimal cell handling. The microtools of index. complex shape are fabricated by two-photon excitation photopolymerization. New 4-beam DIC microscope employs interference of four beams, which optic Appropriate chemical surface functionalization establishes controlled and axes are sheared quadrilateral. The 4-beam DIC creates orientation- strong attachment of the cells. Holographic optical traps are used to precisely independent OPD gradient magnitude image, which can be seen by an eye control the position of the cells in 6D, the positional accuracy is in the order through the ocular in real-time. In order to obtain an OPD map, the microscope of 10 nm. The power of the approach is demonstrated by greatly improved captures a set of images with different biases. The bias control unit can be imaging of live cells. placed anywhere between the polarizers. Therefore the 4-beam DIC is simpler to implement, in comparison to the OI-DIC. 2868-Pos Board B475 Other interference and phase microscopy techniques use modified or Fibrin Network Formation and Thrombolysis using a Birefringence restricted numerical apertures (NA) of the condenser and/or objective lens. Measuring Often times the contrast of their raw images is low and is strongly affected Naoyuki Yokoyama1, Hayata Machida1, Yuito Tsukamoto1, by wavefront aberrations. The OI-DIC and the 4-beam DIC employ: (1) the Shinya Ohkubo2. full NAs of condenser and objective lenses, (2) high-contrast raw images, (3) 1Department of Artificial Organs, National Institute of Technology, Numazu the optical image subtraction, and (4) the computation image subtraction. College, Numazu-City, Shizuoka, Japan, 2Department of Optical information Thus, the OI-DIC and the 4-beam DIC can provide the best resolution engineering, National Institute of Technology, Numazu College, images. Numazu-City, Shizuoka, Japan. Background: Fibrin is a fibrous plasma protein, which is known as precursor 2865-Pos Board B472 of clot. The polymerized fibrin network together with platelets forms a One-Nanometer Steps in the Motion of a Linear Molecular Motor Serratia thrombus which causes fatal thromboembolism especially in the extracorpo- marcescens Chitinase A Resolved by Gold Nanoprobe real circulation using heart-lung machine or dialyzer. Because the length of Ryota Iino1,2, Akihiko Nakamura2. fibrin is shorten than visible ray wavelength, we cannot detect fibrin using 1Institute for Molecular Science, Okazaki, Japan, 2Okazaki Institute for an optical microscope. Objective: To develop early phase thrombus detec- Integrative Bioscience, Okazaki, Japan. tion system, we proposed fibrin imaging method using the birefringence Serratia marcescens chitinase A (SmChiA) is a linear molecular motor mov- properties. In this study, the relationship between fibrin network forma- ing on and hydrolyzing crystalline chitin processively. Here, we directly tion/resolution and optical birefringence properties was evaluated. Method: visualized steps and pauses in the motion of SmChiA. By using gold nano- Birefringence imaging system was consisted of halogen light source, red particle (40- or 20-nm in diameter) as a low-load probe, movement of light interference filter, polarizer, quarter wave plate, optical condenser, SmChiA was observed with total internal reflection dark-field microscopy sample flow pass, 10 objective lens, and CCD image sensor. Experimental at 1000 fps or 2000 fps. Step sizes were 1.1 nm and 1.2 nm for forward plasma, which was kept at temperature of 36C and coagulated gradually,

BPJ 7920_7924 Wednesday, February 15, 2017 583a was set in flow tube and guided by continuous flow pump to the sample tive access to the data via a web browser. An example website has been flow pass. The retardation of the sample plasma was calculated from set up at http://fpb.ceb.cam.ac.uk. In the future, we hope that this changes CCD images which was obtained by system at every 5 seconds. After con- the way research data are shared and suggest that publishers will use the soft- firming sufficient aggregation of fibrin, heparin was injected to the sample ware for online publication. Data can thus be shared in their entirety, moving flow pass so as to activate fibrinolytic system of blood. Result: The retarda- on from the current practice of providing selective views or movies shown tion of plasma increased over time within 5 minutes. In contrast, fibrinolytic from a single perspective, which are not capable of providing a full under- system reduced phase differences by melting aggregated fibrin within standing of the data. 6minutes. 2871-Pos Board B478 2869-Pos Board B476 The Extra Microscope Boundary Effects in FRAP Recovery in the Confined Geometries of Ani- Alberto Diaspro1,2, Luca Lanzano`1, Paolo Bianchini1, mal, Plant and Fungal Cells Giuseppe Vicidomini1, Marti Duocastella1, Francesca Cella Zanacchi1, James K. Kingsley1, Jeffrey P. Bibeau2, Cem Unsal1, Iman S. Mousavi1, Colin J.R. Sheppard1. Zhilu Chen3, Xinming Huang3, Luis Vidali2, Erkan Tuzel1. 1Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy, 2Department of 1Department of Physics, Worcester Polytechnic Institute, Worcester, MA, Physics, University of Genoa, Genova, Italy. USA, 2Department of Biology and Biotechnology, Worcester Polytechnic In the last 40 years, optical fluorescence microscopy, due to its inherent ability Institute, Worcester, MA, USA, 3Department of Electrical and Computer of imaging living systems during their temporal evolution, had a continuous Engineering, Worcester Polytechnic Institute, Worcester, MA, USA. update on three main tracks, namely: three-dimensional (3D) imaging, pene- Fluorescence Recovery After Photobleaching (FRAP) has been an important tration depth at low perturbation and resolution improvements. However, tool used by cell biologists in the past few decades to study the diffusion and computational optical sectioning, confocal laser scanning, two-photon excita- binding kinetics of proteins, vesicles and other molecules in the cytoplasm, tion and super-resolved methods can be considered as milestones in optical nuclei or the cell membrane. FRAP has been particularly useful in the char- microscopy. With the recent Nobel Prize in Chemistry in 2014 for the devel- acterization and development a mechanistic understanding of tip-growth opment of super-resolved fluorescence microscopy, it has been revealed how across many cell types over long time (seconds) and length (microns) scales. the optical microscope can offer unimaginable performances in terms of In FRAP, a high intensity laser beam is applied to a Region of Interest (ROI), spatial resolution.[Diaspro A. 2014. Il Nuovo Saggiatore]. Starting from photo-bleaching its contents, and the brightness recovery associated with the this point, considering important revolutions like the ones of confocal and transport of fluorescent molecules into this area is used to construct a recov- two-photon excitation microscopy coupled to the advent of green fluorescent ery curve, and make quantitative estimates of diffusion coefficient and per- proteins [Diaspro A. and van Zandvoort M.A.M.J. (eds) 2016. Super- centage of bound fraction of molecules. While many FRAP models have resolution Imaging in Biomedicine. CRC press], I will discuss about some been developed over the past decades, the influence of the complex bound- converging and correlative techniques that can be used for what I like to aries of three-dimensional geometries on the recovery curves, in conjunction name ‘‘the extra microscope’’. The meaning is related to series of advances with ROI and optical effects (imaging, bleaching, scanning), has not been and new methods that allow getting information at the nanoscale or preferable well studied in the literature. Here, we developed a three-dimensional at the molecular scale referring to both spatial resolution or structural informa- computational model of the FRAP process that incorporates particle diffu- tion. Three examples are related to resolution improvement by image subtrac- sion, cell boundary effects, and the optical properties of the scanning tion microscopy using the STED donut, volumetric imaging fast-inertia confocal microscope, and validated this model using the tip-growing cells approach and fluorescence lifetime and correlation methods (SPLIT, PLICS) of Physcomitrella patens. This validation also provides an accurate estima- [Scipioni et al. 2016. BJ]. Within this framework second harmonic generation, tion of the diffusion coefficient of 3xmEGFP for moss cells, which allows phase methods and Mueller matrix [Sheppard C.J.R, Castello M. and Diaspro for the calculation of an effective cytoplasmic viscosity for molecules of A., 2016. JOSA A 33 (4), 741-751] signatures based on polarization are open- this length scale in moss cells for the first time. We then show how the ing a new window for multimodal imaging including label free approaches. cell boundary and optical effects confound the interpretation of FRAP recov- The Extra Microscope has tunable and flexible performances depending on ery curves, the bound fraction of fluorescent proteins, and the number of dy- the biological question and reflects the integration in a flexible way of several namic states of a given fluorescent protein across a wide range of cellular approaches [Teodori L. et al. 2016. J.Biophotonics]. It is worth noting that geometries, including the budding yeast, S. pombe, tip-growing plant cells, new correlative approaches coupling optical super resolved methods with nuclei and lamellipodia of cells. Finally, we illustrate how existing theoret- scanning probe microscopes are providing interesting developments that ical and computational models perform in each of these scenarios, provide a will be outlined [Chacko, J.V. et al.2013. Cytoskeleton]. Considering all these computational tool and guidelines on how to use FRAP quantitatively in such aspects we can re-phrase the sentence related to the Galilei’s ‘‘occhialino’’ geometries. into ‘‘microscopium extraordinarium nominare libuit’’.

2870-Pos Board B477 2872-Pos Board B479 First Person Bioimage: An Online Tool for Presentation and Publication of Efficient Parametric Imaging with GPU Computing Volumetric Data Dianwen Zhang1, Xiang Zhu2, Angelo Bifone3, Alessandro Gozzi3, Marcus Fantham, Clemens F. Kaminski. Silvia Capuani4, Marco Palombo4,5. University of Cambridge, Cambridge, United Kingdom. 1Microscopy Suite, Beckman Institute for Advanced Science & Technology, New microscopy techniques (e.g. SPIM, 3D STORM, OPT, etc.) and many UIUC, Urbana, IL, USA, 2China Agricultural University, Beijing, China, other 3D bioimaging modalities (tomographic EM, MRI, CT, etc.) generate 3IIT, Center for Neuroscience and Cognitive Systems UniTn, Rovereto, Italy, terabytes of volumetric data. Whilst highly specialized software packages 4IPCF-UOS Roma, Physics Department, ‘‘Sapienza’’ University of Rome, are available for the analysis and visualization of such data, their use re- Rome, Italy, 5CEA/DSV/I2BM/MIRCen & CEA-CNRS URA 2210, quires specialized skills and they are often costly. Furthermore the capacity Fontenay-aux-Roses, France. to share data and allow interactive exploration by third parties is highly Parametric imaging plays a critical role in modern biophysical, biomedical limited. research and clinical diagnosis. It can provide useful visual representations Here we present FP BioImage, an easy-to-use and powerful open source visu- of a sample with respect to the parameters underlying the mathematical alization tool that permits researchers to share their volumetric image data on- models associated with sample data. For this technique, nonlinear model line and third parties to interact with, and explore, datasets in their entirety. FP fitting optimization is a commonly used approach to estimate the parameters BioImage provides the viewer an immersive experience for the exploration of on a pixel-by-pixel basis to create parametric maps. With the increased so- complex 3-dimensional bioimaging data, and makes use of the latest graphics phistication of modern imaging systems, the amount of data processed capabilities embedded in all modern web browsers so that no software installa- with parametric imaging techniques is exploding and the processing time tion is required. is often limiting the advancement of these technologies for real-time and The tool is fast and user responsive, requires no training for use, and includes automotive applications. However, in many applications, the computation advanced rendering and data manipulation capabilities. Data can be intuitively for each image pixel can be carried out independently of any others, and explored from a ‘first person perspective’, akin to navigating virtual space in such sort of computations can profit tremendously from parallel processing. modern computer games, allowing users to conceptualize and contextualize de- Nowadays, graphics processing unit (GPU) has become a standard tool in tails in the data to aid biological interpretation. high-performance parallel computing. To realize real-time automated image From a researcher’s perspective the tool makes it now possible to easily reconstruction for parametric imaging techniques, such as multi-parametric share volumetric imaging data globally, providing anyone full and interac- microscopy and magnetic resonance imaging (MRI), we have developed a

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GPU-based nonlinear model fitting optimizer called GPU-LMFit. We Monitoring cell culture media conditions is highly important for industries in demonstrate the applications of GPU-LMFit in super resolution localization order to optimize cell proliferation for applications such as regenerative med- microscopy, fluorescence lifetime imaging microscopy, diffusion-weighted icines and protein synthesis. The various chemical components of the medium MRI (DW-MRI) and myocardial longitudinal relaxation time (T1) MRI us- have unique absorbance and fluorescence characteristics and are present in a ing modified Look-Locker inversion recovery (MOLLI) based techniques. wide and dynamic concentration range. Thus we investigated the use of the The results show that the use of GPU-LMFit can readily result in more new simultaneous Absorbance and fluorescence Excitation Emission Matrix than tens of times of speedup of parametric analyses in these techniques, (ABEEM) method for rapid and effective measurements of the varying com- compared with the software using CPU-only processing. An important ponents simultaneously. Importantly, the ABEEM method uniquely facilitates example will be presented that when GPU-LMFit was used with a medium correction of fluorescence inner-filter effects to yield quantitative fluorescence level GPU like Quadro K2000 for a DW-MRI image data set to reconstruct spectral information that is largely independent of component concentration. non-Gaussian diffusion parametric images, the results show that the images This is significant because it allows one to effectively monitor quantitative can be constructed up to 240x faster than with CPU processing alone. In this component changes using multivariate methods to generate and evaluate spec- application, GPU-LMFit helps to reduce the time for DW-MRI processing tral libraries. Here, we applied ABEEM measurements to monitor Chinese from hours to seconds. Our results show the performance of GPU-LMFit Hamster Ovary (CHO) cell culture medium conditions during a 4 day cell pro- is excellent to significantly improve the efficiency of parametric analyses liferation period. The medium comprised Eagle’s minimal essential medium and can thus be a useful tool to enable automated parametric imaging for (E-MEM), Fetal Bovine Serum, Penicillin, Streptomycin and non-essential real-time visualization, analysis and diagnostics. amino acids. The main fluorescent component concentrations were analyzed using Parallel Factor Analysis (PARAFAC). The PARAFAC analysis was Optical Spectroscopy: CD, UV-VIS, Vibrational, validated for four main components, namely, tyrosine, tryptophan, riboflavin and NAD(P)H. The tyrosine, tryptophan and riboflavin components remained Fluorescence II relatively constant during the course of the cell proliferation. Importantly, the PARAFAC score of NAD(P)H increased continuously during cell prolifera- 2873-Pos Board B480 tion to indicate it is a key fluorescence indicator of the culture medium con- Raman Spectroscopy for Prostate Cancer Detection and Characterization dition and cell proliferation. This is significant because NAD(P)H is a very Kelly Aubertin1,2, Vincent Trinh3,4, Michael Jermyn5,6, important factor in cell metabolism. It was concluded that the ABEEM Joannie Desroches6,7, Catherine St-Pierre6, Maria-Claudia Vladoiu2, method and PARAFAC analysis were effective tools for monitoring the Andre´e-Anne Grosset2, Fred Saad2,8, Dominique Trudel2,3, cell proliferation process as an example for other potentially important Fre´de´ric Leblond6,7. applications. 1Imaging & Engineering, CRCHUM, Montreal, QC, Canada, 2Montreal 3 Cancer Institute (ICM), CRCHUM, Montreal, QC, Canada, Department of 2875-Pos Board B482 4 Pathology, CHUM, Montreal, QC, Canada, Department of Pathology and Metabolic Shifts in Huntington Disease Revealed by Fluorescence Lifetime 5 Cellular Biology, University of Montreal, Montreal, QC, Canada, Thayer Imaging Microscopy School of Engineering, Dartmouth College, HANOVER, NH, USA, Sara Sameni. 6 Department of Engineering Physics, Polytechnique Montreal, Montreal, QC, UCI, Irvine, CA, USA. 7 Canada, Imaging and engineering, CRCHUM, Montreal, QC, Canada, Huntington disease (HD) is a progressive autosomal neurodegenerative disor- 8 Department of Surgery, University of Montreal, Montreal, QC, Canada. der due to the expanded CAG or polyglutamine (polyQ HTT) repeats in the Prostate cancer (PCa) is the most frequently diagnosed cancer among men. HD gene mapped into the 4th chromosome. One of the early sign and hallmark PCa diagnosis is currently based on analyses of the morphological organiza- of the individual carrying mutated gene of HD is metabolic dysfunction. HD tion of tissue. The process from tissue collection to diagnosis is time patients suffer from excessive weight loss correlated with progression of the consuming and can be too long for intraoperative diagnosis. Raman spectros- disease despite of adequate calorie intake. However, this effect is not fully un- copy (RS) is a label-free and non-destructive laser-based technique that pro- derstood. In this work, we use fluorescence lifetime imaging microscopy vides real-time molecular information about tissue. Histologically, cancer (FLIM) coupled with phasor analysis to measure metabolic alteration of HD patterns are intermingled with entrapped benign glands. Because of the in living cells and in the Drosophila eye discs model. We used endogenous very high level of heterogeneity exhibited by prostate tissues, it is highly NADH autofluorescence as an intrinsic biomarker and cofactor of meta- important to identify precisely the tissue type measured by RS. To this bolism. We are able to get pixel maps of metabolic alteration in HEK293 cells aim, we developed a method for high-resolution co-registration of RS with and in the eye discs of transgenic Drosophila expressing expanded polyQ PCa diagnosis. HTT. Our results show significant shift toward larger free to bound ratio of Raman spectra are acquired on fresh ex vivo prostates within 2 hours after NADH indicating shift from OXPHOS to glycolytic state in the present of radical prostatectomy using a multi-wavelength hand-held contact Raman expanded polyQ. In addition, in the nucleus compartment further lifetime shift probe. The prostate is then reintegrated to the usual clinical pathological work- toward increased free NADH is a possible indication of synergies between flow for formalin fixation and paraffin embedding (FFPE) and preparation for transcriptional dysregulation and metabolic dysfunction. This can lead to optic microscope histopathological analyses. The precise reconstruction of the oxidative stress and eventual cell death. This powerful FLIM/Phasor method prostate slice with hematoxylin & eosin (H&E) tissue allows the spatial corre- can potentially be applied to screen native HD tissue sample, for drug 2 lation of the measured areas (0.2 mm ) with the correspondent histopatholog- screening, and development of new approaches for the treatments of such ge- ical information, for point-by-point diagnosis. The tissue is then classified into netic disorder. This work is supported in part by NIH grant P41 GM103540 groups (benign/malignant) and subgroups according to the percentage of and NSF BEST IGERT grant. benign glands, stroma or cancer grades. Different machine learning algorithms were tested to classify the spectra with 2876-Pos Board B483 different levels of categorization. Preliminary results showed that RS is capable Lifetime Spectroscopy of Exosomes under Evanescent Field Illumination of detecting PCa with an accuray of 89%. Within the benign tissue group, high Justin Aluko1, James Monypenny2, Simon Poland1, Susan Cox1, Tony Ng2, stroma/glands rations have been correlated with the spectral signature of Simon Ameer-Beg1. collagen, which is the main constituent of stroma. Within the cancer tissue 1Randall Division of Cell and Molecular Biophysics, King’s College London, group, high grade patterns were find to be different from low grade patterns, London, United Kingdom, 2Division of Cancer Studies, King’s College demonstrating that RS is able to distinguish aggressive from less aggressive London, London, United Kingdom. cancers. Understanding the mechanisms driving drug resistance in cancer patients is fundamental for continued progression towards effective treatment. Human 2874-Pos Board B481 epidermal growth factor receptor (HER/ErbB) network rewiring, through re- Effective Monitoring of Cell Proliferation Media Components using ceptor dimerization, is believed to play such a role. Therefore, it is highly Simultaneous Absorbance and Fluorescence Excitation-Emission Matrix desirable to detect and monitor such phenomena non-invasively, in the clinic. Data Analyzed with Parallel Factor Analysis Time-correlated single photon counting fluorescent lifetime imaging (FLIM) Adam M. Gilmore1, Yuichi Kitagawa2, Takuyi Moriyama3, Daisuki Irikura4, is a highly sensitive and non-invasive method to detect protein interactions. Yasushi Nakata4. Modern FLIM systems typically comprise a laser scanning module and point 1Horiba Instruments Inc., Edison, NJ, USA, 2Horiba Techno Service Co., Ltd. detector to analyse the fluorescence of an assay on a pixel-by-pixel basis. Horiba, Kyoto, Japan, 3Horiba Techno Service Co., Ltd., Kyoto, Japan, Confocal sectioning improves the signal-to-noise and resolution of TCSPC 4Horiba Ltd., Kyoto, Japan. data, yet suffers from being particularly slow; typical acquisition times can

BPJ 7920_7924 Wednesday, February 15, 2017 585a take up to five minutes per frame. Our lab has recently significantly improved of AR was extremely short in fluid environments (12 ps in water at room upon this by parallelising up to 1024 confocal points simultaneously, reducing temperature) and increased over 10-fold when dissolved in glycerol (231 acquisition times to seconds. However, for sparse image fields where data is ps). The temperature dependence of AR’s quantum yield in ethanol and glyc- located in a small percentage of pixels, considerable time is wasted scanning erol followed Arrhenius behavior with a lower activation energy in ethanol every pixel in the field-of-view. As such, we introduce a new fluorescence (3.2 kcal/mol) than in glycerol (7.6 kcal/mol), providing additional support lifetime data acquisition technique whereby data is collected sequentially for molecular rotor behavior. AR also showed high sensitivity to the bulk and automatically from features of interest within the field-of-view, greatly rheological properties of gelatin desserts and to changes in consistency of improving the efficiency of data acquisition. We can then reconstruct, for custards during cooking. the first time, lifetime images based on the localization information of such This study makes a significant contribution to the currently incomplete photo- features. Furthermore, the system is flexible such that if the entire frame is physical characterization of this safe-to-ingest fluorophore and its application not necessary, the user can select particular areas within the total field-of- as a sensor in edible products. view to measure, further improving acquisition times. We demonstrate the advantage of the system by imaging human breast cancer exosomes contain- 2879-Pos Board B486 ing the marker GFP-CD63 under evanescent field illumination. Additionally, The Development of a Novel Approach to Oriented Circular Dichroism we show that the system has sufficient resolution to significantly improve the using Magnetically-Aligned Bilayers throughput of a FRET assay detecting association of EGFR complexes on the Luke S. Evans1,2, Rohanah Hussain2, Giuliano Siligardi2, exosome surface. Philip T.F. Williamson1. 1Biological Sciences, University of Southampton, Southampton, United 2877-Pos Board B484 Kingdom, 2Beamline B23, Diamond Light Source, Oxfordshire, United Equilibrium of Two Fluorescence States in the UnaG-Bilirubin Complex Kingdom. Yoh Shitashima, Togo Shimozawa, Toru Asahi. Oriented circular dichroism (oCD) spectroscopy is a challenging but Advanced Science and Engineering, Waseda University, Tokyo, Japan. revealing technique that is used to ascertain the tilt angle of membrane pro- UnaG, a member of the fatty-acid-binding protein (FABP) family, is a teins in oriented lipid bilayers. The preparation of conventional oCD sam- recently discovered fluorescent protein, which have similar fluorescence spec- ples, where lipid bilayers are deposited on a support substrate, is a trum and brightness of green fluorescent protein (GFP). The UnaG has a laborious process which can require extensive repetition and optimisation unique feature that the fluorescence is induced by binding of Bilirubin (BR) to produce well aligned bilayers with good optical properties. Even then un- for its chromophore and the formation of the tight UnaG-BR complex (holoU- certainty over the depth of bilayers deposited and effective path-length re- naG). The advantages of holoUnaG compared to GFP, such as approximately sults in inconsistency between samples and also within different regions of half size of GFP and the oxygen-independent chromophore formation would the same sample. In light of this challenge, we propose a novel approach allow visualization of the targets that has been impossible to label so far. to oCD utilising magnetically-alignable lipid bilayers (or ‘bicelles’) which However, the information of fluorescent properties of UnaG has been not are relatively fast and easy to prepare. The convenience of preparation, enough to correctly analyze and interpret the fluorescence data acquired by and their propensity to orient with respect to a magnetic field, has led to using UnaG. In this study, we focused on the characteristic of fluorescence in- the use of anisotropic bicelles extensively in solid-state NMR studies of tensity (FI) increase after addtion of BR to apoUnaG, which consist of the two membrane proteins. We are exploiting this magnetic alignment for oriented sequential increases, viz. the first rapid and following slow increases. From CD studies using a modified magnetic synchrotron-radiation CD instrument the analysis of the relationship between extent of FI increase and the concen- (beamline B23, Diamond Light Source, UK). In contrast to mechanically trations of UnaG and BR, the first rapid FI increase corresponded to the im- aligned samples, bicelle measurements are conducted in standard cuvettes mediate formation of holoUnaG by binding of BR to apoUnaG, and the where both protein concentration and path-length are well defined. Further following slow FI increase corresponded to the fluorescence enhancement characterisation and validation of alignment has been carried out using of holoUnaG itself by first-order reaction, i.e., from the first formed holoU- both NMR and EPR spectroscopies. naG1 to matured bright holoUnaG2. Moreover, by using the FI distribution We have optimised this technique to the transmembrane domain of the putative analysis method (FIDA), the distribution of two FI components corresponding glycosyltransferase Fukutin-1, whose localisation to the Golgi apparatus is to holoUnaG1 and holoUnaG2 was detected in the solution at fluorescence essential for its function. Oligomerisation and changes in tilt angle, induced steady state. The ratio of the abundance and the brightness of holoUnaG1 by the bilayer lipids, appear to be the driving force for this localisation in the and holoUnaG2 in steady state were 6:4 and 1: 3.9, respectively, resulting Golgi. Using oCD spectroscopy with magnetic alignment of membrane pro- that the unimolecular reaction consist of forward direction at k12 = teins, we have identified changes in tilt angle as a response to changing lipid 3 1 3 1 1.3010 s , and reverse direction at k21 = 1.9810 s . The fact that environments. the reaction rates were affected by ionic strength implied that the bi-phasic switching of amino-acids coordination to the BR chromophore and resulting 2880-Pos Board B487 alteration of the fluorescence efficiency may underlie the mechanism of the Linear Dichroism of DNA: Characterization of the Distribution of transition between holoUnaG1 and holoUnaG2. Orientations Resulting from Hydrodynamic Shear John C. Sutherland. 2878-Pos Board B485 Physics, Augusta University, Augusta, GA, USA. Photophysical Properties of Allura Red for Monitoring Quality of Foods Linear dichroism provides information on the orientation of chromophores and Pharmaceuticals that are components of, or bound to, an orientable macromolecular, such Bogumil Zelent1, Rahul Chib2, Sarah Waxman2, Alexia Ciarfella2, as DNA. For molecular alignment induced by laminar hydrodynamic shear, Maria Corradini3, Richard D. Ludescher2. the two principal axes orthogonal to the direction of alignment are not equiv- 1Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, alent. As a result, the magnitude of the increase or decrease in the flow USA, 2Food Science, Rutgers University, New Brunswick, NJ, USA, 3Food induced change in absorption for light polarized parallel to the direction of Science, University of Massachusetts, Amherst, MA, USA. flow can be more than a factor of two greater than the corresponding Allura Red AC (AR) is a synthetic color approved for use in foods and pharma- decrease or increase for light polarized perpendicular to the plane defined ceuticals in the US. The sensitivity of this dye as a fluorescence probe of micro- by the directions of the flow and the direction of the flow velocity gradient. viscosity has recently been reported. The dye fluorescence is weak, often The ratio of the two flow-induced changes in absorption give the dichroic undetectable, in low viscosity fluids; however, its quantum yield, emission in- increment ratio. This ratio is characterized using the equivalent orthogonal tensity and lifetime increase significantly with an increase in medium viscosity. orientation model that assumes that each absorbing unit is aligned parallel It thus exhibits presumptive molecular rotor behavior. to one of the principal axes of the apparatus. The absorption of the aligned To facilitate the operationalization of AR as a probe of food quality, its pho- molecules is characterized by components parallel and perpendicular to the tophysical properties and performance in model food systems were studied. orientable axis of the molecule. Although determined by measurements of The absorption, emission and excitation spectra of AR was measured in wa- polarized absorption, the dichroic increment ratio is independent of the op- ter, glycerol and their mixtures. Additionally, the fluorescence quantum tical properties of the aligned molecules, and therefore independent of wave- yields and lifetimes were determined as a function of increasing viscosity length. The dichroic increment ratio can be used to determine if the (0.6 to 15 mPa s) and temperature (4to40C) in water, ethanol and glyc- alignment is effectively uniaxial and, if not, the extent to which alignment erol. AR showed a large Stokes shift (~2500 cm1)andamoderatesensi- of molecules parallel to the flow gradient is decreased relative to alignment tivity to polarity. Its sensitivity to molecular crowding was characterized in the direction perpendicular to both the direction of flow and the flow using the Fo¨rster-Hoffmann equation (x ~ 0.40). The fluorescence lifetime gradient. Analysis of published data indicates that for the alignment of

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DNA in rectangular flow cells, where the average shear tends to be low, there We solve the first order reaction-diffusion equations which describes binding- are significant differences between these alignment probabilities, but for the diffusion kinetics using photobleaching scanning profile of confocal laser scan- higher average shear produced in a Couette cell, the alignment can be essen- ning Microscopes approximated by a Gaussian laser profile. We show how to tially uniaxial. The equivalent orthogonal orientation model returns results solve the first order photobleaching kinetics partial differential equations with equivalent to those of more general tensor models provided that only optical prebleach steady state initial conditions using a time-domain method known as properties for light propagation and polarizations parallel to principal axes of a Krylov Subspace Spectral method (KSS method). KSS are explicit methods the experimental system are measured, which is the usual configuration of an for solving time-dependent variable-coefficient partial differential equations linear dichroism experiment. Approaches for measuring the dichroic incre- (PDEs). KSS methods are advantageous compared to other methods because ment ratio with modern dichrometers are discussed. of its high resolution and its superior scalability. We will apply Gaussian Quad- rature rules in the spectral domain developed by Golub and Meurant to solve 2881-Pos Board B488 PDEs. We present a simple rough analytical solution, as well as a computa- Measuring Polycyclic Aromatic Hydrocarbons in Water using the Simul- tional solution that is first-order accurate. We then use this solution to examine taneous Absorbance and Fluorescence Excitation-Emission Matrix short and long time behaviors. Method: Advantages of Classical Least Squares Regression Over Parallel Factor Analysis 2884-Pos Board B491 Boqian (Ben) Yang, Adam M. Gilmore. Estimating FRET Efficiency using Excitation-Scanning Hyperspectral Fluorescence, Horiba Scientific, Edison, NJ, USA. Imaging Many Polycyclic Aromatic Hydrocarbons (PAHs) are carcinogenic and John Robert Griswold, Naga Annamdevula, Joshua Deal, Thomas Rich, hence of important environmental concern in drinking water sources. Silas Leavesley. Most USEPA PAHs of concern exhibit high fluorescence quantum yields University of South Alabama, Mobile, AL, USA. and sub mg/l to mg/l detection limits in neat solutions. However, drinking Fo¨rster Resonance Energy Transfer, or FRET, is an indispensable tool in the water sources commonly contain other natural compounds, including humic biological sciences for measuring intermolecular distances. Current tools that and fulvic acids and proteins, in carbon concentrations of several mg/l that measure FRET suffer from limited signal strength, making it difficult to exhibit significant interfering absorbance and fluorescence backgrounds in observe rapid cell-signaling events. We have developed a unique mode of the PAH detection region. We investigated the use of the new simultaneous hyperspectral imaging which filters the excitation wavelengths and collects Absorbance and fluorescence Excitation-Emission Matrix (ABEEM) method total fluorescence emission. Our prior work has shown that excitation- to measure PAHs in raw water. The ABEEM method corrects fluorescence scanning is an effective tool for detecting fluorophores in cell samples. inner-filter effects to yield quantitative fluorescence spectral information to The purpose of this study was to assess the feasibility of using this be largely independent of component concentrations. We compared Clas- excitation-based technique for FRET measurements. In traditional spectral sical Least Squares Regression (CLS) and Parallel Factor Analysis (PAR- imaging FRET studies, the emission spectra of donor and acceptor are AFAC) in terms of identification and detection limits for the PAHs. The resolved from the emission spectrum of the sample using linear unmixing CLS library was calibrated with respect to concentration for the individual However, since excitation scanning collects the entire sample emission EPA PAHs dissolved in deionized organic-carbon free water. The same data without filtering wavelengths, a new approach is needed to utilize our set was used calibrate a model using the 3-way PARAFAC technique. Vali- method. Here we present data indicating that excitation scanning can be suc- dation of the CLS and PARAFAC models was compared with PAHs added cessfully adapted for FRET studies. Fluorescence excitation-scanning hyper- to natural raw water of varying organic carbon concentrations. Identification spectral images were acquired by sequentially exciting samples with an of individual PAHs and mixtures and detection limits were significantly 18-20 nm bandwidth (FWHM) tunable excitation source, provided by a better with CLS. The evaluation is consistent with the fundamental princi- 300W Xe arc lamp coupled to a Sutter VF-5 tunable filter. Images were ac- ples CLS vs. PARAFAC and primarily because CLS can be distinctly cali- quired from 380-490nm in 5nm increments. Donor and acceptor excitation brated with the individual PAH component spectra. We conclude that the signatures were then separated from the total signal using a spectral library. identification and detection limits of the ABEEM and CLS analysis can Two long-pass dichroic filters were employed, at 458nm and 495nm. FRET be significant enough for environmental contamination monitoring in drink- efficiencies were estimated by comparing donor excitation signal between ing water sources; certainly, as a first screening method before the EPA scans with different dichroic filters. Results indicate that excitation- standard solid-phase extraction and liquid or gas-chromatographic analysis scanning techniques provided accurate estimates of FRET efficiency, and methods. that this method could be a useful tool in FRET-based cell signaling studies. This work was supported by NIH grants P01HL066299, S10RR027535, and the Abraham Mitchell Cancer Research Fund. 2882-Pos Board B489 Frequency Extrapolation of Mechanical Properties Obtained from 2885-Pos Board B492 Brillouin Scattering Measurements in Biological Samples Rapid Spectral Imaging of Very Large Microscopy Images Kareem Elsayad. Yuval Garini. Advanced Microscopy, VBCF, Vienna, Austria. Physics, Bar Ilan University, Ramat Gan, Israel. Brillouin Light Scattering Microspectroscopy (BLSM) is an all optical tech- The spectral content of biological samples reflects on the content and phase of nique that can be used to yield information on the high frequency (GHz) me- the material. By measuring a spectral image, or more specifically, the spectrum chanical properties of materials (Longitudinal Modulus, Shear Modulus). at each point of the sample, it is possible to assess different biological qualities Results from BLSM measurements are thus distinct from those obtained us- and even diagnose the sample in case it is a tissue section or cytological sample. ing lower frequency perturbation or correlation based approaches, often Spectral imaging is already in use for many years, but so far it was not possible yielding their interpretation or direct comparison challenging. Here we to measure large samples in a rather short time. We present a novel method for compare studies of the frequency dependence of the mechanical parameters scanning very large spectral images of microscopy samples in a short time (Shear Modulus) up to the MHz-range - derived from Fluorescence Correla- frame that is more than an order of magnitude faster than all existing systems. tion Spectroscopy (FCS) studies - to the corresponding BLSM obtained The system captures the information ‘on the fly’ while the sample is continu- elastic parameters, for a range of biological samples. We discuss our results ously being scanned. The spectral separation is achieved through Fourier in terms of a scaling theory, which allows us to extrapolate the mechanical spectroscopy where high spectral resolution of ~5 nm at 500 nm is demon- parameters obtained using BLSM to lower frequencies with some additional strated with a diffraction-limited spatial resolution. A brightfield sample of information of the system being studied, and thus offer a means of under- 10X10 mm2 measured with a magnification of 10X with 40 spectral points standing BLSM measurements in light of results obtained from established can be measured within 5 minutes. We demonstrate the use of the system for techniques used for measuring the quasi-static mechanical properties of bio- cancer detection. logical samples. 2886-Pos Board B493 2883-Pos Board B490 Plasmonic Electricity: A Digital Form of Metal-Enhanced Fluorescence Solution of PDEs for First-Order Photobleaching Kinetics using Krylov Chris D. Geddes. Subspace Spectral Methods Institute of Fluorescence, UMBC, Baltimore, MD, USA. Somayyeh Sheikholeslami, James Lambers. Fluorescence technologies are entrenched in the biosciences today. In nearly The University of Southern Mississippi, Hattiesburg, MS, USA. all aspects of fluorescence spectroscopy light is focused and collected by a

BPJ 7920_7924 Wednesday, February 15, 2017 587a detector which converts the photon flux into a digital signal which is then facilitate this dynamic behavior. Using these soft arms, antibodies swivel in displayed. To boost optical signatures many groups have shown that the solution, groping for the antigens at surface, and the flexible nature of hinge close proximity of fluorescent species to fluorophores, significantly amplifies region facilitates the binding to the antigen. We analyzed the Fab swivel the fluorescence signatures many fold, as much as 103 in the near-field, a movements as random walks, and estimated the flexibility of the IgG hinge technology described as Metal-Enhanced Fluorescence by the Geddes labs region (3). Further, we imaged antibody-antigen interaction. The antibodies in years past.1 However, hidden within these close-range near field exhibited random flexible movements, as the two Fab regions bind to the an- fluorophore-metal interactions is an induced plasmonic current, directly pro- tigen surface, leaving the Fc region to swing in all directions. Also, our portional to the excitation irradiance and the concentration of the fluoro- studies exhibited that the binding occurs either in a standing posture, or in phores present in the near-field, < 20 nm. The current can be read a low-lying posture. HS-AFM can directly observe dynamic behaviors of directly, opening up huge opportunities for both the amplification and the biomolecules as movie in solution, and reveal functions in regard to mechan- direct detection of fluorescence, i.e. digital fluorescence, such as in digital ical aspects. Direct and dynamic images have advantages to elucidate the immunoassays DNA detection and in fluorescence microscopy. The direct properties of proteins and revealing the functional mechanisms. 1. T. Ando measurement of fluorescence is likely to find profound applications and im- et al., Proc. Natl. Acad. Sci. U.S.A. 98, 12468-(2001) 2. N. Kodera et al., plications in the biosciences and promises to change both the way we think Nature 468: 72-(2010) 3. J. Preiner et al., Nature Communications 5: and use fluorescence spectroscopy today. 4394-(2014). In this presentation, we subsequently present our recent findings and demonstrate the role of different metallized substrates and the role of elec- 2889-Pos Board B496 tron donors and quenchers on the magnitude of the induced Plasmonic Non-Raster High-Speed AFM Imaging of Biopolymers Current. Brett Hartman1, Sean Andersson1, William Nagel2, Kam Leang2. Reference 1Mechanical Engineering, Boston University, Boston, MA, USA, 2University ‘‘Metal-Enhanced Fluorescence,’’ Edited by Geddes, C.D., John Wiley and of Utah, Salt Lake city, UT, USA. Sons, New Jersey, June 2010, 625 pgs, ISBN: 978-0-470-22838-8. High-speed atomic force microscopy (HS-AFM) has advanced significantly in recent years with a few commercial instruments now able to image at rates Force Spectroscopy and Scanning Probe between one to ten frames per second. These advances have allowed HS- AFM to visualize biological molecules as they move. Despite these ad- Microscopy II vances, however, there are several limitations in terms of utility as well as many systems of interest whose native speeds remain beyond current instru- 2887-Pos Board B494 ments, such as the motion of a cytoplasmic dynein with speeds on the order Development of Temperature-Controlled High-Speed AFM of 800 nm/s. Here, we describe a combination of non-raster scanning algo- Hirohide Takahashi1, Atsushi Miyagi1, Lorena Redondo-Morata2, rithms combined with a new high-speed three-axis scanning stage to achieve Simon Scheuring1. 1 2 fast imaging on biopolymers and similar samples. The method combines a Weill Cornell Medicine, New York, NY, USA, Inserm, Marseille, feedback-based algorithm that uses the data in real time to steer the tip along France. the biopolymer, decreasing imaging time by focusing the measurements High-speed atomic force microscopy (HS-AFM) has contributed important on the interesting areas. The non-raster scanning algorithm is combined novel insights into the dynamics of biological samples such as lipid bilayers, with the new high-speed, dual stage scanning platform. The scanner’s high membrane proteins, molecular motors and living cells at nanometer lateral resonances (in the tens of kHz range) is expected to achieve 30 frames per and sub-second temporal resolution. Lipid bilayers are found in several second or higher imaging rates. different temperature-dependent states, termed phases; the main phases are solid and fluid phases. The transition temperature between solid and fluid phases is lipid composition specific. Under certain conditions some lipid bila- 2890-Pos Board B497 yers adopt a so-called ripple phase, a structure where solid and fluid phase do- AFM Imaging of DNA G-Wires in Solution mains alternate with constant periodicity. Because of its narrow regime of Krishnashish Bose, Anh Tuaˆn Phan. existence and heterogeneity, ripple phase and its transition dynamics remain Division of Physics and Applied Physics, School of Physical and poorly understood. Here, a temperature control device is developed and inte- Mathematical Sciences, Nanyang Technological University, Singapore, grated to high-speed atomic force microscopy (HS-AFM) allowing to observe Singapore. dynamics of phase transitions from ripple phase to fluid phase reversibly in real Short G-rich DNA sequences have been reported to form G-wires, as time. Based on HS-AFM imaging, the phase transition processes from ripple observedbyAFMimagingonMicainair.However,tilldatesuchstructures phase to fluid phase and from ripple phase to metastable ripple phase to fluid have not been observed directly in aqueous solution at the single-molecule phase could be reversibly, phenomenologically, and quantitatively studied. level. Furthermore, the detailed structure of G-wires is not well understood. The results show phase transition hysteresis in fast cooling and heating pro- Understanding and controlling the formation of G-wires would have impli- cesses, while both melting and condensation occur at 24.15 C in quasi- cations in their potential role in diseases and also nanotechnology applica- steady state situation. A second metastable ripple phase with larger periodicity tions. We developed a new sample preparation strategy and used ultra-short is formed at the ripple phase to fluid phase transition when the buffer contains AFM cantilevers to overcome the problem of floating G-wires, which þ Ca2 . The presented temperature-controlled HS-AFM is a new unique experi- occurred with conventional protocols for AFM imaging of DNA in solution. mental system to observe dynamics of temperature-sensitive processes at the Using our new protocol, we could image DNA G-wires in aqueous solution, nanoscopic level. and also resolved structural features down to 1 nm. We imaged G-wires formed by the Tetrahymena telomeric sequence d(G4T2G4) and other 2888-Pos Board B495 related sequences and found that these G-wires have a height of 2.950.3 High-Speed AFM Reveals Advanced Details on Dynamic Behavior of nm, about 30-50% higher than what was reported previously. We also Antibody observed 4 nm periodic features which were either purely left-handed or Norito Kotani, Ramanujam Kumaresan, Yoko Kawamoto-Ozaki, zig-zag. For benchmark and comparison, we resolved the minor and major Takashi Morii, Takao Okada. grooves of linear duplex DNA and obtained a height of 2.150.4 nm, which Biomolecule Metrology, Research Institute of Biomolecule Metrology, is very close to that of solution structure. Our work has opened up new pos- Tsukuba, Japan. sibilities for visualization of DNA G-quadruplexes in aqueous solution, High-Speed Atomic Force Microscope (HS-AFM) can observe the dynamic which could allow investigation of their structure and interaction with other behavior of biomolecules in solution, as a movie (1, 2). HS-AFM also can be molecules. used to image directly with high resolution in nano-scale order, without any chemical fixing of the samples or fluorescence. Antibody IgG and IgM are 2891-Pos Board B498 important proteins in Immune system. We have observed dynamic behavior Extracellular Membrane Potential Measurement of Single Living Cells of IgG and IgM in solution using HS-AFM. ‘‘Y’’ shape of IgG was imaged with Scanning Ion Conductance Microscopy clearly, and Fab and Fc regions were well distinguished. IgM was imaged to Namuna Panday, Jin He. contain five monomers, and each monomer has two antigen binding sites By Physics, Florida International University, Miami, FL, USA. HS-AFM imaging at video rate, we revealed that the Fab regions moved in Recently, the existence of multiple micro-domains of extracellular potential torsional direction like swinging arms, and the soft structure of hinge regions around individual cells have been revealed by voltage reporter dye using

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fluorescence microscopy. One hypothesis is that these long lasting potential 2894-Pos Board B501 patterns play a vital role in regulating important cell activities such as em- Curli Mediate Bacterial Adhesion to Fibronectin via a Tensile Collective bryonic patterning, regenerative repair and reduction of cancerous disorgani- Binding Network zation. We used multifunctional Scanning Ion Conductance Microscopy Yoo Jin Oh1, Michael Hubauer-Brenner1, Hermann Gruber1, Yidan Cui2, (SICM) to study these extracellular potential patterns of a single cell with Lukas Traxler1, Christine Siligan1, Sungsu Park3, peter Hinterdorfer1. higher spatial resolution. With this novel technique, we measured and 1Johannes Kepler University Linz, Linz, Austria, 2Mechanobiology Institute, compared the extracellular potential distributions of living melanocytes National University of Singapore, Singapore, Singapore, 3Sungkyunkwan (Mela-A) and melanoma(B16) cells and found both the mean magnitude University, suwon, Korea, Republic of. and spatial variation of extracellular potential of the Mela-A cells are bigger Escherichia coli (E. coli) cells contain the amyloid curli protein CsgA that than those of B16 cells.We also found the change in membrane potential of bind to surfaces of the extracellular matrix protein fibronectin. Using a nano- B16 cells is small with change of potassium ion concentration in bath solu- mechanical force-sensing approach, we obtained real-time information about tions with respect to mela-A cells. As compared to the voltage reporter dye the distribution of molecular bonds involved in the adhesion of curliated bac- based fluorescence microscope method, SICM can achieve quantitative po- teria to fibronectin. We found that a dense collective network of bonds is tential measurements of non-labeled living cell membranes with higher formed between curli and fibronectin fibers which results in tight bacterial spatial resolution. binding to cell surfaces. Nanomechanical force recognition measurements re- vealed that approximately 10 bonds were disrupted either sequentially or simultaneously. For a single RGD/CsgA bond, we attained a force of 51 2892-Pos Board B499 pN and a short lifetime of about 0.85 s. However, the work required to disso- An Optical Setup for the Study of Mechanotransduction in Living Cells at ciateawholeE. coli cell from the fibronectin surface was 2750 k $T. the Single Molecule Level B Thus amyloid formation of E. coli surfaces leads to multi-bond structural Marios Sergides1,2, Tommaso Galgani1,2, Claudia Arbore1,2, components of fibrous nature that explains the strong mechanical binding Francesco S. Pavone1,2, Marco Capitanio1,2. of curliated bacteria to hosts and unveils the functions of these proteins in 1LENS - European Laboratory for Non-linear Spectroscopy, Florence, Italy, bacterial internalization and invasion. Overall we demonstrate the potential 2Department of Physics and Astronomy, University of Florence, Florence, of single-molecule and single-cell force spectroscopy for revealing accurate Italy. dynamic and statistical information about the nanomechanical behavior of Forces and mechanical stresses acting on biological systems are emerging as multiple bonds involved in collective network formation during cellular regulatory factors essential for cell life. Emerging evidence indicates that adhesion. factors such as applied forces or the rigidity of the extracellular matrix This study was funded by an APART (Austrian Programme for Advanced determine the shape and function of cells and organisms. Mechanotransduc- Research and Technology) fellowship of the Austrian Academy of Science tion, i.e. the conversion of mechanical forces into biochemical and biomol- (to Y. J. O.). ecular signals, is at the basis of many biological processes fundamental for the development and differentiation of cells, for their correct function and for the development of pathologies. Although optical and magnetic tweezers 2895-Pos Board B502 and atomic force microscopes (AFMs) have been extensively used to apply Force Spectroscopy of T4 Bacteriophage Adhesion during Infection mechanical stress on living cells and study their mechanical properties, Cesar A. Quintana-Catan˜o1, Martha J. Vives-Flo´rez2, methods to investigate the conversion of mechanical stimuli into biochem- Manu Forero-Shelton1. ical and biomolecular signals with molecular sensitivity are still lacking. 1Department of Physics, Universidad de los Andes, Bogota´ D.C., Colombia, Here we propose an experimental arrangement combining optical tweezers 2Department of Biological Sciences, Universidad de los Andes, Bogota´ D.C., and fluorescence imaging with the ability to manipulate specific membrane Colombia. receptors and detect intracellular signals, with a sensitivity up to the single In order to achieve a successful infection of their host, E. coli, T4 bacterio- molecule. Optical manipulation is accomplished with a diode laser emitting phages require an initial adhesion-governed mechanism for adsorption to the at 808 nm to minimize photodamage of living cells. Three-dimensional po- cell surface followed by viral DNA ejection into the bacterial cytoplasm. We sition detection combined with nanometric position control of the sample developed an assay to measure single phage-bacterium interactions using allow precise application and measurement of the force applied on cell Atomic Force Microscopy (AFM) in order to probe the initial interactions be- membranes. Fluorescence excitation of organic chromophores and fluores- tween them. Here we report interaction forces of the long tail fibers with the cence proteins is obtained using total internal reflection fluorescence bacterial surface as well as their dynamics. (TIRF) to follow processes occurring near the cell membrane, or with an optimized highly-inclined light sheet to image intracellular or nuclear events 2896-Pos Board B503 with high signal-to-noise ratio, while a sCMOS camera allows single mole- High Fibrinogen Levels Promote Erythrocyte-Erythrocyte Adhesion: A cule sensitivity over a large field of view. The capability of the system is Cardiovascular Risk Factor in Heart Failure and Arterial Hypertension tested on living cells transfected with genetically encoded fluorescence Patients probes. Ana Filipa Guedes1, Luı´s Sargento2, Jose´ Braz-Nogueira3, Nuno Lousada2, Carlos Moreira3, Filomena A. Carvalho1, Nuno C. Santos1. 2893-Pos Board B500 1Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de AFM with a Versatile Optics System for Living Cell Studies Lisboa, Lisbon, Portugal, 2Hospital Pulido Valente, Centro Hospitalar Lisboa Evan Nelsen. Norte, Lisbon, Portugal, 3Hospital Santa Maria, Centro Hospitalar Lisboa Physics and Astronomy, UNC-Chapel Hill, Chapel Hill, NC, USA. Norte, Lisbon, Portugal. Cells exert forces on their environment and respond to mechanical stimuli. Erythrocyte aggregation is an indicator of cardiovascular risk, which is Determining why cells react physically in the manner they do and how cells influenced by plasma fibrinogen concentration. Fibrinogen levels are transform forces into biochemical responses are important for understanding elevated during cardiovascular diseases. Our main goals were to understand cytoskeletal structure and organization, immunity and disease states like how fibrinogen-erythrocyte binding influences erythrocyte aggregation and cancer. The atomic force microscope (AFM) is a tool that can mechanically how it constitutes a cardiovascular risk factor in essential arterial hyperten- stimulate a cell and measure forces on the molecular and cellular scales. Us- sion (EAH) and chronic heart failure (CHF). Fibrinogen-erythrocyte and ing the AFM in conjunction with fluorescence microscopy enables force to erythrocyte-erythrocyte adhesion measurements were conducted by atomic be correlated with specific parts of a cell. A typical optical microscope, force microscopy (AFM)-based force spectroscopy. Upon increasing fibrin- however, does not allow cells to be imaged in a plane parallel to the direc- ogen concentration, there was an increase in the work and force necessary tion of the AFM’s applied force. To solve this problem, our lab developed a for cell-cell detachment, both for healthy donors and EAH patients. Never- technique to image cells from the side called Pathway Rotated Imaging for theless, higher values were obtained for the EAH patients at each fibrinogen Sideways Microscopy with Light Sheet (PRISM-LS). In addition, we have concentration. Fibrinogen-erythrocyte (un)binding forces were higher in added several imaging techniques to the system to complement AFM EAH and CHF patients, when compared with the control group, despite a studies, including software controlled positioning of multicolor light lower binding frequency. Ischemic CHF patients showed increased binding sheet, point and broad illumination, among others. Applications for the forces compared to non-ischemic patients. Differences between CHF pa- system include studying nuclear mechanics, phagocytosis, and filopodia tients and healthy donors, in terms of erythrocyte elasticity (Young’s dynamics. modulus) and AFM tip penetration depth into the cells were also assessed.

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Erythrocytes from non-ischemic patients presented higher stiffness than of IBPs to the basal plane differs from one type of IBP to another. In another those from the other two groups. Nevertheless, a significantly higher cell work we studied the IBP from an Antarctic marine bacterium, Marinomonas penetration depth at the same applied force was observed for ischemic heart primoryensis. We showed that this IBP has a different function than any failure patients. Erythrocyte deformability (assessed as elongation index) re- other known IBP, which is as an adhesin that helps the bacterium stick to sults show that heart failure patients presented higher erythrocyte deform- ice crystals. Antibodies raised against the ice-binding part of the protein ability than the control group at lower shear stresses, and lower abolished the ability of the bacteria to bind ice, while antibodies raised deformability at higher shear stresses. This indicates that patients’ erythro- against other parts of the protein failed to do so. This principle of ‘‘knocking cytes are more deformable than those from healthy donors in blood vessels out’’ the adhesion function can theoretically be used to prevent formation with larger internal diameters; however, in smaller-diameter vessels the of biofilms, thereby combating infections. This is currently the only opposite trend exists. Finally, a 12-month clinical follow-up shows that example known of an adhesin that binds to ice. Funding: ERC, ISF, CHF patients with higher fibrinogen-erythrocyte binding forces, probed by CHIR, NSERC. AFM at the beginning of the assessment, had a significantly higher probabil- References: ity of being hospitalized due to cardiovascular complications on the subse- 1) Bar-Dolev M., Braslavsky I. and Davies P.L., Annl. Rev. Biochemistry quent year. Our results show that AFM can be a promising tool for clinical 2015. prognosis, pinpointing those patients with increased risk for cardiovascular 2) Bar-Dolev M. et al. R. Soc. Interface 2016. diseases. 3) Haleva L., Celik Y. and Bar-Dolev M. et al. Biophysical Journal 2016. Guedes et al. (2016) Nature Nanotechnology, 11:687-92. 4) Lewis J. K., Bischof J. C., Braslavsky I., et al. Cryobiology 2016.

Biosurfaces 2899-Pos Board B506 Aquaporin Biomimetic Membrane for Energy Conservative Water 2897-Pos Board B504 Desalination Graphene-Oxide Gel as Biomimetic Antimicrobial Cloak Ahmed Fuwad1, Hyunil Ryu2, Tae-Joon Jeon2, Sun Min Kim1. 1 Valentina Palmieri1,2, Massimiliano Papi1,2, Francesca Bugli3, Mechanical Engineering, Inha University, Incheon, Korea, Republic of, 2 Maurizio Sanguinetti4, Luca Angelani5, Marco De Spirito1, Claudio Conti5. Biological Engineering, Inha University, Incheon, Korea, Republic of. 1Physics Institute, Universita` Cattolica del Sacro Cuore, Roma, Italy, One of the important issues affecting people around the globe is inadequate 2Institute for Complex Systems, National Research Council (ISC-CNR), access to clean and safe water. Problems with water are expected to increase Rome, Italy, 3Universita` Cattolica del Sacro Cuore, Roma, Italy, in coming decades at an alarming rate, causing water scarcity around the 4Microbiology Institute, Universita` Cattolica del Sacro Cuore, Roma, Italy, world even in water rich areas. To address the problems a novel approach 5Institute for Complex Systems, National Research Council (ISC-CNR), is needed for water purification at low energy consumption, high purity Roma, Italy. and at the same time the chemical treatment and waste production should Antibacterial surfaces have an enormous economic and social impact on the be minimized. Here we present a nature inspired biomimetic water filter con- worldwide technological fight against diseases. However, bacteria develop taining aquaporin protein. Aquaporin is the most efficient water channel in resistance and coatings are often not uniform and not stable in time. The nature due to its high water selectivity and permeability. In spite of its challenge is finding an antibacterial coating that is biocompatible, cost- merits, the manufactural difficulties of aquaporin embedded membranes as effective, not toxic, and spreadable over large and irregular surfaces. well as their fragility preclude their industrial applications. To overcome Here we demonstrate an antibacterial cloak by laser printing of graphene these problems, by crosslinking lipid molecules, we propose a novel method oxide hydrogels mimicking the Cancer Pagurus carapace. We observe up that stabilizes aquaporin embedded membranes using electrostatic interac- to 90% reduction of bacteria cells. This cloak exploits natural surface pat- tions and alternative coating methods. Furthermore, surface morphological terns evolved to resist to microorganisms infection, and the antimicrobial analysis show that the proteoliposomes were successfully immobilized on efficacy of graphene oxide. Cell integrity analysis by scanning electron mi- the substrate and retain their structure. The performance of this aquaporin croscopy and nucleic acids release show bacteriostatic and bactericidal ef- biomimetic membrane was tested for a forward osmosis (FO) based water fect. Nucleic acids release demonstrates microorganism cutting, and purification system. microscopy reveals cells wrapped by the laser treated gel. A theoretical active matter model confirms our findings. The employment of biomimetic 2900-Pos Board B507 graphene oxide gels opens unique possibilities to decrease infections in Assembling Functional Proteins on Gold-Glass Surfaces biomedical applications and chirurgical equipment; our antibiotic-free Timothy Robson1, Deepan Shah2, Luke Clifton3, Becky Welbourn3, approach, based on the geometric reduction of microbial adhesion and the Jeremy Lakey1. mechanical action of Graphene Oxide sheets, less likely induces bacterial 1Newcastle University, Newcastle, United Kingdom, 2Orla Protein resistance. Technologies, Newcastle, United Kingdom, 3ISIS Pulsed Neutron and Muon Source, Harwell, United Kingdom. 2898-Pos Board B505 The generation of functional surfaces using biological molecules is of great in- Ice-Binding Proteins - Not Only for Ice Growth Control terest for cell culture and diagnostic applications. Control of protein orientation Maya Bar-Dolev1, Shuaiqi Guo2, Lotem Haleva1, Yeliz Celik3, is difficult but advantageous in many cases to retain activity. The results from Peter L. Davies2, Ido Braslavsky1,3. simple adsorption methods can be extremely variable and often result in dena- 1Institute of Food Science and Nutrition, The Hebrew University of tured or inactive proteins. The industrial sponsor of this work, Orla Protein Jerusalem, Rehovot, Israel, 2Department of Biomedical and Molecular Technologies, utilise gold-thiol chemistry and the self-assembling nature of a Sciences, Queen’s University, Kingston, ON, Canada, 3Physics and beta-barrel outer membrane protein (OMP) from E. coli to create reproducible Astronomy, Ohio University, Athens, OH, USA. protein arrays on gold surfaces. The E. coli OMP is a robust protein scaffold Ice-binding proteins (IBPs) evolved in organisms living in cold ecosystems that can be engineered to display a wide variety of functional motifs or domains as a protective measure against freezing stresses. Some IBPs prevent their such as, cell adhesion proteins, single chain antibodies or antigens. Sputter hosts from freezing mainly by depression of the freezing point of the body coating is often used to achieve thin and even gold surfaces suitable for most fluids below the melting point. Others help the organism avoid the damage uses but the method requires expensive equipment and specialist training and of freezing by inhibiting ice recrystallization. These functions make IBPs the layers are poorly transparent, limiting their use in cell biology and micro- excellent candidates for ice growth control, such as in cryopreservation of scopy. This has led our group to develop a simple, flexible method for depos- food and tissues. Organ banking, still in its infancy, is becoming a priority iting high densities of gold nanoparticles on to glass substrates. These are cheap field for funding in order to revolutionize transplantation medicine and em- to produce using inexpensive equipment and all steps are carried out at room bryo preservation. To allow the use of IBPs in these applications, more basic temperature without harsh solvents. Moving to a gold-glass platform enables knowledge about their function is needed. Recently we developed a new the more flexible use of microscopy and spectroscopic techniques in cell culture tool, called the mini cold finger, which allows us to grow a single ice crystal and biosensing applications. Characterisation of the new surfaces by electron in a microfluidic channel and inspect the interactions of various IBPs with it. microscopy, fluorescence microscopy and neutron reflection reveals well- By injecting solutions of fluorescently-labeled IBPs into the device with an ordered fields of nanoparticles coated in a functional protein layer. Further ice crystal present, we studied the ability of various IBPs to bind the basal analysis of functionalised nanoparticles in solution has been carried out using plane of ice. Basal plane affinity is a crucial factor that directly affects the UV-Visible spectroscopy, dynamic light scattering, small angle neutron specific activity of IBPs. We further showed that the dynamics of binding scattering and electron microscopy. This shows several proteins bound to the

BPJ 7920_7924 590a Wednesday, February 15, 2017 nanoparticle surface through a gold-thiol bond. Functional motifs are displayed be seen in the distance distribution function obtained by small-angle X-ray away from the surface and retain the ability to bind specifically to antigens in scattering (SAXS). This functionality is transferred to interfacial structures the solution. These new surface topologies open new avenues for applications in this project. Aqueous-solid interfaces are modified by covalent binding of self assembling protein layers. of PEG chains to the solid phase, which are cross-linked with a CaM mutant. The main achievements of this project are the controlled build-up of such 2901-Pos Board B508 interfacial layers and the characterization of the CaM functionality in these layers. Using X-ray reflectometry, we have observed that a PEG-CaM layer Wettability and Carboxyl Functionalization of PLA Films Enhancing by 2þ an Atmospheric Pressure Dielectric Barrier Discharge System is growing in thickness upon rinsing with a TFP/Ca solution and 1 1 1 decreasing in thickness upon rinsing with an EGTA solution, which binds Stephanie Vanslambrouck , Ivan Rodriguez Duran , Pascale Chevallier , 2þ Corinne Hoesli2, Gaetan Laroche1. Ca from CaM. Furthermore, pressure has been applied to probe the 1Laboratoire d’inge´nierie de surface, Laval University, Quebec, QC, Canada, strength of interactions between CaM and TFP in solution and in the 2Stem Cell Bioprocessing Laboratory, McGill University, Montreal, QC, PEG-CaM layer, which are expected to be mainly electrostatic in nature. Canada. By measuring the fluorescence resonance energy transfer (FRET), we see Poly(lactic acid) (PLA), a biodegradable polymer, is one of the most widely a pressure-induced transition of collapsed CaM-TFP to open CaM-TFP used polyesters in the pharmaceutical and biomedical fields due to its me- free in solution. However, this transition cannot be observed in the PEG- chanical characteristics, good biocompatibility, and excellent chemical and CaM-TFP layer suggesting a stabilization of the collapsed state. Overall, thermal stabilities. Despite these excellent properties, PLA suffers from novel biochemically responsive interfacial structures are formed that can some disadvantages, such as strong hydrophobicity, poor cell affinity, and be switched reversibly under ambient conditions, in contrast to well- absence of suitable functional groups for covalent immobilization with known pH or temperature sensitive surface modifications. bioactive molecules. Several methods, including mechanical, chemical and plasma treatments, have been investigated to modify polymer surfaces for Biomaterials tailoring the surface properties of PLA while preserving its bulk characteris- tics. In the present study, we describe an atmospheric pressure dielectric bar- 2904-Pos Board B511 rier discharge plasma treatment in a N2/N2O atmosphere in order to improve Multifunctional Drug Delivery System based on Poly-N-Vinylpyrrolydone the wettability and the carboxyl functionalization of PLA films. This func- Block Copolymer Micelles tionalization method was compared with the common method of PLA Camilla L. Andersen1, Sven B. Romme1, Peter Fojan1, Cristian P. Pennisi2, surface modification, which consists in hydrolysis in alkali solutions. Anna L. Luss3, Pavel P. Kulikov3, Mikhail I. Shtilman3, Leonid Gurevich1. 1 Different concentrations of N2O gas and different treatment time were Department of Physics and Nanotechnology, Aalborg University, Aalborg, used for the plasma treatment. The chemical and physical changes of the Denmark, 2Department of Health Science and Technology, Aalborg treated surfaces were observed by contact angle measurements, X-ray photo- University, Aalborg, Denmark, 3D. Mendeleyev University of Chemical electron spectroscopy and scanning electron microscopy (SEM). Moreover, Technology of Russia, Moscow, Russian Federation. the amount of carboxyl functional groups created on the PLA films was eval- The traditional approach of systemically administering therapeutic agents to uated using the toluidine blue O method. The results showed that the wetta- a patient suffers serious drawbacks, which became particularly apparent in bility of PLA films and the amount of carboxyl functions present on the cancer chemotherapy. Many of these issues, including dose-limiting toxicity, polymer surface significantly increased after the plasma treatments and low bioavailability and short circulation time, can be successfully addressed were very similar than after alkali hydrolysis. Furthermore, SEM images re- by polymeric drug delivery systems based on amphiphilic block copolymers vealed that almost no damages were observed on the PLA films after plasma comprising hydrophobic and hydrophilic segments. Upon drug loading, the treatment in contrast to the films functionalized in alkali solutions. polymer forms micelles with a drug-loaded hydrophobic core (formed by the hydrophobic segments of the polymer) and a hydrophilic corona (formed 2902-Pos Board B509 by the hydrophilic segments). In this report we present a versatile system Effect of High Pressure on Protein Binding to Poly(Acrylic Acid) Brushes based on a biodegradable amphiphilic PVP-acrylic acid copolymer that could Artem Levin, Claus Czeslik. be applied for drug delivery and diagnostic purposes. Using from Department of Chemistry and Chemical Biology, TU Dortmund University, Curcuma longa as a model drug, we demonstrate that sub-100 nm micelles Dortmund, Germany. loaded with a hydrophobic drug can be produced and remain stable under Polyelectrolyte brushes can provide a native-like environment for the immo- physiological conditions. In vitro studies carried out on primary fibroblasts bilization of proteins at aqueous-solid interfaces. In particular, poly(acrylic and a glioblastoma cell line (U87) show that cells readily uptake the obtained acid) (PAA) brushes have been shown to preserve the secondary structure micelles via non-receptor mediated endocytosis. The observed micellar de- of proteins and maintain enzyme activity to a high degree. Moreover, the de- livery is significantly more efficient compared to the cellular absorption of gree of protein adsorption at a PAA brush can be controlled by the ionic the free drug. We show that curcumin-loaded micelles exhibit cytotoxicity strength of the protein solution. Here, we investigate the effect of pressure against the tested cell lines, while empty micelles do not. Due to the presence on the degree of protein binding at a PAA brush. Because this binding is of free carboxylic acid groups we also show that a covalent attachment of largely electrostatic in nature, pressure can be expected to be a further cancer-cell targeting moieties to the polymer can be achieved in order to parameter that controls the protein affinity of a PAA brush. It is well known obtain selective drug delivery. Furthermore, we demonstrate that the PVP- that pressure will break ionic bonds due to electrostriction, generally acrylic acid copolymer is able to chelate Gd(III) ions within the micellar observed at about 500 - 2000 bar, because the hydrating water around the corona making the system suitable for MRI based imaging, and hence emerging charges has a smaller specific volume. Indeed, when positively creating a theranostic platform comprising therapeutic and diagnostic charged alpha-chymotrypsin is adsorbed at a PAA brush, there is a capabilities. pressure-induced partial desorption of the protein. Conversely, when nega- tively charged calmodulin is adsorbed at a PAA brush, there is apparently 2905-Pos Board B512 a pressure-induced enhancement of the degree of protein adsorption. From Structural Investigations of Supercontracted Spider Dragline Silk pH-dependent measurements, a significant role of electrostatic interactions Justine Dionne, Thierry Lefe`vre, Philippe Bilodeau, Miche`le Auger. for this pressure effect is suggested. Overall, the mechanism of protein bind- Chemistry, Universite´ Laval, Que´bec, QC, Canada. ing to a PAA brush is discussed in terms of the new pressure experiments Spider dragline silk is a semicrystalline polymer of great interest in materials presented here. science due to its unique mechanical properties and biocompatibility. Spun from the major ampullate glands, this type of silk possesses an exceptional 2903-Pos Board B510 combination of elasticity, strength and toughness. At the molecular level, Design of Bioresponsive Interfaces using Conformational Transitions of spider silk is composed of fibrous proteins organized into crystalline nanodo- Calmodulin mains embedded in an amorphous matrix. In the presence of liquid water or Suleyman€ Cinar, Claus Czeslik. high humidity, the amorphous phase is plasticized and its hydrogen bonding Department of Chemistry and Chemical Biology, TU Dortmund University, network disrupted, which results in the shrinking of the fiber up to 50% of its Dortmund, Germany. initial length. This phenomenon, known as supercontraction, is triggered by Some proteins undergo major conformational changes upon ligand binding, the entropic folding of the polypeptide chains resulting from the water- such as calmodulin (CaM). When CaM binds trifluoperazine (TFP), there is a induced increase in chain mobility. However, disagreements exist in the marked transition of CaM from an open to a collapsed conformation, as can literature as to whether a change in the secondary structure of the protein

BPJ 7920_7924 Wednesday, February 15, 2017 591a occurs or not during supercontraction. Furthermore, although it has already Electrospinning is a technology for electrostatic formation of polymer nano- been established that supercontraction induces a disorientation of the molec- and micro- fibers. We used this method to prepare blend scaffold from a biode- ular units, this effect has not been quantified yet. Therefore, we will inves- gradable polymer, poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV), and a tigate and quantify, for different species, the conformational and model globular protein, bovine serum albumin (BSA). These polymers were orientational variations of the silk proteins induced by supercontraction. mixed at 70:30 ratio in a common solvent (1,1,1,3,3,3-hexafluoroisopropanol) To achieve our goals, we will use Raman spectromicroscopy, a technique and electrospun. Blend solution-cast film and single-component electrospun that as long been proven to be a useful tool to probe silk. Moreover, the ef- films were used as control ones. We analyzed the film properties: morphology, fects of drawing speed on the magnitude of supercontraction will also be dissolution kinetics, hydrophilicity, cytocompatibility. In contrast to the ordi- examined. nary single-component electrospun films, the blend films consisted of several types of fibers: PHBV round fibers, BSA flat ribbons and the fine network. 2906-Pos Board B513 When the films were immersed in water, BSA dissolution rate from the blend Extracting Information on Molecular Interactions using Data from Binary was lower than from the solution-cast film. Vero cell viability and hydrophilic- Mixtures ity was higher on the electrospun films than on the solution-cast ones. The ob- Simran S. Gurdasani, Ryan Z. Lybarger, Horia I. Petrache. tained electrospun blend materials introduces a new class of polyester-protein Physics, Indiana University Purdue University Indianapolis, Indianapolis, IN, blends. It can be used in tissue engineering. USA. One general question in chemical and biological physics is how mixed molec- 2909-Pos Board B516 ular species behave compared to single-component systems. Here we investi- Role of Charge and Ligand-Receptor Binding in Specific Targeting of gate how binary solution mixtures affect the interaction between lipid Peptide-Tagged Cationic Liposome Nanoparticles for Gene Delivery membranes. Experimentally, by X-ray scattering, it is observed that the equilib- Emily Wonder1, Lorena Simo´n-Gracia2, Pablo Scodeller2,3, rium spacing in multilamellar lipid vesicles although highly regular is very sen- Ramsey Majzoub1,4, Venkata Ramana Kotamraju3, Kai K. Ewert1, sitive to the chemical composition of aqueous solutions. In order to understand Tambet Teesalu2,3, Cyrus Safinya1. the role of various ionic species on membrane equilibrium spacings, one can 1University of California, Santa Barbara, Santa Barbara, CA, USA, consider ‘‘competition’’ measurements in which the fraction of ionic compo- 2University of Tartu, Tartu, Estonia, 3Sanford-Burnham-Prebys Medical nents is varied at constant total concentration. A complete description of Discovery Institute, La Jolla, CA, USA, 4Janssen Research & Development such measurements often requires a complicated theory with a large number LLC, Spring House, PA, USA. of parameters. Here we show that experimental data curves can be parameter- Cationic liposomes (CLs) are a common synthetic carrier of DNA and short- ized with simple power functions using a mixing parameter and two mixing ex- interfering RNA for gene delivery and silencing, with clinical trials ongoing. ponents. These phenomenological parameters are quite general and are a good Optimization of transfection efficiency requires understanding of the interac- measure of the cooperativity or competition of ionic or molecular species in bi- tions between cellular membranes and CL–nucleic acid nanoparticles (CL- nary mixtures. NA NPs), which affect NP binding, uptake, endocytic trafficking, and endoso- mal escape. PEGylation (PEG: polyethylene glycol) sterically stabilizes CL- 2907-Pos Board B514 NA NPs, and the attachment of targeting ligands (e.g. peptides) to the distal Principles and Applications of Functional Lipidic Biomaterials in Molecu- end of the PEG chains may enable targeted gene delivery (Majzoub et al., Bio- lar Recognition, Membrane Protein Crystallization and Drug Delivery materials 2014, 35, 4996; Majzoub et al., Biochim Biophys Acta 2015, 1848, Livia Salvati Manni, Simone Aleandri, Ehud M. Landau. 1308). We used linear and cyclic peptides containing variations of the RGD Department of Chemistry, University of Zurich, Zurich, Switzerland. and C-end rule motifs (e.g. iRGD and RPARPAR) to target integrin and/or We have designed and synthesized a library of lipids with novel functionalities neuropilin-1 receptors, respectively (Ewert et al., Bioorg Med Chem Let in order to correlate lipid molecular structure with the ensuing nanomaterial dy- 2016, 26, 1618;Teesalu et al., Front Oncol 2013, 3, 216; Simo´n-Gracia et al., namics, stability and phase behavior. The designed lipids were assembled as Biomaterials 2016, 104, 247). within lipidic mesophases and their dispersions, forming functional biomate- To achieve specific targeting, one must decouple ligand effects from the non- rials that were employed in areas ranging from molecular recognition, seques- specific electrostatic attraction of our cationic liposome NPs. To this end, we tration and enrichment of nucleic acids to biosensing. An exciting new class of prepared NPs of varied membrane charge densities and charge ratios (resulting biomaterials that are based on cyclopropanated lipids was developed. These in NP surface charges that range from highly positive to slightly negative), form stable lipidic cubic phases (LCPs) at low temperature, opening the way tagged with different ligands at a variety of ligand densities. We evaluated to conduct biophysical and biochemical investigations on temperature sensitive the cell binding, uptake, endocytic trafficking, and transfection efficiency of bio-macromolecules, specifically membrane proteins. Furthermore, pH- our NPs using flow cytometry, colocalization with Rab proteins (markers of sensitive lipidic matrices for hydrophilic as well as hydrophobic drug incorpo- membrane-bound organelles) (Majzoub, Wonder et al., J Phys Chem B 2016, ration and release were designed, as well as stimuli-responsive lipids that can 120, 6439), and gene expression measurements. Preliminary in vivo experi- be incorporated into biomaterials. Efficient pH- and light-induced binding, ments in a mouse model of peritoneal carcinomatosis demonstrated tumor hom- release and sequestration of hydrophilic dyes were demonstrated. Significantly, ing by iRGD-tagged CL–DNA NPs. these processes could be activated sequentially, thereby achieving high degree of temporal and dosage control. The scope of lipidic materials for drug delivery was expanded to azobenzene-containing hexagonal phases, in which ‘‘on de- 2910-Pos Board B517 mand’’ single-step as well as sequential light-triggered release and retention Structured DNA Nanoparticles for Spatially Controlled Antigen of embedded dye molecules were demonstrated. Finally, cubosomes were sta- Presentation bilized and functionalized with a novel, designed biotin-based block copol- Re´mi Veneziano, Mark Bathe. ymer, resulting in dispersed biomaterials that were applied against the human Biological Engineering, Massachusetts Institute of Technology, Cambridge, adenocarcinoma cell line HeLa. These cubosomes are able to simultaneously MA, USA. transport paclitaxel, a potent anti-cancer drug, and a hydrophobic fluorescent Synthetic nanoparticles (NPs) can be designed to organize and display antigens dye in active targeting of cancer cells. Such biotinylated cubosomes are poten- to mimic natural protein architectures such as viruses and toxins. These syn- tially applicable in diagnosis, drug delivery and monitoring of therapeutic thetic lipid- and polymer-based NPs have a range of applications in biophysical response for active targeting versus cancer cells. science, including the controlled display of multivalent antigen assemblies to probe immune cell function. However, existing NPs used for antigen presenta- 2908-Pos Board B515 tion do not allow for precise control over the 3D structural organization and Electrospun Biodegradable Scaffold Made of Poly(Hydroxybutyrate- stoichiometry of surface-displayed molecules. The recent emergence of scaf- Co-Hydroxyvalerate) & Bovine Serum Albumin folded DNA origami NPs represents a promising tool to assemble complex mo- Elizaveta Pavlova1,2, Dmitry Bagrov2,3, Dmitry Shchelokov3, lecular architectures consisting of numerous antigen subtypes organized in 3D Anton Bonartsev3,4, Irina Zharkova3, Dmitry Klinov2. with nanometer-scale structural fidelity that can be chosen to mimic viruses and 1Moscow Institute of Physics and Technology, Dolgoprudny, Russian toxins. For example, DNA-NPs can be used to help investigate the relative roles Federation, 2Federal Research Clinical Center of Physical-Chemical of 3D antigen spacing to stimulate B-cell, T-cell, or dendritic cell activation. Medicine of the Federal Medical and Biological Agency of Russia, Moscow, Here, using a versatile design and synthesis procedure for programming arbi- Russian Federation, 3Lomonosov Moscow State University, Moscow, trary DNA-NPs on the 5-100 nanometer-scale, we control peptide and protein Russian Federation, 4A.N.Bach Institute of Biochemistry RAS, Moscow, antigen presentation for immune cell assays. DNA-NP-protein assembly struc- Russian Federation. ture is characterized using a combination of TEM and cryoEM for up to 60 to

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120 copies, and binding activity of the synthetic supramolecular complex is 2913-Pos Board B520 characterized using surface plasmon resonance. Supramolecular complex sta- Homogalacturonans Illuminate the Role of Counterion Condensation in bility is evaluated in vitro under physiological conditions using a real-time Polyelectrolyte Transport FRET-based degradation assay, with DNA sequence design and modifications Amir H. Irani1, Jessie L. Owen1, Davide Mercadante2, performed to increase complex stability. Finally, we further develop an aPCR- Martin A.K. Williams1. based approach to easily produce large quantities of single-stranded DNA 1Institute of Fundamental Sciences, Massey University, Palmerston North, scaffold required to assemble the NPs. Taken together, synthetic structured New Zealand, 2Heidelberger Institut for Theoretische Studien, Heidelberg, DNA-NPs offer a new molecular platform for the controlled synthesis of com- Germany. plex supramolecular antigen assemblies with full control over stoichiometry Homogalacturonans (HGs) are polysaccharide co-polymers of galacturonic and 3D presentation for the investigation of immune cell response. acid and its methyl-esterified counterpart. The inter- and intra-molecular dis- tributions of the methylesterifed residues are vital behaviour-determining 2911-Pos Board B518 characteristics of a sample’s polymeric fine structure and much experimental Plasma Membrane Vesicles with Engineered Transmembrane Protein effort has been directed to their measurement. While many techniques are Ligands for High-Affinity Cell Targeting able to measure the sample-averaged degree of methyl-esterification (DM), Chi Zhao1, David J. Busch1, Conor P. Vershel1, Hisham A. Ali1, the measurement of inter- and intra-molecular charge distributions are chal- Natalie C. Miroballi1, Jeanne C. Stachowiak1,2. lenging. Capillary electrophoresis has been suggested as a useful technique 1Department of Biomedical Engineering, University of Texas at Austin, for monitoring the intermolecular DM distribution of homogalacturonan Austin, TX, USA, 2Institute for Cellular and Molecular Biology, University samples, by measuring the distribution of electrophoretic mobilities amongst of Texas at Austin, Austin, TX, USA. chains. For samples where the distance between charges is always greater Nanoparticle-based drug delivery systems have the potential to target than the Bjerrium length a simple relationship between chain-averaged diseased cells on the basis of their receptor expression profiles. Toward charge density and electrophoretic mobility is indeed obtained. However, this goal, synthetic materials conjugated with antibodies against specific when either: i) the DM of HGs with randomly patterned charges becomes cellular receptors have been shown to concentrate in solid tumors. However, low enough, or ii) the intramolecular patterning of charged groups becomes chemical conjugation reactions are difficult to control, frequently limiting blockwise enough; counterion condensation is expected to play an important ligand uniformity and complexity. In contrast, the surfaces of living cells role in the electrical transport of these polyelectrolytes. That the resultant are decorated with highly uniform populations of sophisticated transmem- mobilities of HGs in this regime do not scale simply with DM has been brane proteins. To harness cellular capabilities, here we demonstrate that well documented experimentally, but a successful quantitative calculation plasma membrane vesicles (PMVs) derived from engineered donor cells of the expected effects has been lacking. Here, molecular dynamics (MD) can display multifunctional transmembrane proteins that precisely target simulations are used to calculate the electrophoretic mobilities of HGs that cells on the basis of the expression level of a specific cellular receptor. These have different amounts and distributions of charges placed along the back- multifunctional targeting proteins incorporate (i) a protein ligand, (ii) an bone. The simulations are shown to capture experimental results remarkably intrinsically disordered protein spacer to make the ligand sterically acces- well: even for blocky or low-DM samples that possess high local charge sible, and (iii) a fluorescent protein domain that enables quantification of densities. The simulations elucidate the role of charge patterning and coun- the ligand density on the PMV surface. PMVs displaying protein ligands terion condensation in the determination of the electrophoretic mobility of with affinity for epidermal growth factor receptor (EGFR) bound at polyelectrolytes, and more specifically provide a general mechanism for increasing concentrations to breast cancer cells that expressed increasing mapping between homogalacturonan fine structure and measured mobility levels of EGFR. As an example of the generality of this approach, we devel- distributions. oped PMVs displaying a single-domain antibody against green fluorescence protein (eGFP), which bound to cells expressing eGFP-tagged receptors with 2914-Pos Board B521 a selectivity of z50:1. To further demonstrate the versatility of PMVs, we An Electric Eel-Inspired Origami-Enabled Battery that Generates 100 V engineered targeting proteins with serial repeats of the ligand domain to Thomas B.H. Schroeder1,2, Anirvan J. Guha2, Aaron Lamoureux3, mimic the high-affinity binding mechanisms commonly observed in multiva- Jerry Yang4, Max Shtein3, Michael Mayer2. lent biochemical interactions. Collectively, these results demonstrate the 1Chemical Engineering, University of Michigan, Ann Arbor, MI, USA, flexibility and high selectivity of PMVs as a novel biomaterial for cell target- 2Adolphe Merkle Institute, Fribourg, Switzerland, 3Materials Science ing. Moving forward, the use of engineered donor cells to produce PMVs and Engineering, University of Michigan, Ann Arbor, MI, USA, 4Chemistry will enable the development of true multi-functional materials with diverse and Biochemistry, University of California San Diego, San Diego, CA, applications from drug delivery to tissue engineering. USA. Electrogenic fish such as the electric eel and the torpedo ray generate electric 2912-Pos Board B519 discharges of up to 1 kW in order to sense and incapacitate prey. Each electro- Molecular Modeling of Liquid Crystal/Phospholipid Interface as a Label- genic fish contains an array of polarizable cells called the electric organ that can Free Biosensor comprise up to 80% of the organism’s body; these organs have evolved inde- Donya Ohadi, Mark J. Uline. pendently at least six times in natural history. The electric organ’s discharges Chemical Engineering, University of South Carolina, Columbia, SC, USA. are short, on the timescale of single milliseconds; they arise when a complex Ligand-receptor binding has traditionally been monitored by techniques that þ neural architecture stimulates the opening of Na channels on the innervated either require a label to be attached to the analyte or complex optical side of each electrocyte cell simultaneously, leading to transcellular potentials methods to be detected. This has motivated the research for label-free assays that add across a series of cells and an ionic current that scales with the width of to detect interfacial biological interactions without the use of special optical the organ. devices under controlled conditions. Phospholipid monolayers coupled with Here we present a hydrogel-based battery system that mimics the eel’s simul- thermotropic liquid crystals as a responsive support can be used as a label- taneous generation of many small potentials via electrochemical ion gradients free biosensor. The hydrophobic acyl chains of the lipids contact the hydro- across semipermeable hydrogel membranes. This battery creates an ionically phobic liquid crystal surface and the polar lipid head groups are presented to conductive pathway by bringing large arrays of printed hydrogel units into specific binding events involving proteins, enzymatic reactions, viruses, physical contact all at once using stacking and folding geometries with a single bacteria, hybridization of DNA, etc. Because of the long-range orientational degree of mechanical freedom. Printing large and complex gel patterns made it order and the optical anisotropy of liquid crystal molecules, these chemical possible to generate voltages exceeding 100 V. Miura-ori folding of 2-D arrays and biomolecular binding events can be transduced into amplified optical provides self-registered packing into small form factors with improved power signals that can be detected even with the naked eye. The mechanism by characteristics. which lipid anchoring effects the liquid crystal surface remains to be eluci- dated. Therefore, a molecular study of the phospholipid/liquid crystal inter- face to determine the mechanisms by which binding events are transmitted 2915-Pos Board B522 from the analytes to the liquid crystal layer is crucial for design of novel Towards Simulating Large-Scale Self-Assembly of Proteins under Flow sensors. Our model allows us to fully characterize the organizations of phos- Ana Maria Herrera1, Anil K. Dasanna2, Frauke Gr€ater1,3. pholipids within the monolayer and also the orientations of liquid crystals 1Molecular Biomechanics Group (MBM), Heidelberg Institute for in the bulk. Through this molecular model we mimic the experimental Theoretical Studies, Heidelberg, Germany, 2BioQuant, Heidelberg systems by specifying interaction energies and their positional and angular University, Heidelberg, Germany, 3Interdisciplinary Center for Scientific dependences. Computing, Heidelberg University, Heidelberg, Germany.

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Under shear flow, many proteins show an increased tendency to aggregate. Spi- agent. These ultra-small nanoclusters are an order of magnitude smaller than der silk is a prime example, as the microscopic structure and mechanical per- typical nanoparticles, which are generally R10’s of nanometers in diameter. formance of silk fibers depend on the arrangement of the crystalline and Because the antimicrobial activity of silver nanoparticles (AgNPs) is known amorphous phases therein. It is thought that flow is needed for the fiber assem- to increase with decreasing particle size, AgDNAs have the potential to outper- bly, since the stretching and alignment of the protein chains would enhance the form larger commercial nanoparticles. We have developed a method to purify formation of the crystalline regions. However, molecular mechanisms of flow- AgDNAs of known size and concentration, allowing us to demonstrate the induced protein aggregation have remained elusive. broad-spectrum efficacy of a well-characterized AgDNA cluster against several To study the effect of the flow on spider silk assembly we combined Multi- Gram (þ) and Gram (-) bacteria. Furthermore, by using purified AgDNAs we Particle-Collision-Dynamics (MPCD), a mesoscale simulation method, and are able to quantifiably compare their performance to Agþ ions and the smallest atomistic Molecular Dynamics (MD) simulations. The description of the commercially available AgNPs. interaction potentials for the coarse-grained silk peptides is based on the Self Organized Polymer Model with Side-Chains (SOP-SC). Under the 2918-Pos Board B525 assumption that crystal formation drives assembly, we defined attractive C60 Fullerenes as Contrast Agents - Structural, Spectroscopic and Nano- native interactions exclusively within the well-conserved poly-alanine re- toxicity Studies peats of the sequence, which are known to form the beta-sheet crystalline Augustyn Molinski1, Jakub Zareba1, Justyna Izykowska1,2, phases. Michalina Skupin1, Weronika Andrzejewska1, Stefan Jurga1,2, We performed simulations of the fragments of the repetitive region of silk pep- Maciej Kozak1,3. tides under elongational and shear flow conditions on the atomistic and meso- 1Department of Macromolecular Physics, Adam Mickiewicz University in scopic scale. The dependence of the extension of a single peptide on the drag Poznan, Poznan, Poland, 2NanoBioMedical Center, Adam Mickiewicz force closely follows a worm-like chain behavior on both scales, validating University, Poznan, Poland, 3Joint Laboratory for SAXS Studies, Adam our computational model. To analyze the first steps of the assembly pathway, Mickiewicz University, Poznan, Poland. simulations of a set of silk peptides with different starting configurations and Recently, C60 fullerenes have been proposed as contrast agents for MRI varying flow conditions were performed. Our insights into the role of flow method [1]. Especially promising for in vitro and in vivo NMR imaging are on crystallization and assembly can serve as a guide for biological and synthetic their complexes with gadolinum (containing Gd3þ ions entrapped inside the material design. fullerene cage) known also as gadofullerenes [2]. The general problem of carbon nanomaterials is their toxicity towards living 2916-Pos Board B523 cells [3]. The aim of our study is to devise fullerene-based suspensions charac- Ratiometric Tension Probes for Mapping Receptor Forces and Clustering terized with low toxicity, which could later be developed into metallofullerene at Intermembrane Junctions contrast agents. We want to achieve that goal using C60 fullerenes, pluronics to Victor Pui-Yan Ma1, Yang Liu1, Lori Blanchfield2, Hanquan Su1, lower the toxicity and phosphatidylcholine derivatives (DMPC or DPPC) to Brian D. Evavold2, Khalid Salaita1,3. enhance biocompatibility of fullerenes and to stimulate the cellular absorption. 1Department of Chemistry, Emory University, Atlanta, GA, USA, In this work we would like to present results of toxicity studies of C60 fullerene 2Department of Microbiology and Immunology, Emory University, Atlanta, systems pluronic modified C60 fullerene systems towards HeLa cells, along GA, USA, 3Wallace H. Coulter Department of Biomedical Engineering, with results of structural analyses by AFM and SEM microscopies, FTIR and Georgia Institute of Technology and Emory University, Atlanta, GA, USA. SAXS. This study was supported by Ministry of Science and Higher Education Short-range communication between cells is required for the survival of multi- (Poland), within the project, Najlepsi z najlepszych!’’ (DEC - POWR.03.03.00- cellular organisms. One mechanism of receptor-mediated communication be- 00-P001/15). tween two cells employs surface displayed ligands and receptors that only [1] M. Mikawa, et al., Bioconjug. Chem. (2001), 12(4), 510-514. bind when two cells make physical contact with one another. Because of this [2] R.D. Bolskar, Nanomedicine. (2008), 3(2), 201-213. reason, it is likely that cellular forces are generated and transmitted via the [3] A. Magrez, et al. Nano Lett. (2006), 6(6), 1121-1125. ligand-receptor complexes at these intermembrane junctions. The goal of this work is to develop a nanoscale tool that allows one to probe mechanical forces 2919-Pos Board B526 experienced by ligand-receptor complexes within such a fluid intermembrane Scanning He Ion- and Nonlinear Optical Microscopy Combined with junction. To achieve this goal, we develop gold-nanoparticle based ratiometric Force Measurements for the Characterization of Spider Silk molecular force probes to directly map receptor tension, clustering and lateral Irina Iachina1, Jacek Fiutowski2, Serguei Chiriaev2, Per Lyngs Hansen1, transport within a model cell-cell junction in real time. These tension probes Adam Cohen Simonsen1, Jonathan R. Brewer1. consist of two fluorophores that define the relative molecular density of ligand 1University of Southern Denmark, Odense, Denmark, 2University of and the forces, thereby generating tension signals independent of receptor clus- Southern Denmark, Sønderborg, Denmark. tering. Using T-cell as a model system, we show the first direct evidence that Spider silk has many properties that may be of industrial use as the tensile the TCR-ligand complex experiences sustained pN forces within a fluid mem- strength of spider silk is comparable to that of alloy steel and the silk is about brane junction during activation. half as strong as for example Kevlar but has the advantage of being spun at room temperature. The aim of this work is to characterize spider silk from 2917-Pos Board B524 the orb web weaving spider Araneus diadematus by determining the nano- Few Atom Silver Clusters Display Broad Spectrum Antimicrobial Activity and microscopic structures within the silk and couple these to the macro- Danielle Schultz, Nancy Lin. scopic properties such as tensile strength, elasticity, and adhesion properties. NIST, Gaithersburg, MD, USA. In order to image the nanoscopic structures within the silk the technique According to the Center for Disease Control 2013 threat report, at least 2 Scanning He ion Microscopy is used. With this technique it is possible to million people suffer direct infection each year by antibiotic-resistant bacteria, resolve structures of about 1 nm without treating/altering the sample and such as Staphycoccus aureus (e.g., MRSA), resulting in 23,000 deaths in the to mill through the sample in order to visualize the internal structures of United States alone. Healthcare associated infections claim another 75,000 the silk fiber. The silk is then imaged under different tensions using this tech- lives each year, 60% of which are attributed to microbial colonization on im- nique. Using fluorescence microscopy the lipids and proteins of the fiber are planted medical devices. These staggering statistics reveal the urgency in visualized showing the overall structure of the fiber. Force and stress-strain developing new antibiotics and better antimicrobial materials and in re- measurements are performed while imaging the silk using multiphoton- and evaluating older, known antibacterial agents such as silver. widefield microscopy in order to monitor the structural changes of the fiber Few-atom silver clusters stabilized by DNA (AgDNAs), best known for their during stress. The results of these measurements are analyzed within the intriguing optical properties, show promise as a new class of silver antibacterial context of polymer physics.

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