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Symposium: Large Macromolecular Machines in detail. The NPC is surprisingly modular, consisting of only 30 of the nucleoporin family (Nups). These Nups assemble into sub-complexes the that form higher-order structures called spokes. Eight spokes assemble into even larger modules: coaxial outer and inner rings form a symmetric core scaf- 743-Symp fold at the heart of which are sturdy diagonal columns. The scaffold is con- Cryo-EM Structure of Human and New Insight into its Assem- nected to a membrane ring, a nuclear basket and cytoplasmic RNP export bly and platform. These discrete and relatively rigid modules are held together by flex- Kelly (Thi Hoang Duong) Nguyen1, Jane Tam1, Robert Alexander Wu2, ible connector cables, a combination imbuing the structure with both strength Basil J. Greber1, Eva Nogales3, Kathleen Collins1. and flexibility. The scaffold surrounds a central channel that is formed in 1 Molecular and Cell , University of California, Berkeley, Berkeley, part by nucleoporins termed FG Nups, from which multiple intrinsically disor- 2 3 CA, USA, Harvard University, Boston, MA, USA, Howard Hughes Med dered Phe-Gly (FG) repeat motifs project, contributing to an organized central Inst, University of California, Berkeley, Berkeley, CA, USA. density termed the central transporter. These FG motifs mediate selective nu- In eukaryotes, linear ends are capped with telomeres, which pro- cleocytoplasmic transport through specific interactions with nuclear transport tect the against end-joining and end-replication issues. Telo- factors. Taken together, this integrative structure allows us to rationalize the ar- meres are also progressively shortened because of incomplete replication by chitecture, dynamics, transport mechanism, and evolutionary origins of the replisome. Critically short telomere length leads to chromosomal degrada- the NPC. tion and apoptosis. To balance this loss, telomerase ribonucleoprotein (RNP) synthesizes telomeric repeats through its telomerase 746-Symp (TERT) and an integral RNA subunit (hTR) carrying the template for repeat Stepping Through DNA Mismatch Repair Initiation synthesis. In addition to TERT and hTR, the human telomerase holoenzyme Rafael Fernandez-Leiro1,2, Doreth Bhairosing-Kok3, Flora Groothuizen3, consists of a number of other factors required for RNP assembly Laffeber Charlie4, Joyce H. Lebbink4, Peter Friedhoff5, Meindert Lamers2,6, and localization. Telomerase has been shown to play important roles in can- Titia K. Sixma3. cer, aging and a number of human diseases. Due to its cellular scarcity and 1CNIO, Madrid, Spain, 2LMB, Cambridge, United Kingdom, 3Biochemistry, complex RNP assembly, the composition of human telomerase is still a Netherlands Cancer Institute, Amsterdam, Netherlands, 4ErasmusMC, matter of debate and available structural information on human telomerase Rotterdam, Netherlands, 5Giessen University, Giessen, Germany, 6LUMC, is limited to a 30A˚ negative stain reconstruction. Here we present biochemical Leiden, Netherlands. and cryo-EM studies on the human telomerase holoenzyme, revealing the Maintenance of genome stability is dependent on correct action of DNA first detailed architecture of an active telomerase RNP from a multicellular mismatch repair (MMR), to prevent the incorporation of errors dur- and new insight into its assembly and function. Our findings ing replication. In MMR, a MutS ATPase (or eukaryotic MutSalpha) recognizes provide a structural framework for understanding human telomerase disease the mismatch or short insertion/deletion loops and activates a MutL (or eukary- mutations and represent an important step towards telomerase-related clinical otic MutLalpha ATPase by changing its conformation. This enables MutL to therapeutics. activate an endonuclease for cleavage of the newly synthesized strand, so that it can be excised and resynthesized. MMR communicates between replica- 744-Symp tion and other repair processes and has important roles in recombination, triplet High-Resolution Modeling and Simulation of Cellular Structures and Pro- repeat expansion, somatic hypermutation and signaling. Errors in MMR pro- cesses, One at a Time teins lead to a mutator phenotype and a cancer predisposition in humans, called Emad Tajkhorshid. Lynch syndrome. , University of Illinois, Urbana, IL, USA. How information is transmitted from the mismatch to repair action is unclear Cells constitute functional units of biology and . Alongside with more due to a lack of knowledge about the interplay between the different traditional techniques, which continue to provide valuable information components. Structures of MMR complexes could resolve this, but the on ‘‘in vitro’’ systems, the last decade of biophysical research has witnessed transient nature of the interactions has complicated their analysis. Recently, emergence of technologies that have enabled scientists to study biophysical we managed to trap a first partial MutS/MutL complex, by site-specific cross- and biochemical processes within a cellular confinement, or even in living linking, which allowed structure solution by X-ray crystallography and cells, thus providing significantly more relevant information on the single particle cryo-electron (cryo-EM). Here we show how molecular mechanisms underlying biology. Computational has cryo-EM can now be used to study MMR complexes by enriching for specific been no exception to this revolution. Fueled by substantial advances states. This provides detailed insight in multiple states along this transient in structural characterization of cell-scale systems, e.g., owing to the explo- pathway. sion of cryo-electron microscopy (cryo-EM) data, novel computational modeling and simulation technologies have been developed that enable us to construct high-resolution molecular models of and cell- Symposium: Biological Membranes and Vesicles scale systems. Key challenges that are partially overcome include unprece- dented complexity of such molecular systems, their sheer size requiring 747-Symp handling several orders of magnitude larger number of particles, the need Uncovering the Mechanisms of Clathrin-Mediated Endocytosis using for new software design for simulation engines and new file formats that Quantitative Biology Approaches are able to handle such large data sets, and the long timescale of cellular Julien Berro. processes. In this presentation, I will report on some of our novel Department of Molecular Biophysics and Biochemistry, Department of Cell methodologies and algorithms developed over the last few years for auto- Biology, Yale University, New Haven, CT, USA. mated construction of high-resolution models of diverse membrane systems Clathrin-mediated endocytosis (CME) consists of the formation of a vesicle and their applications to a number of key cell-scale systems while inte- out of a flat membrane. When membrane tension and/or turgor pressure are grating various modality of available experimental data. The resulting, high, actin dynamics is required to produce the forces required to invaginate rigorously crafted models provide the most detailed structures that can be the membrane and pinch it off into a vesicle. However, because endocytic now used to begin to address fundamental biophysical questions at a cellular structures are intrinsically transient, out of equilibrium, and diffraction level. limited, how the actin meshwork produces forces at the molecular level has remained elusive. Quantitative microscopy and mathematical modeling 745-Symp have allowed us to determine that actin polymerization alone cannot produce Structure-Function Mapping of the Complex sufficient force to invaginate the plasma membrane in yeast. Using a single Michael P. Rout. molecule approach, we have demonstrated that the endocytic machinery turns Rout Lab, Rockefeller Univ, New York, NY, USA. over several times during CME, which may help enhance force production Despite the central role of Nuclear Pore Complexes (NPCs) as gatekeepers of during CME. In addition, we used mathematical modeling to show that RNA and protein transport between the cytoplasm and nucleoplasm, their large proteins that crosslink actin filaments can store large amounts of elastic size and dynamic nature have impeded a full structural and functional elucida- energy upon binding, and subsequently produce force and torque. Our tion. Here, we have determined a subnanometer precision structure for the studies shed light on new mechanisms for force production by actin mesh- entire 52.3 MDa, 552-protein yeast NPC by satisfying diverse data including works that are not exclusively based on polymerization, and could have stoichiometry, a cryo- map, and chemical cross- applications beyond endocytosis, and other actin-based structures that are links.The structure reveals the NPC’s functional elements in unprecedented diffraction limited.

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748-Symp dration layer Thermal Diffusion Forced Rayleigh Scattering (TDFRS) was Crowding in the Cellular Context: Tales of Clusters and Dynamics used. Michael Feig. QENS results show that the conformational entropy of streptavidin is reduced Biochemistry & , Michigan State University, East upon biotin binding, indicating that the process is enthalpy driven. From liter- Lansing, MI, USA. ature data an increase of the conformational entropy of the hydration layer upon Biological are highly concentrated in biological environments biotin binding is calculated. TDRFS results also indicate an increased entropy giving rise to crowding effects that impact structure, dynamics, and ultimately of the hydration layer upon biotin binding. This indicates that the hydration function. It is increasingly becoming clear that weakly attractive non-specific layer plays an important role in stabilizing the binding of biotin to streptavidin, interactions may be a significant factor in crowding effects. Such interactions its increase in conformational entropy caused by the burial of hydrophobic can lead to the dynamic formation of clusters which impacts diffusive proper- groups compensates the increase of entropy in the protein. The change in ties and may lead to the formation of phase-separated states. Recent results conformational entropy per residue upon biotin binding to streptavidin is of from computer simulations of cellular systems at different the same order of magnitude, as that of the transition from an unfolded scales are presented that relate biomolecular interactions to cluster formation myoglobin to a molten globule. It is an order of magnitude larger than that and altered diffusive behavior. Models of concentrated protein solutions, bac- of other protein ligand binding interactions. This indicates that a significant terial cytoplasms, with and without DNA, and in the presence of membrane sur- change of conformation occurs upon biotin binding to streptavidin, more faces are discussed. The models range from fully atomistic systems to coarse- than the established closing of the binding loop. The internal dynamics grained models derived from the atomistic systems in order to expand to larger show, that the residues of free streptavidin perform a jump-diffusive motion spatiotemporal scales. Where possible, the results from the simulations are in the picosecond timeframe, while the more rigid complex does not show related to experimental data. jump-diffusive motions.

751-Plat Platform: Protein-Small Molecule Interactions Structural and Functional Characterization of Periplasmic Sialic Acid Binding Proteins from Pathogenic 749-Plat Thanuja Gangi Setty, S. Ramaswamy. Mapping Ligand Binding Landscapes using Weighted Ensembles of Institute for Stem and Regenerative Medicine, Bangalore, India. Trajectories Pathogenic bacteria have evolved different strategies to escape the host im- Alex Dickson. mune system. One of the strategies is to scavenge and coat themselves with Biochemistry & Molecular Biology, Michigan State University, East host sialic acids, which allows the bacteria to deceive the host immune system. Lansing, MI, USA. This is also called ‘molecular mimicry’. N-acetylneuraminic acid (Neu5Ac) Protein-ligand interactions involve many conformational states. To bind the and N-glycolylneuraminic acid (Neu5Gc) are the two common sialic acids ligand must search across a rough free energy landscape that contains many and many pathogenic bacteria scavenge these sugars and they are added to metastable minima. A map of this ‘‘ligand binding landscape’’ would be useful the terminus of lipooligosaccharide (LOS) or lipopolysaccharides (LPS) as during drug design, as it contains a set of possible binding modes that could be non-reducing sugars. In addition, part of the scavenged sialic acid is also uti- stabilized by modifications of the ligand. These maps would also include infor- lized by the bacteria as carbon and source. Detailed structural and mation about pose connectivity, both with each other and with the unbound functional studies of sialic acid binding proteins will help in rational design state, which can provide insight into ligand binding kinetics, a useful parameter of drugs against different pathogenic bacteria. for drug design. Unfortunately, creating maps of the ligand binding landscape is Pathogenic bacteria like Haemophilus influenzae (Hi), Fusobacterium nuclea- a great challenge, as binding and release events typically occur on timescales tum (Fn), Pasteruella multocida (Pm) and Vibrio cholerae(Vc) use Sialic acid that are beyond the reach of molecular simulation. The Dickson lab has devel- binding protein (SiaP), a component of the TRAP (Tripartite ATP-independent) oped simulation algorithms (e.g. WExplore, REVO) that can broadly and effi- transport system, to scavenge sialic acid present in the periplasm. Further, SiaP ciently explore free-energy landscapes and are capable of simulating ligand delivers the bound sugar to a membrane protein that transports the sugar from release pathways that occur on timescales as long as minutes. Importantly, the periplasm to the cytoplasm. SiaP’s are two domain proteins, with a very these methods employ only unbiased trajectory segments, allowing for the con- well conserved ligand-binding pocket. Our thermodynamic studies illustrate struction of Markov state models (MSM) and conformation space networks that that the binding of Nue5Ac and Neu5Gc to SiaP is an enthalpically driven pro- combine the results of multiple simulations. In this talk I will describe how cess. The structure of the sialic acid transport protein (SatA), a component of these methods have revealed new insights into long timescale ligand release ABC (ATP binding cassette) transport system, from Haemophilus ducreyi(Hd) pathways, as well as the interconversion pathways between ligand poses. In displays a different sialic acid binding pocket when compared to SiaP. The particular I will focus on the application of WExplore to map the binding land- structural differences reflect in our thermodynamic analysis with SatA binding scapes of two bromodomain-inhibitor systems (Brd4-MS436 and Baz2B- to Neu5Ac and Neu5Gc being an entropically driven process. Altogether, our ICR7), and the use of Markov state models to predict stable poses. In this structural and functional analyses show that, sialic acid binding proteins bind work we find that transitions between deeply bound states convert through to same ligand with similar binding affinities, but by using different binding the unbound state 81% of the time, implying a trial-and-error approach to topologies. ligand binding. This finding has consequences for both the way we think about ligand binding processes, and how molecular dynamics should be used in drug 752-Plat design. Mechanical Strength of Catch Bond Forming FimH and Mannose Laura A. Carlucci, Keith Johnson, Wendy E. Thomas. 750-Plat Bioengineering, University of Washington, Seattle, WA, USA. The Relevance of Conformational Entropy for Protein Ligand Interac- Adhesive interactions must withstand mechanical forces occurring in an - tions: the Case of Biotin and Streptavidin ism. Forces have influenced some proteins such as integrins, clotting factors, Mona Sarter1, Andreas M. Stadler2, Doreen Niether3, Bernd W. Koenig4, and bacterial adhesive proteins to develop catch bond properties, in which Michaela Zamponi5, Lohstroh Wiebke6, Simon Wiegand3, Joerg Fitter7. the lifetime between a and ligand increases with applied force. 1I. Physikalisches Institut/ JCNS-1. ICS-1, RWTH Aachen/ FZ Juelich, One of the most well studied catch bond behaviors is found between the Aachen, Germany, 2ICS-1 & JC, Forschungszentrum Julich€ GmbH, Julich,€ E. coli adhesive protein, FimH, and its ligand, mannose. FimH has two distinct Germany, 3ICS-3, FZ Juelich, Julich,€ Germany, 4ICS-6; Structur, conformations with significantly different mannose affinities. The low affinity Forschungszentrum Julich,€ Juelich, Germany, 5JCNS-MLZ, JCNS at MLZ, conformation predominates the initial FimH-mannose interactions, as this state, Garching, Germany, 6FRM II, TUM, Munchen,€ Germany, 7Inst Biologische characterized with an open binding pocket, facilitates fast kinetics. A force Info, Forsch Juelich, Juelich, Germany. pulling on the FimH-mannose interaction elongates FimH, inducing a transition Molecular dynamics play a vital role for the biological function of proteins. For into the high mannose affinity state, which includes a closed mannose binding protein-ligand interactions changes of conformational entropy of protein and pocket. hydration layer are relevant for the binding process. In an experimental study While in this force-stabilized high affinity conformation, FimH has a remark- we investigated the relevance of conformational entropy for the binding of ably long-lived lifetime that has never been fully measured before. We suspect biotin to the protein streptavidin. Quasi Elastic Neutron Scattering (QENS) the closed binding pocket is responsible for this lifetime, and mannose dissoci- was used to investigate the difference in conformational entropy between the ation occurs when the pocket spontaneously opens. If the opening of this pocket ligand-free and ligand-bound protein. The internal streptavidin dynamics occurs independent of the strength of the applied force, mannose unbinding before and after biotin binding were compared. To gain information on the hy- would thus represent a force independent, or ideal bond, interaction. To explore

BPJ 9328_9339 152a Monday, March 4, 2019 this possibility, we measured the lifetimes of the mannose-FimH interaction at 755-Plat various forces with FimH in the high affinity state. Targeting the mRNAs Process: A Novel Theoretical Based We utilized a magnetic tweezer apparatus, to apply various constant forces to Approach to Design Tailored Anticancer Agents mannose-coated magnetic beads bound to immobilized FimH. While true ideal Daniele Di Marino1, Stefano Raniolo1, Alessandro Gori2, bond behavior cannot be concluded with certainty, the FimH-mannose bond Vittorio Limongelli1. often outlasted the 15-minute collection period, even at high forces relative 1Faculty of Biomedical Sciences, Institute of Computational Science, to other catch bond forming proteins. Understanding the relationship between Universita` della Svizzera Italiana, Lugano, Switzerland, 2Istituto di Chimica force and FimH’s incredibly stable binding pocket, would grant further insight del Riconoscimento Molecolare, ICRM, CNR, Milano, Italy. into the catch bond mechanism associated with FimH. The interaction between the eIF4E and the eIF4G is essential for the transla- tional machinery assembly and, consequently, for the initiation step of mRNAs 753-Plat translation process. High levels of eIF4E in the cell are linked to the cancer Menthol Binds to Extracellular and Transmembrane Domains of the Hu- onset, generally associated with poor prognosis. The inhibition of the eIF4E- man A4b2 Nicotinic Receptor eIF4G interaction represents a concrete strategy to reduce cell proliferation Rezvan Shahoei1,2, Emad Tajkhorshid2,3. with a direct effect on cancer progression. A well-characterized group of pro- 1Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA, teins, called 4E-binding proteins (4E-BPs), is known to inhibit the interaction 2NIH Center for Macromolecular Modeling and , Beckman between eIF4E and eIF4G. The CYFIP1 protein is a peculiar member of the 4E- Institute, University of Illinois at Urbana-Champaign, Urbana, IL, USA, BP family that interacts with eIF4E through a a-short helix. The eIF4E binding 3Biochemistry, Center for Biophysics and Quantitative Biology, University region in CYFIP1 shows low conservation among the other 4E-BPs, therefore of Illinois at Urbana-Champaign, Urbana, IL, USA. assuming a specific binding mode. Here we illustrate a rational approach to Menthol, a small organic compound, has long been used for its flavor as well as design a stapled CYFIP1-derived (SCD) able to interact selectively its local anesthetic and counterirritant properties. In the US, the 2009 Family with eIF4E showing promising anticancer activity. First, we have employed Smoking Prevention and Tobacco Control Act by the Congress banned all ad- classical molecular dynamics simulations (MD) to identify the best position ditives to cigarettes with the exception of menthol. However, an abundance of on the CYFIP1 structure to introduce the staple. Afterwards, we have per- scientific and clinical research characterize menthol as a non-inert molecule in formed 3ms of Funnel-Metadynamics (FM) to characterize the binding free- the body. Smoking menthol cigarettes has been correlated with lower quitting energy surface (FES) of the eIF4E-SCD interaction. The FM results show rates and higher relapse rates and it has been shown that all cigarettes, even if that the SCD peptide binding free energy is 7 kcal/mol and staple modifi- labeled as nonmenthol, contain menthol. Unfortunately, at the molecular level cation stabilizes the active conformation of the peptide. Prompted by the prom- not much is known about menthol’s interaction with its sites of action, most ising theoretical data, we have synthesized the SCD peptide and tested it on prominently nicotinic acetylcholine receptors (nAChRs) in the . The pub- MDA-MB-231 human breast cancer cell line. The experimental results show lished X-ray crystal structure of the human a;4b;2 nicotinic receptor - the most that the SCD peptide is not cytotoxic and shows encouraging anticancer activity abundant nAChR in the human brain - provided an unprecedented opportunity targeting selectively the translational machinery. Our study shows how to for us to perform molecular dynamics simulations to investigate the interaction combine with success standard and cutting-edge computational techniques in between menthol and a heteropentameric nAChR. In this study, we first used drug design, paving the way to further investigations on peptide/protein inter- free energy perturbation calculations as well as flooding simulations to calcu- action in biological systems of pharmacological interest. late menthol’s water-membrane partition coefficient. The results from the flooding simulations were used to determine the penetration depth as well as 756-Plat the preferred orientation of menthol molecules in a POPC bilayer. Um- Protein Databank Survey Hints into the Emergence of Protein-Adenine brella sampling simulations were performed to derive the free energy profile Recognition in Evolution 1 1 1 2 1 of menthol across a POPC . After concluding that menthol mole- Aya Narunsky , Ron Solan , Amir Kessel , Rachel Kolodny , Nir Ben-Tal . 1Dept Biochemistry and Molecular Biology, Tel Aviv Univ, Tel Aviv, Israel, cules partition in mammalian membranes in large numbers we performed 2 flooding simulations in the presence of the human a;4b;2 nicotinic receptor. Univ of Haifa, Haifa, Israel. We demonstrate that menthol molecules bind to both the extracellular and The ability of proteins to recognize small molecules, or ‘ligands’, forms the ba- transmembrane domains of the protein with larger numbers of menthol mole- sis of virtually all protein functions. Proteins interacting with the ancient li- cules binding to the transmembrane domain of the protein after partitioning gands, that were present on Earth since the beginning of life, may hold into the membrane. valuable information about how molecular recognition emerged and evolved. We focus on adenine because it is an integral component of ancient cofactors 754-Plat used by and it serves as a ligand also in its isolated form. To explore Molecular Determinants of Non-Oxime Bispyridinium Activity at Adults the way proteins recognize adenine moieties and interact with them, we devel- Muscle nAChRs oped a computational pipeline that extracts protein-adenine complexes from Max Epstein. the Protein Databank (PDB), structurally superimposes them according to their Biochemistry, University of Oxford, Oxford, United Kingdom. (rigid) adenine fragments, and detects the hydrogen bonds that mediate the Organophosphorus nerve agents (OPNAs) represent the deadliest class of protein-ligand interactions. Adenine has triangular shape that offers three bind- chemical warfare agent known. Since their inception in the 1930’s, OPNAs ing edges: The Watson-Crick edge, the Hoogsteen edge, and the sugar edge. have been used in targeted assassinations, terrorist activities and in asymmetric Our analysis shows that, contrary to previous claims, each of these edges warfare. The standard post-exposure treatment plan for OPNA exposure usu- may participate in adenine recognition by proteins, and that these interactions ally includes a combination of atropine, oxime and a benzodiazepine. Despite may be mediated by water molecules. This in turn suggests that adenine binding this varied therapeutic arsenal, the nicotinic acetylcholine receptor (nAChR) has emerged more than once in evolution. Analysis of the sequence and struc- mediated effects of OPNA have not yet been addressed pharmaco- ture of the adenine binding site shows that binding is often mediated by specific logically. It has been previously shown that removal of the reactive group of segments, referred to as ‘themes’, that occur repeatedly in proteins. oxime compounds does not completely ablate their ability to recover neuro- These themes could be the descendants of ancient mini-proteins that ultimately muscular firing post nerve agent exposure and this was attributed to an interac- evolved to the proteins that we see today. tion with the nAChR. Radioligand binding experiments and structure-activity relationship (SAR) studies on a series of bispyridinium (BPD) compounds sug- Platform: Excitation-Contraction Coupling/ gested that this interaction occurs at the orthosteric binding site of the muscle Cardiac and Skeletal Muscle Electrophysiology II nAChR, consistent with previous studies that revealed the role of cation-p in- teractions for positively charged ammonium compounds within this pocket. 757-Plat It was also noted that increasing the length of the BPD alkyl linker leads to a L-Type Calcium Channels are a Major Source of Plasmalemmel Calcium corresponding increase in antagonist activity. It is thought this reflects an allo- Influx for Drosophila Cardiomyocytes steric interaction of the second pyridinium group with another region of the Worawan B. Limpitikul1, Meera C. Viswanathan2, Brian O’Rourke3, nAChR. Knowing that one pyridinium moiety binds to the orthosteric site, David T. Yue4, Anthony Cammarato5. we have used unbiased molecular dynamics (MD) simulations and two- 1University of California, San Francisco, San Francisco, CA, USA, 2Sch of electrode voltage clamp (TEVC) electrophysiology to investigate which region Med, Johns Hopkins Univ, Baltimore, MD, USA, 3Dept Med, Johns Hopkins of the nAChR might be responsible for the allosteric component of BPD bind- Univ, Baltimore, MD, USA, 4Dept Biomed Eng/Neuro, Johns Hopkins Univ, ing. Our results provide important information for the design of new com- Baltimore, MD, USA, 5Dept Med Div Cardio, Johns Hopkins Univ, pounds for the treatment of OPNA exposure. Baltimore, MD, USA.

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L-type Ca2þ channels (LTCCs) play a major role in shaping cardiac action po- (iPSC-CM) and endothelial cell (human cardiac microvascular endothelial tential and thus, dysregulation of these channels underlies numerous cardiac pa- cell (HCMEC) and human umbilical vein endothelial cells (HUVEC)) co- thologies. Understanding their modulation with high fidelity relies on culture platform. To enhance physiological relevance of this platform, we investigating LTCCs in their native environment with intact binding partners. have used fibrin hydrogels as a 3D scaffold to approximate micro- Such studies would benefit from genetic manipulation of endogenous LTCCs mechanical -like conditions. We have shown by fixed and live cell in cardiomyocytes, which often proves cumbersome in mammalian models. immunofluorescence microscopy that this platform supports spontaneous Drosophila, however, offers a potential alternative as it possesses a relatively cellular reorganisation, allowing biomimetic physical CM-EC association, simple heart, is genetically pliable, and expresses many conserved . Fluo- thus enabling bidirectional biochemical and mechanical modes of cell signal- rescence in situ hybridization performed in fly cardiomyocytes demonstrated an ling. We have observed a significant reduction of iPSC-CM spontaneous abundance of mRNA encoding Ca-a1D and Ca-a1T, which are homologous to beating rate after 1 week 3D co-culture with EC. In electrically paced condi- mammalian LTCCs and T-type Ca2þ channels, respectively. Cardiac-specific tions, optical mapping of a genetically encoded fluorescent Ca2þ reporter, knockdown of Ca-a1D via interfering RNA completely abolished myocyte GCaMP6f, revealed significant prolongation of iPSC-CM Ca2þ transient ki- contraction, suggesting Ca-a1D represents the primary functioning Ca2þ chan- netics while in 3D co-culture with EC, via increased time to 50%, 80% and nel in Drosophila hearts. To accurately probe the transmembrane currents, we 90% relaxation, and a reduced rate of Ca2þ transient decay. Investigation of successfully developed a method to isolate viable single cardiomyocytes and iPSC-CM metabolic activity using TMRM mitochondrial inner membrane recorded Ca2þ currents via patch clamping. The profile of Ca2þ currents in in- dye revealed significantly depolarised mitochondrial membrane potential in dividual cells when Ca2þ channels were hypomorphic or absent, or under selec- iPSC-CM in co-culture with EC. These results highlight the importance of tive LTCC blockage by nifedipine, also confirmed the predominance of Ca-a1D EC influence on excitation-contraction coupling, and the necessity of theirs current across all activation voltages. T-type currents, activated at more nega- and other non-myocyte inclusion in iPSC derived in vitro models in order tive voltages, were also detected. Lastly, Ca-a1D channels displayed calcium- to faithfully replicate physiology and further our understanding of cardiac dependent inactivation, a critical negative feedback mechanism of LTCCs, and function. were augmented by forskolin, an activator of the PKA pathway. In summary, þ the Drosophila heart possesses a conserved compendium of Ca2 channels 760-Plat with similar regulatory mechanisms, suggesting that the fly may serve as a Biochemical and Epigenetic Modifications Occur in Muscles of Patients robust and effective platform for studying the (dys)regulation of cardiac with Selenoprotein N Related Congenital Myopathy þ Ca2 channels. Christoph Bachmann1, Nicol Voermans2, Heinz Jungbluth3, Francesco Muntoni4, Francesco Zorzato5,6, Susan Treves1,7. 758-Plat 1Dept Anesthesiol/Biomed, Basel Univ Hosp, Basel, Switzerland, 2Dept Efficient High-Throughput Screening for Type 1 Ryanodine Receptor Neurology, Radboud University Medical Center, Nijmegen, Netherlands, Inhibitors using ER Ca2DMeasurements 3Dept Pediatric Neurology, King’s College and Evelina Children’s Hospital, Takashi Murayama1, Nagomi Kurebayashi1, Mari Yuasa-Ishigami2, London, United Kingdom, 4Institute of Child Health, UCL, London, United Shuichi Mori2, Haruo Ogawa3, Junji Suzuki4, Kazunori Kanemaru5, Kingdom, 5Dept Life Sci & BioTech, Univ Ferrara, Ferrara, Italy, 6University Masamitsu Iino5, Hiroyuki Kagechika2, Takashi Sakurai1. Hospital Basel, Basel, Switzerland, 7Life Sciences, University of Ferrara, 1Juntendo University, Tokyo, Japan, 2Tokyo Medical and Dental University, Ferrara, Italy. Tokyo, Japan, 3The University of Tokyo, Tokyo, Japan, 4University of Multiminicore Disease is a phenotypically heterogeneous congenital myop- California, San Francisco, CA, USA, 5Nihon University, Tokyo, Japan. athy caused by mutations in several genes. Patients exhibiting the classic Type 1 ryanodine receptor (RyR1) is a Ca2þrelease channel in the sarco- form characterised by spine rigidity, early onset and respiratory insufficiency plasmic reticulum and plays an important role in excitation-contraction are linked to recessive mutations in SELENON (previously known as coupling. Genetic mutations in RyR1 cause various skeletal muscle diseases SEPN1), the encoding selenoprotein N (SelN). The proposed pathome- including malignant hyperthermia (MH). Because the main underlying mech- chanism of disease includes oxidative stress leading to abnormal skeletal anism of the pathogenesis is hyperactive Ca2þrelease by gain-of-function of muscle calcium regulation. To identify pathological pathways activated in the RyR1 channel, inhibition of RyR1 is expected to be a promising treatment the muscles of patients affected by this congenital myopathy, we investigated for these diseases. We here developed a novel high-throughput screening the physiological, biochemical and epigenetic characteristics of skeletal mus- (HTS) platform using time-lapse fluorescence measurement of Ca2þin the cles from patients with SELENON mutations. Muscles from all patients (ER) to identify RyR1 inhibitors. RyR1 carrying an showed considerable biochemical and epigenetic modifications including 2þ MH mutation and R-CEPIA1er, a genetically-encoded ER Ca indicator, depletion of RyR1, Cav1.1 and SERCA1 proteins, increased levels of class were stably expressed in HEK293 cells and time-lapse fluorescence was II deacetylases and altered expression of myomiRs. From a func- measured using a fluorometer. Since MH mutant RyR1 reduces ER Ca2þin tional point of view, depolarization induced Ca2þ transients were signifi- HEK293 cells through Ca2þleakage, specific drugs that inhibit RyR1 increase cantly smaller and slower, in patient’s myotubes though the peak Ca2þ ER Ca2þby preventing Ca2þleakage. By screening 1,535 compounds in a li- transient elicited by direct RyR1 activation was similar in cells from controls brary of well-characterized drugs, we successfully identified four compounds and patients. These findings suggest that SELENON mutations result in com- including dantrolene, a known RyR1 inhibitor, and three structurally-different plex downstream changes in skeletal muscle, similarly to the intricate compounds; oxolinic acid, 9-aminoacridine and alexidine. Interestingly, they secondary changes observed in muscles of patients with congenital myopa- showed different dose-dependencies and isoform specificities. The highly thies caused by different primary genetic defects. Understanding the mecha- quantitative characteristic and good correlation with the channel activity vali- nisms regulating the activation of these epigenetic pathways could lead to the date this HTS platform by ER Ca2þmeasurement to explore novel drugs for development of new drugs aimed at improving the muscle function in RyR1-linked diseases. patients affected by congenital myopathies associated with reduced RyR1 protein content. 759-Plat Endothelial Cell Regulation of Excitation-Contraction Coupling in 761-Plat Induced Pluripotent Stem Cell Derived Myocardium Structural Insights into Recognition of Ryanodine Receptors by PKA Oisı´n King1, Fatemeh Kermani1, Brian Wang1, Warrapong Kit-Anan1, Omid Haji-Ghassemi1, Zhiguang Yuchi2, Filip Van Petegem3. Jerome Fourre1, Anna M. Randi2, Cesare M. Terracciano1. 1Univ British Columbia, Vancouver, BC, Canada, 2Tianjin University, 1Myocardial Function, National Heart and Lung Institute, Imperial College, Tianjin, China, 3Dept Biochem/Molec Biol, Univ British Columbia, London, United Kingdom, 2Vascular Science, National Heart and Lung Vancouver, BC, Canada. Institute, Imperial College, London, United Kingdom. Ryanodine Receptors (RyRs) are large intracellular channels that control Endothelial cells (EC) are active regulators of cardiac function, and bi- the release of Ca2þ from the SR or ER. They are targets for a multitude of reg- directional signalling between EC and cardiomyocytes (CM) is essential for ulatory events, including the by like PKA and maintenance of cardiac tissue . For this reason, cardiac EC CaMKII. Here we provide direct crystallographic insights into the recognition show strong promise as therapeutic targets for the prevention and treatment of RyR2, the cardiac isoform, by PKA. We found an extensive and unusual of heart disease. However, due to the lack of physiologically relevant multi- binding interface that is the target for several disease-associated mutations. cellular in vitro platforms, many mechanisms governing the crosstalk between Calorimetry and kinetic assays show that RyR2 is a better substrate these two cells types are poorly understood. To investigate this relationship, than RyR1. Either phosphorylation or phosphomimetics at nearby target we have developed an induced pluripotent stem cell derived cardiomyocyte sites drastically change the affinity and activity of PKA towards the RyR2

BPJ 9328_9339 154a Monday, March 4, 2019 phosphorylation substrate. Structures show that the PKA-RYR2 interface is mM BTP-2). Taken together, these data indicate that muscles from highly dynamic and subject to change depending on the phosphorylation status. CASQ1-null mice compensate for the lack of CASQ1 and reduced SR Our results directly suggest an interplay between CaMKII and PKA for recog- Ca2þ content by constitutively assembling CEUs to promote Ca2þ entry nizing their target sites in RyR2. via SOCE even in the absence of store depletion.

762-Plat 764-Plat Acute Genetic Ablation of Sodium-Calcium Exchange: of Novel Morphological and Functional Insights in Python Cardiac Biology Excitation-Contraction Coupling and Calcium Regulation Claudia Crocini1, Kathleen C. Woulfe2, Leslie A. Leinwand1. Sabine Lotteau1, Rui Zhang1, Christina Grabar1, Stephan Aynaszyan1, 1BioFrontiers, Univ Colorado Boulder, Boulder, CO, USA, 2Univ Colorado Xin Yue2, Yushun Zhang2, Kenneth D. Philipson3, Michela Ottolia4, Denver, Denver, CO, USA. Joshua I. Goldhaber1. Pythons are infrequent feeders that can ingest meals equal to their own body 1Smidt Heart Institute, Cedars Sinai Medical Center, Los Angeles, CA, USA, mass. The intensive digestive effort is associated with a dramatic increase of 2Department of Structural Cardiology, First Affiliated Hospital of Xi’an heart and cardiac hypertrophy. Rise of heart rate is mediated by reduced Jiaotong University, Xi’an, Shaanxi, China, 3Department of Physiology, parasympathetic tone on the heart and by nonadrenergic, noncholinergic fac- UCLA, Los Angeles, CA, USA, 4Department of Anesthesiology & tors (Skovgaard et al., Am J Physiol Regul Integr Comp Physiol, 2009), and Perioperative Medicine, UCLA, Los Angeles, CA, USA. cardiac hypertrophy is the results of increased cardiomyocytes size (Ri- Previously described murine models of sodium-calcium exchange (NCX) quelme et al., Science 2011). Assessing the mechanisms responsible for ablation exhibit cardiac remodeling that complicates interpretation, likely a this physiological cardiac growth and improved output at cellular level has result of embryonic excision of the NCX gene and chronic over been challenged by the increased collagen content in reptile hearts compared time. To investigate the effect of acute ablation of NCX, we created a cardiac to mammals (Diwan et al., Circulation 2008), that limits the effectiveness of specific NCX KO in adult mice using the tamoxifen-inducible cardiac specific current cardiomyocytes isolation. Here, we have developed a Langendorff- (aMHC-MerCreMer) . We injected tamoxifen (40mg/kg/day IP) for free protocol to enzymatically isolate viable cardiomyocytes from python 5 consecutive days. One week post tamoxifen, NCX protein expression was hearts and an optimized protocol to isolate python myofibrils and measure reduced by 96% in ventricular tissue and NCX current was completely elim- their mechanical properties. Isolated python cardiomyocytes from both inated in 4/9 cells (85% average reduction). LV function by echocardiography ventricle and atria have been employed to study sarcomeric structure by was preserved and, unlike ventricular-specific NCX KO mice produced by the immunofluorescence demonstrating the ability of our isolation protocol to MLC2v promoter, we observed no fibrosis or hypertrophy. We observed a maintain cellular morphology. Tension and kinetics of activation and relax- 12% increase of resting calcium and normal Ca transient amplitudes in iso- ation of isolated myofibrils from python ventricle have been measured using lated cardiomyocytes loaded with fura-2 AM (30 min, 2.5 mM), despite a a custom-built apparatus with fast solution switching. At isometric length, 2 37% reduction in ICa (P < 0.001), a two-fold increase of SERCA2 the resting tension of python myofibril was 2.1 5 0.94 mN/mm . In the pres- 2 (P<0.01) and a two-fold increase of PMCA (P<0.05). Reduced ICa and pre- ence of pCa 4.5, python myofibrils developed 31.5 5 20.0 mM/mm of served Ca release are indicative of increased EC coupling gain, which we force. The duration of linear phase relaxation was 166 5 22.4 ms. The pos- have observed in MLC2v NCX KO mice. However, changes in SERCA sibility to characterize isolated cardiomyocytes and myofibrils from python and PMCA are distinct from the MLC2v NCX KO at 8-12 weeks, where hearts provides us with novel tools to study excitation-contraction coupling resting Ca was similar to wildtype and SERCA and PMCA protein expression machinery that could unveil cellular mechanisms responsible for the extreme were unchanged. 4 weeks after tamoxifen, NCX expression and current were post-prandial cardiac adaptation occurring in reptiles and inform mammals virtually eliminated and ICa was reduced by 47%. SERCA and PMCA cardiac hypertrophy. continued to increase but fibrosis and hypertrophy developed despite normal- ization of diastolic Ca. In conclusion, mice survive after acute ablation of NCX by reducing Ca influx, increasing EC coupling gain, and increasing alternative Ca efflux and SR uptake mechanisms. However, prolonged Platform: Energy Transducing Complexes and absence of NCX results in cardiac remodeling, possibly by activating Ca- Mitochondria in Cell Life and Death dependent gene programming. 765-Plat 763-Plat Structure-Based Change in the Rate-Limiting Step of Photosynthetic Muscles from Calsequestrin-1 Knockout Mice Contain Pre-Assembled Electron Transport Calcium Entry Units that Provide Constitutively Active Store-Operated William A. Cramer1, J. Ness1, S. Saif Hasan1, Katherine Ehringer1, Calcium Entry Sejuti Naurin1, Valentyn Stadnytskyi2, Iskander M. Ibrahim3, Antonio Michelucci1,2, Simona Boncompagni1, Laura Pietrangelo1, Sujith Puthiyaveetil3. Robert Dirksen2, Feliciano Protasi1. 1Dept Biol Sci, Purdue Univ, West Lafayette, IN, USA, 2Lab of Chemical 1University of Chieti, Chieti, Italy, 2Pharmacology and Physiology, Physics, NIDDK, NIH, Bethesda, MD, USA, 3Dept of Biochemistry, Purdue University of Rochester, Rochester, NY, USA. Univ, West Lafayette, IN, USA. 2þ 2þ ˚ Store-operated Ca entry (SOCE) is a ubiquitous Ca influx mechanism The 2.5 A crystal structure (1) of the cytochrome b6f complex obtained þ triggered by depletion of intracellular Ca2 stores. We recently discovered from the cyanobacterium Nostoc sp. PCC 7120 (pdb 4OGQ) was used that acute treadmill exercise of wild type mice drives the formation of as a guide for modification by site-directed mutagenesis in the cyanobacte- new intracellular junctions between the sarcoplasmic reticulum (SR) and rium Synechococcus sp. PCC 7002 of the rate-limiting step in the central transverse-tubules (TTs), referred to as Calcium Entry Units (CEUs). electron transport/proton translocation chain of oxygenic photosynthesis. CEUs contain STIM1 and Orai1, the two essential proteins of SOCE. Skel- This step is associated with the oxidation and deprotonation of plastoquinol etal muscle fibers from mice lacking calsequetrin-1 (CASQ1-null), the pri- on the electrochemically positive (p) side of the membrane. The mutagen- 2þ mary Ca -binding protein in the lumen of SR terminal cisternae, exhibit esis strategy is based on structure studies of the b6f complex in the þ reduced SR Ca2 content and massive SR depletion when stimulated at absence and presence of quinol analogue inhibitors which bind and inhibit high frequency. Here we report that CEUs are constitutively assembled in electron transport on the p-side of the thylakoid membrane. The strategy extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles focused on two conserved prolines located on the p-side of the F-helix, of CASQ1-null mice, even in the absence of acute treadmill exercise. The proximal to the C-helix, in subunit IV of the seven subunit cytochrome increased number/area of CEUs in muscles from sedentary CASQ1-null b6f complex. These prolines, residues 105 and 112 in the F-helix, cause a þ mice directly correlated with: i) enhanced rate of Mn2 quench in single bend in this helix away from the C-helix in the cytochrome b subunit. FDB fibers, both in the absence of and after store depletion; ii) higher ability Thus, they are predicted to increase the portal aperture for the plastoquinol þ to maintain peak Ca2 transient amplitude in FDB fibers during repetitive, generated in the photosystem II reaction center complex that serves as the high-frequency stimulation (40 successive 500 ms 50Hz stimuli delivered electron-proton donor to the [2Fe-2S] iron-sulfur protein and the p-side b- every 2.5 seconds); and iii) increased capability of EDL muscles to maintain heme. Changing the two prolines to resulted in a decrease of 30- 2þ contractile force in the presence of 2.5 mM external Ca during repetitive, 50 % in: (i) the initial growth rate of the cellculture; (ii) the rate of O2 evo- high-frequency stimulation. This enhanced resistance to fatigue was abol- lution, and (iii) reduction of photo-oxidized cytochrome f. 1Hasan, S. S. and þ ished by experimental interventions that inhibit Ca2 influx via SOCE W. A. Cramer (2014) Structure, 22:1008-1015; research support: DOE DE- þ þ (i.e., equimolar replacement of external Ca2 with Mg2 or addition of 10 SC0018238.

BPJ 9328_9339 Monday, March 4, 2019 155a

766-Plat chondria, to parameterize IM structure for use in computer simulations of Chemomechanical Coupling of Mitochondrial Complex I mitochondrial activities, and to look for clues about the nature of IM hetero- Chitrak Gupta1, Umesh Khaniya2, Chun Kit Chan3, Marilyn Gunner2, geneity. Concurrently, we also are developing computational tools to expe- Christophe Chipot3, Francois Dehez4, Abhishek Singharoy1. dite the extraction from tomograms of 3D membrane surfaces to use in the 1Biodesign Institute, Arizona State University, Tempe, AZ, USA, simulations. We have discovered that lamellar cristae contain numerous 2Department of Physics, City College of New York, New York, NY, USA, roughly circular fenestrations (diameters 20-70 nm) that may facilitate ma- 3Department of Physics, University of Illinois at Urbana-Champaign, trix diffusion between adjacent cristae. In a few tomograms, swollen lamellar Champaign, IL, USA, 4University of Lorraine, Nancy, France. and tubular cristae coexist, with fenestrations aligned in a way suggesting The mitochondrial respiratory chain comprising of five protein complexes uti- their involvement in a transition between the two topologies. (Crista swelling lizes energy from catabolic processes to synthesize ATP. Complex I, the first is associated with oxidative stress in some cells.) Computer simulations em- and the largest complex of the chain, harvests electrons from NADH to reduce ploying idealized and extracted IM surfaces suggest that cristae may function quinone while pumping protons across the membrane. The working-principle to sequester or buffer key metabolites such as ADP and affect the rate of up- of such charge-transfer processes remains fragmentary due to bottlenecks take of calcium released from nearby internal stores. (Supported by NIH arising from coupled conformational transitions and solvation dynamics. A grant U01HLI16321.) recent study has reported the X-ray structure of Complex I from Thermus ther- mophilus containing 16 subunits along with 9 iron-sulphur clusters[1], which 769-Plat are reduced by electrons from NADH. Here, we have used large-scale molec- Human VDAC3 Forms VDAC1-Type Anionic Channels that are ular dynamics simulations to study chemomechanical coupling in Complex I. High-Conducting, Permeable to Metabolites, and Regulated by Cytosolic First, we identify the switches within Complex I that couple the dynamics Proteins of the quinone binding pocket to the site of NADH reduction. Second, our free- Maria Queralt-Martin1, Lucie A. Bergdoll2, Jeff Abramson2, energy simulations reveal that the affinity of quinone for the binding-site is Daniel Jacobs1, Oscar Teijido Hermida3, David P. Hoogerheide4, higher than that of quinol - a design essential for quinol release upon reduction. Sergey M. Bezrukov1, Tatiana K. Rostovtseva1. Third, Monte Carlo Continuum Electrostatics calculations detect a network of 1Section on Molecular Transport, Eunice Kennedy Shriver National Institute residues underlying charge-transfer from the quinone-binding site to the trans- of Child Health and Human Development, National Institutes of Health, membrane subunits responsible for proton pumping. Finally, Implicit Ligand Bethesda, MD, USA, 2Department of Physiology, David Geffen School of Sampling has been used to show an increase in oxygen-access of the reduced Medicine, University of California Los Angeles, Los Angeles, CA, USA, iron-sulphur complexes, possibly leading to creation of oxygen free radicals. 3Medical Laboratory, EuroEspes Biomedical Research Center, This finding hints at a role played by complex I in aging, which is influenced Bergondo, La Corun˜a, Spain, 4National Institute of Standards and by generation of such reactive oxygen species. [1] Baradaran et. al., Nature, Technology, Gaithersburg, MD, USA. 2013. The voltage-dependent anion channels (VDACs) are the most abundant channel-forming proteins in the mitochondrial outer membrane, controlling 767-Plat mitochondria permeability and function. Among the three known isoforms A New Synthetic FRET Sensor to Analyze Allosteric AMPK Activation in mammals, VDAC1 is the most abundant and best characterized one, and and Cellular Energy State is known to be involved in various mitochondria-associated . Uwe Schlattner1, Martin Pelosse1, Imre Berger2. VDAC1 forms highly conductive anionic channels open to metabolites and 1LBFA, Inserm U1055, Univ. Grenoble Alpes, Grenoble, France, 2School of respiratory substrates, such as ATP and ADP. VDAC3 is the least abundant Biochemistry, University of Bristol, Bristol, United Kingdom. isoform in most cell types, poorly characterized, and of almost unknown AMP-activated protein AMPK senses and regulates cellular energy physiological function. Despite high sequence similarity with VDAC1 sug- state. AMPK activation by increasing AMP and ADP concentrations involves gesting a similar pore-forming ability, VDAC3 reconstituted in planar lipid a conformational switch within the heterotrimeric complex. This is exploited membranes (PLMs) was reported to form low-conducting, unstable channels. here for the construction of a synthetic sensor of cellular energetics and allo- Here we report that recombinant human VDAC3 reconstituted into PLMs steric AMPK activation, AMPfret. Based on engineered AMPK fused to fluo- forms highly conductive (G = 3.9 5 0.4 nS in 1 M KCl), weakly anion- rescent proteins, the sensor allows direct, real-time readout of the AMPK selective (PCl-/PKþ = 1.3 in 1/0.2 M KCl), and voltage-gated channels. These conformational state by fluorescence resonance energy transfer (FRET). AMP- features, typical of VDAC1, indicate that the VDAC3 high-conducting state fret faithfully and dynamically reports the binding of AMP and ADP to the is permeable to metabolites. We show that the known cytosolic regulators of þ AMPK g-CBS sites, competed by Mg2 -free ATP. The FRET signal correlates VDAC1, tubulin and alpha-synuclein, interact with VDAC3 channel, with activation of AMPK by allosteric mechanisms and protection from inducing characteristic (millisecond) blockages with 60% reduction of the dephosphorylation, attributed here to specific CBS sites, but does not require open-state current. Notably, the interaction on-rates are reduced about 100 AMPK activation loop phosphorylation. Moreover, AMPfret detects binding times compared to VDAC1, suggesting a differentiated physiological role of pharmacological compounds to the AMPK a/b-ADaM site thus enabling of VDAC3. Markov state modeling of the interaction dynamics indicates activator screening. Cellular assays demonstrate that AMPfret is applicable qualitatively similar behavior to VDAC1, with some isoform-specific varia- in vivo for spatiotemporal analysis of energy state and allosteric AMPK tions. Cysteine residues in VDAC3 were hypothesized to account for protein activation. inability to form typical VDAC open channels; however, we found that an engineered cysteine-less hVDAC3 mutant forms functional channels with 768-Plat similar basic properties as VDAC3-WT, but with a higher insertion rate How the Nanoarchitecture of Cardiac Muscle Mitochondria Affects Func- into PLM and a higher interaction on-rate with alpha-synuclein. Thus, our tion: Lessons from Computer Simulations results ‘‘rehabilitate’’ a previously overlooked role of VDAC3 in mitochon- Carmen A. Mannella1, Zheng Liu2, Chyongere Hsieh3, Nasrin Afzal4, drial physiology. Raquel A. Adams4, M. Saleet Jafri1,4, W. Jonathan Lederer1. 1Ctr Biomed Eng/Tech, Univ Maryland, Baltimore, MD, USA, 2Tongji Univ 770-Plat Sch Med, Shanghai, China, 3DEM Facility, Wadsworth Center, Albany, NY, Molecular Link between MCU and MRS2P Channels for Mitochondrial USA, 4Sch Syst Biol, George Mason Univ, Fairfax, VA, USA. Ion Homeostasis and Energy Mitochondria share a fundamental design: an outer membrane (OM) sur- Shanmughapriya Santhanam1, Xueqian Zhang1, Jianliang Song1, rounding a topologically complex inner membrane (IM) that in turn encloses Joseph Y. Cheung1, Peter Basile Stathopulos2, Muniswamy Madesh3. a dense matrix. Folds in the IM define compartments (cristae) that are con- 1Center for Translational Medicine, Temple Univ, Philadelphia, PA, USA, nected to the peripheral IM (adjacent to OM) by narrow junctions. Cristae 2Department of Physiology and , Western University, London, can vary greatly in number and shape, sometimes between mitochondria ON, Canada, 3Department of Medicine, University of Texas Health San within the same cell. For example, in cardiac myocytes interfibrillar mito- Antonio, San Antonio, TX, USA. chondria contain closely packed lamellar cristae, while most mitochondria Ca2þ has been shown to modulate mitochondrial metabolism through the acti- 2þ 2þ on the cell periphery have tubular cristae. Whether this structural heteroge- vation of Ca -dependent dehydrogenases. [Ca ]m uptake is facilitated by the neity has a functional basis (adaptation of the IM to stimuli such as local en- large electrochemical gradient across the inner mitochondrial membrane and ergy demand or oxidative stress) is unknown, as is whether the extreme mediated by the Mitochondrial Calcium Uniporter (MCU). MCU is a hetero- topologies are related by remodeling or formed de novo. We are using elec- oligomeric complex and known to be regulated by several of its interacting tron microscopic tomography to create a database of mouse cardiac mito- partners MICUs, MCUR1, EMRE and MCUb. But there is a lack of knowledge

BPJ 9328_9339 156a Monday, March 4, 2019 on the exact molecular mechanism of MCU regulation. Our recent structural Energy-starvation and redox imbalance are two characteristics of heart fail- insight of the N-terminal domain revealed a b-grasp-like fold containing the ure (HF), but it is not clear whether these characteristics are related and, if MCU regulating acidic patch (MRAP) that binds Mg2þ/Ca2þ with mM affin- so, by what common molecular/metabolic mechanism. Distinct creatine ki- ity, destabilizes MCU, shifts self-association equilibrium to monomer and at- nase (CK) isoforms occur in the cytosol and the mitochondria and they 2þ 2þ tenuates [Ca ]m uptake. The weak binding affinity for Mg is well suited both generate ATP by transferring the phosphoryl group between creatine for the regulation of MCU as mitochondrial matrix have higher Mg2þ concen- and ADP. The CK reaction is impaired in human and experimental HF where tration (0.2-2 mM). Our recent identification of Mg2þ as regulator of MCU reduced CK activity, creatine and creatine phosphate levels are observed. channel, intrigued us to explore the molecular link between MCU and the un- However, it is not known whether the impaired CK system in HF contributes studied Mg2þ selective transporter, Mrs2p for mitochondrial ion homeostasis to increased oxidative stress. Here, we tested whether over-expressing mito- and bioenergetics. To establish the link between MCU and Mrs2p, we gener- chondrial CK (Mt-CK) that sits at the intersection of ATP and reactive oxy- ated a CRISPR/Cas9-mediated Mrs2p global model. Mito- gen species (ROS) production attenuates alterations in cardiac energy 2þ chondrial Mg channel activity (IMrs2p) was measured in mitoplast obtained metabolism and redox balance in mice prone to pressure overload-induced from cardiomyocytes isolated from WT and Mrs2p-/- mice by adopting our HF. Following transverse aortic constriction (TAC) to generate HF, WT well devised patch-clamp experiments. Knocking out Mrs2p significantly abla- mice displayed a marked rise in ROS (þ93% vs sham, n=13, p<0.01) as 2þ ted IMrs2p validating Mrs2p as an authentic mammalian mitochondrial Mg measured by electron paramagnetic resonance (EPR) in isolated left ventri- 2þ tg channel. Additionally, simultaneous measurement of [Ca ]m uptake and cles (LVs). This effect was significantly attenuated in TAC Mt-CK mice DJm was performed in cardiomyocytes isolated from WT and Mrs2p-/- (þ32% vs sham, n=13, p=NS). In WT hearts, Mt-CK transgenesis upregu- 2þ mice. In line with ablated IMrs2p MCU-mediated Ca uptake was increased lated antioxidant enzymes, such as thioredoxin reductases (TrxR). When sub- in Mrs2p-/- myocytes, reinforcing the concept of Mg2þ-dependent MCU regu- jected to TAC, Mt-CKtg mice showed significantly enhanced TrxR activity lation. Conversely, loss of MCU did not alter the IMrs2p, suggesting a cation compared to WT (0.6350.02 vs. 0.5150.01 mU/ml, p<0.001). This dependent MCU regulation that is consistent with other Ca2þ channels biochemical/molecular adaptation is functionally relevant because LV myo- tg including L-type, RyRs, IP3Rs, and CRAC. cytes isolated from Mt-CK were more resilient to either H2O2 or isoproter- enol (ISO)-induced irreversible arrhythmias/function cessation than WT 771-Plat cells, both at baseline and after TAC. Thus, the Mt-CK system is a novel Mitochondrial Megachannel Resides in Monomeric ATP Synthase node whereby cardiac energetic and redox regulation intertwine. This evi- Nelli Mnatsakanyan1, Han-A Park2, Wu Jing1, Marc C. Llaguno1, dence offers a new platform for treating energy-deprived and redox- Besnik Murtishi1, Maria Latta1, Ellie Davis1, Paige Miranda1, imbalanced failing hearts. Youshan Yang1, Fred Sigworth1, Elizabeth A. Jonas1. 1Yale University, New Haven, CT, USA, 2The University of Alabama, Tuscaloosa, AL, USA. Platform: Microtubules, Structure, Dynamics Mitochondrial ATP synthase has been shown recently to be vital not only for cellular energy production but also for energy dissipation and cell death. We and Associated Proteins identified and characterized a large non-selective uncoupling channel within 773-Plat the ATP synthase c-subunit ring, the persistent opening of which initiates Severing Enzymes Amplify Microtubule Arrays through Lattice cell death. We have continuing evidence for the crucial role of this channel GTP-Tubulin Incorporation in mitochondrial permeability transition (mPT). We have now purified ATP Annapurna Vemu1, Ewa Szczesna1, Elena A. Zehr1, Jeffrey O. Spector1, synthase from porcine heart mitochondria and performed single-channel Nikolaus Grigorieff2, Alexandra M. Deaconescu3, Antonina Roll-Mecak1,4. studies. Excised proteoliposome patch-clamp recordings demonstrate that 1 National Institute of Neurological Disorders and Stroke, NIH, Bethesda, highly pure and fully assembled ATP synthase monomers form large conduc- 2 2þ MD, USA, Howard Hughes Medical Institute, Janelia Farm Research tance, Ca -sensitive and voltage-gated channels. We confirmed the mono- 3 Campus, Ashburn, VA, USA, Department of Molecular Biology, Cell meric state of ATP synthase by cryo-electron microscopy studies of ATP Biology, and Biochemistry, Brown University, Providence, RI, USA, synthase reconstituted into proteoliposomes. We have also heterologously 4 National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA. overexpressed and purified human ATP synthase c-subunit from E. coli plasma Spastin and katanin sever and destabilize microtubules. Their mutation causes membranes. We show that human c-subunit purified from bacteria forms large debilitating diseases. Paradoxically, despite their destructive activity they in- conductance channels identical to those purified from HEK-293 cells. The crease microtubule mass in spindles, plant cortical arrays and neurons. channel is gated by polar amino acid residues situated at the mouth of the Combining electron and single-molecule TIRF microscopy we show that pore and by the hydrophilic F portion of ATP synthase. We find that dissoci- 1 the elemental step in microtubule-severing is the generation of nanoscale ation of ATP synthase F from F occurs when we expose primary hippocam- 1 O damage throughout the microtubule by active extraction of tubulin hetero- pal neurons to glutamate toxicity, suggesting that the non-reversible dimers. These damage sites are repaired spontaneously by GTP-tubulin incor- dissociation of F from F is pathological. We have successfully knocked 1 O poration. As a result, the microtubule shaft is rejuvenated with GTP-tubulin out five of the six alleles of the three genes encoding ATP synthase c-subunit islands that stabilize it against depolymerization and newly severed ends in mouse embryonic stem cells by CRISPR-Cas9, which resulted in only emerge with a high-density of GTP-tubulin that protects against depolymer- 10% of the total c-subunit expression. Patch-clamp recordings of mitoplasts ization. Consequently, spastin and katanin increase microtubule rescue fre- isolated from these cells demonstrate low conductance activity that is poorly quency without affecting growth rates and catastrophe. The stabilization of calcium responsive. These findings confirm that the largest of all inner mito- the newly severed plus-ends and the higher rescue frequency synergize to chondrial membrane conductances resides within the ATP synthase monomer, amplify microtubule number and mass. Thus, severing enzymes regulate more specifically within its membrane-embedded c-subunit ring. microtubule architecture and dynamics by promoting GTP-tubulin incorpora- tion within the microtubule shaft. 772-Plat Mitochondrial Attenuates ROS Emission and Improves 774-Plat Myocyte Survival after ROS in the Failing Heart Direct Observation of Individual Tubulin Dimers Binding to Growing Gizem Keceli1, Joevin Sourdon1, Ashish Gupta1,2, Carlo G. Tocchetti3, Microtubules Bongsoo Park4, Jacopo Agrimi1, Michelle Leppo1, Keith J. Mickolajczyk1,2, Elisabeth Geyer3, Tae Kim3, Luke Rice3, Genaro A. Ramirez-Correa5, Shyam S. Biswal4, Nazareno Paolocci1,6, William O. Hancock2. Robert G. Weiss1,2. 1The Rockefeller University, New York, NY, USA, 2Biomedical 1Division of Cardiology, Johns Hopkins School of Medicine, Baltimore, MD, Engineering, Penn State Univ, University Park, PA, USA, 3Biophysics, USA, 2Department of Radiology, Division of Magnetic Resonance Research, University of Texas Southwestern Medical Center, Dallas, TX, USA. Johns Hopkins School of Medicine, Baltimore, MD, USA, 3Department of The biochemical basis of microtubule growth has remained elusive for over Translational Medical Sciences, University of Naples Federico II, Naples, thirty years despite being fundamental for both and asso- Italy, 4Department of Environmental Health and Engineering, Johns Hopkins ciated chemotherapy strategies. Here, we combine interferometric scattering Bloomberg School of Public Health, Baltimore, MD, USA, 5Department of microscopy with recombinant tubulin to monitor individual tubulins binding Pediatrics, Division of Cardiology, Johns Hopkins School of Medicine, to and dissociating from growing microtubule tips. We make the first Baltimore, MD, USA, 6Department of Biomedical Sciences, University of direct, single-molecule measurements of tubulin on- and off-rates. We detect Padua, Padua, Italy. two of transient dwell times, and determine via binding-interface

BPJ 9328_9339 Monday, March 4, 2019 157a mutants that they are separated by the formation of inter-protofilament 777-Plat bonds. Applying a computational model, we find that slow association ki- Active Fluctuations of Microtubule Networks Facilitate Faster Motility of netics with strong interactions along protofilaments best recapitulate our Dynein data, and furthermore predict plus-end tapering. Overall, we provide Yasin Ezber. the most direct and complete quantification of how microtubules grow to Physics, UC Berkeley, Berkeley, CA, USA. date. Dyneins are molecular motors that produce force and motility towards the microtubule (MT) minus-end for essential cellular processes, such as cargo 775-Plat transport, cell division and ciliary beating. Dynein favors release from MT un- Computational Modeling and Cryo Electron Tomography Reveal a New der assisting forces and resists MT detachment under hindering force. It re- Mechanism for Microtubule Assembly and Dynamics mains unclear whether this asymmetric force-detachment affects dynein Nikita B. Gudimchuk1,2, Evgeni V. Ulyanov1, Eileen O’Toole3, motility under active fluctuations of the MT network. Here, we used optical Dmitrii S. Vinogradov2, Fazly I. Ataullakhanov1,2, J. Richard McIntosh3. trap to exert oscillatory forces on yeast cytoplasmic dynein as it transports 1Physics Department, Lomonosov Moscow State University, Moscow, cargo beads and glide MTs in vitro. Without ATP, force oscillations drive Russian Federation, 2Center for Theoretical Problems of Physico-Chemical minus-end directed motility even when the motor was pulled stronger towards Pharmacology, Russian Academy of Sciences, Moscow, Russian Federation, the plus-end. Dynein moves faster as the amplitude of force oscillations was 3Molecular, Cellular and Developmental Biology Department, University of increased. With ATP, force oscillations increase the speed of cargo transport Colorado, Boulder, CO, USA. by single dynein and MT gliding by multiple dyneins. We conclude that active Microtubules (MTs) are tubulin whose dynamic instability is essen- fluctuations in the cytoplasm facilitate faster motility of dynein towards the tial for many cellular processes. The propensity of tubulins to polymerize minus-end. into MTs depends on an associated : GTP vs. GDP. However, despite decades of research, it remains unclear how and to what extent the 778-Plat affect tubulin conformation and/or inter-tubulin bonds. Using Oxidative Stress Restructures the Cellular Microtubule Cytoskeleton via cryo electron tomography, we have recently shown that the tips of growing Repair-Mediated Rescue Events and shortening MTs both exhibit curved protofilaments (PFs) of very similar Rebecca R. Goldblum1, Kyle White1, Mark McClellan1, curvatures. Based on that observation, we have proposed a Brownian dy- Joseph M. Metzger2, Melissa K. Gardner3. namics model and a new mechanism for MT assembly in which MT elonga- 1Biochemistry/Biophysics, Univ Minnesota, Minneapolis, MN, USA, tion results from the straightening of curved GTP-bound PFs due to thermal 2Department of Integrative Biology and Physiology, Univ Minnesota, fluctuations and the formation of lateral bonds between tubulins in adjacent Minneapolis, MN, USA, 3Genetics, Cell Biology, and Development, Univ PFs. Here we provide both experimental and theoretical evidence supporting Minnesota, Minneapolis, MN, USA. that model. We have examined model behavior as a function of its key pa- Microtubules are cylindrical cytoskeletal polymers composed of a/b-tubulin rameters, such as tubulin bending stiffness, the strengths of lateral and lon- heterodimers that make up an ordered tubulin lattice. Microtubules display gitudinal bonds, and the concentration of soluble tubulin. Calibrated, the dynamic length changes, termed ‘‘dynamic instability’’, in which slow elon- model correctly describes the amount of force that can be generated by a sin- gation phases are followed by rapid shortening. In cells, microtubules form a gle MT and the dependence of MT assembly rate on soluble tubulin concen- network that is a key component of the cellular cytoskeleton. Under patho- tration. The shapes and lengths of PFs at growing MT tips are strikingly logical conditions of oxidative stress, we and others have found that cardio- similar at different tubulin concentrations - a model prediction that we myocytes display a denser microtubule cytoskeleton, which may lead to the have verified by cryo-electron tomography. Modest changes in lateral bonds progressive structural and functional cellular changes associated with between tubulins in simulations are sufficient to drive a switch from myocardial ischemia and systolic dysfunction. This reorganization of the assembly to disassembly, whereas the longitudinal bonds largely determine microtubule network occurs despite only small increases in tubulin expres- the lengths of curved PFs at the MT tip. This revised view of growing MT sion, suggesting that alterations to microtubule length regulation are tip structure and MT dynamics has important implications for our under- involved. Using biophysical reconstitution experiments and live-cell imag- standing of MT regulation and the mechanics of MT attachment to ing, we found that oxidative stress directly provokes microtubule ‘rescue,’ kinetochores. the transition from rapid microtubule shortening to slow elongation, while it has little effect on other microtubule dynamic instability parameters or 776-Plat the microtubule nucleation rate. To explore a mechanism for this observa- Dynamic Instability and Treadmilling Coexist for In Vitro Microtubules tion, we used electron microscopy, and observed structural damage, consist- Goker Arpag, Marija Zanic. ing of holes and sheet-like structures, under conditions of moderate oxidative Cell and Developmental Biology, Vanderbilt University, Nashville, TN, stress. Further, using a quantitative two-color tubulin ‘‘repair’’ assay (Reid et USA. al, 2017), we found that repair of structural defects within the microtubule Microtubules are cytoskeletal polymers that play essential roles during multi- lattice via the incorporation of free tubulin was 133% higher under condi- ple cellular processes. Dynamic nature of these polymers allows them to tions of oxidative stress compared to controls. Such repaired regions have remodel the microtubule network spatially and temporally to fulfill their spe- been termed ‘rescue islands’ because they facilitate rescue events, and thus cific duties. Dynamic instability is a well known microtubule behavior in promote net microtubule elongation (Dimitrov et al., 2008). We conclude which both ends independently switch between phases of growth that microtubule structural damage may explain our observations of oxida- and shrinkage. Earlier studies observed that microtubules can exhibit another tive stress-mediated increases in microtubule density in cardiomyocytes, mode of dynamics, a phenomenon called treadmilling, in which the growth potentially providing insight into the progressive myocardial changes that rate at one end is comparable to the shrinkage rate of the other end. While accompany Ischemic Heart Disease. microtubule dynamic instability has been widely studied, the conditions that lead to microtubule treadmilling are not well understood, and the relations be- 779-Plat tween these two modes of microtubule dynamics are not well characterized. Tau’s Proline Rich Region Dominates Tubulin Binding Here, we show that the dynamic instability and treadmilling of individual Kristen McKibben1, Elizabeth Rhoades2. polymers coexist in vitro around 6uM tubulin in solution in the absence of 1Biochemistry and Molecular Biophysics Graduate Group, Univ microtubule associated proteins. We investigate different time scales to char- Pennsylvania, Philadelphia, PA, USA, 2Department of Chemistry, Univ acterize the dynamic behavior of microtubules by calculating the net growth/ Pennsylvania, Philadelphia, PA, USA. shrinkage rate within the given time window and find that the microtubules Tau is an intrinsically disordered, microtubule associated protein best known exhibit treadmilling episodes in either direction, as well as growth or for its role neurodegenerative tauopathies such as Alzheimer’s disease. Tau’s shrinkage at both ends. The treadmilling behavior arises due to the bias in physiological roles include stabilizing microtubules and regulating microtubule the dynamic instability for a given time window, resulting in net shrinkage dynamic instability. Considerable attention has been given to tau’s microtubule at one end and net growth at the other end. We find that, with tubulin alone, binding region (MTBR), and more recently to its MTBR-flanking R’ region, treadmilling towards the minus ends is more likely than towards the plus ends and their role in tau binding to both microtubules and soluble tubulin. N-termi- within time segments varying from 45 seconds to 10 minutes. By focusing on nally flanking the MTBR is the proline rich region (PRR), which is widely the microtubule dynamics at different time scales, this study provides a deeper accepted as critical for tau-mediated polymerization of microtubules and also understanding of the relations between dynamic instability and microtubule cited as demonstrating weak microtubule binding. Here, we investigate the treadmilling. role of the PRR in tau binding to soluble tubulin. We find that the PRR

BPJ 9328_9339 158a Monday, March 4, 2019 dominates binding of tau to soluble tubulin. The PRR has two equivalent bind- centration of macromolecules can exceed 300 g/L. One such interaction that ing sites for soluble tubulin dimers, and surprisingly, competes with the occurs along the cellular membrane, is between a globular 7-kDa SH3 domain MTBR-R’ for binding to soluble tubulin suggesting at least partial overlap and a 25-kDa intrinsically disordered region of Son of Sevenless (SOS). of the binding sites in these separate regions. In the absence of the MTBR, Despite its importance as a mediator in the mitogen-activated PRR is capable of binding microtubules and polymerizing tubulin, albeit signaling pathway present in all eukaryotic cells, most biophysical character- weakly. Collectively, our data suggests the PRR region is an important izations of this complex are performed in dilute buffered solutions where coso- binding motif for the soluble tubulin pool. Interestingly, this domain is not lute concentrations rarely exceed 10 g/L. We are investigating the effects of visible in the recent cryo-EM structure of microtubule-bound tau. Future crowding, or high concentrations of physiologically-relevant cosolutes, on work will focus on describing the relationship between PRR and MTBR-R’ the equilibrium thermodynamics and kinetics of binding between SH3 and on the microtubule. SOS. The SH3 domain is labeled with a fluorine atom on its sole , allowing the interaction to be monitored with 19F NMR. Subsequent NMR line- 780-Plat shape analysis allows quantification of both rate constants, and hence the equi- Mechanisms of Bidirectional Transport of Misaligned Chromosomes in librium constant. Two systems are used. The first system, a control, comprises Mitosis the SH3 domain and a SOS-derived peptide. Experiments using a range of Saad Ansari1, Zachary Gergely2, Christopher Edelmaier1, biologically-relevant molecules reveal that the kinetics are affected more Nicolas Santander1, Patrick Flynn1, Adam Lamson1, Matthew A. Glaser1, than the stability of the complex. The origins of these effects are likely J. Richard McIntosh2, Meredith D. Betterton1. increased viscosity and electrostatic interactions with crowder molecules. 1 2 Physics, University of Colorado Boulder, Boulder, CO, USA, Molecular, The second system is that between SH3 and the full-length disordered region Cell and Developmental Biology, University of Colorado Boulder, Boulder, of SOS. Crowding effects are expected to be larger in magnitude than those CO, USA. observed for the peptide studies based on hypotheses from traditional crowding In eukaryotic mitosis, chromosomes must align in the center of the spindle for theory. These experiments are the first to analyze the effects of crowding on a accurate chromosome segregation. Across many , lost or poorly posi- protein-intrinsically disordered and have immediate implica- tioned chromosomes are transported first toward the spindle pole, then away tions for understanding the effects of the cellular environment on protein- from the pole toward the midplane of the spindle. This bidirectional chromo- protein interactions. some transport is essential for proper congression; it depends on microtubule dynamics and kinetochore motors and proteins. For bidirectional transport, 783-Plat the cells need to know when to move chromosomes towards or away from A Conserved Asparagine in a Ubiquitin Conjugating Enzyme Promotes a the spindle poles. In vertebrates, phosphorylation gradients and tubulin post- Reactive Substrate Geometry translational modifications may provide directional cues, but fission yeast Isaiah Sumner1, Walker M. Jones2, Aaron G. Davis1, R. Hunter Wilson3, that lack these processes bidirectionally transport their chromosomes. Katherine L. Elliott1. Fission-yeast chromosome recapture depends on microtubule dynamics and 1Dept Chem/Biochem, James Madison Univ, Harrisonburg, VA, USA, the Dam/DASH complex, but the regulation of bidirectional movement prior 2Chemistry, University of Georgia, Athens, GA, USA, 3Chemistry, to biorientation is not well understood. We investigate bidirectional chromo- University of Minnesota, Minneapolis, MN, USA. some movements on fission-yeast monopolar mitotic spindles using quantita- Cells can rapidly respond to their environments by modifying proteins tive imaging and computational modeling to identify the contributions of involved in the relevant regulatory pathways. In one such modification, ubiq- kinetochore motors, kinetochore proteins, and microtubule dynamics to bidi- uitination, the small protein ubiquitin is attached to a lysine sidechain of a rectional chromosome transport. target protein. Lysine ubiquitination is important in DNA repair and inflam- mation response. It happens in three steps and is catalyzed by a ubiquitin acti- vating enzyme (E1), a ubiquitin conjugating enzyme (E2) and a ubiquitin Platform: Protein Assemblies/Enzyme Function, ligase (E3). The E2 enzyme catalyzes the transfer of the ubiquitin to its target Cofactors and Post-translational Modifications I protein. Here, we use three computational techniques - Born-Oppenheimer Molecular Dynamics (BOMD), single point Quantum Mechanics/Molecular 781-Plat Mechanics energies (QM/MM), and classical Molecular Dynamics (MD) - Measuring Macromolecular Size-Distributions and Interactions at High to probe the mechanism used by the ubiquitin conjugating enzyme, Ubc13. Concentrations by Sedimentation Velocity These simulations support a long-held hypothesis that Ubc13 uses a step- Sumit K. Chaturvedi1, Jia Ma1, Patrick H. Brown1, Huaying Zhao1, wise, nucleophilic attack mechanism. They also show that the formation of Peter Schuck2. a tetrahedral, zwitterionic intermediate is rate limiting. However, simulations 1NIBIB NIH, Bethesda, MD, USA, 2Lab Cell Imaging/Macro Biophys, of the N79A Ubc13 mutant contradict another popular assertion that a highly NIBIB NIH, Bethesda, MD, USA. conserved asparagine (Asn79 in Ubc13) stabilizes the charged intermediate to In concentrated macromolecular solutions, weak physical interactions control enhance the rate. In fact, calculated reaction profiles of the N79A mutant the solution behavior including particle size distribution, aggregation, liquid- show that stabilizing the intermediate actually increases the barrier to product liquid phase separation, or crystallization. This is central to many fields formation. Finally, comparisons of wild-type, N79A, N79D and H77A Ubc13 ranging from colloid chemistry to cell biology and pharmaceutical protein en- show that Asn79 helps position the substrate for nucleophilic attack and may gineering. Unfortunately, it is very difficult to determine macromolecular as- also help position a proton acceptor, allowing for the substrate lysine to sembly states and polydispersity at high concentrations in solution when become deprotonated. average interparticle distances are comparable to macromolecular dimensions, since the macromolecular distance-distribution is correlated and all motion is 784-Plat coupled through long-range hydrodynamic, electrostatic, steric, and other in- Direct Observation of Protein Translocation by the 26S teractions. Here we present a sedimentation velocity technique that, for the Erik Jonsson, Jared Bard, Erika M. Lo´pez-Alfonzo, Ellen Goodall, first time, can resolve macromolecular size-distributions at high concentra- Ken Dong, Andreas Martin. tions, by simultaneously accounting for average mutual hydrodynamic and Dept Molec/Cell Bio, Univ Calif Berkeley, Berkeley, CA, USA. thermodynamic interactions. It offers high resolution and sensitivity of protein As with the , the proteasome is a motor that shapes the of other solutions up to 50 mg/ml, extending studies of macromolecular solution state proteins in the cell. It handles all degradation flux from the ubiquitin protea- closer to the concentration range of therapeutic formulations, serum, or intra- some system. The precise interplay between degradation signal (ubiquitin cellular conditions. code) and motor function has remained elusive. Here we employ single mole- cule FRET to observe the interactions between an acceptor labeled proteasome 782-Plat and a donor labeled substrate. The proteasome is reconstituted from a heterol- Physiologically-Relevant Crowding Effects on the SH3-Son of Sevenless ogous expression system with non-standard amino acid reprogramming for Interaction fluorescent dye labeling. Ubiquitin chains of defined length and linkage type Samantha S. Stadmiller, Jhoan Sebastian Aguilar, Gary J. Pielak. are enzymatically loaded onto fluorescently labeled substrates that we then Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, observe engage with and translocate through the proteasome. We find that dif- USA. ferential ubiquitin linkage types confer variance in the kinetics of interaction Nearly all biological processes, including tightly regulated protein-protein in- with the proteasome, which we call an engagement profile. These engagement teractions implicated in cell signaling, occur inside living cells where the con- profiles offer us the opportunity to begin to decipher the ubiquitin code in

BPJ 9328_9339 Monday, March 4, 2019 159a addition to understanding how the motor operates in reading this code. They The surface layer (S-layer) is a crystalline protein structure surrounding the also give us glimpses of force production by the 26S proteasome, a molecular cell envelope of a variety of bacteria and nearly all archaea. While mecha- machine tuned by billions of years of evolution. nisms of production and maintenance for microbial surface structures, including lipid and polymer layers, have been described, an in vivo under- 785-Plat standing of S-layer biogenesis and growth has yet to be elucidated. Here, Characterization of the Assembly and Disassembly of Proteins we provide the first look at S-layer biogenesis in living, crescent-shaped cells Derived from Hepatitis B using two-color STimulated Emission Depletion fluorescence microscopy. Maelenn Chevreuil1, Sonia Fieulaine2, Laetitia Poncet1, Karen Perronet3, By specifically labeling the S-layer protein from C. crescentus, RsaA, we Thomas Zinn4, Eric Jacquet5, Naima Nhiri5, Stephane Bressanelli2, found that in vivo biogenesis occurs preferentially at the cell pole, the base Guillaume Tresset1. of the stalk, the division plane, and in crack-like features along the cell 1 2 Laboratoire de Physique des Solides, Orsay, France, Inst Integrative Bio body. S-layer biogenesis patterning occurs independent of the site of RsaA 3 Cell, CEA CNRS, Gif Sur Yvette, France, Institut d’optique graduate school, secretion, but requires the production of new cellular surface area. Growing 4 5 Palaiseau, France, ESRF, Grenoble, France, ICSN, CNRS, Gif-sur-Yvette, a new S-layer on cells lacking the S-layer is not spatially controlled and re- France. veals that RsaA self-associates on the cell surface and forms patches that , described as ‘‘organisms at the edge of life’’, do not have their own grow by protein addition at their perimeter. Single molecule tracking mea- metabolism and require a host cell to create multiple copies of themselves. surements indicate that RsaA monomers can diffuse on the lipopolysaccha- Despite a significant amount of work dedicated to the viral life cycles, there is ride layer to which they are non-covalently anchored until incorporated currently no physical model accounting for the dynamical pathways along which into immobile clusters. These observations suggest that protein crystalliza- proteins self-assemble around a genome to form the viral capsid. The main goal tion drives construction and maintenance of the S-layer lattice in C. cres- of this project is to elucidate the dynamical pathways for the assembly and disas- centus. As such, protein self-assembly is a generalizable mechanism for sembly of hepatitis B virus (HBV) . The wild type HBV capsid protein unsupervised extracellular surface production and maintenance across any (HBV Core) is a 183-residue polypeptide comprising an assembly domain microbial cell type. As an evolutionary driver, 2D protein self-assembly ra- (NTD, residues 1-149) and a -binding domain (CTD, residues tionalizes the exceptional sequence, structure, and species diversity observed 150-183). The NTD is necessary and sufficient for self-assembly into icosahedral for S-layers. 120-subunit capsids where the subunit is a dimer of Core. The CTD is necessary and sufficient for interaction with RNA and packaging into the capsid. Using 788-Plat Small angle X-ray scattering (SAXS), we have been probing the assembly and Quenchable Probes for Imaging Oxidative Stress in Vivo disassembly processes of empty capsids (NTD only) with salinity and chaotropic Oshini Ekanayake, Sam Scinto, Joseph Fox, Sharon Rozovsky. agent as variables. Using total internal reflection fluorescence microscopy Chemistry and Biochemistry, University of Delaware, Newark, DE, USA. (TIRFM) we study RNA packaging with fluorescently-labelled full-length Cellular oxidative stress is a result of elevated levels of reactive oxygen species Core (NTDþCTD) while using fluorescence thermal shift assay we probe the (ROS). In the cell, ROS are converted to less harmful forms such as hydrogen interaction strength between proteins. Taken together, our data allow us to char- peroxide (H2O2). H2O2 leads to protein sulfenylation, where cysteine residues acterize the transition states for self-assembly and disassembly of empty or are transiently oxidized to sulfenic acid (-SOH). Additionally, an increase in loaded viral capsids. It would be remarkable to unveil which way HBV has adop- ROS is implicated in a variety medical conditions including cancer, neurodeve- ted to ensure its survival. Understanding its mechanisms of survival is a key step lopmental diseases, cardiovascular diseases and neurodegenerative diseases. to spotting its weaknesses in order to promote the development of inhibitors of Therefore, protein sulfenylation is not only an indicator of a signaling event un- pregenomic RNA packaging against HBV. der oxidative stress but identifies the onset of said disease states. A number of compounds targeting sulfenic acids have been identified over the years 786-Plat including 5,5- dimethylcyclohexane-1,3-dione or dimedone and more recently Assembly Mechanism of Farnesylated hGBP1 Studied by Time-Resolved 9-hydroxymethylbicyclo[6.1.0]nonyne (BCN). Unfortunately, dimedone suf- Saxs and Electron Microscopy fers from slow reaction rates, while BCN lacks specificity towards sulfenic Charlotte Lorenz, Andreas M. Stadler. acids. Here we describe the use of a transcyclooctenol (TCO) as a sulfenic JCNS-1 & ICS-1, Forschungszentrum Julich,€ Julich,€ Germany. acid reactive molecule, which renders a stable and irreversible thioether adduct Proteins are optimized for interactions with other molecules and proteins, with reactive sulfenic acids. We show TCO’s ability to enter live cells. Impor- thereby forming the fundamental basis for all kind of functions in organisms. tantly, by exploiting the rapid TCO-tetrazine reaction, we can quench excess Self-assembly as in aggregation or protein phase-separation is becoming a probe in a bio-orthogonal fashion. This will prevent erroneous detection of ar- research focus as malfunctioning can have a severe impact on our lives like tefactual sulfenylation caused by any unnatural stress that might occur during in the case of Alzheimer’s disease. Our protein of interest is the human Guany- sample processing. Thus, TCO holds great promise for enabling the study of late Binding Protein 1 (hGBP1) that is known for homo-oligomerization and ROS and their implications within the cell with a variety of spectroscopic polymerization upon nucleotide activation. After post-translational attachment studies. of a farnesyl lipid as physiologically present in cells, the farnesylated hGBP1 was shown to be involved in immune responses like antimicrobial and antiviral functions by forming ‘vesicle-like structures’ in vivo that cannot be observed in Platform: Biomolecular Methods In and Out of absence of the farnesyl modification [1]. Using time-resolved ultra-small angle X-ray scattering (TR-USAXS), the polymerization in presence of nucleotides Cells was studied covering a broad size and time range at the beamline ID02 (ESRF). For cross-validation, time-resolved electron microscopy was per- 789-Plat formed at different stages of the polymerization process. The obtained real Atomic Structure of Nearly Indestructible Pili from a Hyperthermophilic space distances for the nucleation cores were used for a model refinement of Acidophile 1 2 3 the small angle scattering data. With a final model of scattering contributions Fengbin Wang , Virginija Cvirkaite-Krupovic , Joe S. Wall , 2 2 1 of monomeric proteins, nucleation cores, an interaction term and polymer scat- David Prangishvili , Mart Krupovic , Edward H. Egelman . 1Biochemistry and Molecular , Univ Virginia, Charlottesville, VA, tering, the time-dependent change of the different species are extracted and 2 3 analyzed in terms of a kinetic model for the protein polymerization. USA, Institut Pasteur, Paris, France, Dept Biology, Brookhaven Natl Lab, [1] N. Britzen-Laurent, et al., PloS one, Vol. 5 (12) 2010. Upton, NY, USA. The mechanism by which viruses infect hyperthermophilic archaeal acido- 787-Plat philes has been poorly understood. Quemin et al. (2013) showed by transmis- Protein Self-Assembly Drives Surface Layer Biogenesis and Maintenance sion electron microscopy and whole-cell electron cryo-tomography that SIRV2 in C. crescentus rod-shaped virions specifically recognize the tips of highly abundant pilus-like Jonathan Herrmann1, Colin Comerci2, Joshua Yoon3, filaments on the surface of S. islandicus. However, the identity of these pili was Fatemeh Jabbarpour1, Lucy Shapiro4, Soichi Wakatsuki1, unknown. All approaches to biochemically analyze the filaments failed, as the William E. Moerner5. filaments could never be degraded or depolymerized under a variety of extreme 1Structural Biology, Stanford Univ, Stanford, CA, USA, 2Biophysics conditions: boiling in SDS, boiling in 5M guanidinium chloride, or incubating Program, Stanford Univ, Stanford, CA, USA, 3Applied Physics, Stanford with a high concentration of pepsin for a week. We therefore were never able to Univ, Stanford, CA, USA, 4Developmental Biology, Stanford Univ, Stanford, analyze the filaments by mass spectrometry to determine the identity of the pro- CA, USA, 5Chemistry, Stanford Univ, Stanford, CA, USA. tein composition of the pili. We have been able to use cryo-EM to generate

BPJ 9328_9339 160a Monday, March 4, 2019 a 4.1 A˚ resolution reconstruction of the pili, which, combined with bioinformat- tools for cryo-EM structure comparison and assessment with ics, has allowed us to determine its amino acid sequence and build an atomic participation (challenges.emdataresource.org). The scientific community model de novo. The N-terminal portion of the protein is a homolog of the bac- responded enthusiastically with more than 90 scientists worldwide partici- terial Type 4 pilin (T4P) N-terminal domain, but in contrast to bacterial T4P, pating. Following a participants’ workshop held in October 2017 at Stanford the helix does not partially melt and is packed in the pili in a very different University/SLAC National Accelerator Laboratory, key results and recom- manner. The globular domain is mainly b-stranded, with a largely hydrophobic mendations are now collated in a 2018 special issue of the Journal of surface. Glycosylation of several surface residues can be seen in the cryo-EM . In this presentation we will describe how this round of map, and STEM mass analysis suggests a significant amount of additional Cryo-EM Challenges has stimulated important discussions that will help glycosylation that must be largely disordered and therefore not seen in the propel this field forward. reconstruction. The factors that cause these pili to be nearly indestructible will be discussed. 793-Plat A Complete Atomic Model for Flight Muscle Myosin Filament 790-Plat Hamidreza Rahmani1, Nadia Daneshparvar2, Zhongjun Hu2, Characterizing Functional States of a Model Ligand-Gated Ion Channel by Dianne Taylor3, Robert J. Edwards4, Kenneth A. Taylor3. Cryo-Electron Microscopy 1Physics, Florida State Univ, Tallahassee, FL, USA, 2Florida State Univ, Urska Rovsnik, Rebecca Howard, Bjorn Forsberg, Marta Carroni, Tallahassee, FL, USA, 3Inst Molecular Biophysics, Florida State Univ, Erik Lindahl. Tallahassee, FL, USA, 4Cell Biology, Duke University Medical Center, Biochemistry & Biophysics, Stockholm Univ, Solna, Sweden. Durham, NC, USA. Pentameric ligand-gated ion channels are important mediators of electro- The structure of muscle thick filaments is essential to muscle physiology and chemical signal transduction in the brain and other systems. Recent progress understanding different inherited myopathies. Recently, the relaxed thick fila- in protein biochemistry and structural biology has shed light on topology ment structure from the large waterbug Lethocerus indicus was reported at a and possible gating mechanisms in these channels, yet the functionally rele- resolution of 5.5 A˚ confirming a model for myosin rods packing into ribbons vant endpoints of activation and modulation remain unclear. In order to and observing four non-myosin densities ‘‘threaded’’ among the myosin obtain alternative structural insights, we optimized protein purification, rods. A larger data set taken on the DE64 detector, as well as novel efforts reconstitution, and grid preparation conditions for structure determination on sample preparations and data analysis have led to a new structure of with of a prokaryotic ion channel using cryo-electron microscopy (EM). We pre- the resolution of 4.1 A˚ in the backbone. Although we cannot resolve every sin- pared samples at various presumed distributions of active and inactive gle side-chain and the alpha-helix completely, sharpening tools like local de- states, with the goal of quantifying distinct classes within individual cryo- blur have helped us locating most of the larger side chains. We used these EM grids. Modification of carbon support, glow-discharging vapor, and side chains as guides to manually build an atomic structure without using blotting time were critical to improving particle distribution and orientation, any coiled-coil constraints, unlike the previous model. This is the first atomic and molecular simulations were harnessed to validate structural models. structure of the complete myosin molecule in its native structure, resolved This work promises to inform methods development in cryo-EM of mem- thanks to the helical symmetry of thick filaments in flight muscle. After brane proteins, and to populate the gating landscape in this important ion building this model, we placed myosin molecules back in the structure to be channel family. able to look at their interactions, specifically the known mutations on the pre- served sites between Lethocerus and Human cardiac myosin. Lethocerus flight 791-Plat muscle myosin II and human beta-cardiac myosin have substantial similarities Cryo-EM Structures Reveal Mechanisms of Activation and Inactivation in both in sequence and arrangement. Even though our model needs additional re- Bestrophin Channels finements, we looked at 200 mutation sites (150 preserved) in the myosin Alexandria N. Miller, George Vaisey, Stephen B. Long. backbone and observed that 40 relate to coiled-coil interaction and 50 mu- Memorial Sloan Kettering Cancer Center, New York, NY, USA. tations happen where two myosin molecules are interacting, and both sites are Bestrophin (BEST1-4 in humans) channels are ligand gated chloride (Cl-) preserved. Therefore, a complete structure of the Lethocerus myosin filament þ channels that are activated by calcium (Ca2 ). Mutations in BEST1 cause can entail substantial information relevant to the cardiomyopathy mutations. retinal degenerative diseases. Partly because BEST channels have no Supported by NIH. sequence or structural similarity to other ion channels, the molecular mecha- nisms underlying gating in this family of channels are unknown. Here, we 794-Plat present multiple cryo-electron microscopy (cryo-EM) structures of chicken Computational Assessment of Distance Restraint Requirements for Accu- BEST1, determined at 3.1 A˚ resolution or better, that reveal the conforma- rate Protein Structure Determination by MAS NMR tional changes that underlie the processes of Ca2þ dependent activation Ryan W. Russell1,2, Matthew Fritz1,2, Jodi Kraus1,2, Caitlin M. Quinn1, and inactivation gating. Opening of the pore is due to the repositioning of Angela M. Gronenborn2,3, Tatyana Polenova1,2. tethered pore-lining helices within a surrounding protein shell that dramati- 1Department of Chemistry and Biochemistry, University of Delaware, cally widens the ‘‘neck’’ of the pore. The neck serves as both the activation Newark, DE, USA, 2Pittsburgh Center for HIV Protein Interactions, and inactivation gates and prevents ion flow by hydrophobic block in the University of Pittsburgh School of Medicine, Pittsburgh, PA, USA, deactivated and inactivated states. The open pore comprises a 90 A˚ -long 3Department of Structural Biology, University of Pittsburgh School of aqueous vestibule and a cytosolic ‘‘aperture’’ constriction. The aperture func- Medicine, Pittsburgh, PA, USA. tions as a size-selective filter and controls anion permeability. Ca2þ binding Magic angle spinning (MAS) NMR is a powerful approach to to a ‘‘Ca2þ clasp’’ instigates pore opening and a Ca2þ-free structure reveals probe the structure and dynamics of non-crystalline proteins and large protein that, unlike voltage-gated Naþ and Kþ channels, similar molecular rearrange- assemblies at atomic resolution. However, the requirements for an accurate ments are responsible for inactivation and deactivation. The structures and structure derived from MAS NMR spectroscopy have not been systematically accompanying electrophysiological studies define the principal gating transi- quantified. Except for SH3, all MAS NMR structures reported to date were tions of BEST1 and reveal a new molecular paradigm for gating in ligand- determined using torsion angles (4/c) generated from a qualitative interpreta- gated ion channels. tion of backbone chemical shifts. To fill this gap, we have pursued MAS NMR structure calculations using synthetic distance restraints, for several test pro- 792-Plat teins with known high-resolution structures and possessing different secondary Outcomes of the Cryo-EM Map and Model Challenges structures. Our results establish the requirements for accurate structure determi- 1 2 3 Catherine L. Lawson , Andriy Kryshtafovych , Grigore Pintilie , nation with respect to the total number and type of restraints and highlight the Helen M. Berman1,4, Wah Chiu3,5. 1 essential role of chemical shift-based torsion angle predictions in generating Institute for Quantitative Biomedicine, Rutgers University, Piscataway, NJ, tighter structural ensembles. USA, 2University of California Davis, Davis, CA, USA, 3SLAC National Accelerator Laboratory, Stanford University, Stanford, CA, USA, 795-Plat 4Chemistry & , Rutgers University, Piscataway, NJ, USA, Intracellular Metal Speciation in Streptococcus sanguinis is Pivotal for 5Department of Bioengineering, Stanford University, Stanford, CA, USA. Redox Maintenance The next step in enabling cryo-EM to continue its growth as a leading Cody Murgas, Ashley K. Forney, Shannon Baker, Seon-Sook An, method for structure detemination is to create best practices in reconstruction Todd O. Kitten, Heather R. Lucas. and model building and robust community-approved structure validation Virginia Commonwealth University, Richmond, VA, USA. methods. We have sponsored two challenges to highlight the need for map Streptococcus sanguinis is an opportunistic bacterium living beneficially in the and model validation standards, and to expedite development of quantitative human mouth but forming heart valve vegetations when in the blood stream,

BPJ 9328_9339 Monday, March 4, 2019 161a leading to infective endocarditis. It has been suggested that Mn and Fe are co- based on the predictive ability of long-range pi contact frequencies mined factors in the enfeeblement of this bacterium. To delineate the abundance of from folded protein structures. Building on this, we are developing a more available metal ions relative to the intracellular protein content, we developed comprehensive predictor of IDR phase separation including contributions a protocol involving two spectroscopic techniques: Inductively Coupled from other energetic feature, such as solvation and protein cross-beta, electro- Plasma-Optical Emission Spectrometry (ICP-OES) and Electron Paramagnetic static, cation-pi, and hydrophobic contacts. Consistent with this, phase separa- Resonance (EPR) spectroscopy. Our strategy facilitates direct measurement of tion is highly sensitive to post-translational modifications that change these intact whole cell samples for metal speciation. ICP-OES enables quantification contacts. In addition, biochemical assays show significant differences in of the total metal concentration, whereas EPR allows for differentiation and RNA processing in dilute vs. condensed protein conditions. These results characterization of Fe and Mn oxidation states. Addition of the cell- provide insights into specificity in formation of biological condensates and permeable biological siderophore, deferoxamine, shifts the metal oxidation underscore the significance of phase separation in regulation of biological or- state equilibrium (from reduced to oxidized) to aid in the quantification of intra- ganization and function. cellular metal pools. Through this methodology, the influence of exogenous metals on intracellular metal speciation and oxidative maintenance in S. san- 798-Symp guinis can be determined. Previously, it was demonstrated that the SsaB lipo- A Protein Condensate Drives Actin-Independent Endocytosis 1 1 2 protein facilitates intracellular Mn accumulation, which can be correlated with Stephen Michnick , Louis-Philippe Bergeron-Sandoval , Rohit Pappu , 3 4 4 aerobic S. sanguinis virulence. We have now established that SsaACB is a Mn/ Paul Franc¸ois , Adam G. Hendricks , Allen J. Ehrlicher , Hossein Khadivi Heris4. Fe transport system. We have prepared an ATP-binding cassette transporter 1 2 knockout (DssaACB) in order to clarify the mechanism of Mn/Fe import and Biochemistry, University of Montreal, Montreal, QC, Canada, Washington University, St. Louis, MO, USA, 3McGill University, Montreal, QC, Canada, the effect of metal ion availability on the intracellular redox environment. 4 Our research indicates that SsaACB is responsible for both Fe and Mn trans- Dept BioEng, McGill Univ, Montreal, QC, Canada. port, and that Mn is pertinent for oxidative maintenance, highlighted through Endocytosis underlies intra- and extracellular material trafficking in eukary- complementary studies conducted under anaerobic conditions. Advanced otes, and is essential to protein metabolism, intercellular signaling, membrane studies analyzing SsaB structural changes and competitive metal binding are remodeling and other cell regulatory processes. Although endocytosis is usu- also under investigation to elucidate the role of SsaB and metals in the viru- ally driven by F-actin polymerization in yeast cells, membrane invagination lence of S. sanguinis which will provide insight into the health of the oral can also occur through a yet unknown actin-independent mechanism when cavity. turgor pressure is relieved. Here, we demonstrate that membrane invagination can arise from liquid-liquid phase separation (demixing) of proteins with 796-Plat prion-like domains (PLD) from the cytosol. Demixing of these proteins results Reliable Biomolecular Structural Modelling with Small-Angle Scattering in the formation of a protein condensate, which, by virtue of its composition Jill Trewhella. and viscoelastic properties, binds to and deforms plasma membrane and School of Life and Environmental Sciences, The University of Sydney, cytosol. Demonstration that phase separated condensates can perform me- Camperdown, NSW 2006, Australia. chanical work expands the repertoire of known functions of protein conden- The small-angle scattering (SAS) of X-rays or neutrons from sates to include the ability to do work at soft interfaces such as between in solution can provide (1) unique structural insights, (2) information on the the condensate and the membrane. Similar mechanisms may govern or range of potential conformations present, (3) and powerful restraints for contribute to other membrane shaping, invagination and budding processes integrative/hybrid structural modelling (IHM). The rotational and ensemble that are involved in cellular material uptake, secretion, and cell shape averaging of the signal presents multiple challenges to the reliable utilisa- remodeling. tion of SAS data: (1) demonstrating SAS profile represents the species of 799-Symp interest, (2) reliably evaluating model fits, and (3) over-interpretation and Making and Breaking the Symmetry between Sequence-Specific Confor- over-fitting of data. A community driven project has been underway for mational and Phase Behaviors of Disordered Proteins more than a decade now to define the information that should be made avail- Rohit V. Pappu. able regarding sample quality, data acquisition and reduction, presentation Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, of scattering data and validation, and modelling for biomolecular SAS ex- USA. periments. These guidelines (Trewhella et al. (2017) Acta D73, 710) provide Intrinsically disordered proteins (IDPs) feature prominently as drivers of intra- a reporting framework so that ‘‘readers can independently assess the quality cellular phase transitions that give rise to an assortment of membraneless or- of the data and the basis for any interpretations presented’’ concerning the ganelles. This talk will highlight emerging insights regarding the sequence results of bimolecular SAS experiments, including the generation or testing determinants of driving forces of IDP phase separation. The talk will focus of 3D models. The validation task forces for SAS (Trewhella et al. (2013) on the sequence encoded symmetry between the conformational properties Structure 21, 875) and for IHM (Sali et al. (2015) Structure 23, 1156) of and phase behavior of IDPs and demonstrate how this symmetry can be broken. the world-wide Protein Data Bank are additionally addressing the complex These findings necessitate theoretical advances that account for the linkage be- issues concerning the archiving and validation of IHM models that depend tween conformational fluctuations and concentration fluctuations that can be upon computational methods and disparate data from multiple techniques engendered by IDPs. (Burley et al. (2017) Structure 25, 1317). Finally, the question of how many parameters are justified by the model evidence inherent in the 800-Symp SAS profile is an important question to answer when modelling conforma- The Role of Phase-Separation in Heterochromatin tional ensembles (Potrzebowski, Trewhella and AndrQ (2018) in review Geeta Narlikar1, Serena Sanulli2, John D. Gross3, Patrick Griffin4, PLOS ). This presentation will address the recom- Mike Trnka2. mended reporting framework and its rationale, the reliable incorporation 1Biochemistry and Biophysics, University of California San Francisco, San of SAS data in IHM, and the strengths and limits of SAS in ensemble Francisco, CA, USA, 2UCSF, San Francisco, CA, USA, 3Dept Pharm Che, modelling. Univ Calif San Francisco, San Francisco, CA, USA, 4Scripps Res Inst, Jupiter, FL, USA. The gene silencing function of heterochromatin is proposed to arise in part from Symposium: Phase Separations in the Cell the ability of HP1 proteins to promote chromatin compaction. We have recently found that the heterochromatin protein HP1 promotes chromatin self- 797-Symp association and liquid droplet formation. To understand how HP1 action at Phase Separation: Prediction and Role in Biological Regulation the level regulates higher order chromatin organization we are us- Robert M. Vernon1, Brian Tsang1,2, Tae Hun Kim1, Andrew Chong1, ing hydrogen-deuterium exchange, NMR, and crosslinking mass-spectrometry Julie D. Forman-Kay1,2. to assess changes in nucleosome dynamics and conformation. We find the 1Molecular Medicine, Hospital for Sick Children, Toronto, ON, Canada, S. pombe HP1 protein, Swi6, substantially increases the accessibility and dy- 2Biochemistry, University of Toronto, Toronto, ON, Canada. namics of buried core . Restraining these dynamics impairs Swi6 medi- There is a growing appreciation of the role of phase separation of protein and ated segregation of chromatin into phase-separated droplets. We propose that nucleic acid in responsive cellular organization and formation of biomaterials. exposed nucleosomal regions participate in multi-valent interactions to drive Dynamic, multivalent interactions drive phase separation, often involving the higher-order chromatin organization of heterochromatin. Our results raise intrinsically disordered protein regions (IDRs). We have shown the signifi- the possibility that histone core dynamics more generally regulate higher- cance of planar pi:pi contacts in the propensity for IDR phase separation, order chromatin assemblies.

BPJ 9328_9339 162a Monday, March 4, 2019

organize to activate INCX. The resultant induction of slow membrane depolari- Symposium: Regulation of Cardiomyocyte þ zation activates low-voltage-activated Ca2 channels that continues the feed- Beating forward driven self-organization of coupled-clock functions to further accel- erate the rate of diastolic VM depolarization. When the extent of depolarization 801-Symp of the system reaches a critical level, L-Type Ca2þ channels activate, effecting Multimeric Protein Complexes in Regulation of Cardiomyocyte Calcium an abrupt and marked acceleration of VM depolarization that forms the rapid up- Cycling and Survival stroke of the spontaneous AP. Concurrent influx via Ca replenishes intracellular 2þ Litsa Kranias. Ca to balance Ca efflux via NCX; VM depolarization and an increase in intra- 2þ Pharmacology and Systems Physiology, University of Cincinnati College of cellular Ca during the AP activate a family of K channels, leading to VM repo- Medicine, Cincinnati, OH, USA. larization, which, in turn increases availability of NCX and HCN channel Depressed sarcoplasmic reticulum (SR) Ca-cycling, leading to decreased func- molecules for subsequent reactivation. Following re-achievement of the MDP, tion is a clinical hallmark of human and experimental heart failure. Thus, ef- self-organization of functions within the coupled-clock system is reiterated to forts have concentrated on elucidating the mechanisms contributing to drive ensuing AP cycles. Thus, your heartbeat occurs when self-organization aberrant SR Ca-homeostasis to uncover new therapeutic targets. Studies indi- of a system of chemical and current oscillators that slowly depolarized VM cate that both SERCA and phospholamban (PLN) may represent attractive tar- achieves criticality, i.e. the VM required to launch the next AP. gets. However, the initial simple view of the PLN/SERCA regulatory complex has been modified by our identification of new proteins associated with regula- 804-Symp tion of SR Ca-transport and overall Ca-homeostasis in the cardiomyocyte. One Invited Speaker: Mechanobiology of Engineered Hipsc Cardiomyocytes of them is HAX-1, the anti-apoptotic protein, which interacts with PLN and al- Beth L. Pruitt. ters the PLN inhibitory function. Acute or chronic overexpression of HAX-1 ME/BMSE/MCDB, UCSB, Santa Barbara, CA, USA. promoted PLN inhibition on the Ca-ATPase and decreased cardiomyocyte cal- Basic life sustaining functions such as breathing, circulation, and digestion are cium kinetics and contractile parameters. Accordingly, ablation of HAX-1 driven autonomously by coordinated contraction of specialized muscle cells, significantly enhanced SERCA activity and calcium kinetics. Furthermore, yet how these functions incorporate active feedback via force sensing at the the HAX-1/PLN interaction appeared to also regulate cardiomyocyte survival. cellular level is an area of active study. Meanwhile, a variety of specialized The other regulatory protein is the SR intraluminal -rich Ca-binding stretch activated receptors and mechanically mediated biochemical signaling protein (HRC), which interacts with SERCA and regulates the enzyme’s pathways have been identified. Defects in proteins of these mechanically medi- maximal Ca-transport velocity. Thus, HAX-1 and HRC may represent potential ated pathways and receptors have been implicated in disease states spanning therapeutic targets in correcting the depressed SR Ca-cycling in heart failure. cardiovascular disease, cancer growth and metastasis, neuropathy, and deaf- Furthermore, PLN activity is regulated by two phosphoproteins, the ness. Thus, understanding the mechanical basis of homeostasis (health) and inhibitor-1 of protein phosphatase 1 and the small heat shock protein 20, which defective cell renewal function (disease) increasingly requires us to consider affect overall Ca-cycling and contractility. Interestingly, recent studies indicate the role of mechanics. To study how cells and tissues integrate mechanical sig- that this multimeric SERCA/PLN-ensemble is involved in heart failure and ar- nals, we and others have developed specialized cell cultures systems and micro- rhythmias, as well as apoptosis and cell death. Thus, targeted approaches to cor- machined tools to stimulate and measure forces and displacements at the scale rect abnormalities at the SERCA/PLN complex may hold therapeutic promise of proteins and cells. Using induced pluiripotent stem cell derived cardiomyo- in heart failure. In addition, we have identified human variants in these calcium- cytes, we observe cell outputs such as morphological changes, protein expres- cycling genes that affect their ‘‘activity’’, reflecting aberrant Ca-handling and sion, electrophysiological signaling, force generation and transcriptional increased cell death. Therefore, these mutations may be used as prognostic activity in response to mechanical stimuli with an eye to understanding how or diagnostic markers for heart failure and arrhythmia development. mechanics influences progression of a genotype with known point mutations into dysfunctional phenotypes. 802-Symp Slow and Fast Time Scales in Cardiomyocyte Beating Platform: Protein Dynamics and Allostery I Ohad Cohen, Samuel Safran. Chemical and Biological Physics, Weizmann Institute of Science, Rehovot, 805-Plat Israel. Targeting Conformational Entropy to Modulate Binding Affinity Calcium oscillations are ubiquitous in many biological systems (cardiac cells, Jose A. Caro, Shannen Cravens, Kathleen G. Valentine, A. Joshua Wand. hair cells, migrating cells etc.). However, it is difficult to obtain from previous Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, models, that comprise many coupled kinetic rate equations, physical intuition USA. as to why oscillations occur and how these scale with cellular properties. We Molecular recognition by proteins is fundamental to biology. Enthalpy, the hy- show how spontaneous calcium oscillations ( 1 sec) in heart cells can be un- drophobic effect and specific interactions at the interface are often assumed to derstood in simple physical terms from a theory that coarse-grains many of the govern binding energetics, but alone cannot explain the observed consequences molecular details (time scales of milliseconds). The separation of fast and slow of ligand binding. Without knowledge of protein entropy, the thermodynamic time scales in the system can map many (non-linear) kinetic rate equations to a picture of molecular recognition is incomplete. Recent developments in nuclear single one for the slowly varying degree of freedom. We further show, both magnetic resonance (NMR) spectroscopy have provided an experimental way experimentally and theoretically, that a much slower time scale (minutes to to quantify the contribution from protein entropy to binding. We apply this hours) is hidden in the noisy dynamics of beating, and can be extracted with approach to barnase-barstar, one of the strongest protein-protein interactions careful analysis. This long time scale can be theoretically explained in terms known. Both proteins become dynamically more rigid upon binding, indicative of a beating homeostasis that works to preserve oscillations with a relatively of a large entropic penalty. Our strategy was to attempt to reduce this entropic fixed frequency over long times. penalty by introducing an intra-molecular disulfide bond far from the interface to suppress the change in dynamics. We characterized the variant protein struc- 803-Symp turally (X-ray crystallography, NMR spectroscopy), dynamically (NMR relax- Why and When Your Next Heartbeat Will Occur ation), thermodynamically (ITC, DSC), and kinetically (stopped-flow). The Edward G. Lakatta. results indicate that the engineered disulfide produces no significant structural Laboratory of Cardiovascular Science, Intramural Research Program, changes or changes in the enthalpy of binding. Instead, a quenching of side National Institute on Aging, NIH, Baltimore, MD, USA. chain dynamics and a concomitant increase in the binding free energy is Cardiac pacemaker cell automaticity is regulated by molecular self-organization observed that increases the affinity of the complex. Our work illustrates how within a coupled system of a chemical oscillators (‘‘Ca2þ clock’’), comprised of 2þ protein entropy is a fundamental determinant of molecular recognition and a Ca and other ion cycling molecules, and ion current oscillators, comprised of variable that can be manipulated to alter the affinity of complexes. Supported an ensemble of electrogenic molecules (surface membrane or ‘‘M clock’’). Both by the NIH and the Mathers Foundation. Ca2þ and M clock molecules are activated by Ca2þ and voltage cues, that also oscillate within an AP cycle. CAMP, cAMP-mediated PKA and CAMKII- 806-Plat dependent phosphorylation modulate the availability of clock molecules to E. Coli Exhibits Inter-Domain Coupling þ respond these activation cues. A crucial Ca2 clock member, the sarcoplasmic Joseph E. Rehfus, Vincent J. Hilser. þ þ reticulum (SR), functions as a Ca2 capacitor, pumping Ca2 via SERCA2 Dept Biology, Johns Hopkins Univ, Baltimore, MD, USA. and releasing Ca via ryanodine receptors, (RyR). Spontaneous RyR activation Protein molecules are dynamic and conformationally heterogeneous, and becomes evident near the time at which maximum diastolic membrane potential these properties contribute to their functions. Ensemble-based equilibrium þ (MDP) is achieved, and generates sub-sarcolemmel local Ca2 releases that measurements are not always sensitive to low- conformations or

BPJ 9328_9339 Monday, March 4, 2019 163a the kinetics that govern their fluctuations. We have applied single-molecule The proposed model spotlights the specific roles of structural fluctuations in force spectroscopy to study the free energy landscape of adenylate kinase allosteric signaling, and also explains experimental findings on engineered (AK), a three domain protein known to visit excited states defined by local un- mutations. folding of its peripheral domains (LID and AMPbd). These states have previ- ously been shown to modulate both the binding affinity and catalytic rate of 809-Plat this essential enzyme. Using entropy-enhancing mutations, we selectively Single-Molecule Fluorescence Measurements of Transient Protein Com- lower the free energy of locally unfolded states and find that LID stability is plexes Determined via Diffusion-Independent Microfluidic Mixing Johann Thurn1, Bjorn Hellenkamp2, Thorsten Hugel1. highly correlated with AK’s mechanical stability. Our results reveal tight 1 2 coupling between the LID and CORE domains. What’s more, the coupling is Dept Physical Chem, Univ Freiburg, Freiburg, Germany, Columbia LID domain specific, as destabilizing the AMPbd results in no detectable effect. University, New York, NY, USA. Our results further suggest that AK exists in at least two states, with very The dynamic assembly and disassembly of multi-protein complexes is an inte- different mechanical stabilities, interconverting with very slow kinetics. The gral part of many signaling processes. While thermodynamic parameters like heterogeneity in AK’s conformational landscape is greater than currently the equilibrium rate constants, KD’s, are relatively easily determined, the full appreciated and may play additional uncharacterized roles in the protein’s func- kinetics is much harder to access. This is particularly the case for fast kinetics tion and regulation. What’s more, these adaptations are likely not restricted to of protein complexes consisting of proteins with different diffusion coeffi- AK, and may reflect a more general mechanism of evolutionary tuning. In the cients. Commonly used diffusion based mixing approaches in microfluidics future, they may be leveraged in new strategies for the altering of protein func- are not ideal for this, because their readout is dominated by the already disso- tion via novel therapeutics or through de novo design. ciated fast diffusing species. In contrast, our microfluidic device utilizes a different, fast and homogeneous mixing approach independent of diffusion. 807-Plat Fast mixing and a long measurement channel enable us to measure dissociation A Thermodynamic View of Dynamic Allostery in a PDZ Domain Protein between timescales of ms to min. Due to the nature of the confocal single mole- Amit Kumawat1, Suman Chakrabarty2. cule method, we can also follow conformational changes during dissociation. 1Physical and Materials Chemistry Division, CSIR-National Chemical The function of this device is first shown by measuring structural propertiesand Laboratory, Pune, India, 2Chemical, Biological & Macromolecular Sciences, dissociation rates of heat shock protein 90 (Hsp90) dimers. The full potential is S. N. Bose National Centre for Basic Sciences, Kolkata, India. then demonstrated by measuring the fast dissociation of the much smaller D D Cell signaling involves a fascinatingly complex network of interacting protein model client protein 131 and the slow dissociation of the cochaperone molecules, where information might flow through protein-protein interactions Sba1 from Hsp90. or remote signaling within a single protein (allostery). We have used molec- 810-Plat ular dynamics simulations in conjunction with energy decomposition analysis Structural Dynamics Couple Substrate-Induced Allosteric Responses with to elucidate the thermodynamic basis of ‘‘dynamic allostery’’ observed in a Domain Communication in Nonribosomal Peptide Synthetases PDZ3 domain protein present in a large number of signaling protein Subrata H. Mishra1, Aswani K. Kancherla1, Kenneth Marincin1, complexes. Santrupti Nerli2, Nikolaos Sgourakis2, Daniel Dowling3, Allostery without structural changes has often been attributed to dynamics Dominique P. Frueh1. and/or entropic effects. Here we demonstrate that a ‘‘population shift’’ of 1Dept Biophys/Biophys Chem, Johns Hopkins Sch Med, Baltimore, MD, highly coordinated hydrogen bonds and salt bridges might lead to the allo- USA, 2Department of Chemistry and Biochemistry, University of California steric modulation in this protein. We also find that compared to structure Santa Cruz, Santa Cruz, CA, USA, 3Department of Chemistry, UMass based parameters like contact map, non-bonded interaction energy provides Boston, Boston, MA, USA. a more sensitive probe to subtle allosteric modulation in conformational Timely control of protein interactions is central to modulate biological activ- ensemble. We propose that dynamical effects are consequences of modi- ity such as gene activation, cell signaling or metabolism. Here, events such fication in the underlying energy landscape dominated by electrostatic as post-translational modifications and substrate or protein binding affect interactions. proteins so as to govern their interactions with partner proteins and repress Comparing the features of allosteric network due to ligand binding and evi- or promote activity. In spite of progress in identifying protagonists of molec- dences of pH dependent dynamic allostery in this protein, we envisage presence ular communication and predicting the outcome of molecular events, it has of a universal response system. This suggests that different types of external proven more challenging to determine how molecular responses within pro- perturbation might excite different allosteric pathways, but the overall mode teins translate into responses between proteins, particularly when remote of communication seems to be governed by population shift in specific interac- sites are involved within proteins. Such challenges are inherent to under- tions like hydrogen bonding and salt bridges. standing microbial, multi-domain enzymatic systems called nonribosomal peptide synthetases (NRPSs). NRPSs employ repeats of domains to cova- 808-Plat lently tether substrates onto carrier proteins (CP) and assemble them into Structural Fluctuations are Key to Allosteric Stimulation of NDV Hemag- complex products through intervening condensation or cyclization domains. glutinin-Neuraminidase The pharmaceutical properties of NRPS products (e.g. antibiotics) have Nalvi D. Duro, Sameer Varma. spurred attempts at engineering NRPSs, but dynamics between domains Cell/Micro/Molec Bio, Univ South Florida, Tampa, FL, USA. have hampered progress, as it remains unclear whether and how synthesis Newcastle disease virus (NDV) entry into host cells is facilitated by the drives domain communication. Using NMR, we demonstrate that large scale concerted action of two viral membrane proteins: Hemagglutinin- dynamics in a 52 kDa cyclization domain sense substrates tethered to partner neuraminidase (HN) that binds sialic acids (SIAs) on host cells, and a fusion CPs to promote binding and remodel binding sites for downstream partners, protein that fuses host-virus membranes. Here we examine the molecular thus revealing a path for allosteric communication involving three partner mechanism of how HN is stimulated by SIA to activate fusion proteins. domains. We provide a 3D structural description of the enzyme dynamics Comparison of the receptor-free structure of the dimeric form of HN’s recep- and demonstrate that mutations that impede injure the same dy- tor binding domain (RBD) against that bound to SIA-derivatives suggests namic allosteric network, suggesting that catalysis efficiency may be stream- that SIA will likely induce only minor structural changes within individual lined with productive domain communication and substrate recognition HN RBDs. Consequently, understanding allosteric stimulation of HN can through dynamic responses. Our results demonstrate and describe substrate be expected to require simultaneous consideration of induced changes in driven allosteric responses in NRPSs, and they exemplify how structural dy- both structure and dynamics. To accomplish this, we use molecular dynamics namics within proteins can couple substrate recognition with both local and simulations and generate conformational ensembles of HN’s RBD dimer in remote protein communication. its receptor-free and SIA-bound states. Comparison of the two ensembles shows that although SIA induces only minor changes in individual RBDs, 811-Plat it reorients the RBD-RBD interface by about 10. This reorientation is Markov State Model of Influenza Hemagglutinin Reveals Structural Basis accompanied by a constriction of the SIA binding site, an enhancement of for Group 1 Influenza Inhibition by Arbidol structural fluctuations of interfacial hydrophobic clusters, and the creation Sarah E. Kochanek, Rommie E. Amaro. of new inter-RBD salt bridges. To understand the relationship between these Chemistry and Biochemistry, UC San Diego, La Jolla, CA, USA. events, we adapt Dice, our machine learning method (Dutta et al. BJ 2016, Influenza virus infection continues to be a major healthcare issue and the com- Botlani et al. JCP 2018), to track ensemble changes during transition of bination of the strain-specificity of vaccines with the increased circulation of the SIA-bound state into the receptor-free state. Taken together, these results therapeutic resistant strains drives the need for new approaches for the preven- yield a molecular model of the initial events of allosteric stimulation of HN. tion and treatment of infection. Arbidol is a broad-spectrum antiviral with

BPJ 9328_9339 164a Monday, March 4, 2019 demonstrated activity against both group 1 and 2 influenza hemagglutinin sub- 814-Plat types. While the Arbidol binding site has been determined for group 2 hemag- Distribution of Mechanical Stress in the Cell Envelope glutinins, it remains unidentified for group 1 hemagglutinins. Further, based on Sunny Hwang1, Nicolo Paracini2, Jerry M. Parks3, Jeremy H. Lakey4, differences between group 1 and group 2 hemagglutinins, it is unknown James C. Gumbart5. whether or not Arbidol binds in a group-specific manner. Integrative modeling 1MSE, Georgia Institute of Technology, Atlanta, GA, USA, 2Newcastle techniques combining cryo-electron tomography with x-ray crystallography, University, Newcastle upon Tyne, United Kingdom, 3Oak Ridge National homology modeling, and protein-protein docking have led to the construction Laboratory, Oak Ridge, TN, USA, 4MSE, Newcastle University, Newcastle of an all-atom model of the 2009 H1N1 influenza viral coat. Using the petascale upon Tyne, United Kingdom, 5Physics, Georgia Institute of Technology, computing machine Blue Waters, we have performed molecular dynamics sim- Atlanta, GA, USA. ulations of this model. From these simulations, individual hemagglutinin trajec- The cell envelope in Gram-negative bacteria comprises two distinct mem- tories have been extracted and analyzed with the construction of a Markov state branes with a cell wall between them. There has been a growing interest in model. From our Markov state model, we present evidence of a druggable the mechanical adaptation of this cell envelope to the osmotic pressure (or pocket that is able to accommodate Arbidol. Further, this druggable site is turgor pressure), which is generated by the difference in the concentration located in an analogous position to the known group 2 Arbidol binding site, of solutes between the cytoplasm and the external environment. However, it suggesting that the binding of Arbidol to influenza hemagglutinin is not group remains unexplored how the cell wall, the inner membrane (IM), and the outer specific. Structural insights obtained here will facilitate the development of the membrane (OM) effectively protect the cell from this pressure by bearing the next generation of influenza therapeutics. resulting surface tension, thus preventing the formation of inner membrane bulges, abnormal cell morphology, spheroplasts and cell lysis.In this study, 812-Plat we have used molecular dynamics (MD) simulations combined with experi- A General Method to Design Allosteric Conformational Switches ments to resolve how and to what extent models of the IM, OM, and cell Ronald L. Koder, Peter J. Schnatz, Joseph Brisendine, Craig Liang, wall respond to changes in surface tension. We calculated the area compress- Bernard H. Everson, Cooper French. ibility modulus of all three components in simulations from tension-area iso- Dept Physics, City Coll New York, New York, NY, USA. therms. Experiments on monolayers mimicking individual leaflets of the IM Greatly increasing the magnitude of a protein’s net charge using surface and OM were also used to characterize their compressibility.While the mem- supercharging transforms that protein into a ligand-gated or counterion- branes become softer as they expand, the cell wall exhibits significant strain gated conformational molecular switch. To demonstrate this we first modi- stiffening at moderate to high tensions. We integrate these results into a model fied the designed helical bundle hemoprotein H4 using simple molecular of the cell envelope in which the OM and cell wall share the tension at low modeling, creating a highly charged protein which both unfolds reversibly turgor pressure (0.3 atm) but the tension in the cell wall dominates at high at low ionic strength and undergoes the ligand-induced folding transition values ( > 1 atm). commonly observed in signal transduction by intrinsically disordered pro- teins in biology. Due to the high surface charge density, ligand binding to 815-Plat this protein is allosterically activated up to 1300-fold by low concentrations Microdomain Structure and Mechanical Properties of Lipid Monolayers of divalent cations and the polyamine spermine. To extend this process Mimicking Red Blood Cell Membranes Under Oxidative Stress further using a natural protein, we similarly modified E. coli cytochrome Bob-Dan Lechner1, Paul Smith2, Peter C. Winlove1, Chris D. Lorenz2, 1 B562 and the resulting protein behaves in a like manner. These simple model Peter G. Petrov . systems allow us to derive and then experimentally validate a mass-action 1School of Physics, Exeter University, Exeter, United Kingdom, 2Department model for the coupled folding, binding and allosteric activation behavior of Physics, King’s College London, Strand, United Kingdom. of ligand-gated conformational switches, establishing a set of general engi- Red blood cells (RBCs) have remarkable mechanical properties that are major neering principles which can be used to convert natural and designed soluble determinants of blood flow. This is made possible by the unique structure of the proteins into allosteric molecular switches useful in biodesign, sensing, and erythrocytes. The lipid membrane accounts for the bending rigidity of RBCs . We further show that such supercharged proteins have a whereas the membrane skeleton, built mainly of a spectrin dimer network built-in ‘dynamics dial’, allowing us to manipulate the degree of disorder sparsely anchored to the lipid bilayer, regulates the shear elasticity.[1] Defor- in the protein by changing the counterion concentration. This manipulation mation of RBC is a key property to ensure efficient passage through the micro- enables to examine in unprecedented detail the effect that dynamic disorder circulation. A number of diseases such as diabetes may cause changes in the has on fundamental protein enzyme functions such as ligand binding and cat- RBC’s deformability and the physical properties of the erythrocyte plasma alytic action. membrane due to oxidative stress.[2] To reduce the complexity of the system, model membranes and lipid monolayers have been used to determine mem- brane properties.[3] We investigate the microdomain structure of synthetic Platform: Membrane Structure lipid monolayers mimicking the composition of the inner or outer leaflet of erythrocyte plasma membranes by means of the Langmuir trough technique 813-Plat coupled with fluorescence microscopy, gracing incidence X-ray diffraction Lipid Bilayer Structure Refinement with Saxs/Sans Based Restrained (GIXD) and X-ray reflectivity (XRR) to study domain structures. Surface relax- Ensemble Molecular Dynamics ation (dilational rheology) is used to quantify the dynamic interfacial rheolog- Yevhen K. Cherniavskyi1,2, D. Peter Tieleman1,2. ical properties of the lipid film and Molecular Dynamics (MD) simulations 1Biological Sciences, University of Calgary, Calgary, AB, Canada, 2Centre reveal details in monolayer organization at molecular length scales. In partic- for Molecular Simulation, University of Calgary, Calgary, AB, Canada. ular, we investigate the effect of oxidation of key membrane constituents to Small angle scattering is a powerful technique that can probe the structure of clarify the role of oxidative stress on the plasma membrane organisation and lipid bilayers on the nanometre scale. Retrieving the real space structure of lipid physical properties. We show that replacement of cholesterol by 7-ketocholes- bilayers from the scattering intensity can be a challenging task, as the fluid na- terol as main oxidation product leads to significant stiffening of the monolayer ture of lipid bilayers results in a liquid-like scattering pattern which is hard to at surface pressures above 20 mN/m, in the range of the bilayer equivalence interpret. The standard approach to this problem is to describe the bilayer struc- surface pressure (p z 30 mN/m). Fluorescence imaging was used to reveal ture as a sum of density distributions of separate components of the lipid mole- crystallite-like microdomain structures of the monomolecular layers containing cule and then fit the parameters of the distributions against experimental data. 7-ketocholesterol whereas the monolayer with non-oxidized cholesterol fea- The accuracy of the density-based analysis is partially limited by the choice of tures more circular domains. the functions which are used to describe component distributions, especially in the case of multicomponent bilayers. The number of parameters in the model is 816-Plat balanced by the need for an accurate description of the underlying bilayer struc- Phase Partitioning of Peptide Anchors in Plasma Membrane Vesicles Pre- ture and the risk of overfitting the data. Here we present an alternative method dicts Their Recruitment to B Cell Receptor Clusters in Live Cells for the small angle scattering intensity interpretation for lipid bilayers. The Sarah A. Shelby1, Ivan C. Serrano2, Kandice R. Levental2, Ilya Levental2, method is based on restrained ensemble molecular dynamics simulations, Sarah L. Veatch1. that allow direct incorporation of the scattering data into the simulations in 1Biophysics, University of Michigan, Ann Arbor, MI, USA, 2Integrative the form of a restraining potential. This approach combines the information Biology and Pharmacology, University of Texas Health Science Center, that is implicitly contained in the force field used for the simulation with struc- Houston, TX, USA. tural data from the scattering intensity and is free from prior assumptions It has long been appreciated that cell membranes are heterogeneous on length- regarding the bilayer structure. scales relevant to the protein and lipid interactions that comprise biochemical

BPJ 9328_9339 Monday, March 4, 2019 165a networks at the cell surface. Lipid bilayer membranes, including plasma mem- amino acids. We will discuss these physical properties and their consequences branes isolated from mammalian cells, are capable of separating into distinct on the gating of channels in the presence of phosphatidic acid, cholesterol and liquid phases. It is proposed that fluctuations associated with these phases bilayer asymmetry. Overall, the study sheds light on mechanisms by which comprise one mode of protein organization at the cell surface. Recent work can potentially regulate protein functioning in other interfacial signaling from the Veatch group has shown that several peptide markers of ordered events. phases in isolated giant plasma membrane vesicles (GPMVs) are also enriched in B cell receptor (BCR) clusters in living B lymphocytes imaged using super- 819-Plat resolution localization microscopy. Meanwhile, efforts in the Levental group The Importance of Glycolipid Crosslinking in Altering the Membrane have quantified the partitioning of a broad assortment of membrane anchored Curvature between liquid-ordered and liquid-disordered phases in isolated Abir Kabbani1, Krishnan Raghunathan2, Anne Kenworthy3, Wayne Lencer4, GPMVs. Here, we join forces to quantify the correspondence between peptide Christopher V. Kelly5. partitioning with respect to membrane phases in GPMVs and BCR clusters in 1Biophysics, University of Michigan, Ann Arbor, MI, USA, 2University of intact cells. To date, we observe a remarkable qualitative correspondence when Pittsburgh, Pittsburgh, PA, USA, 3University of Virginia School of Medicine, comparing the behavior of a small panel of distinct peptides probed in each sys- Charlottesville, VA, USA, 4Biophysics, Harvard University, Cambridge, tem: peptides which favor the liquid-ordered phase in GPMVs are enriched in MA, USA, 5Physics, Wayne State University, Detroit, MI, USA. BCR clusters, while peptides that prefer the liquid-disordered phase in GPMVs The AB5 toxins crosslink their membrane anchors before the complexes trans- are depleted from BCR clusters. Ongoing work is expanding to include addi- locate into highly organized heterogeneous microdomains and are trafficked. tional peptides in both systems. Overall, our studies demonstrate a correspon- Glycolipid crosslinking has been demonstrated to be a critical factor in deter- dence between phases in GPMVs and phase-like domains surrounding BCR mining the toxin phase partitioning preference. In this study, we decipher the clusters in intact cells. This work extends our previous efforts to describe the role of this pentameric binding in driving membrane structure alteration. Pre- local membrane environment that is the context for BCR signaling and suggests viously, we demonstrated that Cholera Toxin Subunit B (CTxB) can drive that partitioning in GPMVs is a valuable experimental tool for predicting nanoscale membrane budding on a quasi-single component POPC bilayer by domain composition in intact cellular membranes. Polarized Localization Microscopy (PLM). Our current work investigates the effects of lipid crosslinking and lipid phase on the membrane budding process 817-Plat driven by CTxB. First, the concentration of CTxB or its ganglioside receptor Membrane Topography Creates the Appearance of Anomalous Diffusion GM1 was varied in a POPC membrane to modulate the number of cross- Ingela Parmryd1, Jeremy Adler2, Ida-Maria Sintorn3, Robin Strand3. linked GM1s per CTxB. The induced membrane buds were large with an 1Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden, average radius of 116 þ/- 70 nm under high GM1 concentration (1%) or low 2Department of , Genetics and Pathology, Uppasla University, CTxB concentration (0.01 mg/mL). In contrast, smaller buds were detected Uppsala, Sweden, 3Department of Information Technology, Uppsala with an average radius of 50 þ/- 20 nm under low GM1 concentration University, Uppsala, Sweden. (0.3%) or CTxB concentration (0.25 mg/mL). Furthermore, no membrane topo- Cells are neither flat nor smooth, which has serious implications for prevailing logical change was detected with monovalent CTxB mutant under similar con- plasma membrane models. Several features of the models are based on interpre- centrations conditions. These results strongly implicate the multivalent binding tations of single particle tracks (SPT) on live cells. SPT reports that diffusion in of GM1s by CTxB as an important factor for the physical properties of the the plasma membrane is much slower than in artificial membranes and that the induced curvature structure. To study the role of lipid phase in the budding pro- motion is non-Brownian (anomalous): i.e. change the rate of movement over cess, we utilized a panel of synthesized GM1s with different saturation levels of time. SPTs are generally analysed using the mean squared deviation of their ceramide tails. Slight variations in the size of the induced buds were de- Euclidean distance measurements, but only with a flat and horizontal mem- tected for membranes containing 0.3% of GM1 C18:1, C18:0, or C16:1, with brane does the straight-line distance between successive locations measure average bud radii of 155 þ/- 50, 90 þ/- 40,and 85 þ/- 20 nm, respectively. the net movement. Microscopy suggests plasma membranes are folded, so a These results suggest a key dependence of the membrane curvature on the 2D analysis requires that molecules leave and rejoin the surface. That lipids toxin’s intrinsic properties. and membrane proteins leave the membrane to enter an aqueous environment is highly unlikely. 3D localizations are better, but still require molecular depar- 820-Plat ture from the membrane. To establish the magnitude of the problem we used Dyslipidemia Induced Endothelial Stiffening is Accompanied by Increased diffusion simulations to produce probability distributions. We demonstrate Membrane Tension that 2D and 3D Euclidean distances substantially underestimate diffusion on Manuela A. Ayee1,2, Irena Levitan2. non-flat surfaces and mistakenly report anomalous diffusion, resembling hop 1Chemical Engineering Department, Dordt College, Sioux Center, IA, USA, diffusion. We find that the shortest within surface distance (SWSD), the 2Department of Medicine, University of Illinois at Chicago, Chicago, IL, geodesic distance, is perfect for foldable surfaces but still underestimates USA. movement on deformed (unfoldable) surfaces. The unexpected reason is that Dyslipidemia involving an elevation in circulating levels of is a topographical features can produce both super- and subdiffusion, i.e. the key risk factor for disorders such as heart disease, diabetes, and stroke. In appearance of anomalous diffusion. Even using the SWSD, topography- the endothelium, membrane/cytoskeleton are believed to uncorrected diffusion measurements are unable to correctly report the interac- play an important role in multiple endothelial functions such as permeability tion between a diffusing species and a non-flat surface. We conclude that differ- and angiogenic potential. One effect of elevated levels is modi- entiating between topography-induced and genuine anomalous diffusion fication of the lipid composition of endothelial membranes, resulting in requires characterising the surface by simulating Brownian motion on high res- changes to membrane biomechanics. In this study, we quantify changes to olution cell surface images, followed by a comparison with the experimental the mechanical properties of the aortic endothelial layer of dyslipidemic data. Our findings challenge membrane models, regardless what methodology mice as well as the membranes of cultured human aortic endothelial cells underpin them, that fail to consider topography. (HAECs) using atomic force microscopy (AFM). This technique provides estimates of two important parameters: (i) the elastic modulus, which repre- 818-Plat sents the stiffness of the membrane/cytoskeleton complex, and (ii) the force Interaction of Lipids with Voltage-Gated Ion Channel Proteins required for membrane tether formation, reflecting membrane tension and Nidhin Thomas1, Kranthi Kiran Mandadapu2, Ashutosh Agrawal1. membrane/cytoskeleton attachment. We find significant stiffening of the 1Dept Mech Eng, University of Houston, Houston, TX, USA, 2Univ aortic endothelial layer in dyslipidemic mice as compared to wild type con- California Berkeley, Berkeley, CA, USA. trols. This result parallels our observation of significant membrane stiffening Voltage-gated channels regulate the action potential in neurons. In contrast to in HAECs exposed to pathological levels of combined low density lipopro- the traditional views, recent experimental studies reveal that in addition to the teins (LDL) and oxidized LDL. The observed stiffening in both tissues and ionic concentrations, voltage-gated ion channels are sensitive to the lipid cells is accompanied by a corresponding increase in the force required for composition. However, the physical underpinnings of these unconventional ob- membrane tether formation, signifying a change in membrane tension and servations remain poorly understood. In this study, we systematically analyzed cytoskeleton adhesion under dyslipidemic conditions. Our results suggest the interactions of the amino acids of the voltage-gated channels with bilayers that dyslipidemic conditions both alter membrane lipid composition and of different compositions to investigate the consequences of mechanical and fundamentally disrupt the biomechanical integrity of the endothelial layer, electrostatic interactions. Our study reveals that both the geometric and the including changing the /cytoskeleton attachment and the electrostatic properties synergistically regulate the local forces acting on the membrane tension.

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Platform: Computational Methods and conformations and their general 3D shapes, transition probabilities, and equi- librium probabilities. When a protein is rigid except for two loops transiting be- Bioinformatics tween opened and closed states, SHHMM would describe each state of each loop as a cluster of sub-conformations. The transition probabilities describe 821-Plat the correlation between the current state of one loop to the future state of Membrane Permeability from Conventional MD Simulations: Counting another or the same loop. Transitions vs. Bayesian Analysis Both methods can augment other techniques for studying the function of sub- Andreas Kramer€ 1, Richard M. Venable1, Eric Z. Wang2, cellular processes and highlight the role of local configurations in biomolecular Bernard R. Brooks1, Richard W. Pastor1. systems. 1NHLBI NIH, Bethesda, MD, USA, 2Bioengineering, University of Maryland, College Park, MD, USA. 824-Plat The permeation of small molecules through membranes can presently be Surface-Free Protocol for Computing PKA’s (DelPhiPKa): Applications to observed in conventional (i.e., non-enhanced) molecular dynamics simulations. Protein-Protein Interactions This contribution focuses on three important aspects of such calculations. (1) Swagata Pahari, Lexuan Sun, Emil Alexov. The advantages and disadvantages of calculating permeability by direct count- Physics and Astronomy, Clemson University, Clemson, SC, USA. ing of transition events versus Bayesian analysis based on the inhomogeneous DelPhiPKa is widely used for accurate prediction of pKa’s of ionizable resi- solubility diffusion model. (2) A new Python/Cþþ tool that speeds up a previ- dues in proteins, and . DelPhiPKa approach is unique because it ous implementation of the Bayesian analysis by two orders of magnitude and does not define the molecular surface, rather it uses a smooth Gaussian-based allows permeabilities to be extracted in a matter of seconds from a previously dielectric function throughout the space. The new development considers the generated trajectory. (3) Simulated and permeabilities of water, oxygen, and presence of salt in the modeling protocol in a non-trivial manner via desolva- ethanol through various homogeneous bilayers. The results fall short of the tion penalty term within the Boltzmann factor of Poisson-Boltzmann equation. experimental values, clearly demonstrating the requirement for accurate polar- This factor penalizes the salt ions to enter in the solute interior in a smooth izable force fields. manner without requiring sharp border between solute-solvent. Therefore, the presence of salt near is determined by a combination of 822-Plat favorable electrostatic interactions and desolvation penalty. The DelPhiPKa- Computing Long Time Dynamics using Dynamically Corrected Kinetic computed pKa’s are in very good agreement with the experimentally deter- Monte Carlo (DC-KMC) mined pKa, having RMSD of 0.74. The unique approach of Gaussian-based Animesh Agarwal, Cesar A. Lopez, Nicolas W. Hengartner, smooth dielectric results in improved pKa’s predictions compared to the stan- Sandrasegaram Gnanakaran, Arthur F. Voter. dard method of two dielectric regions. DelPhiPKa also enables the calcula- Los Alamos National Laboratory, Los Alamos, NM, USA. tions of pKa’s of polar residues such as serine, cysteine, tyrosine and There have been numerous methodologies developed over the years to . It has been demonstrated that DelPhiPKa outperforms all the other address the time scale problem associated with molecular dynamics (MD) existing methods, even the explicit water models, while benchmarking the simulations of complex biological systems. In recent years, Markov State calculated pKa’s against experimental pKa’s of cysteine. Furthermore, we Models (MSM’s) have gained prominence in computing long-time dynamics calculated the pKa’s shifts due to the mutations in the protein-protein com- from a pool of short MD simulations. However, the predicted dynamics plexes taken from the SKEMPI database. We demonstrate that significant is prone to errors since MSM attempts to model a non-Markovian jump fraction of mutations listed in SKEMPI database results in unusual pKa’s process by a Markov chain. In this work, we propose a new approach, for introduced mutation or induce pKa’s shifts of neighboring titratable dynamically corrected kinetic Monte Carlo (DC-KMC), that propagates a groups. This is discussed in term of altering pH-dependence of the binding complex system from state to state with arbitrary accuracy on both short free energy. and long-time scales, irrespective of the definition of the metastable state boundaries, and irrespective of the basis set on which the states are defined. 825-Plat This method builds on concepts introduced in accelerated MD approaches Structural Topology of Glycoprotein Surface Networks using High and multistate dynamical corrections to transition state theory. We demon- Throughput Atomistic Modeling and Graph Theory strate the robustness of our approach by reproducing the folding dynamics Srirupa Chakraborty1, Zachary Berndsen2, Cesar Lopez3, Andrew Ward2, of villin headpiece and the conformational transitions of membrane associ- Bette Korber3, Nicolas Hengartner3, S. Gnanakaran3. ated Kras-4B protein. 1Theoretical Biology and Biophysics/ Center for Nonlinear Studies, Los Alamos National Laboratory, Los Alamos, NM, USA, 2The Scripps Research 823-Plat Institute, San Diego, CA, USA, 3Theoretical Biology and Biophysics, Los Dynamic Combinatorial Analysis of Local Configurations in Molecular Alamos National Laboratory, Los Alamos, NM, USA. Dynamics Simulation: Frequent Itemset Mining and Hierarchical Hidden Glycoproteins form an important class of biomolecules, playing critical func- Markov Model tions in a wide range of biological processes, such as protein folding and Ka Chun Ho, Donald Hamelberg. immuno-regulation. They are also known to be involved in a number of dis- Department of Chemistry, Georgia State University, Atlanta, GA, USA. ease conditions, including cancer, rheumatoid arthritis and viral infections. An automatic, multi-scale, and three-dimensional (3D) summary of local con- Experimental analyses of these glycan modifications remain extremely chal- figurations of the dynamics of proteins can help to discover and describe the lenging due to their structurally heterogeneous and dynamic nature. More- relationships between different parts of proteins across spatial scales, over, conventional all-atom molecular dynamics simulations can provide including the overall conformation and 3D configurations of side chains and spatio-temporally localized dynamics but suffer from sampling issues. domains. These discoveries can improve understanding of the function and Here, we have developed a high throughput pipeline to generate extensive allosteric mechanism of proteins. Current methods are unable to effectively number of glycoprotein conformations in atomistic detail, by homology summarize 3D shapes or dynamics of local configurations across multiple modeling of the protein and de novo modeling of the glycans, using using spatial scales. the ALLOSMOD program of MODELLER suite. This template-free glycan We propose Frequent Substructure Clustering (FSC) and Subconformational modeling method optimizes an energy function given by a combination of Hierarchical Hidden Markov Model (SHHMM) to fill this gap. FSC of the CHARMM36 force-field terms and spatial harmonic restraints in Cartesian Cb of the GB3 protein identifies six clusters of co-occurring local config- space. Next, we make use of graph theory to capture the precise topological urations. The clusters localize at different regions, contribute to the overall network, pinpoint potential interaction pathways, and identify concerted conformation, and form two anti-correlating groups. The results suggest FSC behavior of the glycans. This integrated approach has been implemented to could describe dynamical relationships between different parts of proteins profile the glycan shield in the HIV envelope glycoprotein (Env), which through 3D descriptions of the frequently occurring local configurations at has one of the highest known surface glycan densities. We first validate different spatial resolutions. this integrated approach by generating a robust ensemble of soluble BG505 SHHMM consists of three layers describing transition between conformations, Env with varying levels of glycosylation depending on the processivity, conformations as combinations of sub-conformations, and sub-conformations and quantitatively comparing to cryo-EM maps, using progressive low-pass as pairwise distances. Each sub-conformation describes the 3D configurations filtering. Quantification of relative centrality of different glycan positions, of part of a molecular system. Two sub-conformations may describe distinct subnetwork features, and analyses of local communities within the network, shapes formed by the same set of atoms. SHHMM extracts clusters of sub- have aided in comprehensive global characterization of the glycan shield.

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Specifically, glycan clustering geometry and network attributes have been uti- available analytical pipelines are inadequate for handing and analyzing the re- lized to define regions of relative vulnerability on the sugar shield, which can sulting gigabytes and terabytes of high-content imaging data. We expressed be harnessed for potential vaccine design. fluorescent protein fusions of clathrin and dynamin2 at endogenous levels in genome-edited human embryonic stem cells, which were differentiated into 826-Plat hESC-derived intestinal epithelial organoids. Lattice light-sheet imaging with A Computational Human Whole-Cell Model Reveals the Effects of Spatial adaptive optics (AO-LLSM) allowed us to image large volumes of these orga- Organization on RNA Splicing noids (70 60 40 mm xyz) at 5.7 s/frame. We developed an open-source data Zhaleh Ghaemi, Zaida Luthey-Schulten. analysis package termed pyLattice to process the resulting large (60 Gb) Dept Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, movie data sets and to track clathrin-mediated endocytosis (CME) events. USA. CME tracks could be recorded from 35 cells at a time, resulting in 4000 pro- Spatial organization is a fundamental characteristic of cells, achieved by uti- cessed tracks per movie. On the basis of their localization in the organoid, we lizing both membrane-bound and non-bound . We construct a classified CME tracks into apical, lateral, and basal events and found that CME spatially resolved human whole-cell (HeLa) model from experiment-based dynamics is similar for all three classes, despite reported differences in mem- structural, morphological and reaction network data to describe the mRNA brane tension. pyLattice coupled with AO-LLSM makes possible quantitative splicing process and dynamics of splicing particles. We performed stochas- high temporal and spatial resolution analysis of subcellular events within tic simulations for up to 15 minutes of biological time of the entire cell. tissues. We find that the number of nuclear pore complexes controls the number of assembled splicing particles; that even a slight increase of splicing particle localization in nuclear speckles (non-membrane-bound organelles) Platform: Protein Structure and Conformation II leads to a disproportionate enhancement of mRNA splicing and reduction in the transcript noise; and that compartmentalization is critical for the 829-Plat yield of correctly assembled splicing particles. Our model also predicts Single-Molecule Conformational Analysis of Apolipoprotein E that the distance between genes and speckles has a considerable effect on Melissa D. Stuchell-Brereton, Berevan Baban, Greg DeKoster, Carl Frieden, the mRNA production rate, further emphasizing the importance of genome Andrea Soranno. organization around speckles. The HeLa cell model, including organelles Dept Biochem & Mol Biophys, Washington Univ St Louis, St Louis, MO, and subcompartments, provides an adaptable foundation to study many USA. cellular processes which are strongly modulated by spatio-temporal The e4-allele isoform of apolipoprotein E (ApoE4) plays a key-role in Alz- heterogeneity. heimer’s disease and cardiovascular pathologies. A large body of evidence supports that conformations of the protein are instrumental in its contribution 827-Plat to function and disease; yet, a comprehensive structural picture of ApoE4 is The Role of Cellular Replicative Lifespan and Stem Cell Dynamics on still missing, largely due to the strong aggregation/oligomerization propensity Corneal Epithelium Homeostasis and Pattern Formation of the protein, which complicates accessing the monomeric form. Further- Lior Strinkovsky, Evgeni Havkin, Yonatan Savir. more, ApoE4 contains several segments that are intrinsically disordered Faculty of Medicine Dept. of Physiology, Biophysics and , and, therefore, may be invisible to conventional structural biology methods. Technion, Haifa, Israel. In our lab, we have overcome these complications by harnessing state-of- The anterior outer transparent part of the eye, the cornea, acts as a lens that the-art single-molecule fluorescence spectroscopy and, for the first time, we focuses light into the eye. In addition, it serves as a barrier that protects the are able to access the structural ensemble of the monomeric full-length eye against external hazards and injury and thus maintaining its integrity and ApoE4 (free in solution, embedded in oligomers, and bound to lipids). Inter- its continuous regeneration is crucial for proper vision in vertebrates. Stem estingly, under native conditions, ApoE4 adopts multiple conformations - cells reside in niches at the circumference of the cornea, the limbus, which previously unidentified - that coexist in equilibrium. These conformations separates the cornea from the conjunctiva replenish and maintain corneal are highly dynamic and malleable to oligomerization and lipid binding. homeostasis. Recent lineage tracing experiments resulted in spike-like Our experiments provide a new perspective on the mechanism of lipid bind- patterns that extended from the limbus to the center of the cornea over ing and set the stage for understanding the interaction with Alzheimer’s dis- time. The mechanism that underlies these centripetal dynamics is not fully ease factors. understood. We developed a novel mathematical model that capture the stochastic dynamics of epithelial cells and pattern formation in the cornea. 830-Plat Our model capture both short and long interaction range between cells. A New Step to Elucidate Molecular Mechanisms Involved in Caries For- Moreover, we take into account two opposing models of stem cell dynamics mation: NMR Characterization of the C3 Domain from Streptococcus Mu- that have been proposed: The Hierarchical model where stem-cells are tans Adhesin P1 rare long-lived, slow-dividing cells and the Stochastic model where stem- Gwladys Riviere1, Joanna Long2, Jeanine Brady2. cells are abundant equipotent cells that divide frequently and their loss 1Biochemistry and Molecular Biology Department, University of Florida, is dictated by neutral drift. We show that the replicative life-span of the Gainesville, FL, USA, 2Department of Oral Biology, University of Florida, cells and the spatial correlation between replication and removal from Gainesville, FL, USA. the cornea play a major role in whether homeostasis can be maintained Adhesin-P1, is secreted from and attaches to the Streptococcus mutans cell without symmetry breaking signals. We derive the conditions that allow ho- wall as a key virulence factor in establishing dental caries. Recent AFM, meostasis that is consistent with biological timescales and mutants dy- ITC, solid-state NMR studies highlighted that the C-terminal domain namics. We show how the conditions for self-organizing homeostasis (C123), naturally found in saliva, interacts specifically with intact adhesin- depend on the stem cell dynamics and provide an experimental prediction P1 on the cell surface and forms amyloid fibrils. These results indicate that to discriminate between the models. The result of this study can be extended the C-terminal domain plays a vital role in cellular adhesion and in biofilm to any cellular system in which spatial homeostasis is maintained through formation. cell division. However, with sizes of 51 kDa and 185 kDa, respectively, the C123 domain and intact Adhesin-P1 are too large to achieve high-resolution data for full struc- 828-Plat tural analysis by NMR. Here, we instead report on biologically relevant inter- 4D Cell Biology: Big Data Image Analytics and Lattice Light-Sheet Imag- actions for the smaller domain of C3 with the C12 and A3VP1 domains of ing Reveal Dynamics of Clathrin-Mediated Endocytosis in Stem Cell- Adhesin P1. The binding of C3 to A3VP1 is a likely initial step in biofilm for- Derived Intestinal Organoids mation and may enable us to understand the virulence properties of S. mutans. Johannes Scho¨neberg1, Daphne Dambournet1, Tsung-Li Liu2, Using solution-NMR, we are able to characterize the structure and binding of Ryan Forster1, Dirk Hockemeyer1, Eric Betzig3, David G. Drubin1. the C3 domain to both C12, and A3VP1. NMR titration experiments with 1Dept Molec & Cell Biol, Univ Calif Berkeley, Berkeley, CA, USA, 2Howard 15N-enriched C3 domain allowed us to observe the specific binding of Hughes Medical Institute, Janelia Research Campus, Ashburn, VA, USA, A3VP1 to C3 and identify the biologically relevant binding site. We also 3Univ Calif Berkeley & Janelia Research Campus, Berkeley, CA & Ashburn, observed a specific interaction of resveratrol with C3. Resveratrol is a natural VA, USA. product known to inhibit the formation of beta-amyloid fibrils. From these ex- New methods in stem cell 3D organoid tissue culture, advanced imaging, and periments, we can propose an inhibition mechanism for the A3VP1/C3 big data image analytics now allow tissue-scale 4D cell biology, but currently interaction.

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We have also collected preliminary MAS-DNP-NMR data for C3 amyloid fi- Antigen epitope mapping provides critical information for structure-based brils, an important first step in characterizing structural transitions important therapeutic antibody design and rational vaccine design. Hydrogen exchange to biofilm formation. mass spectrometry (HX-MS) is a fast way to perform high-throughput epitope mapping – it reveals which residues on the antigen potentially interact with 831-Plat the antibody by measuring mass shift. However, unlike X-ray crystallography, Fibril Structure of Abeta40 Visualized by Cryo-Electron Microscopy and HX-MS data alone cannot provide atomic level resolution. Computational Solid-State NMR modelling is compliment to HX-MS that can help achieve high-resolution Ujjayini Ghosh, Robert Tycko. epitope mapping. Here, we incorporated HX-MS data into the Rosetta Laboratory of Chemical Physics, NIDDK NIH, Bethesda, MD, USA. antibody-antigen docking protocol as constraints. We tested HX-MS con- Amyloid fibrils are highly ordered protein aggregates, formed due to protein strained docking on four single-chain camelid antibody-antigen complexes misfolding. A striking feature of the amyloid fibrils is that these single fibril that have crystal structures available. We found that using HX-MS data signif- structure is formed by a wide variety of proteins which are associated with icantly improved the docking performance compared with no constraints. The several functions - both beneficial and pathological. The amyloid-b protein HX-MS docking protocol discriminated between native-like and non-native fibrils are found in the plaques of the Alzheimer’s disease (AD) patients. models and identified atomic resolution of epitope residues. Together with AD is a fatal neurodegenerative disease and is the main cause of dementia HX-MS data, the Rosetta-based tool we developed here can be used for in the aging population. The Ab40 peptide is the key component of the extra- high-resolution high-throughput epitope mapping. cellular deposition in brain. These structures may damage the brain irrevers- ible, give rise to neuronal dysfunction and cellular toxicity. Therefore, it is 834-Plat b interesting to study the molecular structure of A fibrils to further our under- Revealing Multiple Conformations of Proteins at Long Distances by b standing of how amyloid-like fibrils are formed on the molecular level. A - using Singular Value Decomposition Method in Pulsed Dipolar ESR fibrils are highly insoluble and they rapidly aggregate. They are also not Spectroscopy crystallizable. Therefore, solid-state NMR (ss-NMR) is suitable for deter- Madhur Srivastava, Jack H. Freed. mining the structure of amyloids. In fact, most of our understanding about Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA. amyloids till today came from ss-NMR. But how accurate these experimen- Revealing protein conformations are essential for determining protein struc- tally derived models are? To test this, we used cryo-electron microscopy to ture and dynamics, including its function and interaction with the environ- b determine the structure of a specific A 40 polymorph, whose structure has ment, for understanding disease mechanisms. However, it is challenging for already been determined by ss-NMR[1]. Cryo-EM does not require crystalli- biophysical methods to resolve multiple conformations, especially at longer zation and hence suitable for amyloids. However, cryo-EM has limited suc- distances, limiting their application to few biological systems. Pulsed Dipolar cess in case of amyloids because amyloids are structurally heterogeneous. Electron Spin Resonance Spectroscopy (PDS) is powerful method for obtain- From ssNMR, an atomic structure is determined whereas from cryo-EM a ing conformations between the distance ranges of 1 to 10 nm. In PDS, a global structure is determined. Here we have used cryo-EM to determine dipolar signal is acquired from the interaction between a pair of spin labels, b the structure of A 40 fibril, complemented with additional ss-NMR from which the distance distribution between them, P(r) may be obtained. experiments. However, due to the ill-posed nature of the inversion of the dipolar signal Molecular structural basis for polymorphism in Alz- 1. Paravastu, A.K., et al., to yield the P(r), one must resort to Tikhonov regularization or model fitting heimer’s beta-amyloid fibrils. Proceedings of the National Academy of Sci- methods to obtain reasonable results. They either compromise between good ences of the United States of America, 2008. 105(47): p. 18349-18354. resolution and stability of the P(r) or require a priori model functions to es- timate P(r), which may not accurately represent the actual distance distribu- 832-Plat tions. This is especially true if the P(r) is multimodal. We developed a new Molecular Modeling of the Alpha Globin/eNOS Complex via Crosslinking and objective approach based on singular value decomposition that yields Mass Spectrometry 1 1 1 2 an optimum approximate solution. Instead of solving for the complete dis- T.C. Stevenson Keller , Brant E. Isakson , Linda Columbus . tance distribution all at once, the method finds the optimal distribution value 1Molecular Physiology and Biological Physics, University of Virginia, 2 at each distance or distance range by determining each of their different sin- Charlottesville, VA, USA, Chemistry, University of Virginia, gular value cut-offs.2 The new method ensures optimal convergence at all dis- Charlottesville, VA, USA. tance ranges, accurately retrieving high resolution multimodal distributions Nitric oxide (NO) signaling is a key regulator of blood pressure homeostasis, representing multiple conformations.1,2 inducing vasodilation and reducing peripheral resistance. We have shown (1) Srivastava, Freed (2017) J. Phys. Chem. Lett.8, 5648 (2) Srivastava, Freed that inhibiting the alpha globin/eNOS interaction is a target for increasing (under revision) J. Phys. Chem. A NO availability, leading to blood vessel dilation. A small, ten-residue peptide mimicking an alpha globin sequence competes for eNOS binding with the full-length alpha globin, disrupting complex formation through direct binding 835-Plat to eNOS. Continued development of therapeutic small molecules targeting Coevolutionary Landscapes of Kinase Family Proteins Allan Haldane1,2, Peng He2,3, William F. Flynn2,4, Ronald M. Levy2,3. this critical protein-protein interaction will be aided by a structural model of 1 2 the binding interface of alpha globin and eNOS. Additionally, a molecular Department of Physics, Temple University, Philadelphia, PA, USA, Center model of the interaction informs the mechanism of NO signaling inhibition for Biophysics and Computational Biology (CBCB) and Institute for by alpha globin. Fluorescence polarization binding assays were used to deter- Computational Molecular Science (ICMS), Philadelphia, PA, USA, 3Department of Chemistry, Temple University, Philadelphia, PA, USA, mine the binding affinity of the alpha globin/eNOS complex. To determine the 4 eNOS motif that interacts with alpha globin, crosslinking mass spectrometry Department of Physics and Astronomy, Rutgers University, New was used. Recombinant eNOS oxygenase domain and the alpha globin mimetic Brunswick, NJ, USA. peptide were crosslinked covalently and proteolyzed with multiple proteases. The co-variation of pairs of mutations in multiple sequence alignments High resolution LC-MS is used to determine the modified residues to recon- (MSAs) of protein families can be used to build statistical ‘‘Potts’’ models struct the interaction computationally. Using recombinant full-length alpha of the sequence patterns. These models have been found to accurately pre- globin, we can validate results from the peptide/eNOS interaction and under- dict contacts in protein structures, illustrating how evolutionary fitness land- stand the molecular determinants of this protein-protein interaction. Overall, scapes of protein families can be connected to corresponding conformational the molecular understanding of this complex could lead to future therapeutics free energy landscapes. We show how a Potts model, parametrized on the to increase NO signaling for hypertensive pathologies. protein-kinase family MSA, can further be used to predict the propensity of particular kinase sequences to assume a ‘‘DFG-out’’ conformation, impli- 833-Plat cated in the susceptibility of some kinases to type-II inhibitors. We also Hydrogen Exchange Mass Spectrometry Guided Docking Generates investigate the model’s ability to describe kinase mutational statistics. We Atomic Resolution Epitopes for Single-Chain Camelid Antibody-Antigen show how the pairwise (residue-residue) interaction terms of the model Complexes are necessary and sufficient to capture the higher-than-pairwise mutation Jing Zhou1, Jeliazko Jeliazkov1, Yuqi Shi2, David Weis2, Jeff Gray1. patterns of natural kinase sequences, and can indicate which physical inter- 1Chemical and Biomolecular Engineering, Johns Hopkins University, actions contribute to the fitness and conformational preference of individual Baltimore, MD, USA, 2Department of Chemistry, The University of Kansas, sequences, and discuss the number of sequences necessary for model Lawrence, KS, USA. inference.

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836-Plat derived from an ancestral anion transport mechanism. We previously identi- Structure Based Search for Multiple Binding Sites of SOS1 PR Domain fied the structural architecture of prestin as a 7 transmembrane domain in- Recognizes an Uncovered Motif Favoring GRB2-SOS1 Association verted repeat architecture, a layout shared with SLC4 (e.g., AE1) and Tsung-Jen Liao1, Hyunbum Jang2, Ruth Nussinov3, David Fushman4. SLC23 transporters. To address the molecular mode of action of anions in 1Biophysics Program, Univ Maryland, College Park, MD, USA, 2Frederick electromechanical function, we conducted a glutamate scan of the putative Natl Lab, Leidos Biomed Res Inc, Frederick, MD, USA, 3Ctr Cancer Res anion binding site. While introduction of glutamate at most positions probed Nanobio, Natl Cancer Inst, Frederick, MD, USA, 4Department of Chemistry was disruptive for protein function, glutamate at a central position (S396E) and Biochemistry, Univ Maryland, College Park, MD, USA. was tolerated. Moreover, S396E rendered prestin anion-independent and Grb2 is an adaptor protein, containing an SH2 domain in the middle and insensitive to competitive anionic blockers. We conclude that S396E reveals nSH3/cSH3 domains at both ends. The SH2 domain recognizes a phosphory- location and coordination of substrate anions. Anion binding may enable the lated tyrosine in epidermal growth factor receptor (EGFR), and both nSH3/ voltage-dependent conformational transition by perturbing a local hydrogen cSH3 domains associate with SOS1, enhancing Ras activation. SOS1 is a gua- or electrostatic network. To gain insights into the conformational dynamics nine nucleotide exchange factor (GEF) of Ras, which exchanges GDP by underlying electromechanical activity we performed cysteine accessibility GTP. Ras mutants commonly occur in cancer. Grb2 associates with SOS1 scanning mutagenesis of transmembrane segments 3 and 10, which mainly C-terminal proline-rich (PR) domain, translocating the SOS1’s catalytic form the binding site as defined by glutamate substitution. Accessibility domain close to Ras at the plasma membrane. Previous studies suggested was essentially restricted to intracellular reagents. However, a position within that SOS1 PR domain provided only one strong binding site TM3 near the substrate binding site was accessible both from the inside and (PVPPPVPPRRRP motif) for Grb2. However, the PR motif of SOS1 does from the outside - contrary to the crystal structure of the bacterial homolog not support the simultaneous association of nSH3/cSH3-SOS1. Here, we SLC26Dg predicting inaccessibility to extracellular solutes in the inside- collaborate NMR experiment with MD simulation, uncovering a new motif open conformation. Two-sided accessibility thus indicates movement of the at the C-terminal PR domain, which favors cSH3 instead of nSH3 of Grb2. rigid TM bundle comprising TMs 3 and 10 towards the extracellular side, The structure based analysis gives an insight of searching uncovered PR bind- possibly in an elevator-like movement. We speculate that this partial move- ing motifs. ment constitutes the conformational transition underlying prestin’s electrome- chanical activity. Platform: Membrane Pumps, Transporters, and 839-Plat Mechanics of an Nramp-Family Transition Metal Transporter Exchangers Aaron T. Bozzi1, Christina M. Zimanyi1, Lukas B. Bane1, John M. Nicoludis2, Brandon K. Lee1, Casey H. Zhang1, Rachelle Gaudet1. 837-Plat 1Dept of Molecular and Cellular Biology, Harvard University, Cambridge, D On the Nature of the High-Affinity Iodide Binding Site of the Na /I Sym- MA, USA, 2Dept of Chemistry and Chemical Biology, Harvard University, porter (NIS) Cambridge, MA, USA. Silvia Ravera1, Juan P. Nicola1, Xuelang Mu1, Ignacia Echeverrı´a2, The Nramp (natural resistance associated macrophage protein) family of trans- Yuly E. Sanchez2, L. Mario Amzel2, Nancy Carrasco1. porters, found in all kingdoms of life, enables the uptake of a range of essential 1Cellular and Molecular Physiology, Yale School of Medicine, New Haven, divalent transition metals. Two mammalian Nramp paralogs are required for CT, USA, 2Hopkins School of Medicine, Baltimore, MD, USA. the dietary uptake and endosomal recycling of non-heme iron, as well as in The sodium/iodide symporter (NIS), the key plasma membrane protein that the innate immune response to intracellular pathogens. Nramps show pH actively transports iodide (I-) into the thyroid gland, couples the ‘‘uphill’’ dependence and co-transport protons, and thus have been labeled as proton- inward transport of I- against its electrochemical gradient to the ‘‘downhill’’ coupled transition metal transporters. inward translocation of Naþ down its electrochemical gradient. NIS- We have determined crystal structures of the Deinococcus radiodurans mediated I- transport is the first step in thyroid hormone biosynthesis. The Nramp homolog in four different conformations: apo inward-facing, apo - high affinity of NIS for the halide I (KM for transport 10-30 mM) is in occluded, and apo or metal-bound outward-facing. Using biochemical exper- many respects surprising, as the affinities of Cl-utilizing transport proteins iments to probe a panel of conserved protonatable polar residues, we fully for their anion are in the mM range, and it is generally accepted that proteins delineate the Nramp proton transport pathway, which originates from a bind halides with low affinity. How can the affinity of NIS for I- be so high? metal-binding aspartate and leads through a second conserved aspartate into It is clear that high-affinity binding of I- to its transporter is an evolutionary a salt-bridge network leading to the cytoplasm. We also show that the adaptation that was necessary to make it possible to capture the anion at the membrane potential affects transporter function. The combination of very low concentrations of I- found in the environment. Nevertheless, a substrate gradients and physiological voltage enforces unidirectional substrate question remains as to what are the characteristics of the site that can movement. achieve this feat. Using these structures, and results from computational approaches and þ Also, the highest affinity of NIS for I- is only achieved when Na is already biochemical and cell-based functional assays, we arrive at a working bound to the transporter (a 10-fold increase, from 220 to 22 mM), which model for the conformational change mechanism and metal selectivity of brings up a second question: how does Naþ binding increase the affinity of Nramps. Our model provides a structural basis for how separate ion pathways NIS for I-? solve the electrostatic dilemma of using a cation co-substrate to drive a These questions were addressed by identifying possible I- liganding residues cation primary substrate, while also providing further evidence of the mech- with molecular dynamics simulations, making amino acid substitutions at anistic diversity within the broader LeuT-fold family of structurally-related those positions, and investigating NIS function to elucidate the molecular transporters. requirements for I- binding and/or translocation. The picture that emerges þ from these studies is one in which I- is a hydrophobic anion, and a Na 840-Plat ion provides the charge compensation necessary to increase the affinity of A Multidrug and Toxin Efflux (MATE) Transporter Involved in NIS for I-. Aluminum Resistance is Modulated by a CBL5/CIPK2 Calcium Sensor/ Funding: GM114250 Protein Kinase Complex Julia Miller1, Alison Coluccio2, Jan Niklas Offenborn3, Anette M€ahs3, 838-Plat Jo¨rg Kudla3, Leon Kochian4, Miguel Pin˜eros2. An Electrostatic Switch for Gating the Electromechanical Activity of 1School of Integrative Plant Science, Cornell University, Ithaca, NY, USA, SLC26A5 (prestin) 2Robert W. Holley Center for Agriculture and Health, USDA-ARS, Cornell Dominik Oliver, Dmitry Gorbunov, Julia Hartmann, Dominik Lenz, University, Ithaca, NY, USA, 3Westf€alische Wilhelms-Universit€at Munster,€ Vijay Renigunta. Institut fur€ Biologie und Biotechnologie der Pflanzen, Munster,€ Germany, Department of Physiology, Philipps University Marburg, Marburg, Germany. 4Global Institute of Food Security, University of Saskatchewan, Saskatoon, SLC26 comprises a family of multifunctional anion transporters that mediate SK, USA. passive or coupled transport of small anions. SLC26A5 (prestin) stands out Members of the ALMT (Al-Activated Malate Transporter) and MATE (Multi- by functioning as a voltage-driven mechanical actuator in sensory outer drug And Toxin Efflux) families confer plant aluminum resistance on acid soils hair cells. The quasi-piezoelectric function of prestin depends on binding by mediating organic acid anion efflux, thereby immobilizing toxic aluminum of intracellular anions, suggesting that the underlying dynamics may be (Al3þ) ions in the rhizosphere. Although similar in function, ALMT and MATE

BPJ 9340_9351 170a Monday, March 4, 2019 structure and regulation are remarkably different: while ALMT transport activ- resistance to chemotherapy, and are associated with drug resistance in ity in heterologous systems is dependent on and enhanced by extracellular microorganisms. Al3þ, the MATE transporter mediates constitutive and Al3þ-insensitive trans- in planta 843-Plat port, suggesting that the MATE transport activity is modulated by D additional cellular mechanisms associated with an upstream Al3þ signaling Vacuolar H -ATPase in the Nuclear Membranes Regulate Nucleo- cascade. Functional (i.e. electrophysiological) approaches allowed us to screen Cytosolic Proton Gradients 1 2 3 for and identify the calcineurin B-like (CBL5)/protein kinase (CIPK2) complex Raul Martinez-Zaguilan , Juliana Santos , Arnoldo Facanha , Souad R. Sennoune4. as a modulator of AtMATE1 activity. Bimolecular fluorescence complementa- 1 tion analysis in Xenopus oocytes and in planta validated the specificity of the Dept Cell Physiol/Molec Biophys, Texas Tech Univ Hlth Sci Ctr, Lubbock, TX, USA, 2Mechanical Engineering, Texas Tech Univ, Lubbock, TX, USA, protein-protein interactions among CBL5, CIPK2 and AtMATE1, and suggests 3 a mechanism by which the CBL5/CIPK2 complex alters the trafficking of At- Lab. Biologia Celular e Tecidual Centro de Biosciences and Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Rio de Janeiro, MATE1 into and out of the plasma membrane. We identify CBL5-CIPK2 as 4 part of a calcium-regulated pathway involved in aluminum resistance re- Brazil, Internal Medicine, Texas Tech Univ Hlth Sci Ctr, Lubbock, TX, sponses, by which phosphorylation of the downstream target protein (At- USA. MATE1) limits unnecessary carbon loss via unregulated citrate exudation, The regulation of the luminal pH of each organelle is crucial for its function and thereby regulating the abiotic stress tolerance response in a temporal and spatial must be controlled tightly. Nevertheless, it has been assumed that the nuclear manner. pH is regulated by the cytoplasmic proton transporters via the diffusion of Hþ across the nuclear pores because of their large diameter. However, it has 841-Plat been demonstrated that ion gradients exist between cytosol and nucleus sug- The New ‘‘Patch Clamping’’ for Transporters: Oscillating Perfusion and gesting that the permeability of ions across the nuclear pores is restricted. V- Quantitative Functional Imaging of Biosensors for Transporter Functional Hþ-ATPase is responsible for the creation and maintenance of trans- Measurements membrane electrochemical gradient. We hypothesize that V-Hþ-ATPase Jacob P. Keller. located in the nuclear membranes functions as the primary mechanism to regu- Janelia Research Campus, HHMI, Ashburn, VA, USA. late nuclear pH and generate Hþ gradients across the nuclear envelope. We Transmembrane transporter proteins allow the passage of essentially all biolog- studied the sub-cellular heterogeneity of Hþ concentration in the nucleus and ically important molecules across the lipid membranes of cells and organelles cytosol using ratio imaging microscopy and SNARF-1, a pH indicator, in pros- and are therefore of central importance to all forms of life. Current methods of tate cells. Our results indicated that there are proton gradients across the nuclear transporter measurement, however, are lacking in several dimensions. Herein, a membranes that are generated by V-Hþ-ATPase located in the outer and inner method is described in which oscillating perfusion stimuli are presented to nuclear membranes. We further demonstrated that these gradients are mostly transporter-expressing cells or tissues, and activity is measured through imag- dissipated by inhibiting V-Hþ-ATPase with bafilomycin. Immunoblots and ing the corresponding oscillating fluorescence responses of intracellular fluo- V-Hþ-ATPase activities corroborated the existence of V-Hþ-ATPase in the nu- rescent sensors. This approach yields continuous temporal readouts of clear membranes. This study demonstrates that V-Hþ-ATPase is functionally transporter flux and can therefore be used to discover and measure time- expressed in nuclear membranes and is responsible for nuclear Hþ gradients dependent responses to drugs and other stimuli. This technique, called the that may promote not only the coupled transport of substrates, but also most Oscillating Stimulus Transporter Assay (OSTA), should greatly facilitate electrochemically driven events across the nuclear membranes. This study rep- both functional characterization of transporters as well as high-throughput resents a paradigm shift that the nucleus can regulate its own pH micro- screening of drugs for transporters of particular pathophysiological interest. environment, providing new insights into nuclear ion homeostasis and As such, OSTA may do for transporters what patch clamping did for ion signaling. channels. Since its initial publication in 2016, OSTA has been extended and improved in 844-Plat many respects. These include ‘‘SPR-like’’ kinetics-of-inhibition measurements Structural Insights into the Function and Auto-Regulation of Lipid of transporters in situ, space-time-variant responses of whole drosophila CNS Flippases 1,2 1,2 3 1 explants, neurotransmitter release phenomena from the serotonin transporter Joseph A. Lyons , Milena Laban , Dovile Januliene , Jakob Ulstrup , 4 4 4 SERT by releasing agents like MDMA, and others. These extensions have Cedric Montigny , Thibaud Dieudonne , Valentine Guinot , Werner Kuehlbrandt3, Guillaume Lenoir4, Arne Moeller3, Poul Nissen1,2. been forthcoming and not yet met with any roadblocks, suggesting the tech- 1 nique is robust, versatile, and extensible. Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark, 2Dandrite - Nordic-EMBL Partnership for Molecular Medicine, 842-Plat Aarhus University, Aarhus, Denmark, 3Department of Structural Biology, Allosteric Modulation of ATP Hydrolysis of the Mouse P-Glycoprotein by Max Planck Institute of Biophysics, Frankfurt, Germany, 4CEA, Saclay, Substrates and Inhibitors France. Reza Dastvan1, Smriti Mishra1, Yelena B. Peskova2, Robert K. Nakamoto2, Biological membranes are composed of bilayers, typically with highly asym- Hassane S. Mchaourab1. metric distributions of lipids that are of key importance for biomembrane func- 1Molecular Physiology and Biophysics, Vanderbilt University, Nashville, tion and dynamics, such as in secretory pathways, lipid signaling, cell-cell TN, USA, 2Molecular Physiology and Biological Physics, University of interactions, and motility. The asymmetric distributions are actively maintained Virginia, Charlottesville, VA, USA. by so-called lipid flippases and floppases that also counteract the passive scram- Efficient substrate extrusion by ATP binding cassette (ABC) efflux trans- bling of lipid gradients. Dominating contributions in eukaryotes come from porters entails coupling of substrate binding in transmembrane domains lipid flippases of the P4 subfamily of P-type ATPases, where the majority of (TMDs) to the transduction of ATP energy by protein conformational motion lipid flippases form a binary complex with a member of the Cdc50 family. in nucleotide binding domains (NBDs). Despite recent progress in deter- These transporters are involved in the inward active translocation, hence ‘‘flip- mining the structural dynamics of ABC transporters, the central question of ping’’, of phospholipids across the membrane bilayer. Central questions on P4 how substrates and inhibitors allosterically modulate the ATP hydrolysis re- ATPase lipid flippases address i) their overall architecture and function in the action has not been elucidated. Here, we use Double Electron Electron Reso- take up and flipping of lipids in membranes, and ii) how they are able to accom- nance (DEER; also called PELDOR) spectroscopy to uncover the basis of modate and transport a much larger substrate than other P-type ATPases, which substrate stimulation of ATP turnover for the mouse ABC transporter P- usually transport small cations. Additionally, some P4-ATPases are subjects to glycoprotein (ABCB1, Pgp) and illuminate the mechanism by which the auto-inhibition mediated through extended cytosolic termini, a feature that is transporter discriminates between substrates and inhibitors. Our results reveal shared with other P-type ATPases, like the plasma membrane calcium ATPase. that rate acceleration of ATP turnover by distinct transport substrates is asso- Here we present the first structures of a yeast lipid flippase, the phosphatidyl- ciated with the stabilization of a catalytically and structurally asymmetric serine (PS) specific flippase Drs2p/Cdc50p, with and without the auto- transition state of Pgp. In contrast, this transition state is negligibly populated regulatory C-terminus, thus sampling inhibited and uninhibited conformations. in the basal cycle and inhibited by binding of high affinity Pgp inhibitors. In- Comparison of an autoinhibited and a truncated, PI4P activated state show re- tegrated with a wealth of biochemical, structural and dynamic data, our re- arrangements of the cytosolic domains which transmit into flexible changes in sults lead to mechanistic principles that describe substrate coupling and the transmembrane domain. From these observations we propose a lipid sub- inhibition of Pgp. Our approach outlines a general strategy to evaluate the ef- strate transport pathway. Our results provide a structural framework to under- ficacy of allosteric modulators for multidrug ABC exporters which control stand lipid translocation between membrane leaflets and the auto-regulation of the pharmacokinetics of xenobiotics in humans, are implicated in cancer lipid flippases.

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Symposium: Chromatin Organization and 847-Symp How to Read and Write Mechanical Information in DNA Molecules Regulation: From Physical Principles to Helmut Schiessel. Lorentz Institute for Theoretical Physics, Leiden University, Leiden, Biological Phenomena Netherlands. DNA molecules contain a second layer of information on top of the classical 845-Symp genetic information. This second layer is geometrical/mechanical in nature DNA Shape Shifting as a Gene Therapy Tool and guides the folding of DNA molecules inside cells. With the help of a Jonathan M. Fogg1, Qian Wang2, Allison Judge3, Erik Stricker4, new Monte Carlo technique, Mutation Monte Carlo, we demonstrate that the B. Montgomery Pettitt5, Lynn Zechiedrich4. two information layers can be multiplexed (as one can have two phone conver- 1Baylor College of Medicine, Houston, TX, USA, 2Rice University, Houston, sations on the same wire). This allows, for instance, to guide on top of genes the TX, USA, 3Biochemistry and Molecular Biology, Baylor College of packaging of DNA into with single base-pair precision. By map- Medicine, Houston, TX, USA, 4Molecular Virology and Microbiology, ping our model onto a probabilistic model we achieve a speed-up that allows for Baylor College of Medicine, Houston, TX, USA, 5SCSBMB, Univ Texas genome-wide calculations. I will show results for genome-wide multiplexing Med Branch, Galveston, TX, USA. for baker’s yeast. In addition, genome wide studies of DNA mechanics for DNA supercoiling affects DNA metabolism yet much about how it does so is 50 different organisms taught us a simple general rule: around unknown. Using 336 bp DNA minicircles covering a range of positive to start sites DNA is stiff for unicellular life and soft for multicellular life. The negative supercoiling, we unveiled the first three-dimensional structures of reason for this difference is surprising. supercoiled DNA using cryo-electron tomography. With supercoiling, DNA can form far more bent and contorted shapes than predicted. We sought to understand how the interplay of DNA sequence and supercoiling drives Symposium: Synthetic Biology the formation of these shapes using coarse-grained simulations and biochem- ical probing. disruptions indicate regions of high bending, as local- 848-Symp ized denaturation (from base flipping) creates flexible hinges. At the same Synthetic Electrophysiology time, sharp bending at the apices of highly writhed DNA circles leads to Harry McNamara, Adam Cohen. broken base pairs. Probing with nuclease Bal-31 revealed exposed bases as Harvard University, Cambridge, MA, USA. a function of supercoiling. Bal31 cleaved all the negatively supercoiled Most research in electrophysiology focuses on identifying the roles of 336 bp minicircles but the rate increased beyond a distinct negative super- component ion channels in governing the electrical dynamics of complex tis- coiling threshold. This threshold shifted to more negative supercoiling for sues such as heart or brain. We are exploring a synthetic approach: starting 672 bp minicircles with inherently less curvature, demonstrating the relation- with electrically inert cells, what collective behaviors emerge as we intro- ship between bending and base accessibility. A sharp positive supercoiling duce specific ion channels, one at a time? We find that even with simple threshold was required for Bal-31 cleavage to even occur. We mapped combinations of ion channels, surprisingly rich and complex dynamics can Bal-31 cleavage sites and, using coarse-grained simulations, determined emerge. In HEK293 cells expressing an inward-rectifier potassium channel, the DNA register of our cryo-electron micrograph images. Our data reveal Kir 2.1, electrical polarization occurs via a stepwise jump. In a confluent two hotspots of Bal-31 cleavage located 180 apart along the DNA circum- monolayer of these cells, electrical polarization spreads via collective ference, suggesting that exposed bases are predominantly located at superhe- bioelectric domain wall. Simple numerical simulations reproduce these ef- lical apices and that a particular three-dimensional conformation is dominant. fects. Cells expressing Kir 2.1 and a voltage-gated sodium channel, NaV The relative probability of Bal-31 cleaving at either site varied as a function 1.5, produce action potential-like spikes. When these cells are grown into of supercoiling. Together these data reveal the interplay among sequence, a confluent monolayer, the spikes propagate as collective waves. We use supercoiling, and shape, resulting in conformational changes that should pro- patterned optogenetic stimulation to excite these cells, and wide-field voltage foundly influence DNA interactions with proteins. Understanding these imaging to map the voltage dynamics. Cells grown on different-shaped changes could facilitate the design of supercoiling-dependent DNA nano- islands show geometry-dependent transitions from regular beating, to period structures for gene therapy. doubling, to arrhythmia. These experiments illustrate how qualitatively new and surprising phenomena can emerge from the many-body behavior of elec- 846-Symp trically coupled cells, even when the electrophysiology of the individual cells Chromosome Organization by Loop Extrusion and Phase Separation is understood exhaustively. Leonid Mirny. MIT, Cambridge, MA, USA. 849-Symp Inferring principles and mechanisms of 3D organization of chromosomes from Mechanisms, Diversity and Optogenetic Applications of Channelrhodop- Hi-C and imaging data is a challenging biophysical problem. Our works sug- sins from Cryptophyte Algae gest that chromosomes are organized by a process of loop extrusion. We pro- Elena G. Govorunova, John L. Spudich. posed that loop extrusion is a universal mechanism responsible for formation Biochemistry & Molecular Biology, University of Texas Health Science of domains in interphase [1,2], and chromosome compaction and segregation Center at Houston McGovern Medical School, Houston, TX, USA. in metaphase [3]. I will review recent experimental studies [3-5] that provide Channelrhodopsins are the only so far known light-gated ion channels, and strong support to loop extrusion as a universal mechanism of chromosome their use for optical control of cellular excitability (optogenetics) has revolu- folding. These experiments have also showed that spatial segregation of tionized neuroscience. Channelrhodopsins were first discovered in green euchromatin and heterochromatin is achieved by a different mechanism. To algae, where they function as phototaxis receptors. We have found two addi- elucidate mechanisms of this spatial segregation, we examined Hi-C and micro- tional channelrhodopsin families in phylogenetically distant cryptophytes. scopy for cells with inverted nuclei and conventional nuclear architecture [6]. Proteins from the first family passively conduct cations as their counterparts Polymer model base on these data show that attraction between heterochromat- from green algae, but their primary sequences are closer to those of rhodopsin ic regions drive the phase separation, while interactions with the lamina central ion pumps. Members of the second family share some key residues with green for spatial positioning of phases in the nucleus. Taken together our results algae channelrhodopsins, but exhibit anion selectivity and therefore are demonstrate two major processes shape chromosome organization through called ‘‘anion channelrhodopsins’’ or ACRs. We have identified 30 native the cell cycle: heterochromatin-driven phase segregation and SMC-driven ACR variants highly variable in conductance, current kinetics and spectral loop extrusion. 1. Fudenberg G, Imakaev M, et al. Formation of Chromosomal sensitivity. Channel gating in ACR1 from Guillardia theta (GtACR1) com- Domains by Loop Extrusion. Cell Rep (2016) 2. Goloborodko A, Imakaev MV, prises two kinetically distinct mechanisms, each of which is controlled by Marko JF, Mirny L. Compaction and segregation of sister chromatids via active specific amino acid residues. Analysis of photochemical conversions has re- loop extrusion. Elife. May 18;5 (2016) 3. Nora EP, et al. Targeted degradation vealed that fast and slow channel closing temporally correlate with de- and of CTCF decouples local insulation of chromosome domains from higher-order reprotonation of the Schiff base, respectively. GtACR1 and several other genomic compartmentalization Cell (2017) 4. Schwarzer W, Abdennur N, et cryptophyte ACRs generate large hyperpolarizing currents and are potent al., Two independent modes of chromosome organization are revealed by co- silencers of neuronal spiking, successfully used in worms, flies, zebrafish, fer- hesin removal Nature (2017) 5. Gibcus J, Samejima K, Goloborodko A, et rets and mice. Additionally, we have shown that GtACR1 enables optically al., Mitotic chromosomes fold by condensin-dependent helical winding of controlled shortening of action potentials in cardiomyocytes. We have ob- chromatin loop arrays Science (2018) 6. Falk M, Feodorova Y Naumova N, tained a high-resolution X-ray crystal structure of GtACR1 dark state and et al., Heterochromatin drives organization of conventional and inverted nuclei found an intraprotomer disulfide bridge in the N terminus, disruption of https://www.biorxiv.org/content/early/2018/01/09/244038 which slows current kinetics, an intramolecular tunnel from the extracellular

BPJ 9340_9351 172a Monday, March 4, 2019 surface to a large cytoplasmic cavity, and a unique configuration of the photo- 852-Plat active site likely responsible for the delayed Schiff base deprotonation. Using Fenestrations Control the Resting State Block of a Voltage Gated Sodium this structural information, we have identified residues that determine absorp- Channel tion wavelength and current-voltage dependence. Our results provide clues Tamer M. Gamal El-Din, Michael J. Lenaeus, Ning Zheng, for rational engineering of ACR molecules further to increase their optoge- William A. Catterall. netic utility. Sch Med, Univ Washington, Seattle, WA, USA. At the molecular level, potency and efficacy of drug action are thought to be determined primarily by drug binding to a specific receptor site on the target pro- Platform: Ion Channels, Pharmacology and tein. In contrast to this conventional paradigm, we show here that the potency of local anesthetics (LAs) and antiarrhythmic drugs (AADs) that block sodium Disease channels is controlled by fenestrations in the walls of the pore that control drug access to the drug receptor site directly from the membrane phase. LAs 850-Plat and AADs selectively block sodium channels in rapidly firing nerve and muscle TMC1 Forms the Pore of the Mechanosensitive Transduction Channels in cells to relieve these conditions. Ancestral bacterial sodium channels are also Inner Ear Hair Cells blocked by LAs and AADs, and the structure of the bacterial sodium channel Nurunisa Akyuz1, David P. Corey2. Na Ab revealed fenestrations connecting the lipid phase of the membrane to 1Harvard Medical School, Boston, MA, USA, 2Dept Neurobiology, Harvard V the central cavity of the pore, which are conserved in vertebrate sodium chan- Med Sch, Boston, MA, USA. nels. We co-crystallized lidocaine and flecainide with Na Ab, which revealed The TMC1 and TMC2 proteins are critical components of the mechanotrans- v strong drug-dependent electron density in the central cavity in the pore near duction complex in vertebrate inner-ear hair cells. To understand TMC pro- amino acid residues important for drug binding. Mutation of the contact residue teins, we sought insight into the structural properties of TMC1. First, we T206 greatly reduced drug-binding affinity, confirming this binding site as the performed a variety of tests—including size-exclusion chromatography, chem- receptor for LAs and AADs. The sidechain of F203 restricts the size of fenestra- ical crosslinking, multi-angle light scattering and cryo electron microscopy— tions in wild-type Na Ab. The mutation F203A increased and the mutation all of which suggest that TMC1 assembles as a dimer. Further, TMC predicted V F203W decreased the size of the fenestrations. These mutations had graded ef- secondary structure and hydrophobicity suggest an architecture with ten trans- fects on resting-state block by flecainide, lidocaine, and benzocaine, altering membrane domains. The dimeric stoichiometry, and topology similarity be- drug affinity from 51-fold to 2.6-fold in order of their molecular sizes. These re- tween TMCs and the TMEM16 family of anion channels and lipid sults show that conserved fenestrations in the pores of bacterial and eukaryotic scramblases, suggest that TMCs may have a similar fold. We used I-TASSER sodium channels are crucial pharmacologically and determine the level of to align TMC1 with TMEM16A, for which the atomic structure has been resting-state block by these major classes of widely used drugs. Fine-tuning solved. TMEM16A dimerizes at an interface involving the tenth transmem- drug access through fenestrations provides an unexpected new avenue for brane domain, and each subunit of the dimer has an separate ion conducting structure-based design of ion channel-blocking drugs. pore, bounded by transmembrane domains S4-S7—a configuration strikingly different from well-known ion channels with a central pore bounded by three 853-Plat to six subunits. To ask whether S4-S7 of TMC1 might also enclose a functional Does Disruption of the E873-R933 Salt Bridge in CFTR and Alteration of pore, we designed and prepared AAV constructs of TMC1, bearing cysteine the Membrane Bilayer Around It Play a Biological Role? mutations, for expression in inner ear hair cells of Tmc1/2-null mice. Through Emily Langron1, Valentina Corradi2, Peter D. Tieleman2, Paola Vergani1. our collaboration with Holt lab, we found that cysteine-modification reagents, 1NPP, UCL, London, United Kingdom, 2Dept Biol Sci, Univ Calgary, when applied acutely, altered mechanosensory currents through these mutant Calgary, AB, Canada. channels in expected ways. Our data together provide evidence that TMC1 CFTR is an anion channel which plays an important role in regulating transe- forms the pore of sensory transduction channels in auditory hair cells with a pithelial fluid movement. In recent cryo-EM CFTR structures, transmembrane permeation pathway lined by residues on S4-S7. helix 8 (TM8) was found to be partially unwound. Because this is one of the main structural differences between CFTR and close relatives which function 851-Plat as pumps, the unwound helix is thought to play a part in CFTR’s ion channel Inhibition of TMEM16A by Docosahexaenoic Acid Plays a Crucial Role in function. Molecular dynamics simulations have detected a salt bridge between Blood Vessel Relaxation E873 (in TM7), and R933 (in TM8), both in regions thought to be embedded Kathryn E. Acheson, Paolo Tammaro. within the hydrophobic core of the membrane. In most simulations, the salt Department of Pharmacology, University of Oxford, Oxford, United bridge screens the charged residues from the hydrophobic environment. How- Kingdom. ever, in some simulations using the ATP-free structure, the two side chains lose Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated with contact, and a membrane defect is formed, with lipid headgroups and water beneficial cardiovascular effects, such as vasodilation. The mechanism under- molecules penetrating deep into the bilayer core. We have investigated the lying this vasodilation, however, is not fully defined. In arterial smooth mus- E873-R933 interaction using thermodynamic mutant cycles, and newly devel- cle, TMEM16A chloride channels promote membrane depolarisation and oped fluorescence assays that allow simultaneous estimates of CFTR conduc- vessel contraction. Cloned TMEM16A channels are inhibited by DHA. tance and membrane density. Our preliminary results suggest that the salt Here, we examine whether inhibition of native TMEM16A by DHA plays a bridge is important for both processing and gating: while E873R reduces ion role in aortic relaxation. Patch-clamp electrophysiology and isometric tension channel function and R933E decreases membrane density, the double mutant recordings were utilised to study the effect of DHA on cloned TMEM16A (E873R/R933E) rescues both defects. To investigate the functional significance channels and aortic contractility. DHA inhibited TMEM16A currents in a of the membrane defect we are currently studying the Y919F mutant. Y919 dose dependent manner, IC50=15.451.5 mM (n=10). To study the regulation þ (also in TM8) is in the immediate proximity of E837 and R933 in ATP-free of TMEM16A by DHA in response to agonist-induced increase in [Ca2 ] , i but not in ATP-bound structures. The hydroxyl group might allow the unusual TMEM16A was co-expressed with the a adrenoreceptor and currents re- 1A- positioning of water and lipid headgroups in the ATP-free structure only. An corded in response to 1 mM phenylephrine (PE). In the absence and presence effect of the Y919F mutation on the E873-R933 coupling described above of DHA (10 mM) the TMEM16A current increased by 7.651.8 fold (n=12) would be consistent with the membrane defect playing an important role in and 2.850.5 fold (n=6), respectively (p<0.05). In aortic rings, inhibition of CFTR biosynthesis and/or function. TMEM16A with either 70 mM DHA or 1 mM 2-(4-Chloro-2-methylphe- 0 noxy)-N -(2-methoxybenzylidene)acetohydrazide (Ani9) reduced PE-induced 854-Plat aortic contraction by 1.950.4 fold (n=9) and 2.050.4 fold (n=7), respec- Generating Potent and Selective Inhibitors of Kv1.3 Ion Channel by Fusing tively. Inhibition of the NKCC1 transporter with 40 mM bumetanide, to lower Knottins (Venom Derived Mini Proteins) into Peripheral CDR Loops of intracellular Cl- concentration ([Cl-]), reduced aortic response to 1 mMPEby Antibodies 3.350.4 fold (n=10). Conversely, lowering extracellular [Cl-] increased Aneesh Karatt-Vellatt1, Damian C. Bell1, Sachin B. Surade1, the aortic response to 1 mM PE by 9.353.4 fold (n=5); this effect was Tim Luetkens2, Ed W. Masters1, Alice M. Luther1, Naja Møller M. Sørensen3, reduced to 2.851.0 fold (n=5) in the presence of 70 mM DHA (p<0.05), Neil J. Butt1, John McCafferty1. implicating TMEM16A channels in the response. DHA-mediated inhibition 1IONTAS, Cambridge, United Kingdom, 2Internal Medicine, University of of TMEM16A channels reduces aortic contractility. This study highlights Utah, Salt Lake City, UT, USA, 3Sophion BioScience, Copenhagen, TMEM16A as a target for modulating vascular tone. Identification of Denmark. the TMEM16A DHA binding site may enable development of new small mol- Pathogenic T-cell effector memory (TEM) cells drive many autoimmune disor- ecules aiming to reduce blood pressure. ders and are uniquely dependent on the Kv1.3 ion channel. A number of knottin

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(venom derived cysteine-rich mini-proteins) inhibitors of Kv1.3 are being but showed strongly reduced currents with slower kinetics of activation; instead developed as potential drug candidates, but often suffer from manufacturing the CLC-7 heterozygous mutation showed a behavior similar to the WT. We are difficulties, short half-lives and lack specificity. currently studying whether the Cl-/Hþ coupling is affected by the mutations us- IONTAS have developed a novel molecular format wherein a peripheral CDR ing an optical assay that employs the E2GFP/DsRed Cl2/pH sensor fused to the loop of an antibody has been replaced by a knottin - dubbed a KnotBody. In this C-terminus of CLC-7. novel KnotBody format, the knottin benefits from the increased in vivo half-life of an antibody and the antibody gains additional functional diversity by the 857-Plat insertion of a knottin scaffold which evolved in venomous species to inhibit Inhibition of HCN Channels by Beta-Blocker Carvedilol ion channels. We have used this format to develop a panel of low-nM IC50 Pingzheng Zhou, Ying Cao. Kv1.3 inhibitors with selectivity exceeding 400-fold versus Kv1.1 currents, a Ion Channels Pharmacology, Southern Medical University, Guangzhou, closely related Kþ channel. Finally, selecting knottins from the vast, diverse China. evolutionary tool-box we have developed different families of KnotBodies Carvedilol is a clinically broadly used and effective beta-blocker for treatment that inhibit Nav1.7 or ASIC1a channels towards therapeutic development for of congestive heart failure (CHF) and several clinical trials have demonstrated the treatment of chronic pain or stroke. that carvedilol shows a favorable effect compared to other beta-blocking agents in patients with CHF. The mechanism underlying the advantage of carvedilol 855-Plat over other beta-blocking agents is not clearly understood. Besides beta- Is the Hydrophobic Gasket a Secondary Selectivity Filter in the Human blockers, inhibitors of hyperpolarization-activated cyclic nucleotide-gated Voltage Gated Proton Channel HHV1? (HCN) channels, which play a critical role for spontaneous rhythmic activity Richard Banh1, Kethika Kulleperuma1, Vladimir V. Cherny2, Deri Morgan2, in heart, have also been proposed to be suitable in reducing heart rate and Boris Musset3, Sarah Thomas4, Susan M.E. Smith4,Regis Pome`s1, thereby beneficial for treatment of CHF. In the present study, we investigated Thomas E. DeCoursey2. the effect of carvedilol on HCN channels. We found that carvedilol is the only 1Biochemistry, University of Toronto, Toronto, ON, Canada, 2Physiology & beta blocker tested showing inhibitory effects on the major sinoatrial isoform Biophysics, Rush University, Chicago, IL, USA, 3Institut fur€ Physiologie und HCN4 channel in the present study. Carvedilol inhibited HCN4 in a € € Pathophysiologie, Paracelsus Medizinische Privatuniversitat, Nurnberg, concentration-dependent manner with IC50 value of 4.4 mM. Besides, carvedi- Germany, 4Biology and Physics, Kennesaw State University, Kennesaw, GA, lol also inhibited HCN1 and HCN2 channel with relatively weaker affinity USA. compared to HCN4. Carvedilol blocked HCN channels by decelerating the A large family of membrane proteins, the voltage gated ion channels, regulate a channel activation and hyperpolarizing shift the voltage-dependent of activa- vast array of physiological functions in essentially all life forms. The mecha- tion. Our data also shows carvedilol is a non use-dependent HCN4 inhibitor, nism by which these molecules sense membrane potential and respond by which is different compared to previous reported inhibitors, e.g., ivabradine creating ionic conduction is incompletely understood. These channels contain or ZD7288. In conclusion, carvedilol is a negative gating modulator of HCN a ring of hydrophobic amino acids near the center of the voltage sensing domain channels. It represents a novel structure for future HCN channel inhibitors in the membrane, the ‘‘hydrophobic gasket,’’ HG, which is anchored by a high- drug design. ly conserved Phe (part of the Charge Transfer Center) in nearly all voltage- gated ion channels and voltage-sensing phosphatases. Various functions of this structure have been suggested in addition to its evident role of separating Platform: Optical Microscopy and internal and external aqueous solutions. During gating, the periodic cationic residues in the S4 helix are thought to ratchet past the HG. Here we identify Superresolution Imaging II the HG in voltage-gated proton channels and test the hypothesis that it func- tions as a secondary selectivity filter. Selectivity is ensured primarily by the 858-Plat interaction of Asp112 and one or more Arg in S4. However, molecular dynamics Single Particle Trajectories Reveal Active Endoplasmic Reticulum simulations indicate that cation exclusion may not be complete at the Asp-Arg Luminal Flow 1 2 3 3 selectivity filter. Potential of mean-force calculations show that the top of the Pierre Parutto , Joseph E. Chambers , Marcus Fantham , Laurence Young , 2 3 2 1 free energy barrier opposing Naþ permeation coincides with the most hydro- Stefan Marciniak , Clemens F. Kaminski , David Ron , David Holcman , Edward Avezov2,4. phobic region of the pore, corresponding with the HG region in other 1 ´ 2 voltage-sensing domains. We replaced amino acids of the HG with less hydro- Institut de Biologie, Ecole Normale Superieure, Paris, France, Institute for phobic or hydrophilic ones, both individually and in groups, and measured Medical Research, University of Cambridge, Cambridge, United Kingdom, þ 3Dept ChemEng & BioTech, Univ Cambridge, Cambridge, United Kingdom, reversal potentials at various pH and in the presence of Na . All mutants tested 4 þ þ 7 Clinical Neurosciences, UK Dementia Research Institute at University of were selective for H and did not conduct Na , with PH/PNa > 10 as a lower limit. We conclude that proton selectivity is accomplished by the primary selec- Cambridge, Cambridge, United Kingdom. tivity filter, comprising the interaction of Asp and Arg. The Endoplasmic Reticulum (ER) is a contiguous membrane-bound network of sheet-like reservoirs and tubules extending throughout the cell. This 856-Plat morphology is maintained by membrane shaping proteins and supports the dis- Investigating Functional Consequences of Novel Disease-Causing Muta- tribution of ER luminal content to distant sites. Motion of ER-luminal proteins tions of CLCN7 Gene was generally considered as passive, although measurements of protein Eleonora Di Zanni, Alessandra Picollo. mobility using fluorescence recovery after photo-bleaching (FRAP) have pre- Institute of Biophysics IBF, National Research Council CNR, Genova, Italy. viously uncovered an energy dependence that is difficult to reconcile with pas- CLC-7 is an intracellular chloride-proton antiporter of the CLC protein family. sive diffusion. In complex with its accessory protein Ostm-1, it localizes to the lysosomes Based on recent advances in super-resolution microscopy, allowing to image where it is important for protein degradation and to the ruffled border of oste- individual ER-luminal proteins, we present a finer view of the ER luminal mo- oclasts, where it is thought to play a critical role in the acidification of the tion at the single molecule level. Using the Langevin equation together with resorption lacuna and in the osteoclast-mediated bone resorption. ClC-7 KO local estimators of motion parameters, we found that luminal motion can be mouse phenotype revealed severe osteopetrosis, retinal degeneration and neu- divided in two spatially separated categories: a slow diffusive-like motion at rodegeneration associated with lysosomal storage. Consistently several muta- tubule junctions and a fast and directed motion in the tubules. The latter is pre- tions in the human CLCN7 gene were identified in patients with sent in multiple cell types, disappears after applying ATP-depletion treatments, osteopetrosis, a disease characterized by dense and fragile bones associated is not detected when imaging ER membrane proteins and is coherent with trans- with highly heterogeneous clinical symptoms. We investigated the effects of versal tubule contractions observed in fast Structured Illumination Microscopy two undescribed CLC-7 mutations identified in patients diagnosed for osteopet- (fSIM) experiments. rosis: a novel homozygous missense mutation associated with a clinical pheno- Furthermore, this specific jump-diffusion motion allowed us to develop an type manifesting a severe neurodegeneration and a heterozygous missense algorithm, based on local trajectory density, to reconstruct the ER network mutation in a patient with a milder phenotype. We verified by confocal micro- of a cell from individual trajectories. Studying the characteristics of these scopy that the lysosomal co-localization of CLC-7 and Ostm1 was preserved networks from multiple cells, over the 36sec duration of the recordings, re- for both CLC-7 mutants. Moreover, to determine the effects of CLC-7 muta- vealed that they form a main strongly connected component allowing redis- tions on transporter activity we performed patch-clamp recordings on a tribution of the luminal content over the entire ER. Finally, an analysis of plasma-membrane-targeted CLC-7 that overcomes the limit of the lysosomal the transient dynamic of the network showed that luminal content redistribu- localization of the protein. Preliminary results surprisingly reveal that the tion supported locally by alternating periods of uni-directional flows in CLC-7 homozygous mutation did not completely abolish chloride currents, tubules.

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Overall this analysis highlights the capacity of super-resolved single particle 861-Plat trajectories analysis to provide new insights about the dynamics of cellular Active Feedback Tracking of Single Viruses and Fluorophores in Solution structures. Kevin D. Welsher. Dept Chemistry, Duke University, Durham, NC, USA. 859-Plat Single molecule spectroscopy across multiple timescales is typically restricted Chromatin Nanoscale Organization Investigated by FLIM-FRET and to immobilized proteins on surfaces due to rapid three-dimensional diffusion of STED Superresolution Microscopy unbound probes. A potential solution to ‘‘un-tether’’ surface-bound single 1,2 1 1,2 1,2 Simone Pelicci , Michele Oneto , Melody Di Bona , Isotta Cainero , molecule methods is real-time 3D single particle tracking (RT-3D-SPT), which 3 1,2 01 Paola Barboro , Alberto Diaspro , Luca Lanzano . uses active feedback to lock onto a freely diffusing probe’s position in real- 1Nanoscopy, Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy, 2 3 time, enabling continuous observation of dynamic processes with photon- Department of Physics, University of Genoa, Genoa, Italy, Academic Unit limited temporal resolution. However, RT-3D-SPT methods have yet to trans- of Medical Oncology, IRCCS Ospedale Policlinico San Martino, Genoa, late to real-time single molecule tracking due to the requirement of high emis- Italy. sive rates. This work describes a new advance in RT-3D-SPT called 3D Chromatin organization plays an essential role in the regulation of gene activ- Dynamic Photon Localization Tracking (3D-DyPLoT) which enables active ity, which involves the packaging of the genome into transcriptionally active feedback tracking of rapidly diffusing and lowly emitting fluorescent particles. and inactive sites. is generated by the recruitment of nu- 3D-DyPLoT uses a 3D flying laser focus to tag each arriving photon with 3D clear enzymes for a variety of nuclear processes. For instance, chromatin archi- position information and a piezoelectric nanopositioner to lock the diffusing tecture is highly reorganized during DDR (DNA damage response) to promote object in the objective focal volume, at diffusive speeds exceeding 10 mm2/ accurate repair of DNA lesions[1]. However, how the higher-order chromatin sec and emission rates of 10 kHz or less. Here we demonstrate the application structures are formed and then behave in various cellular processes in live cells of 3D-DyPLoT to a range targets, from single virus-like particles and quantum remains unclear. dots in water, all the way down to single fluorophores in viscous solution. This Here, we adopt a double strategy based on a novel FLIM-FRET assay and new single molecule tracking can be applied to continuously monitor single super-resolution microscopy to study the nanoscale chromatin organization proteins and nucleic acids, including real-time measurement of transcription in intact cells nuclei. on a freely diffusing, single-dye labelled DNA strand. We further describe The FRET assay is based on the staining of the nuclei with two DNA-binding the application of 3D-DyPLoT as an adaptive tracking method, which adjusts dyes and the frequency-domain detection of FLIM. We show that the FRET the feedback parameters according to the real-time diffusion coefficient, level strongly depends on the relative concentration of the two fluorophores. trading tracking speed for spatial precision on demand in processes involving We describe a method to correct the values of FRET efficiency and demonstrate heterogenous diffusive regimes. We demonstrate the application of this adap- that, with this correction, the FLIM-FRET assay can be used to quantify vari- tive microscope to observe the initial binding events of vesicular stomatitis ations of nanoscale chromatin compaction in live cells. virus-like particles to the surface of live cells. Super-resolution microscopy is used to investigate, in fixed cells, the nanoscale distribution of specific proteins, like poly(ADP-ribose) polymerase 1 (PARP1), 862-Plat implicated in various DNA repair pathways and in the maintenance of genomic Super-Resolution Microscopy as a Tool for Counting Proteins in a Sub- stability[2]. In particular, we apply our recently developed method based on the Cellular Environment modulation of the STED power[3]. We show that, by using the phasor Francesca Cella Zanacchi1,2, Raffaella Magrassi1,2, Carlo Manzo3, approach, we obtain a significant improvement of spatial resolution. Nathan Derr4, Alberto Diaspro1,5. We use this double strategy for monitoring changes in nanoscale chromatin or- 1Nanoscopy and Nikon Imaging Center, Istituto Italiano di Tecnologia, ganization during the DDR. Genoa, Italy, 2Biophysics Institute, CNR, Genova, Italy, 3Universitat de Vic – [1] Hauer HM, Gasser SM, Genes Dev (2017); Universitat Central de Catalunya, Genoa, Spain, 4Department of Biological [2] Chaudhuri AR, Nussenzweig A, Nature Reviews (2017); Sciences, Center for Microscopy and Imaging, Smith College, Northampton, [3] Sarmento MJ et al, Nat. Commun., (2018); MA, USA, 5Physics Department, University of Genoa, Genoa, Italy. Super-resolution microscopy, and in particular single molecule localization mi- 860-Plat croscopy (such as stochastic optical reconstruction microscopy, STORM), of- Single Nitrogen-Vacancy Imaging in Nanodiamonds for Multimodal fers a unique opportunity for quantifying protein copy numbers with Sensing nanoscale resolution [1,2]. 1 1 1 2 2 Maabur Sow , Horst Steuer , Barak Gilboa , Laia Gines , Soumen Mandal , The development of methods able to access a precise molecular counting of 3 3 2 Sanmi Adekanye , Jason M. Smith , Oliver A. Williams , protein copy numbers is essential, clearing the way to address several biolog- 1 Achillefs N. Kapanidis . ical questions using super-resolution microscopy. The challenges of molecular 1Physics, University of Oxford, Oxford, United Kingdom, 2School of Physics 3 counting using super-resolution [3,4] can be addressed using calibration stan- and Astronomy, Cardiff University, Cardiff, United Kingdom, Materials, dards such as DNA origami, since they provide direct characterization for University of Oxford, Oxford, United Kingdom. quantitative imaging when immunofluorescence is used. The fluorescent nitrogen-vacancy (NV) defect in diamond has remarkable - Here we propose to use quantitative approaches based on DNA origami calibra- tophysical properties, including high photostability and unique sensing proper- tion to study the distribution of adhesion proteins and membrane proteins. First, ties; as a result, there has been much interest in using nanodiamonds (NDs) for we focus on quantitative studies of the stoichiometry of membrane proteins (i.e bio-imaging. A facile route for sensing using NDs containing single or multiple membrane channels and their accessory subunits). Furthermore, we apply this NVs is via measuring changes in the NV emission spectra due to chemical (or method for average protein copy number estimation of adhesion proteins (i.e. structural) events on the ND surface, since the NV neutral state (NV0)is60nm - vinculin) in mammalian cells and neurons. blue-shifted versus the negative form (NV ). Bio-functionalised NDs have been 1. Durisic, N., et al., Single-molecule evaluation of fluorescent protein photo- implemented before in single-molecule imaging; however, the effect of func- activation efficiency using an in vivo nanotemplate. Nat Methods, 2014. 11(2): tionalisation on ND’s photophysics has been poorly studied due to a lack of p. 156-62. high-throughput characterisation methods. Such effects are important to under- 2. Ulbrich, M.H. and E.Y. Isacoff, Subunit counting in membrane-bound pro- stand and control, since biomolecules may affect the NV charge, thus causing teins. Nat Methods, 2007. 4(4): p. 319-21. spectral shifts and possibly blinking. Here, we report a high-throughput anal- 3. Jungmann R. et al. Quantitative superresolution imaging with qPAINT Na- ysis of NDs using wide-field epifluorescence dual-colour imaging. We have ture methods doi:10.1038/nmeth.3804 (2016) measured the proportion of single-NVs in fluorescent NDs of different sizes 4. Cella Zanacchi F. et al., DNA Origami: Versatile super-resolution calibration (10 - 100 nm) by analysing their intensity distribution, and found that it was standard for quantifying protein copy-number, Nature Methods 14, 789–792 in the 50-80% range. Using the ratio of green and red intensities, we have (2017) (2017) directly observed the charge conversion of single NVs, and, for the first time, we are able to measure the lifetimes and transition rates between charge states 863-Plat in NDs. We also show that the NV’s charge changes in a pH-dependent fashion Molecular Counting with DNA Origami - Verification and Validation in uncoated NDs (<20 nm diameter), opening the possibility of using NDs for Towards Biological Applications intracellular pH sensing and mapping. Finally, we investigate the photophysics Daniel F. Nino1, Daniel Djarkarsana1, Anton Zilman2, Joshua Milstein1. of NDs conjugated to double-stranded DNA and to proteins involved in bacte- 1Department of Chemical and Physical Sciences, Univ Toronto, Mississauga, rial transcription with a view to developing assays for transcriptional studies. ON, Canada, 2Dept Physics, Univ Toronto, Toronto, ON, Canada. Our results provide us with a new insight into the ND photophysics and their Single-molecule localization microscopy (SMLM) has been a powerful tool for nanoscale sensing capabilities. expanding our understanding of cell biology. By taking advantage of the

BPJ 9340_9351 Monday, March 4, 2019 175a stochastic blinking that fluorophores naturally exhibit and controlling the dy- and Center for Nanoscience, Ludwig Maximilian University, Munich, namics of this process, SMLM pushes the level of resolution an order-of- Germany, 3Max Planck Institute of Biochemistry, Martinsried near Munich, magnitude beyond the diffraction limit allowing light microscopy to visualize Germany. cellular components with improved resolution. There is also tremendous interest Receptor tyrosine kinases (RTKs) contribute to cell proliferation and motility. in using the technique to count single molecules. The main challenge of molec- They react to a variety of ligands, recruit various cellular proteins, and initiate ular counting in SMLM is largely a result of the multiple and random blinking of complex signaling cascades. Dysregulation is found in various diseases fluorophores, which leads to over-counting the number of molecules. In [1], we including cancer. Successful therapy demands detailed knowledge of receptor proposed a method for counting biomolecules based on the blinking statistics of activation, cross-reactivity, and molecular interactions. Single-particle fluorophores and taking into account the labeling efficiency to the target mole- tracking (SPT) provides the required sensitivity to study these transient but cule. We show how our theory may be used by analyzing simulated data [1], and crucial cellular mechanisms at the level of single cells and single membrane in vitro data based on DNA origami structures [2]. Accurately determining the receptors. Hepatocyte growth factor receptor (HGFR, also MET) and number of proteins or nucleic acids in a cell has wide-ranging applications, epidermal growth factor receptor (EGFR) are two prominent members of from systems biology, to /genomics, to fundamental cell biology. two different RTK subfamilies that were previously reported to cross- As a potential application, we’ve begun to apply this technique to the problem interact in lung cancer cells. We profiled the diffusion behavior of these of accurately determining plasmid copy number in individual bacteria. two receptors in living cells using SPT, before and after stimulation either with one or a combination of both cognate ligands, human growth factor 864-Plat (HGF) and epidermal growth factor (EGF). We found that both receptors Structural Contributions to Hydrodynamic Size of Quantum Dots for are cross-activated, which was not shown in living cells so far. We determined In-Vivo Single Molecule Tracking the extent of activation and cross-activation from the relative change of diffu- 1 2 3 4 Janet Y. Sheung , Pinghua Ge , Sung Jun Lim , Sang Hak Lee , sion coefficients after ligand stimulation. Experiments were performed in two Andrew Smith2, Paul R. Selvin2. 1 cancer cell lines with different expression levels of EGFR and MET. We Physics and Astronomy, Vassar College, Poughkeepsie, NY, USA, discuss the influence of EGFR:MET ratios on the occurrence of cross- 2University of Illinois, Urbana, IL, USA, 3DGIST, Daegu, Republic of Korea, 4 interaction and activation as a possible indicator for the success of cancer Pusan National University, Busan, Republic of Korea. therapies with RTK inhibitors. Quantum dots are fluorescent with desirable spectral properties for in-vivo single-molecule imaging. However typical formulations used for imag- ing proteins in cells are hydrodynamically much larger than the protein targets, 867-Plat so it is critical to assess the impact of steric effects deriving from hydrodynamic A Thermodynamic Framework for Understanding RTK Interaction Net- size. We analyze a new class of quantum dots that have been engineered for works Michael D. Paul1, Kalina Hristova2. minimized size specifically for imaging receptors in narrow synaptic junctions 1 2 between neurons. We use fluorescence correlation spectroscopy and transmis- Biophysics, Johns Hopkins University, Baltimore, MD, USA, Dept Matl sion electron microscopy to calculate the contributions of the crystalline core, Sci/Eng, Johns Hopkins Univ, Baltimore, MD, USA. organic coating, and targeting proteins (streptavidin) to the total hydrodynamic Receptor tyrosine kinases (RTKs) are the second largest class of membrane diameter of the probe, using a wide range of core materials with emission span- proteins, and there are 58 of them in humans divided into 20 subfamilies. ning 545 - 705 nm. We find the contributing thickness of standard commercial They play important roles in many biological processes, including cell amphiphilic polymers to be 8to14 nm, whereas coatings based on the growth, differentiation, and division. The prevailing model of RTK activa- tion is that homodimerization allows for autophosphorylation which triggers compact ligand HS-(CH2)11-(OCH2CH2)4-OH contribute 6to9 nm, reducing the diameter by 2to5 nm, depending on core size. By minimizing downstream signaling, although it is appreciated that intra-subfamily hetero- the number of streptavidins for protein targeting, the total diameter can be dimerization can also occur. However, there is growing evidence that many further reduced by 5to11 nm, yielding a diameter of 13.8 18.4 nm. These RTKs have inter-subfamily hetero-interactions as well, although there is little findings explain why access to the narrow synapse derive primarily from the pro- understanding of how this affects RTK signaling in general. We demonstrate tein functionalization of commercial variants, rather than the organic coating the commonality of these inter-subfamily hetero-interactions using quantita- layers. They also explain why those quantum dots with size around 14 nm tive FRET spectroscopy. Moreover, we provide a thermodynamic explana- with only a few streptavidins can access narrow cellular structures for neuronal tion of some of the observed effects of RTK inter-subfamily hetero- labeling, whereas those > 27 nm and a large number of streptavidins, cannot. interactions and the potential ways such interactions can affect general RTK signaling. 865-Plat The Observation Protein Position and Orientation Dynamics using an 868-Plat Unbleachable Probe Functional and Structural Analysis of Cell-Free Synthesized Membrane Paul A. Wiggins. Proteins Dept Physics, Univ Washington, Seattle, WA, USA. Belay Gessesse1, Takuya Ueda2, Yoshihiro Shimizu1. We develop a novel sensor (MagPI) for biophysical applications that can simul- 1Laboratory for Cell-Free Protein Synthesis, RIKEN Center for Biosystems taneously measure the three-dimensional position and orientation of a nanopar- Dynamics Research, Osaka, Japan, 2Department of Computational Biology ticle probe. This probe is unbleachable and unblinking and therefore is ideally and Medical Sciences, Graduate School of Frontier Sciences, The University suited to many in vitro biophysical applications, where precision tracking of of Tokyo, Kashiwa, Chiba, Japan. probe 3D-position and orientation could provide significant new insights, but Membrane proteins play a crucial role in human physiology and pathology long-timescale and high-temporal resolution imaging is required which pre- of disease, and currently, they are targets of nearly 50% of pharmaceutical cludes the use of single-molecule fluorescent probes. Orientation and position drugs. However, due to their hydrophobic nature, membrane proteins are sensing are performed by imaging the magnetic dipole-field generated by the notoriously difficult to produce and are less accessible for functional and nano-particle probe. The physical mechanism for magnetic-field imaging is structural analysis. In our study, we produce G-protein coupled receptors the B-field-induced shift of the photo luminescence intensity of nitrogen- (GPCRs) by a cell-free system supplemented with detergents, micelles, vacancy centers in a diamond sensor. We demonstrate MagPI imaging in a and lipid bilayer mimetics such as nanodiscs and giant unilamellar vesicles tethered-particle-motion analysis of Lac- induced DNA looping. (GUVs). We succeeded in producing GPCRs up to 0.1 mg per ml of the cell- free reaction mixture which is sufficient for biochemical and biophysical studies. Hence, we measured the secondary structure of cell-free synthesized Platform: Membrane Receptors and Signal GPCRs by circular dichroism and obtained typical characteristics of folded Transduction a-helix proteins. Similarly, we determined the ligand binding affinity con- stant by surface plasmon resonance and obtained an affinity similar to 866-Plat previous reports indicating that GPCRs synthesized by the cell-free system Activation and Cross-Interaction of Receptor Tyrosine Kinases Studied by is active. In addition, we found a homogenous nanodisc-GPCR complex as it Single-Particle Tracking was probed by transmission electron microscopy that will be essential Marie-Lena I.E. Harwardt1, Sebastian Strauss2,3, Ralf Jungmann2,3, for structural analysis. In conclusion, the synergy of cell-free systems Marina S. Dietz1, Mike Heilemann1. and the nanodisc technology have a tremendous potential for investi- 1Single Molecule Biophysics, Institute of Physical and Theoretical gating the functions and structures of membrane proteins in a native-like Chemistry, Goethe-University, Frankfurt, Germany, 2Department of Physics environment.

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869-Plat a robust molecular biology pipeline for production and sample preparation of Sponge Model of G-Protein Binding and Unbinding in Membranes stable-isotope labeled receptors, yielding reproducible high-quality NMR Anna R. Eitel1, Steven D.E. Fried1, Suchithranga M.D.C. Perera1, data. From these data, assignment of endogenous residues provided numerous Udeep Chawla1,2, Nipuna Weerasinghe1, Carolanne E. Norris1, probes distributed over the receptor intracellular surface, extracellular surface, 1,3 1,4 Andrey V. Struts , Michael F. Brown . and transmembrane domain, enabling a global characterization of A2AAR re- 1Department of Chemistry and Biochemistry, The University of Arizona, sponses to variable efficacy of bound drugs. The NMR probes also permitted Tucson, AZ, USA, 2Department of Molecular and Cellular Biology, novel insights into changes in the local conformation of conserved GPCR acti- University of Kentucky, Lexington, KY, USA, 3Laboratory of Biomolecular vation motifs. In addition, we observed multiple local efficacy-related con- NMR, St. Petersburg State University, St. Petersburg, Russian Federation, formers at the intracellular surface, which we relate directly to G protein 4Department of Physics, The University of Arizona, Tucson, AZ, USA. signaling. The distributed NMR probes provide novel information on the role Rhodopsin, a prototypical G-protein coupled receptor (GPCR), functions as of a highly conserved GPCR allosteric center, permitting us to study the effect a sensitive photon detector in retinal rod cells. Signal transduction by of this center on signaling pathways in the receptor. Amino acid replacement of rhodopsin is achieved through the binding and release of the transducin the allosteric center caused striking changes in conformational polymorphisms G-protein. Despite the numerous recent structures of complexes with effector at the intracellular surface but did not alter response to drug efficacy at the proteins, the mechanism underlying G-protein release from an activated extracellular surface. We correlated changes at the intracellular surface to GPCR remains elusive [1]. Here we propose that rhodopsin hydration/dehy- structural rearrangements at the toggle switch tryptophan and a conserved acti- dration cycling facilitates transducin binding and release. The dissociation vation motif, providing novel insights into a widespread signaling mechanism constant (Kd) between photoactivated rhodopsin and the high-affinity C-ter- in class A GPCRs. The presented work opens the door to exciting future studies minal analog of transducin (GalphaCT) was measured in response to osmotic of GPCR function, including studies of GPCRs in native environments. stress. The active metarhodopsin-II (MII) fraction of rhodopsin was obtained using UV-visible spectrophotometry, and osmotic stress was generated using 872-Plat various molecular weight polyethylene glycol (PEG) polymers. Preliminary A Conditional Transmembrane Peptide Inhibits Cell Migration by Activa- data indicate that large molecular weight polymers, such as PEG1500, are tion of the EphA2 Receptor unable to enter the protein interior, and dehydrate the rhodopsin. The oppo- Daiane S. Alves1, Justin M. Westerfield1, Xiaojun Shi2, Vanessa P. Nguyen1, site effect is observed for small molecular weight polymers, which enter the Katherine M. Stefanski3, Adam W. Smith2, Francisco N. Barrera1. protein core and have a hydrating effect. Large molecular weight polymers 1Department of Biochemistry & Cellular and Molecular Biology, University 2 reduce the binding affinity for GalphaCT to rhodopsin, while small molecular of Tennessee, Knoxville, TN, USA, Department of Chemistry, University of weight polymers increase this binding affinity. Our results indicate that Akron, Akron, OH, USA, 3Graduate School of Genome Science and rhodopsin hydration is necessary for transducin binding. We therefore pro- Technology, University of Tennessee, Knoxville, TN, USA. pose the sponge model for GPCR signaling, in which an influx of water dur- We have transformed the isolated transmembrane sequence of EphA2 into a ing rhodopsin activation is necessary to stabilize the active MII conformation pH-controlled conditional TM domain. The resulting peptide, TYPE7, is highly and to facilitate transducin binding. Formation of the high-affinity rhodopsin- soluble, interacts with the surface of lipid membranes, and upon acidification tranducin complex displaces water from the protein interior, destabilizing the inserts and folds into a TM helix. The receptor tyrosine kinase EphA2 plays receptor. Nucleotide exchange further dehydrates the receptor, causing it to an important role in cell-cell contacts, cell migration, and cancer metastasis. return to the inactive MI conformation, thus releasing transducin in the pro- Importantly, when TYPE7 inserts into membranes, it binds to endogenous cess. This wet/dry cycle can repeat many times for one photoexcitation EphA2, as shown in liposome and cellular experiments. This new interaction event, explaining how many molecules of transducin can be activated by specifically results in the phosphorylation of the key catalytic residue one molecule of rhodopsin. [1] U. Chawla, et al. (2016) Angew. Chem. Tyr772, present at the activation loop of the intracellular kinase domain. Bind- Int. Ed. 55, 588-592. ing of TYPE7 to EphA2 also causes a downstream signaling change that in- volves de-phosphorylation of Akt, a central regulator of cell migration. In 870-Plat fact, TYPE7 inhibits cell migration as efficiently as an EphA2 ligand in several Mechanism of Specific G Protein Coupling to Adenosine Receptors cell lines. We characterized that the mechanism TYPE7 uses to activate EphA2 Jinan Wang, Yinglong Miao. involves promotion of EphA2 self-assembly into oligomers. Our work high- Center for Computational Biology and Department of Molecular Biosciences, lights the use of biophysical approaches to develop a new method to create syn- University of Kansas, Lawrence, KS, USA. thetic ligands for receptor tyrosine kinases. These new conditional Four subtypes of adenosine receptors (ARs), the A1AR, A2AAR, A2BAR and transmembrane ligands do not only provide new mechanistic information on A3AR, mediate a broad range of physiological functions. They have emerged the target receptor, but also serve as potential targeted therapeutics. as important therapeutic targets for treating human diseases including cardiac ischemia, neuropathic pain and cancer. The odd-numbered subtypes prefer to 873-Plat couple with the Gi/o proteins, while the even-numbered subtypes predominantly Towards Molecular Simulations of Juxtaposed Biomembranes 1 2 3 couple with the Gs proteins. Despite extensive experimental and computational Elizabeth E. Jefferys , Bart Bruininks , Paulo Cesar T. Souza , studies, the exact determinants of ARs-G protein coupling specificity remain Siewert-Jan Marrink4, Mark S. Sansom5. unclear. Here, all-atom enhanced sampling simulations using a robust Gaussian 1Biochemistry, Univ Oxford, Oxford, United Kingdom, 2Univiersity of accelerated molecular dynamics (GaMD) method have been applied to deter- Groningen, Groningen, Netherlands, 3Univ Groningen, Groningen, 4 mine the mechanism of specific coupling between the A1AR and A2AAR and Netherlands, Dept Biophys Chem/Molec D, Univ Groningen, Groningen, 5 the Gi and Gs proteins, respectively. The GaMD simulations have revealed Netherlands, Dept Biochemistry, Univ Oxford, Oxford, United Kingdom. differentiated fluctuations and distinct low-energy conformational states of The importance of membrane-membrane interfaces throughout biology has the ARs upon binding of decoy versus cognate G proteins. Important motifs prompted a multitude of studies investigating the structures and dynamic prop- that are responsible for the receptor-G protein coupling specificity have been erties of their various protein and lipid components. Although such work has also identified. In addition, our GaMD simulation findings are consistent provided considerable insight into the activities of the interaction constituents, with recent experimental data. The GaMD simulations have provided important relatively little is currently understood regarding how they operate together in insights into the mechanism of specific coupling between ARs and the G pro- the context of the juxtaposed membranes to create a functional system. Using teins at an atomistic level, which will facilitate future computer-aided design of molecular dynamics (MD) simulations, it is possible to obtain insight into the selective drug molecules targeting the ARs. dynamics of biological assemblies at the molecular level. In recent years, a combination of forcefield improvements and advances in both hardware and 871-Plat software has allowed MD to access increasingly biologically relevant systems, Studying Structural Plasticity Underlying GPCR Function in terms not only of length- and timescales, but also complexity. MD is Matthew T. Eddy. therefore well poised to act as a powerful bridge between structural studies Chemistry, University of Florida, Gainesville, FL, USA. and classical biochemistry. We are developing the tools needed to apply The encodes for 826 G protein-coupled receptors (GPCRs), MD simulations to study membrane-membrane interactions bridged by cell sur- which are involved in nearly every physiological process and comprise the face glycoproteins and/or glycolipids, with the view of applying these tech- largest class of ‘‘druggable’’ proteins. Using nuclear magnetic resonance niques to study systems such as neuronal connections, the immunological (NMR) in solution, we provide a global view of structural plasticity underlying synapse, and virus-host cell attachment. Here we present our progress in this the function of the human A2A adenosine receptor (A2AAR), a representative methodological development, and provide an example of their application to class A human GPCR. To apply NMR to study a human GPCR we developed a model system.

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Platform: Myosin and Skeletal/Smooth Muscle ently, the spatial arrangement of these two hydrolytic states at the sarcomere level is unclear, although it has been reported that Myosin Binding Protein C Mechanics, Structure, and Regulation (MyBP-C) may stabilize the SRX state, which implies possible localization to the C-zone where MyBP-C exists. Here, we imaged single molecules of flu- 874-Plat orescently labeled ATP being hydrolyzed by myosin in relaxed myofibrils from Macromolecular Crowding Affects the Rate of ADP Release from Actomy- rat soleus skeletal muscle. First, we showed that BODIPY-ATP supports acto- osin myosin activity comparable to non-fluorescent ATP in an in vitro motility Jinghua Ge1, Akhil Gargey2, Yuri Nesmelov3,4. assay. Then, we estimated the myosin BODIPY-ATP hydrolysis rate by 1Department of Cellular and Molecular Physiology, Pennsylvania State measuring the lifetimes of individual BODIPY-ATP molecules binding to University College of Medicine, Hershey, PA, USA, 2Department of Biology, myofibrils followed presumably by the release of BODIPY-ADP. Lifetimes University of North Carolina at Charlotte, Charlotte, NC, USA, 3Department were well fit as two populations with lifetimes of 33.554.1s and 181542s; of Physics and Optical Science, University of North Carolina at Charlotte, consistent with reported kinetic rates for DRX and SRX myosin in skeletal Charlotte, NC, USA, 4Center for Biomedical Engineering and Science, muscle. Each individual ATP binding event was then mapped to its distance University of North Carolina at Charlotte, Charlotte, NC, USA. from the center of the sarcomere (M-line), as imaged by fluorescent antibody We have used macromolecular crowding agent Ficoll PM 70 to mimic the labeling of myomesin. Interestingly, ATP hydrolysis lifetimes within the C- intracellular environment and study the effect of crowding on the kinetics zone (200 - 500nm from the M-line) were significantly longer than those of of actomyosin dissociation and ADP release. Our previous studies of the the flanking non-MyBP-C containing regions. Within the C-zone, hydrolysis heavily mutated D. discoideum myosin S1 construct showed that the effect lifetimes were well fit as two equal sized populations, with lifetimes of of macromolecular crowding results in a more compact state of the myosin 34.4514.2s and 178554s, while in the non-MyBP-C containing regions, head. In the current study, we have used the wild-type rabbit skeletal myosin 95% of the events were well fit as a single population with a lifetime of S1. Addition of 10% w/v Ficoll led to the faster rate of ATP induced actomy- 34.655.2s. Our data suggest that the strongly inhibited SRX myosins exist pre- osin dissociation, in agreement with the excluded volume theory. The kinetics dominantly within the C-zone. of ADP dissociation from actomyosin is best fitted to the two-exponential function. One component of the kinetics transient reflects ADP dissociation, 877-Plat which was slower than the rate of ADP dissociation without Ficoll in solu- The Long Glu-Rich Segments of Troponin T in Flight Muscles of Birds and tion. Our data show that ADP binds actomyosin stronger in the presence of Ficoll. The rate of the second component of the transient does not depend Tianxin Cao, J.-P. Jin, Hanzhong Feng, Deena Damschroder, on the concentration of nucleotides in solution. This component of the Robert Wessells. observed transient can be interpreted as the kinetics of myosin conformational Physiology, Wayne State University, Detroit, MI, USA. þ change, leading to the opening of the active site for nucleotide to bind. We The troponin complex plays a central role in the Ca2 -regulation of conclude that macromolecular crowding not only increases the chemical po- striated muscle contraction and relaxation. Molecular evolution and compar- tential of solutes, but affects protein structural state due to change in the os- ative structural studies have identified Glu-rich segments specifically ex- motic pressure. pressed in the T subunit of troponin (TnT) in diverse flying species, such as birds and insects. Avian pectoral muscle TnT has a Glu-rich segment 875-Plat in the N-terminal variable region and insect TnT has one in the C-terminal The Muscle Myosin Essential Light Chain is Not Essential for Muscle extension. To understand these structural traits of non-homologous evolu- Function tionary origins, which may have been selected for analogous functions Douglas M. Swank, Bernadette M. Glasheen. of flight muscles, will help to understand the mechanisms of striated Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, muscle contraction for the development of therapeutic improvement of NY, USA. skeletal and cardiac muscle functions. We previously demonstrated a po- Myosin, the molecular motor of muscle, is composed of two identical heavy tential function of the N-terminal Glu-rich segment of avian pectoral chains and two different light chains, the essential light chain (Mlc1) and the muscle TnT as a myofilament-associated Ca2þ-reservoir (Zhang et al., regulatory light chain. The light chains are part of the myosin lever arm, which Biochemistry 43:2645-55 2004). To investigate a novel hypothesis that is critical for generating force and motion. Drosophila has a single Mlc1 gene this function reduces the work load of the Ca2þ handling system in myo- that is alternatively spliced into two isoforms. To investigate the function of cytes thus saving energy for sustaining the work of muscle especially during these Mlc1 isoforms in muscle mechanical properties, such as stretch activation long distance flight, we developed a Drosophila line with the Glu rich C-ter- and in enabling the fastest known frequency of muscle contraction, we created a minal segment of TnT deleted to test its requirement for muscle function Drosophila null for Mlc1 using the CRISPR/Cas9 system. To our surprise, we and flight capacity. Engineered drosophila TnT proteins are expressed found that Mlc1 homozygous null embryos hatched, crawled and molted. in E. coli for the characterization of changes in biochemical activities. Sequencing confirmed that we had targeted Mlc1. The resulting Mlc1 null The results showed that the C-terminal truncated Drosophila TnT retains line contains a two-nucleotide deletion, which truncates Mlc1 after 85 amino the capacity of binding to troponin I, troponin C and tropomyosin. acids. Mlc1 mutant larvae crawling velocity was reduced by 50% compared Tpnt gene-modified flies expressing the Glu-rich C-terminal segment 10 to wild-type. Approximately 50% of Mlc1 larvae pupariate, of these at least deleted TnT have been generated for functional studies. Together with 50% progress through metamorphosis, but only 8% eclose and survive to adult- the development and characterization of genetically modified mice express- hood. Fiber level IFM and TDT muscle structure appeared normal in Mlc1 null ing an insect muscle-like TnT in the heart, our study aims at establishing a adults, but they were unable to fly or jump. Skinned IFM fibers isolated from 1 novel mechanism to improve cardiac efficiency for the treatment of heart or 3 day old null flies did not activate or produce power. Transformation with a failure. 4.099 kb DNA fragment containing the genomic of Mlc1 with 5’ and 3’ UTR rescued adult viability, crawling velocity, and IFM power production to 878-Plat wild-type levels. We are currently performing experiments to determine if Thin Filament Regulation Blends Thermodynamic and Mechanical another protein is replacing Mlc1 on the myosin lever arm in the null line. Mechanisms Mlc1 Henry G. Zot1, P. Bryant Chase2, Javier E. Hasbun3, J. Renato Pinto4. We conclude that is not essential for embryonic and larval muscle devel- 1 2 opment and function. Dept Biology, Univ West Georgia, Carrollton, GA, USA, Dept Biology, Florida State Univ, Tallahassee, FL, USA, 3Dept Physics, Univ West 876-Plat Georgia, Carrollton, GA, USA, 4Dept Biomed Sci Col, Florida State Univ, The Skeletal Muscle Super Relaxed State (SRX) is Localized to the C-Zone Tallahassee, FL, USA. Shane R. Nelson1, Amy Li1, Guy Kennedy2, Samantha Beck-Previs1, A simple thermodynamic mechanism predicts that if chemical potential is David M. Warshaw1. constant, transitions between runs and pauses of gliding thin filaments will 1Molecular Physiology & Biophysics, University of Vermont, Larner College occur at constant rate as given by a Poisson process. However, we find that of Medicine, Burlington, VT, USA, 2Instrumentation and Model Facility, frequencies of run time intervals do not support a simple exponential distribu- University of Vermont, Burlington, VT, USA. tion expected for the time-dependent Poisson probability of an uninterrupted Under relaxing (Low/No Calcium) conditions, ATP hydrolysis by myosin in run. Each assay consists of fluorescently labeled native thin filaments (TF) on striated muscle shows two distinct subpopulations: a faster ‘‘Disordered a surface of skeletal muscle HMM in a flow cell containing fixed -log free cal- Relaxed’’ (DRX) population, and a strongly inhibited ‘‘Super Relaxed’’ cium (pCa). Whereas unregulated actin filaments move continuously, TF (SRX) population. This SRX state contributes to the low metabolic rate of alternate randomly between time intervals of runs and pauses. We construct resting muscle and SRX dysregulation may underlie some myopathies. Pres- histograms of run times estimated visually from video recordings of assays

BPJ 9340_9351 178a Monday, March 4, 2019 with the most frequent events (pCa: 6.6, 6.2, 5.8, and 5.4) by unbiased ob- Finally, we reveal the presence of a novel actin binding site within the CBD, servers. Whereas, at low calcium (pCa 6.6), the distribution appears indistin- which coupled to the well-established motor-based actin binding, can guishable from exponential, short run times are increasingly suppressed as contribute to the anchoring and/or actin crosslinking functions of MVI. pCa is decreased in the assay. The pCa 5.4 histogram most resembles gamma rather than exponential distribution. Goodness of fit of the expected and 881-Plat observed frequencies using chi square statistic confirms the calcium- Functional Implications of Dcm End-to-End Bond Mutation in A-Tropo- dependent trend from exponential to gamma distribution. Contrary to our ex- myosin 1 1 1 pectations, adding tropomyosin-troponin complex to same flow cell decreases Alice Ward Racca , Nicholas LaFave , Stephanie Jones , 2 2 1 rather than increases the proportion of short run times, thereby shifting the Michael J. Rynkiewicz , William Lehman , Jeffrey R. Moore . 1Biological Sciences, University of Massachusetts, Lowell, MA, USA, distributions away from exponential and toward gamma at all pCa. We can 2 explain these observations mathematically if events during the run delay the Physiology & Biophysics, Boston University School of Medicine, Boston, rate that pause events arrive by a thermodynamic process. Given the MA, USA. TPM1 constraint of constant free energy, we propose that the delay events arise Mutations in the gene cause both hypertrophic and dilated cardiomyop- a by changes in mechanical force acting on the position of tropomyosin during athy. Deleterious polymorphisms are found throughout -tropomyosin, and the run. These results motivate further investigation of motion-dependent fluc- those within the end-to-end bond regions are poised to drastically change the tuations and run-lengthening agents. Work supported by NIH R01-HL128683 ability of tropomyosin to bind to actin, affecting the balance of the blocked- (JRP) and leave supported by UWG (HGZ). closed-open paradigm of muscle contraction regulation. The M8R mutation is found in both a and g tropomyosins, causing dilated cardiomyopathy and 879-Plat nemaline myopathy respectively. Previous work on pseudo-acetylated and Elucidating Principles of Molecular Elasticity in Muscle Filament Pro- non-acetylated M8R in a-tropomyosin has shown that the mutation causes dras- teins: from High-Resolution Structure to In Vivo Proof tically reduced actin-binding, increased velocity of actin-tropomyosin fila- Philipp Hornburg, Spyros D. Chatziefthimiou, Matthias Wilmanns. ments on myosin, and reduced myosin S1 ATPase. Here we determined the Hamburg Unit, EMBL, Hamburg, Germany. effects of the M8R mutation on the calcium dependence of troponin/tropomy- The protein folding universe has been well understood by more than 100,000 osin regulated in vitro motility of F-actin, its actin-binding at steady state via structures deposited in the Protein Data Bank. However, mechanistic insight co-sedimentation, and its structural stability using circular dichroism. The cal- how proteins react in response to external forces that are universally present cium sensitivity of cardiac thin filaments regulated with M8R was increased in living cells is largely lacking. To investigate such properties, we have chosen compared to WT, as was the cooperativity with no change in maximum actin the M-band of sarcomeric units that are found in striated skeletal and cardiac filament velocity. The denaturation of a-tropomyosin followed a sigmoidal muscles. The M-band is thought to ensure packing regularity and uniform dis- relationship, showing that M8R unfolded more gradually when exposed to tribution of substantial tension across the myosin filaments during contraction/ increasing urea than did WT, but was more sensitive to the presence of urea. relaxation. The M-band protein myomesin, which is considered to act as strain Finally, M8R a-tropomyosin binding of F-actin was, as previously reported, absorber keeping the thick filaments in register during muscle contraction, is extremely low in the absence of S1. The altered regulatory and biochemical composed of 13 domains that form an end-to-end C-terminal dimeric filament properties are consistent with decreased end-end bond stiffness. Using molec- about 40 nm in length. Previously, we showed that the C-terminal myomesin ular dynamics simulations, mutation-induced alterations in tropomyosin struc- domains 9-13 are arranged by a highly repetitive topology, in which an array ture around the end-to-end bond are being characterized. Support by HL123774 immunoglobulin-like domains is connected by highly exposed alpha-helical to JRM. linkers each [1]. When exposed to external forces < 10 pN, the myomesin fila- ment can be reversibly extended by about 2,5-fold in length based on single molecule measurements. Additional structures of myomesin domains 7 and 8 Platform: Intrinsically Disordered Proteins (IDP) reveal an unrelated organization, confining molecular elasticity to the C-termi- and Aggregates II nal part of myomesin. In our present work, we have designed experiments that allow validating our findings on molecular elasticity by two complementary ap- 882-Plat proaches, including super-high resolution imaging and molecular FRET. Changes to the Intracellular Milieu Control the Population and Residual Further details on this will be presented at the meeting. We believe that this Structure of Unfolded Proteins methodology could be applicable to a broad range of related protein filament Yuhang Wang1, Caitlin Davis2, Alex S. Holehouse3, Martin Gruebele4, structures, with great potential to advance our general understanding on molec- Shahar Sukenik5. ular mechanisms generating elastic properties in protein structures. References: 1Biophysics, UIUC, Urbana, IL, USA, 2Univ Illinois, Urbana, IL, USA, [1] Pinotsis et al. (2012) PLoS Biol. Feb;10(2):e1001261. [2] Pernigo et al. 3Biome, Washington University in St. Louis, St Louis, MO, USA, 4Dept (2017) Structure 25(1):107-120. Chemistry, Univ Illinois Urbana Champaign, Urbana, IL, USA, 5Chemistry and Chemical Biology, UC Merced, Merced, CA, USA. 880-Plat Proteins lacking a well-defined fold play important roles in cell signaling, pro- Novel Tales About the Myosin VI Tail liferation, and disease. Due to their lack of tertiary structure, such proteins can Natalia Fili, Alexander Cook, Yukti Hari Gupta, Christopher P. Toseland. be highly sensitive to the surrounding environment. The cellular environment is School of Biosciences, University of Kent, Canterbury, United Kingdom. in constant flux: ionic strength, solute composition, crowding conditions, and Myosin VI (MVI) is an unconventional myosin with the unique ability to move other factors routinely change in response to both external and internal stimuli. towards the minus end of actin filaments. Due to this property, MVI is involved Yet how these changes affect unfolded proteins in vivo is poorly understood. To in a wide range of cellular processes, including endocytosis, exocytosis, cell understand the response of unfolded proteins to changes in the cellular milieu, migration and transcription. This multi-functionality is achieved through the we study the structure of the folded and unfolded state of a FRET-labeled tightly regulated interaction of MVI with various binding partners, which con- glycolytic enzyme in as we perturb the intracellular environment. Live cell trols the conformation and dimerization of the protein, and defines its intracel- data is compared with in vitro measurements, and a computational model helps lular localisation and function. Therefore, not surprisingly disruption in MVI reveal the sensitivity of unfolded proteins to changes in crowding and solute function and regulation leads to various diseases, including aggressive cancers. concentrations in the cell. We show that changes to the intracellular environ- Although MVI is an extensively studied molecular motor, many aspects of its ment can control not only the population of the unfolded state, but that the regulation remain elusive. Deciphering the regulatory events within the tail is structure of the unfolded ensemble can itself be altered in the cell. We propose therefore critical to fully understand how the function of MVI is controlled that unfolded proteins, and especially certain intrinsically disordered proteins, within the cell and how its dysregulation leads to disease. Here, we present are designed to sense and respond to intracellular environment changes by novel insights into how this multipotent myosin is regulated. We present altering their residual structure. how isoform splicing can dictate the intracellular localisation of MVI. In addi- tion, we show how the presence of high and low affinity binding motifs within 883-Plat MVI cargo binding domain (CBD), coupled to a splice variant blocking access Evolution of the Intrinsically Disordered Activation Domain in a Hox to the high affinity site, results in differential regulation of MVI by its binding Transcription Factor partners. We therefore suggest that the difference in affinity between the bind- Ying Liu1, Annie Huang1, Rebecca Booth1, Gabriela Mendes1, ing motifs is a regulatory mechanism, whereby the intracellular function of the Zabeena Merchant2, Kathleen S. Matthews2, Sarah E. Bondos1. non-insert isoform of MVI, in which both binding motifs are accessible for in- 1Dept Molec/Cell Med, Texas A&M Univ Hlth Sci Ctr, College Station, TX, teractions, can be tuned by the relative expression levels of different partners. USA, 2Department of BioScience, Rice University, Houston, TX, USA.

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Linking changes in the amino acid sequence to the evolution of transcription result, NCBD switches on a time scale of tens of seconds between two pop- regulatory domains is often complicated by the low sequence complexity and ulations that differ in their affinities to ACTR by about an order of magni- high mutation rates of intrinsically disordered protein regions. For the Hox tude. Molecular dynamics simulations indicate as a cause reduced packing transcription factor Ultrabithorax (Ubx), conserved motifs distributed of the complex for the cis isomer. Peptidyl-prolyl cis/trans isomerization throughout the protein sequence enable direct comparison of specific protein may be an important previously unidentified mechanism for regulating regions, despite variations in the length and composition of the intervening se- IDP interactions. quences. The Drosophila melanogaster Ubx transcription activation domain can be subdivided into an ‘‘ region’’ that increases activation efficacy 886-Plat and a ‘‘core region’’ required for activation. In cell culture, the strength of tran- Sequence-Based Fingerprinting of Intrinsically Disordered Regions scription activation by Drosophila Ubx correlates with the presence of a pre- Garrett M. Ginell, Megan C. Cohan, Alex S. Holehouse. dicted helix within the core region. Mutations in this helix alter Ubx activity Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, in the developing wing, demonstrating its functional importance in vivo. Curi- USA. ously, this helix is not preserved in species more divergent than flies, suggesting Sequence conservation and the high fidelity of sequence-to-structure rela- the nature of transcription activation may have evolved in these Ubx ortho- tionships enable the identification of protein sequence families. This is espe- logues. A yeast one-hybrid assay demonstrated that the ability of full-length cially true of proteins that are capable of folding as autonomous units. Ubx orthologues to activate transcription varies substantially in Ubx ortho- However, over a third of the eukaryotic proteome consists of intrinsically logues representing 540 million years of evolution. The sequence compositions disordered proteins and regions (IDRs) that either fold upon binding into and the locations of the activation domains also differ. Furthermore, the func- distinct context-dependent structures or persist in disordered albeit functional tion of analogous regions of the Ubx protein may vary from being required for states. Freed from the constraint of autonomous folding, the sequences of transcription activation, enhancing activation efficiency, having no activity, or IDRs are frequently less well conserved as assessed by standard even inhibiting transcription activation. We conclude that, unlike alignment-based metrics. Accordingly, traditional methods for sequence homeodomain-DNA binding, the nature of transcription activation by Ubx classification often fail when identifying sequence families that are defined has substantially evolved. by IDRs. The apparent lack of sequence conservation could be taken to mean that random sequences are interoperable with the sequences of IDRs, 884-Plat yet several recent studies show that this is clearly not the case. Biophysical Conformational Effects of a Disease-Associated Hydrophobic-to-Hydro- studies have helped identify the existence of sequence-to-conformation rela- phobic Substitution and Histidine Protonation State Located at the tionships for IDPs. These relationships, in turn, govern the molecular func- Midpoint of the Intrinsically Disordered Region of proBDNF tions of IDRs and by extrapolation we propose that there is an amino-acid Ruchi Lohia, Grace Brannigan. based grammar that underlies sequence-to-function relationships as well. Rutgers Univ, Camden, NJ, USA. Sequence complexity, amino acid composition, and the linear patterning of Val66Met substitution at the midpoint of the prodomain of precursor brain- different types of amino acids are among the main determinants of derived neurotrophic factor (proBDNF) has been widely studied for its associ- sequence-to-conformation relationships and the consequences for functions ation with increased susceptibility to neuropsychiatric disorders including and phenotypes. We have used these sequence features as fingerprints to schizophrenia and unipolar depression. Previously, NMR studies demonstrated develop ways to delineate sequence families for IDRs. These measures are that the prodomain is disordered with different secondary structure preferences designed to enable the effective classification and comparison of IDRs, for V66 and M66 (Anastasia et al. 2013). with applications towards sequence design, evolutionary analysis, and ulti- We carried out 128ms of molecular dynamics (MD) simulations with tempera- mately the prediction of molecular functions of IDRs. These efforts are ture replica exchange (64replicas, 2ms per replica) of the 90-residues BDNF likely to enable an improved understanding of the evolutionary determinants prodomain with and without Val66Met substitution to investigate its conforma- of IDRs versus intrinsically foldable regions. tional effects. We observe that Val66Met increases the formation of long heli- ces and decreases the formation of long beta sheets structure around residue 66. 887-Plat Secondary structure coupling is found in V66 at residue 66 and 92. Differential Functional Adaptation Mutations Alter Propensity for Alpha-Helical secondary structure in V66 and M66 leads to different pairs of dominating salt Conformation in the Intrinsically Disordered Glucocorticoid Receptor bridges which introduces slight differences in radius of gyration of the two Tau1Core Activation Domain forms. Lennart Nilsson1, Anthony Wright2, Kyou-Hoon Han3. HIS65 is located in a negatively charged region of the prodomain 1Dept Biosci/Nutr, Karolinska Inst, Huddinge, Sweden, 2Dept Lab Medicine, sequence and can exist in protonated form. We further probe the effects Karolinska Inst, Huddinge, Sweden, 3Dept of Nano and Bioinformatics, of protonated HIS65 on the conformational ensemble of V66 and M66 University of Science and Technology, Daejeon, Republic of Korea. with MD simulations (128ms). Consistent with the previous simulations Adaptive mutations that alter the functionality of proteins are enriched within we find that Val66Met increases the formation of long helices. Addition- intrinsically disordered protein regions (IDRs), thus conformational flexibility ally, protonated histidine increases the formation of long helices and appears to translate to evolvability. Pre-structured motifs (PreSMos) with eliminates the beta structure tendency around residue 66 in both V66 and relatively low propensity for secondary structure conformation are believed M66 forms. to be important for IDR function, particularly coupled binding and folding These results indicate that the neutral substitution may exert its effects by crit- protein interactions. A set of 60 change-of-function mutants affecting ically adjusting the entropic cost of local secondary-structure elements, which, the intrinsically disordered 58-residue glucocorticoid receptor tau1core in turn, affects the conformational ensemble via differential salt bridging pat- transcriptional activation domain (GRtau1core) were studied using disorder terns. Additionally, histidine protonation acts as a conformational switch be- prediction algorithms, NMR spectroscopy and molecular dynamics simula- tween helix and beta sheet structures for V66 forms. tions. Overall we conclude that changes in PreSMo stability account for the effects of a subset of change-of-function mutants affecting the 885-Plat GRtau1core IDR. A Proline Switch Explains Kinetic Heterogeneity in a Coupled Folding and Binding Reaction 888-Plat Franziska Zosel1,2, Davide Mercadante2, Daniel Nettels2, The Disordered Protein Bugz Conserves Mitotic Function and Liquid- Benjamin Schuler2. Liquid Phase Separation across 1.6 Billion Years of Evolution 1Novo Nordisk A/S, Maaloev, Denmark, 2Dept Biochemistry, University of Alexander F. Chin1, Vincent J. Hilser1, Yixian Zheng2. Zurich, Zurich, Switzerland. 1Biology, Johns Hopkins University, Baltimore, MD, USA, 2Embryology, The interactions of intrinsically disordered proteins (IDPs) with their molec- Carnegie Institution for Science, Baltimore, MD, USA. ular targets are essential for the regulation of many cellular processes. IDPs How do disordered, liquid-liquid phase-separating protein domains evolve? We can perform their functions while disordered, and they may fold to struc- establish that apparent orthologs of the mitotic-spindle-regulatory protein tured conformations on binding. Here we show that the cis/trans isomeriza- BuGZ conserve biophysical and cellular functions across their 1.6 billion years tion of peptidyl-prolyl bonds can have a pronounced effect on the of divergence since their last common ancestor. Each ortholog contains a interactions of IDPs. By single-molecule spectroscopy, we identify a highly-diverged disordered domain necessary for phase separation, and all or- conserved proline residue in NCBD (the nuclear- binding domain thologs undergo phase separation both in a eukaryotic cell culture system and in of CBP) whose cis/trans isomerization in the unbound state modulates the vitro, where the separation depends on concentration, temperature, salt, and association and dissociation rates with its binding partner, ACTR. As a aliphatic . Importantly, all orthologs rescue the mitotic function of

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BuGZ in a heterologous siRNA knockdown system. Together these results sup- 891-Plat port the hypothesis that the phase-separating and cell-biological functions of Catching HIV in the Act of Fusion: Insight from Cryo-Et Intermediates of the BuGZ disordered domain share true homology despite their sequence dis- HIV Membrane Fusion similarities, and are an example of ancient eukaryotic conservation of phase Amanda E. Ward1, Kelly A. Dryden2, Lukas K. Tamm1, separation and biological function of a disordered protein. Barbie K. Ganser-Pornillos1. 1Molecular Physiology and Biological Physics, University of Virginia School 889-Plat of Medicine, Charlottesville, VA, USA, 2Molecular Electron Microscopy Probing Specificity in Disordered Protein Interactions with Small Mole- Core, University of Virginia School of Medicine, Charlottesville, VA, USA. cules using Integrative Methods The rise of HAART-resistant HIV and the growing HIV-infected patient pop- Gabriella T. Heller1, Francesco A. Aprile1, Massimiliano Bonomi1, ulation necessitates the search for new antivirals. Structural and mechanistic Carlo Camilloni2, Alfonso De Simone3, Michele Vendruscolo1. 1 studies of HIV membrane fusion could yield novel therapeutic targets that Department of Chemistry, University of Cambridge, Cambridge, United block the first step of the viral lifecycle, similar to the licensed drugs Enfuvir- Kingdom, 2Department of Biosciences, University of Milano, Milano, Italy, 3 tide and Maraviroc. HIV’s membrane fusion is mediated by a series of confor- Dept Life Sci, Imperial Coll London, London, United Kingdom. mational changes in the viral glycoprotein, Env, that draw the viral and cell Intrinsically disordered proteins are highly prevalent in many diseases membranes together. Previously, technical limitations have prevented high res- including cancer, cardiovascular disease, diabetes, and neurodegeneration. olution observation of this process and its mediators in vitro. We have devel- Despite the enormous therapeutic potential of drug binding to these proteins, oped an assay to observe intermediates of HIV membrane fusion to giant however, their binding mechanisms with small molecules remain poorly un- plasma membrane vesicles (blebs) by cryo-electron tomography (cryo-ET). derstood. For this reason, disordered proteins are often considered ‘untarget- By synchronizing HIV fusion to CD4 and CCR5 containing blebs before plunge able’. More specifically, one of the main concerns with targeting disordered freezing, we are able to capture multiple intermediate membrane fusion states proteins with small molecules is whether sufficient specificity in the interac- which enabled the following observations: 1. After receptor binding but before tion can be achieved to avoid side effects in drug development. In order to fusion, there often is a broad contact area between viral and target membranes understand the nature of specificity in the interactions between small mole- 2. Env redistributes and reorients in the viral membrane to accommodate fusion cules and disordered proteins, we employ a multidisciplinary approach and 3. A wide fusion pore allows an intact capsid to exit the virus. We are combining NMR spectroscopy, statistical inference, and molecular dynamics currently using this assay to investigate perturbations of HIV membrane fusion simulations. We apply this approach to probe the interaction of one small by viral restriction factors, broadly neutralizing antibodies and pharmaceuti- molecule, 10058-F4 with a disordered region from the oncogene protein c- cals. Our observations help build a model for HIV membrane fusion and cell myc. We thus observe a highly dynamic, entropic interaction that exhibits entry while helping answer specific questions about lipid membrane intermedi- sequence specificity. In particular, we provide a quantitative measure of this ate fusion structures and the fate of the viral capsid. sequence specificity and compare it to the case of the interaction of urea, which is diffuse but not specific. To investigate whether this ‘specific-diffuse’ 892-Plat binding can generally modify intermolecular interactions, we show that it in- Probing Membrane Fusion Intermediates using Bilayer Coated Nanopar- hibits the aggregation of the peptide. These results suggest that this previously ticles unknown binding mechanism may create novel opportunities to discover Ana Villamil1, Peter Kasson2. drugs that target disordered proteins in their monomeric states in a specific 1Cell and Molecular Biology, Uppsala University, Uppsala, Sweden, 2Dept manner. Molec Physiol, Univ Virginia, Charlottesville, VA, USA. Viral membrane fusion is believed to involve highly curved lipid intermediates, but the kinetic role of these intermediates has been difficult to probe directly. Platform: Macromolecular Interactions and We have recently developed methods to measure fusion kinetics of individual influenza virions to liposomes of variable size. In order to probe the role of Effects on Membranes membrane deformation in viral fusion kinetics, we have now also developed supported lipid bilayers on small hydrophilic nanoparticles as a substrate for 890-Plat fusion. As assessed by cryo-electron microscopy, this approach yields tight The Structural Basis of a Membrane-Bound ESCRT-III Helical Assembly control of target membrane curvature; the solid support also reduces membrane Henry C. Nguyen1, Nathaniel Talledge2, John McCullough2, deformation. As expected, influenza fusion to these nanoparticles is reduced Wesley I. Sundquist2, Adam Frost1. compared to liposomes of comparable size. This approach permits the use of 1Dept of Biochemistry and Biophysics, UCSF, San Francisco, CA, USA, different-sized nanoparticles to tune the energetic penalty associated with 2Dept of Biochemistry, University of Utah, Salt Lake City, UT, USA. membrane rearrangements and analyze its impact on the kinetics and efficiency The Endosomal Sorting Complexes Required for Transport (ESCRT) mediate of membrane fusion. In such a manner, we can dissect the impact of membrane critical membrane remodeling events throughout the mammalian cell cycle, size and deformability on viral membrane fusion, comparing with previous including, but not limited to, HIV budding, cytokinetic abscission, and sealing theoretical predictions. of the nuclear envelope. ESCRT-III proteins polymerize into membrane- binding filaments to catalyze these reactions, but the structures and functions 893-Plat of these assemblies remain poorly understood. Our collaborative team recently Bacterial Outer Membrane Vesicle Interaction with Plasma Membranes: determined the first atomic-resolution structure of an ESCRT-III filament - a Insights from Molecular Simulations hetero-polymer consisting of IST1 and CHMP1B. Our previous structure Damien F. Jefferies1, Anna L. Duncan2, Syma Khalid1. demonstrated how one of these subunits, CHMP1B, transitions from a 1Dept Chemistry, Univ Southampton, Southampton, United Kingdom, 2Dept ‘‘closed’’ to an ‘‘open’’ state to form an interlocked and domain-swapped fila- Biochemistry, Univ Oxford, Oxford, United Kingdom. ment. Moreover, we and others have shown that the IST1-CHMP1B copolymer Outer membrane vesicles (OMVS) are released from the cell envelopes of all participates in non-canonical, positive-curvature membrane fission pathways. Gram-negative bacteria studied to date. They are nano-sized (20-200 nm) pro- Very recent work on other ESCRT-III proteins indicated that the mechanisms teoliposomes with the ability to deliver a range of molecular cargo into host of opening and assembly we reported are conserved, but also raised questions cells, but the molecular details of delivery and indeed the processes that pre- regarding membrane binding and remodeling activities. To address these gaps cede delivery are poorly understood. Experimental studies have shown that in our understanding, we have determined the high-resolution structure of a OMVs can to bind to, and bypass plasma membranes via a number of membrane-bound IST1-CHMP1B assembly by cryo electron microscopy (cry- different routes, often depending upon the composition of the OMV. Recently oEM). We find that CHMP1B induces a high degree of curvature alone, and it has been shown that OMVs containing O-antigen level lipopolysaccharide that deposition of the IST1 strand further constricts the membrane by more (LPS) can enter host cells independent of caveolin or dynamin mediated endo- than 2-fold - almost to the fission point. Notably, the distance between outer cytosis. It is thought that instead they exploit specific lipidic regions of the leaflet lipid headgroups is 10 nm and the distance between inner leaflet lipid plasma membrane.To explore this hypothesis we have performed a series of headgroups is reduced to only 4 nm. Conserved residues along helix a1of coarse-grained molecular dynamics simulations of OMVs with and without CHMP1B serve as the major membrane binding surface and exploit both elec- O-antigen level LPS. Our model OMVs are the same size as the smallest trostatic as well as hydrophobic interactions with the convex leaflet of the mem- OMVs produced by E.coli and our plasma membrane models contain a range brane tubule. Our atomic-resolution cryoEM study reveals the structural of lipids type. We have accumulated microseconds of simulations to gain in- mechanisms governing ESCRT-III assembly, membrane-binding, and sights into a process that is now known to be of key importance for bacterial- positive-curvature membrane deforming activities. host interactions.

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894-Plat Fluorescent dyes are widely used to visualize biological targets, as well as Lipid Sponge-Phase Nanoparticles as Enzyme Carriers - Structure and to monitor biological activity, such as viral entry. While powerful tools Intermolecular Interaction Controlling the Enzyme Inclusion for insight, such probes also have the potential to perturb the process under Maria Valldeperas1, Najet Mahmoudi2, Susana C.M. Teixeira3, study in non-obvious ways. Here, we show how dyes used to membrane Martynas Talaikis4, Ieva Matulaitiene4, Gediminas Niaura4, fusion in viral entry can alter both the kinetics and the efficiency of viral Justas Barauskas5, Tommy Nylander1. membrane fusion in a fashion that depends on illumination intensity and 1Physical Chemistry Dept. of Chemistry, Lund University, Lund, Sweden, dye concentration. We demonstrate this using single-virus fusion measure- 2ISIS, STFC, Rutherford Appleton Laboratory, Didcot, United Kingdom, ments. R18, a dye that is commonly used to monitor lipid mixing between 3NIST Center for Neutron Research, Gaithersburg, MD, USA, 4Vilnius membranes, is particularly prone to these effects, while Texas Red is some- University, Vilnius, Lithuania, 5Camurus AB, Lund, Sweden. what less sensitive. These results demonstrate how fluorescent probes can Non-lamellar lipid aqueous phases, such as reverse cubic or hexagonal phases, perturb measurements of biological activity and provide both data and can be used to entrap smaller biomolecules. The curvature of these lipid phases a method for assessing determining minimally perturbative measurement and hence the size of the aqueous cavities depends on the composition, water conditions. content and temperature. The challenge is to encapsulate proteins, such as large enzymes due to the limited size of these cavities. Here, we will present a lipid 897-Plat system, based on mixtures of acylglycerides and acyldiglycerides, which are The SARS-CoV Fusion Peptide Forms an Extended Bipartite Fusion Plat- able to form highly swollen sponge phases (L3), with aqueous pores up to 13 form that Perturbs Membrane Order in a Calcium-Dependent Manner nm of diameter. The structure and composition of the particles were revealed Alex L. Lai1, Jean K. Miller2, Susan Daniel3, Gary R. Whittaker2, by using small angle neutron scattering (SANS), light scattering, cryo-TEM, Jack H. Freed1. size exclusion chromatography and Raman spectroscopy. The Raman spectros- 1Dept Chem/Chem Bio, Cornell Univ, Ithaca, NY, USA, 2Dept Microbiology copy results for the sponge phases are compared with data for lamellar and and Immunology, Cornell Univ, Ithaca, NY, USA, 3School of Chemical and reverse bicontinuous cubic phase in the same lipid system and show large sim- Biomolecular Engineering, Cornell Univ, Ithaca, NY, USA. ilarities in lipid chain confirmation and head group interactions as all three Coronaviruses are a major infectious disease threat, and include the pathogenic structures are formed by lipid bilayers, albeit of different curvature. The L3 human pathogens of zoonotic origin: SARS-CoV and MERS-CoV. Entry of co- structure is preserved even in excess aqueous solution, where they form ronaviruses into host cells is mediated by the viral spike (S) protein, which is sponge-like nanoparticles (L3 NPs). We investigate encapsulation of two key structurally categorized as a class I viral fusion protein, within the same group types of enzymes of different sizes, used in food processing, namely Aspartic as influenza virus and HIV. However, S proteins have two distinct cleavage protease (34 KDa) and Beta-galactosidase (460 KDa). They are today delivered sites that can be activated by a much wider range of proteases. The exact loca- into the process as solutions with a considerable amount of preservatives and tion of the coronavirus fusion peptide (FP) has been disputed. However, most still with limited shelf-life and limited control of the enzyme activity. The evidence suggests that the domain immediately downstream of the S2’ cleav- SANS results reveal differences in the L3 NPs with and without enzyme that age site is the FP (amino acids 798-818 SFIEDLLFNKVTLADAGFMKQY can be interpreted as inclusion of the protein in the liquid crystalline phase. for SARS-CoV, FP1). In our previous ESR spectroscopic studies, the mem- These findings are verified by size exclusion chromatography and the enzy- brane ordering effect of influenza virus, HIV and Dengue virus FPs have matic activity of the encapsulated enzyme, which surpasses the enzymes stabil- been consistently observed. In this study, we used this effect as a criterion to ity in solution. identify and characterize the bona fide SARS-CoV FP. Our results indicate that both FP1 and the region immediately downstream (amino acids 816-835 895-Plat KQYGECLGDINARDLICAQKF, FP2) induce significant membrane Preparing Endosome-Derived Supported Membranes to Study Ebola ordering. Furthermore, their effects are calcium-dependent, which is consistent Virus GP-Mediated Membrane Binding and Fusion with in vivo data showing that calcium is required for SARS-CoV S-mediated Laura Odongo. fusion. Isothermal titration calorimetry showed a direct interaction between Univ Virginia, Charlottesville, VA, USA. calcium cations and both FPs. This Ca2þ-dependency membrane ordering Ebola virus (EBOV) is a highly virulent pathogen that causes a severe hemor- was not observed with influenza FP, indicating that the coronavirus FP exhibits rhagic fever with case fatality rates ranging from 50% to 90%. EBOV poses a a mechanistically different behavior. Membrane ordering effects are greater serious threat to global human health due to its high pathogenicity, high mor- and penetrate deeper into membranes when FP1 and FP2 act in a concerted tality and lack of approved vaccines or specific treatment. EBOV entry into manner, suggesting that they form an extended fusion ‘‘platform’’. cells is mediated by the viral glycoprotein (GP) located on the surface of the virus. Following attachment to the cell, EBOV is trafficked to endolysosomes where proteolytically primed GP binds to its intracellular receptor Niemann Awards and 2019 Biophysical Society Lecture Pick type C1 (NPC1) and is exposed to factors that induce it to change confor- mation. This results in fusion of viral and host cell membranes thereby 898-Natl releasing EBOV genetic material into the host cell. The cellular factors that From Peripheral Proteins to Membrane Motors - Mass Spectrometry act on GP to induce these conformational changes remain unknown, as low Comes of Age pH and interaction with the GP binding domain of NPC1 appear insufficient Carol Robinson. to trigger fusion. Identifying the elusive trigger has proven difficult as there University of Oxford, Oxford, United Kingdom. is no in vitro fusion assay for Ebola virus and full-length NPC1 in a closely What can we learn from the study of a protein complex in the gas phase of the native setting. We are developing such an assay using total internal reflection mass spectrometer? Aside from the compositional information that is avail- fluorescence microscopy to visualize and distinguish between binding, hemifu- able from obtaining the molecular mass of an intact protein complex, many sion, and fusion of pseudoviruses, containing EBOV GP, with endosome- additional properties can be deduced: these include polydispersity and hetero- derived planar supported membranes. To this end, we have purified endosomes geneity, which are some of most challenging properties of protein complexes, from HEK-293T cells, transformed them into planar supported membranes, and making them almost impossible to study by conventional structural biology identified the presence of critical endosomal markers. As proof of concept, we approaches. Mass spectrometry is providing significant insight into protein are also using pseudoviruses bearing Lassa virus GP. Like EBOV, Lassa virus composition within assemblies by uncovering the effects of post- employs an intracellular receptor, in this case lysosomal-associated membrane translational modifications, the dynamics of subunit exchange and changes protein 1 (LAMP1). Lassa virus requires both low pH and LAMP1 for effective in interactions in response to small molecule binding. Recent breakthroughs, entry into cells via the endolysosome, and these two factors are sufficient to which combine the study of intact membrane complexes with knowledge of trigger fusion in vitro. lipid binding sites, is informing both the oligomerisation and downstream coupling of GPCRs. While the majority of these studies of membrane proteins 896-Plat have been performed in detergent micelles very recently we have successfully Detecting and Controlling Dye and Illumination Effects in Single-Virus employed a new means of ionizing proteins directly from membranes. Fusion Experiments Following sonication of lipid vesicles and application of high electric fields, Robert J. Rawle1, Steven G. Boxer2, Peter M. Kasson1,3. proteins are effectively drawn out of their native membrane locations into 1Dept of Molecular Physiology, Univ Virginia, Charlottesville, VA, USA, the gas phase of the mass spectrometer. This new approach is revealing 2Dept Chemistry, Stanford Univ, Stanford, CA, USA, 3Dept of Cell and many new interactions that have been lost following disruption in detergent Molecular Biology, Uppsala University, Uppsala, Sweden. micelles.

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Posters binding cleft. Effect of changes in pH, ionic strength and the state of iron on iron affinity are also investigated. We believe that the study on the dynamics of Fbp addresses us to further Posters: Protein Structure and Conformation II biosensor applications to determine elevated levels of iron in individual cells and targeted drug design practices. 899-Pos Molecular Basis of the Marburg Virus Protein VP24 Interactions with Human Keap1 902-Pos Nisha Bhattarai, Prem P. Chapagain, Bernard Gerstman. Designing Superhydrophilic, Disordered Peptides to Improve the Stability Physics, Florida International Univ, Miami, FL, USA. and Efficacy of Protein Therapeutics The binding of the Marburg virus protein VP24 to the human Keap1 protein Joshua Smith, Patrick McMullen, Zhefan Yuan, Shaoyi Jiang, allows the nuclear accumulation of Nrf2, activating cytoprotective antioxidant Jim Pfaendtner. response pathways during the viral life cycle. We investigate the molecular Dept Chem Eng, Univ Washington, Seattle, WA, USA. level details of VP24-Keap1 interactions for both Marburg and Ebola The therapeutic potential of protein drugs (biologics) has been hindered by VP24. Our results show that the presence of the cysteine residues in the difficulties with long-term storage and rapid clearance from the body. Re- K-loop region of mVP24 provides strong interfacial interactions with combinant fusion proteins provide a scalable synthesis platform for engi- Keap1, including hydrogen bonding and S-Hp interactions, facilitating neered biologics, whereby a polypeptide domain can be appended to alter the formation of a stable complex with Marburg VP24. These cysteine resi- the physical characteristics of a therapeutic protein and enhance its pharma- dues are not present in eVP24, which does form a stable complex with ceutical viability. Two simple design principles, based on the physical prop- Keap1. These results provide insights into how the Marburg virus, but not erties of the polypeptide domain, have been separately applied to address Ebola, is able to activate the antioxidant response pathways through direct in- issues with the storage and delivery of biologics. Superhydrophilic peptides, teractions with Keap1. exemplified by the alternating-charge peptide poly(EK), have been shown to increase the thermostability of proteins in vitro. ‘‘Conformationally disor- dered’’ peptides, exemplified by the homo amino-acid peptide polyG, have 900-Pos been shown to increase the circulation half-life and bioactivity of protein Solution NMR Structure of the Gtpase Activating (GAP) Domain of Vope, therapeutics in vivo. The combination of superhydrophilicity and conforma- A Vibrio Cholerae T3Ss Effector Protein tional disorder in a single fusion peptide may be necessary to simultaneously 1 2 2 3 Kyle P. Smith , Woonghee Lee , Marco Tonelli , Priscilla S-W Yeung . address concerns regarding the storage and therapeutic lifetime of biologics. 1Medicine, Northwestern University, Chicago, IL, USA, 2Biochemistry, 3 In the current work, we use enhanced sampling molecular dynamics (MD) Wisconsin University, Madison, WI, USA, Pharmacology, Northwestern simulations to investigate the conformational ensemble of poly(EK) and University, Chicago, IL, USA. glycine-substituted poly(EK) variants, and validate our structural predictions Vibrio cholerae uses a type III secretion system to inject effector proteins with circular dichroism (CD). We find the (EK)15 peptide exhibits a high into a host cell. Recently, a putative GTPase Activating Protein (GAP) propensity for forming anti-parallel beta strand secondary structures, which called VopE was identified as a pathogenic protein that affected host mito- are stabilized by extensive salt-bridging of the positive and negative side- chondrial dynamics and immune response. However, biophysical and struc- chains. MD simulations predict that limited glycine substitutions effectively tural characterization has been missing. We describe solution NMR disrupt the secondary structure and promote disordered conformations at structure of the putative GAP domain (73-204) of VopE. Using SEC- physiologically relevant temperatures. We conclude that conformational dis- SAXS data, we improved the quality of the output calculated structures. order should be rationally programmed into alternating-charge peptides to Structural comparison with other ToxGAP’s revealed a conserved overall improve their suitability for drug delivery applications. We also contribute fold with several unique features to VopE. By examining the conservation a computational approach to quantify conformational disorder in polypep- of residues at putative binding interface, we again find features conserved tides, which should facilitate the de novo design of more effective fusion toallToxGAP’sandsomeonlyobservedinVopE.BasedonNMRdata proteins. with its putative GTPase target, we hypothesize on other potential target mitochondrial GTPases. 903-Pos 901-Pos Systematic Analysis of Internal Hydration in a Protein Characterization and Iron Binding Dynamics of Haemophilus Influenzae Anne M. Rice1, Jaime L. Schlessman2, Bertrand Garcia-Moreno1. Ferric Binding Protein 1Department of Biophysics, Johns Hopkins, Baltimore, MD, USA, 2US Naval Goksin Liu1, Ezgi Altun2, S. Mert Unal2, Canan Atilgan3, Zehra Sayers4. Academy, Annapolis, MD, USA. 1Material Science and Engineering, Sabanci University, Istanbul, Turkey, Water is an essential component of the structure and function of most biolog- 2Molecular Biology, Genetics and Bioengineering, Sabanci University, ical macromolecules. In the crystal structures of globular and membrane pro- Istanbul, Turkey, 3Fac Eng/Nat Sci, Sabanci Univ, Istanbul, Turkey, 4Dept teins water molecules are often found buried in the hydrophobic interior of the Bio Sci, Sabanci Univ, Istanbul, Turkey. protein. Some of these water molecules are required for stabilization of the Iron is a fundamental metabolite for the survival of all biological systems folded state. Others are essential for function, for example, in catalysis, Hþ because of its ability to function as both electron donor and acceptor. or ion transport. In general, the structural factors that govern internal hydra- In unbound form, Feþ3 is not soluble in water whereas; Feþ2 is highly tion of proteins are poorly understood. Internal water molecules are to be ex- toxic in free form. Different organisms have developed specific mecha- pected in internal cavities and in regions with low packing-density, especially nisms to transport iron. In humans transferrin is the responsible protein if unsatisfied hydrogen bonds are present; however, this alone does not guar- for carrying iron, whereas in bacteria Ferric Binding Protein (Fbp) antee the presence of internal water molecule. We examined patterns of inter- performs this function. Pathogenic bacteria have evolved to sequester nal hydration in a set of 280 high resolution structures of staphylococcal iron from human transferrin through an exceptional mechanism, where nuclease (SNase). This is an especially rich set of proteins for this purpose one of the two irons of transferrin is detached, transported through as many of these proteins have buried polar atoms or artificial cavities, where the periplasmic space and captured by Fbp. In this study, our main internal waters would be favored. Internal water molecules buried deeply in focus is to monitor conformational changes of Fbp from Haemophilus the hydrophobic interior were found in only 24 of these 100 crystal structures. Influenzae, during iron release and binding in alternating environmental These internal water molecules are clustered in 8 specific locations. Water conditions. molecules at each of these internal locations are stabilized by different factors Fbp was expressed and purified by using recombinant DNA technology. Size (e.g. void volume large enough to house water molecules, presence of unsat- Exclusion Chromatography (SEC), Dynamic Light Scattering (DLS), UV-Vis isfied hydrogen bonds of the peptide backbone, of buried polar side-chains Absorption Spectroscopy (AS), Circular Dichroism (CD) and Small Angle and of other internal water sites). Accurate prediction of internal hydration X-ray Scattering (SAXS) are used for detecting structural changes. Addition- is important in a variety of situations (e.g. ab-initio protein structure- ally, features of the 480 nm ligand-to-metal charge transfer absorption peak prediction, structure-based drug design, molecular dynamic simulations, þ of FBP-Iron complex was monitored during iron release/binding process for studies of H transport or catalysis). This set of crystal structures was also both Feþ3 and Feþ2 in different pH and ionic strength conditions. Our results used to benchmark the ability of the DOWSERþþ algorithm for in silico hy- confirm that Fbp changes its conformation to bind iron; hydrodynamic radius dration of proteins to reproduce the patterns of hydration observed in the crys- decreases when it captures the ligand because of the closing up of the metal- tal structures.

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904-Pos 906-Pos Markov Models of Functional Dynamics of Histone Lysine Methyltrans- Classifying of Arrhythmogenic Cardiomyopathy-Linked Desmoplakin ferases by Millisecond-Timescale Molecular Simulation and Chemical Variants through Molecular Mechanisms of Pathogenicity Probing Tyler L. Stevens1, Heather Manring1, Taylor Albertelli2, Ronald Ng3, Rafal P. Wiewiora1,2, Shi Chen1,3, Minkui Luo3, John D. Chodera2. Nathan T. Wright2, Stuart Campbell3, Maegen Ackermann1. 1Tri-Institutional PhD Program in Chemical Biology, Memorial Sloan 1Physiology and Cell Biology, The Ohio State University, Columbus, OH, Kettering Cancer Center, New York, NY, USA, 2Computational and Systems USA, 2Chemistry and Biochemistry, James Madison University, Biology Program, Memorial Sloan Kettering Cancer Center, New York, NY, Harrisonburg, VA, USA, 3Biomedical Engineering, Yale University, New USA, 3Chemical Biology Program, Memorial Sloan Kettering Cancer Center, Haven, CT, USA. New York, NY, USA. Arrhythmogenic Cardiomyopathy (ACM) is a disease that affects 1 in 2000 Histone lysine methyltransferases (HKMTs) are a family of epigenetic en- Americans yearly and segregates with sudden cardiac death (SCD). Up to zymes responsible for catalyzing the methylation of lysines in histones 60% of ACM cases are associated with variants in desmosomal genes, however and other proteins. During the past decade, the understanding of HKMTs the molecular mechanisms associated with ACM remain unclear. Of those 60% has significantly advanced from structural and biochemical perspectives, of cases, up to 40% are linked to the desmosomal protein desmoplakin (DSP). laying foundations for potential therapeutics against multiple diseases, DSP is a structural protein responsible for linking intermediate filaments to the in particular cancer. However, while evidence shows that HKMTs are intercalated disc membrane and contributes to the structural integrity between conformationally dynamic in the context of protein methylation and inhibi- myocytes. Within its NH2-terminus DSP contains a hotspot region for ACM- tor binding, limited knowledge is available about their conformational linked variants. Of the 20 variants within the hotspot region, 9 are pathogenic ensembles. but the remaining 11 are classified as unknown pathogenicity. Our team has In this work, multiple approaches were combined to explore the conformational previously examined the biomolecular properties of the pathogenic hotspot- dynamics of SETD8, which is responsible for the methylation of H4K20 and variants and identified that select variants increased susceptibility to calpain- p53, and implicated in a number of cancers, such as neuroblastoma. A series dependent degradation. Additional pathogenic hotspot-variants however, do of X-ray structures of SETD8 in previously unseen conformations were solved not exhibit enhanced calpain cleavage, indicating that other mechanisms exist with various ligands. Molecular dynamics simulations were conducted to leading to disease. Herein, we sought to test the hypothesis that additional DSP generate thousands of trajectories and analyzed by Markov state models to hotspot-variants of unknown pathogenicity will demonstrate increased suscep- reveal the free energy landscapes, hidden conformations, and kinetic pathways tibility to calpain-mediated degradation. By comparing hotspot-variants of between different conformations. known and unknown pathogenicity, we aim to categorize different variants ac- This approach was extended to groups of HKMTs (such as NSD1/2/3, EZH1/ cording to unique ACM pathomechanisms, i.e. enhanced protein degradation, 2, ASH1L, SETD2) at different evolutionary distances to comparatively altered ligand binding, etc. Using biochemical assays and structural molecular model the dynamics of the protein family and their functional impact. The dynamics we have identified at least 12 of the 20 hotspot-variants result in models are analyzed to understand the free energy profiles along transitions increased calpain-mediated degradation. To understand the direct physiological between active and inactive conformations, the of those implications of a DSP hotspot-variant with increased calpain susceptibility, we activation profiles by protein-protein binding partners (e.g. of EZH2 in the engineered a mutant mouse line harboring the R451G-variant using CRISPR. PRC2 complex), and the role of flexible activation loops in the process. Surprisingly, DSP R451G homozygous mice show lethality by embryonic The plasticity of known binding pockets and the openings of putative allo- day 8. Physiological measurements are underway to assess DSP-R451G hetero- steric pockets are analyzed, with the goal of accelerating the rational design zygous mice at baseline and following cardiac stress. These mice provide a of HKMT chemical probes through capturing unique conformations of indi- novel tool to study the unique ACM-pathomechanism of enhanced calpain- vidual enzymes. mediated degradation.

907-Pos 905-Pos Characterization of Predicted Small Proteins Structural Modulation of RyR1 by MgATP and Free Mg2D in Lipid Mem- Allison Whited1, Christina Cleveland1, Jeffre Allen1, Irwin Jungreis2, brane using CryoEM John Rinn1, Loren Hough1. Ashok R. Nayak1, Alex H. Will1, Joshua Lobo2, Pablo C. Hartmann3, 1Univ Colorado Boulder, Boulder, CO, USA, 2CSAIL, MIT, Cambridge, Montserrat Samso1. MA, USA. 1Department of Physiology and Biophysics, Virginia Commonwealth With the recent improvements in sequencing and bioinformatics techniques, University, Richmond, VA, USA, 2Diamond Light Source, Didcot, United there has been an increase in the number of annotated small (< 100 aa) open Kingdom, 3ThermoFisher Scientific, Cambridge, United Kingdom. reading frames encoding for functional proteins. These proteins have been Mg2þ is the most abundant divalent cation in living cells. In the skeletal mus- found in several species, including humans, and are associated with critical cle cytosol, out of the total Mg2þ concentration of 8 mM, around 1 mM is free cellular functions. A conservative search through the human genome (Human Mg2þ and around 5 mM exists in the form of MgATP, the biological active GENCODE v27) resulted in 168 potential proteins ranging in size from 20-70 form of cellular ATP. Mg2þ is constitutively bound to RyR1 in its free and amino acids. We categorized these proteins into three categories: proteins that complexed forms. In fact, preferential binding of Mg2þ to the acidic side are well annotated, proteins that are annotated based only on homology, tran- chains with similar coordination geometry as Ca2þ, allows it to be a potential scriptional evidence, or computational analysis, and otherwise uncharacterized. antagonistic cation of Ca2þ, which has much lower cytoplasmic concentra- We characterized known and predicted properties of these proteins, including tions (<1 mM - 100 mM). Free Mg2þ is thought to inhibit RyRs through its primary and secondary structural aspects and their micro- and macroscale association with Ca2þ binding sites at cytoplasmic or luminal regions, while localization and functionality. The characterized small proteins in our study the effect of MgATP per se is novel in the context of excitation-contraction have widespread distribution and functionality in the cell and are ubiquitously coupling. expressed throughout the human body. The genes encoding for these proteins, We studied the effect of free Mg2þ and MgATP on RyR1 bound to a POPC including some that are uncharacterized, have also been found in GWASs of bilayer enclosed by membrane scaffold protein (nanodisc). CryoEM recon- several disease or conditions, further underlining their importance. In this structions of RyR1 nanodiscs complexed with MgAMPPCP at 3.8 A˚ resolu- work, we show that while typically uncharacterized, functional small proteins tion show that in the absence of any detergents or FKBP, the RyR1 assumes a have the potential to play important roles in a variety of cellular functions. closed conformation with a pore radius that makes impermeant to Ca2þ ions. Discernible densities for nanodisc and MgATP were observed. Binding of 908-Pos MgATP involves global motion of the central and cytoplasmic domains to- Simulation Guided Design of Spectroscopy Experiments via Maximizing wards the T-tubule and the fourfold axis with reorganization of the P2 domain. Kinetic Information Gain Local motion of the TM helices, U-motif, VSC and EF hands were also Shriyaa Mittal1, Diwakar Shukla2. observed with concomitant closure of the ATP cavity at the intersubunit inter- 1Center for Biophysics and Quantitative Biology, University of Illinois at face. With the cryoEM structure of RyR1-MgATP, we seek to explain the Urbana-Champaign, Urbana, IL, USA, 2Department of Chemical and mechanistic basis of RyR1 inhibition during the physiological resting state of Biomolecular Engineering, University of Illinois at Urbana-Champaign, skeletal muscle. Urbana, IL, USA. Supported by grants AHA 14GRNT19660003, MDA352845, NIH R01 We present a computational tool called Optimal Probes that uses protein molec- AR068431, and NIH U24GM116789. ular dynamics (MD) simulation datasets to address a systematic issue in

BPJ 9352_9354 184a Monday, March 4, 2019 designing spectroscopic experiments. A common problem faced in experi- is that amino acids in a protein coevolve to maintain structural and functional ments that measure relaxation of a pair of residues is narrowing down the integrity. However, most of existing methods remain restricted to protein set of residue pairs to site-specifically label among a large pool of possibil- native structure prediction. Here, we investigate whether and how ECs alone ities. Our method ranks every possibility based on a quantitative measure, can be used in reinforcement learning (RL) to uncover the entire protein Generalized Matrix Rayleigh Quotient (GMRQ) score for a Markov state conformational ensemble. In a recent paper, we evaluated the optimal number model (MSM) of protein conformational dynamics[1]. We optimize the of ECs required to characterize protein conformational dynamics by analyzing choice of residue pairs in the protein to build an MSM that can achieve a 10 diverse proteins. We have shown that (1) few ECs (1-10% of all residue high GMRQ score, thus capturing slow motions of the protein’s dynamics. pairs) suffice to capture the complex dynamics involved in protein folding Once all residue pair sets are scored, the highest scoring inter-residue pairs and conformational changes, and (2) different conformations of the same pro- can be probed to investigate the protein experimentally. We show that incor- tein have unique sets of ECs. Our findings indicate that the structures of porating protein dynamics from simulations in such a manner maximizes in- different protein conformations can be predicted if the set of constraints spe- formation gain from experiments. We illustrate that Optimal Probes can make cific to the structure can be identified. However, it is currently challenging to better choices than experimental choices based on human intuition or limited differentiate shared ECs and conformational specific ECs (30%). Here, we structural information such as distance difference between two known stable propose an automated prediction of protein conformational ensemble from structures of the protein. The tool can also be used to predict optimal exper- sequence coevolution using reinforcement learning. Preliminary results for iment choices considering previous experiments that have already been per- the membrane protein Beta-2 adrenergic receptor (target of many cardiovas- formed on the protein of interest. We demonstrate the use of Optimal cular drugs) show that our proposed approach successfully reveals all three Probes as an intuitive and user-friendly tool on cytoplasmic and transmem- important conformations: inactive, active and intermediate structure. The pro- brane proteins for four experimental techniques, Double Electron-Electron posed method is broadly applicable to many protein families with sufficient Resonance[2], Trp-Tyr quenching, Luminescence Resonance Energy Trans- sequence information and has direct implications in protein structure determi- fer, and Triplet-Triplet Energy Transfer. In conclusion, Optimal Probes pro- nation and drug discovery. vides an automated prediction of residue pairs for biophysical experiment techniques. [1] Mittal and Shukla. J Phys Chem B, 2017 DOI: 10.1021/ 911-Pos acs.jpcb.7b04785 [2] Selvam, Mittal, and Shukla. ACS Central Science, Bioinformatics of New Selective Insecticide Target in Insect Species 2018 DOI: 10.1021/acscentsci.8b00330. Hassan M. Younis. Dept Pestic Chem and Membrane Bio-energetics, Alexandria University, 909-Pos Alexandria, Egypt. Structural Basis Underlying Autoinducer Activation of the Vibrio cholerae Identification of selective and vital protein targets in insect species at the mo- VqmA Quorum-Sensing Receptor lecular level and knowledge about these targets is the prime and crucial step Jon Paczkowski. in the comprehensive strategy of knowledge-based, target-oriented design of Molecular Biology, Princeton University, Princeton, NJ, USA. selective and efficient insecticides. In this context, our research has identified Quorum sensing (QS) is a cell-cell communication process that bacteria use to the insecticide DDT target as the OSCP (Oligomycin sensitivity conferring orchestrate group behaviors. QS is mediated by signal molecules called auto- protein) and subunit ‘‘d’’ of the insect (Apis mellifera) ATP synthase. These . The major human pathogen Vibrio cholerae possesses multiple, two subunits of the insect enzyme differ in their content of amino acids from converging quorum-sensing pathways. At high cell density, when autoinducer that of bovine heart enzyme, which is insensitive to DDT. The OSCP of the concentrations are high, biofilm formation and virulence are repressed. V. bee’s ATP synthase contained 207 acids compared to 190 in bovine and the cholerae uses two transmembrane histidine sensor kinase quorum-sensing re- identities were only 47%. Subunit ‘‘d’’ of the bees had no counterpart in ceptors called CqsS and LuxPQ, which respond to the autoinducers called the bovine. Both subunits, the OSCP and subunit ‘‘d’’ are components of CAI-1 and AI-2, respectively. A new third QS pathway functions in parallel the peripheral stalk (the stator) of the ATP synthase. Interaction of DDT with these two systems. This new QS circuit is composed of a transcription with OSCP and/or subunit ‘‘d’’ would interrupt such structure, freeze the me- factor receptor, VqmA, which activates the expression of a gene encoding a chanical rotation of the enzyme and block synthesis of ATP. The DDT pocket small regulatory RNA called VqmR. VqmR represses biofilm and pathogen- ‘‘niche’’ may include a structure provided by the two protein subunits. Sub- esis genes. The autoinducer that drives VqmA activity is 3,5-dimethylpyra- unit ‘‘d’’ and the ‘‘OSCP’’. The two proteins are rich in hydrophobic amino zin-2-ol (DPO), which is synthesized from threonine and alanine. The acids, which comprise about 30% of subunit ‘‘d’’ and 50% of the OSCP, requirement for threonine is particularly interesting because mucin, found in (amino acid sequences will be provided) that would facilitate their association the gut of the human host, is especially rich in threonine, and DPO can be with the hydrophobic structure of DDT. Identification of this essential made from mucin, suggesting a link between the eukaryotic niche of patho- biochemical target in insects is the first step to design selective and efficient genic V. cholerae and activation of VqmA. Unlike other cytoplasmic insecticides. quorum-sensing receptors, VqmA can function without its autoinducer, albeit with reduced efficacy. To understand the structural basis underpinning DPO- 912-Pos enhancement of VqmA activity, we determined the crystal structure of full- Disassembling and Reassembling Complex Structure of Glutamate Dehy- length VqmA bound to DPO. The VqmA N-terminal ligand-binding region drogenase 1 (DGH-1), Monitored by Tryptophan and 1-Anilinonaphtha- contains a PAS domain and represents the dimerization interface. The lene-8-Sulfonate (ANS) Fluorimetry VqmA DNA-binding domain is a canonical helix-turn-helix fold. Mutational Bogumil Zelent, David F. Wilson, Franz M. Matschinsky. and biochemical analyses of VqmA reveal that large scale macromolecular Dept Biochem/Biophysics, Univ Pennsylvania, Philadelphia, PA, USA. changes occur upon ligand binding. We suggest that basal, DPO- GDH-1 which converts glutamate to alpha-ketoglutarate and ammonia while independent VqmA activity ‘‘primes’’ this pathway to respond to an influx reducing NAD(P) is constitutively expressed in all nucleated cells of mam- of threonine in the gut when biofilm formation needs to be repressed to enable mals. Encoded by a nuclear gene, its mitochondrial matrix concentration is V. cholerae to disperse from the host. high (5-10 mg /ml). Turnover is also high considering a mitochondrial t 1/2 of 1-2 days. The MW of GDH-1 subunits is 56K. Newly synthesized enzyme 910-Pos precursor subunits are transported into mitochondria, are rapidly cleaved and Reinforcement Learning of Protein Conformational Ensemble assemble there to the enzymatically active homo-hexamer of 336K MW. Jiangyan Feng. Furthermore, bovine GDH-1 does form complexes of about 1.5 Mill University of Illinois at Urbana Champaign, Urbana, IL, USA. at concentration above 2-3 mg/ml. Activating point mutations (e.g H454Y) Proteins are dynamic entities with functions governed essentially by their cause life threatening hyperinsulinism. To better understand the biology of conformational dynamics. Unfortunately, prediction of protein conformational this protein, essential for life and of high medical relevance, we are using ensemble is challenging both experimentally and computationally. Experi- tryptophan and ANS fluorescence to study (in model experiments) the process mental techniques can only identify the most stable conformational states, of disassembly caused by urea and reassembly after urea removal by dilution and computational approaches are generally limited by accessible timescales, or dialysis. An extensive literature beginning with the report by Olson and uncertainties in protein model, and insufficient computational resources to Anfinsen in 1952 provides useful background information. We show by achieve proper sampling. monitoring tryptophan and ANS fluorescence that the trimer of the protein, One approach to confront this challenge is to use evolutionary couplings formed at 3M urea, can be readily reassembled to the original higher molec- (ECs), correlations between co-evolving residue pairs. The biological premise ular forms but that smaller structures, formed when exposed to urea

BPJ 9352_9354 Monday, March 4, 2019 185a exceeding 3 M, cannot be reassembled to the original multimeric forms. At 915-Pos this juncture we focus on the reversible process between 0 and 3 M urea, Divulging Characteristic Features of the Novel A-Synuclein Oligomers determining the rates of interconversion of multimeric forms as influenced Augmenting Parkinson’s Disease by pH, temperature, the enzyme’s substrates and its known allosteric modi- Animesh Mondal, Nakul Chandra Maiti. fiers using tryptophan fluorescence. We are testing a hypothesis that intercon- Structural Biology and Bioinformatics, CSIR-Indian Institute Of Chemical version rates between trimers, hexamers and poly-hexamers contribute to the Biology, Kolkata, India. regulation of GDH-1 which controls glutaminolysis in secreting beta- Medical disorders that are allied with disorder proteins appear as a result of cells and cancer cells. accumulation of intracellular or extracellular protein aggregates. Parkinson’s Disease (PD) is such a kind of neurodegenerative disorders that is associated with old aged people. In global scenario more than 10 million people are 913-Pos affected with PD. The clinical symptoms of PD are manifested by toxic effects In-Cell Fast Photochemical Oxidation of Proteins for Proteome Wide of a-Synuclein oligomers that selectively eliminate dopamine producing neu- Structural Biology rons in the substantia nigra of affected persons. However, the real cause behind Emily E. Chea, Lisa M. Jones. the neuronal loss is still unknown. On the basis of recent investigation a-Syn- Pharmaceutical Sciences, University of Maryland Baltimore, uclein aggregates are thought to be the main wrongdoer in PD. Some studies Baltimore, MD, USA. also indicated that a prion like transformation of toxic a-Synuclein oligomer Measuring a drug’s target engagement in cell is critical for drug discovery. could augment the disease propagation. Our research interest intended to anal- Drug binding studies with purified proteins in vitro may not display interac- yse these possibilities by understanding the structural features of toxic a-Syn- tions that are present in the complex environment of cells. Cellular thermal uclein oligomers. Fibrill formation of a-Synuclein is not a simple transition shift assay (CETSA) has previously been used as an in-cell method to study from monomer to fibrils but rather a multistep process that involves intermedi- changes in protein melting temperature due to drug binding. Many proteins, ates of oligomers. The structure and composition of these oligomeric aggre- including membrane proteins, are not detectable by CESTA, limiting this gates observed as an intermediate during the transition period remain ill method to study drug engagement. To alleviate some of these drawbacks, defined. Here the conformation of a-Synuclein oligomers has been character- we proposed to use in-cell fast photochemical oxidation of proteins (IC- ized in different environments to understand its behavioral traits.These oligo- FPOP) for Proteome-Wide Structural Biology (PWSB) to study the effect mers showed less hydrophobic exposure and more compact structure of Gleevec on breast cancer cells. Gleevec was tested on two breast cancer compared to the oligomers formed by conventional incubation pathway. cell lines, triple negative breast cancer (TNBC) from African ancestry From circular dichroism and Raman spectroscopic analysis we have unveiled (TNBC-AA) and from European ancestry (TNBC-EA), which are both nega- existence of a-helical signature. By revealing its character, the multiplicity tive for estrogen receptors, progesterone receptors, and human epidermal of protein misfolding in different stages of assembly will highlight the patho- growth factor receptor. Control cells, which are not treated with Gleevec, physiological nature of oligomers. and sample cells, which are treated with Gleevec, were subjected to FPOP us- ing a flow system allowing for single cell flow past the laser irradiation and 916-Pos collected in a dimethylthiourea and N-tert-Butyl-a-phenylnitrone (PBN) Atomistic Mechanisms Underlying the Activation of G Protein-Coupled quench solution. Immediately following FPOP, all cellular proteins were iso- Sweet Receptor Heterodimer Mediated by Sugar Alcohol Recognition lated and digested with trypsin. After bottom-up LC-MS/MS, significant Thanyada Rungrotmongkol. changes in FPOP modifications between the control and sample cells were Biochemistry, Chulalongkorn University, Pathum Wan, Thailand. observed proteome wide demonstrating multiple proteins were affected by The human sweet taste receptor (T1R2-T1R3) plays an important role in Gleevec. recognizing various low-molecular-weight sweet-tasting sugars and proteins, resulting in the release of intracellular heterotrimeric G protein, which leads 914-Pos to a sweet taste perception. Xylitol and sorbitol, which are sugar Explore the Binding of Hepatitis B Virus Core Protein Peptides with HLA- naturally found in many fruits and vegetables, exhibit the potential caries- A2.1 by Molecular Modeling Methods reducing effect and are widely used for diabetic patients as low-calorie sweet- Lianhua Piao1, Zhou Chen2, Shan Chang1, Jian Li3, Ren Kong1. eners. In the present study, the computational tools are applied to investigate 1Institute of Bioinformatics and Medical Engineering, School of Electrical the structural details of binary complexes formed between sugar alcohols and and Information Engineering, Jiangsu University of Technology, Changzhou, T1R2-T1R3 heterodimeric receptor. Principle component analysis reveals that China, 2School of Chemical and Environmental Engineering, Jiangsu the ligand-binding site in T1R2 domain is adapted by the induced-fit mecha- University of Technology, Changzhou, China, 3The Key Laboratory of nism to accommodate the focused sugar alcohols, in which the residues 233- Developmental Genes and Human Disease, Institute of Life Sciences, 268 are significantly located closer to stabilize ligand. The finding likely sug- Southeast University, Nanjing, China. gests that these structural transformations might be the important mechanisms Hepatitis B virus (HBV) is a DNA virus affecting about 250 million popula- underlying ligand-protein recognitions. The calculated free energies also sup- tions worldwide, especailly in China. Among them, about 1 million people port the aminoterminal domain of T1R2 monomer as the preferential binding died due to the followed complications caused by HBV, such as liver cirrhosis site for such sugar alcohols rather than T1R3 domain, in a correspondence and hepatocellular carcinoma (HCC) [1]. HBV core 18-27 peptide (HBcAg18- with relatively less water molecules accessible into the T1R2 pocket. 27) is a well studied epitope detected in more than 90% patients with human The T1R2 E302 residue is found to be the important recognized residue for leukocyte antigen (HLA)-A2.1, helping the CD8þ T-cell reponse to elimiate sugar alcohol binding through a strongly firmed and electro- the virus [2, 3]. The binding between HBcAg18-27 and HLA-A2.1 determines static attraction. Additionally, the binding affinity of xylitol toward T1R2 the HLA-class I-restricted CD8þ T-cell response to HBV, which is critical in domain is significantly higher than that of sorbitol, making it a sweeter taste the disease progress. Here, flexible peptide docking was used to deduce the molecule. binding mode of wild type and mutations of HBcAg18-27 peptides with HLA-A2.1. Then molecular dynamics simulations were performed to system- 917-Pos atically investigate the binding of HLA-A2.1 with different peptides. Compar- Investigating the Response of Type IV Pilins and Type IV Pilus Filaments ison of MMGBSA free energy showed that the compuational results were well to Applied Force using All-Atom Steered Molecular Dynamics Simulations consistent with the experimantal binding affinities. Per residue energy decom- Maria N. Fairfield1, Stephen J. Jones1, Nicolas Biais2,3, Joseph L. Baker1. position revealed that the anchor residue (LEU19) contributed most to the to- 1Chemistry Department, The College of New Jersey, Ewing, NJ, USA, tal free energy. Mutations in position 21 resulted in few energy changes when 2Biology Department, CUNY Brooklyn College, Brooklyn, NY, USA, 3The compared to mutations in position 27 due to the difference in the buried res- Graduate Center of CUNY, New York, NY, USA. idue area. The modeling results provide atomatic detaileds and dynamic in- Type IV pili (T4P) are long protein filaments which protrude from the surface sights into the binding of HBcAg18-27 with HLA-A2.1, which could benift of prokaryotic cells and are assembled from many copies of a monomer sub- the design of theraputic peptide or diagnosis of HBV. [1] WHO, Global Hep- unit called ‘‘pilin.’’ Pilin is a ladle shaped protein consisting of a helical atitis Report 2017, World Health Organization 2017. [2] Penna, A., Chisari, F. domain attached to a globular domain. Thousands of pilin subunits pack V., Bertoletti, A., Missale, G., Fowler, P., Giuberti, T., Fiaccadori, F. et al., J. together to comprise a single T4P filament, which can grow to be many micro- Exp. Med. 174, 1565-1570 (1991). [3] Sette, A., Vitiello, A., Reherman, B., meters in length. The function of T4P filaments include surface adhesion, Fowler, P., Nayersina, R., Kast, W. M., Melief, C. J. et al., J. Immunol. DNA uptake, biofilm formation, and bacterial motility. They are very strong 153, 5586-5592 (1994). and flexible , able to sustain against large forces and undergo a

BPJ 9352_9354 186a Monday, March 4, 2019 polymorphic transition in which they stretch to three times their original 920-Pos length. In this work we investigate the response of individual T4P monomers High Yield Production of Functional HIF Prolyl Hydroxylase Domain and short T4P filaments (10-18 monomers) to external force using the steered Proteins from Inclusion Body Expression in E. coli molecular dynamics method as implemented in the AMBER molecular dy- Nakoa K. Webber, Thomas J. Fasano, Jacob T. Zangaro, Pamela N. Gallo, namics software package. The starting structure for T4P filament simulations Kayla A. Schardien, Michelle M. Currie, Nathaniel V. Nucci. is based on the recent cryo-EM structure of the T4P from Neisseria gonor- Department of Molecular & Cellular Biosciences, Rowan University, rhoeae in which the helical domain of pilin subunits was found to be partially Glassboro, NJ, USA. ‘‘melted.’’ Our simulations have been designed to directly test the hypothesis Hypoxia-inducible factor (HIF) prolyl-hydroxylase domain-containing proteins that extension of T4P filaments under force involves additional elongation of (PHDs) are 2-oxoglutarate-dependent hydroxylases that regulate HIF turnover the ‘‘melted’’ region of the helical domain of each pilin monomer. Specif- in the cell, thereby controlling the cellular response to hypoxic conditions. ically, we monitor changes of pilin secondary structure over time under There are three PHD isoforms which are promising targets for treatment of applied force in both monomer and filament simulations. We also determine heart attack, stroke, and other ischemic events. Current PHD inhibitors lack iso- which pilin-pilin interactions are disrupted in the T4P filament during the form specificity, thereby limiting their therapeutic potential. We are working initial stages of the force-induced conformational change of this system. toward structural characterization of all three isoforms to inform more effective Finally, we compare the conformational changes observed for individual pilin development of isoform-specific inhibitors. Toward this goal, we have devel- subunits under force with the changes observed for pilin subunits within the oped a method for high-yield recombinant production of the catalytic domain T4P filament environment. of PHD2 (trPHD2) and full-length PHD3 from inclusion bodies in E. coli. We have optimized refolding of the PHDs to minimize aggregation and pre- serve enzymatic activity. Activity is assessed by a fluorescence assay via deriv- 918-Pos atization of 2-oxoglutarate. These efforts provide the needed groundwork for Probing the Polymorphic Transition of Type IV Pilus Filaments Under future assessment of protein structures by NMR and subsequent isoform- Force using Coarse-Grained Molecular Dynamics Simulations selective drug design. This work was supported by a research grant from the 1 2 3,4 Bryan A. Bogin , Christopher A. Lovenduski , Nicolas Biais , New Jersey Health Foundation. Joseph L. Baker1. 1Chemistry Department, The College of New Jersey, Ewing, NJ, USA, 921-Pos 2Physics Department, The College of New Jersey, Ewing, NJ, USA, 3Biology Studying the Dynamic Motions of Water Surrounding the Ice-Binding Department, CUNY Brooklyn College, Brooklyn, NY, USA, 4The Graduate Face of M1.1 Antifreeze Protein Center of CUNY, New York, NY, USA. Joseph C. Iovine1, Pamela N. Gallo1, Kayla A. Callaway2, Peter L. Davies3, Type IV pilus (T4P) filaments are biological polymers composed of many sub- Nathaniel V. Nucci2. units of a protein called pilin. T4P filaments serve a variety of functions for pro- 1Department of Molecular & Cellular Biosciences, Rowan University, karyotic organisms, including adhesion, cell signaling, and ‘‘twitching’’ Glassboro, NJ, USA, 2Department of Physics & Astronomy, Rowan motility. It is known experimentally that T4P filaments can undergo a large- University, Glassboro, NJ, USA, 3Dept Biochemistry, Queen’s Univ, scale polymorphic transition in which they stretch to three times their initial Kingston, ON, Canada. length under hundreds of pN scale forces. Recent cryo-electron microscopy Antifreeze proteins (AFPs) are a family of ice-binding proteins found in based models of T4P have shed new light on the structure of these filaments, aquatic life, insects, and microorganisms that live in areas where temperatures and specifically have revealed the presence of a ‘‘melted’’ region of helix drop below zero degrees celsius . AFPs have an ice-binding face that inhibits within each pilin subunit’s alpha-helical domain. This ‘‘melted’’ region is the growth of ice crystals in vivo. This binding keeps ice crystals small thought to contribute to the remarkable elasticity of T4P filaments, however, enough to prevent cellular damage, thereby protecting the organism. Due to the nature of the polymorphic transition, and the molecular scale rearrange- AFP’s ability to specifically bind to ice crystals in a sea of liquid water, these ments of pilin subunits that occur within the T4P filament, cannot be deter- proteins have become an important model system for understanding molecular mined from this static experimental data alone. In this work we use the recognition. The present study focuses on M1.1 AFP, a 7.1 kD type III AFP GoMARTINI coarse-grained (CG) model, which allows for the study of found in fish, such as arctic pout. By encapsulating the protein in a reverse conformational changes in MARTINI CG proteins, to investigate the response micelle, comprehensive measurement of the water dynamics across the pro- of long T4P filaments to external forces using a steered molecular dynamics tein surface may be performed using nuclear magnetic resonance (NMR). protocol. Simulations are run using the GROMACS software package. The Studying the movement of water near the ice-binding face is crucial for un- recently solved structure of the T4P filament from Neisseria gonorrhoeae is derstanding the mechanism of action of these proteins. Such insight will used as the starting model for CG simulations. Several pulling velocities are inform development of real world applications, such as biocompatible addi- explored, including 10, 1 and 0.1 A˚ /ns. Force versus extension profiles are tives to extend the lifespan of blood samples or protect transplant organs measured, and the filament Young’s modulus, a measure of the elasticity of from environmental damage. the T4P filament, is calculated. Conformational changes in T4P filament qua- ternary structure are monitored, in addition to conformational changes of indi- 922-Pos vidual pilin subunits. Finally, in addition to the native filament, several mutants Single-Molecule Force Spectroscopy of M-Value Mutants of Staphylo- are studied to determine the impact of point mutations on T4P filament coccal Nuclease Indicates Complex Protein Folding Landscape properties. James Rives1, Joseph Rehfus2, Vincent J. Hilser3. 1The Johns Hopkins University, Baltimore, MD, USA, 2Johns Hopkins Univ, Baltimore, MD, USA, 3Dept Biology, Johns Hopkins Univ, Baltimore, MD, 919-Pos USA. Molecular Dynamics Investigations of Enzyme Conformational Changes Point mutations have commonly been utilized to investigate the underlying Prabin Baral, Nisha Bhattarai, Rudramani Pokhrel, Bernard Gerstman, thermodynamic characteristics of proteins. While denaturant-induced unfold- Prem P. Chapagain. ing can provide a quantitative measure for the stability of a protein, interpreta- Physics, Florida International Univ, Miami, FL, USA. tion of the results depends on the model with which the equations are built. We used Steered Molecular Dynamics (SMD) computations to investigate Derived from the analysis, a m-value relates the susceptibility of a protein to the conformational changes involved in the functioning of the enzyme a denaturant and can be used as a proxy to the change in solvent-accessible sur- Peroxidase (HRP). The activity of HRP is modulated by the face area between the native and denatured states. In the case of the model pro- opening of its heme pocket, which is necessary for the binding of amplex tein Staphylococcal Nuclease (SNase), many mutants that alter the m-value red to the heme. AFM experimental investigations have shown that surpris- have been identified. There is a long-standing debate as to whether the muta- ingly weak applied forces can have a noticeable effect on the binding rate. tions favor an intermediate state in the unfolding pathway or perturb the resid- To understand the atomic-level details of how these weak forces can have ual structure of the denatured state of the protein. Bulk experiments have such a noticeable experimental effect, we applied different strength, small historically been used to try to discriminate between the various models, but forces (2PN, 10PN) on atoms at opposite ends of the molecule. The residues these methods are insensitive to finer details of the unfolding/folding path- that we chose to apply forces were the same residues that were used in the way(s) due to signal averaging. Single-molecule force spectroscopy via optical AFM experiments. We discuss how applied forces that are weaker than tweezers in the presence of chemical denaturant offers an alternative approach many of the internal interactions are nevertheless able to facilitate large by following individual trajectories of the unfolding/folding pathway(s). Our conformational changes in the enzyme that have a significant effect on the force ramp experiments have revealed the presence of an intermediate in enzyme’s functioning. both the wild-type and mutants of SNase, which was confirmed in subsequent

BPJ 9352_9354 Monday, March 4, 2019 187a force clamp experiments. Further experimentation using optical tweezers al- play a role in tuning biological function, but this would only be possible if lowed the determination of unfolding rates and contour length changes associ- proteins behaved as materials with low dielectric constant, as assumed in ated with the folding/unfolding pathway of SNase. many continuum electrostatics models. Buried ion pairs are of interest then not only for their functional roles, but also because they can be used to 923-Pos examine the balance between Coulomb and hydration energies in the dry, hy- Computational and Spectroscopic Investigation of Communication Mech- drophobic interior of proteins. As the distance between a pair of internal ioniz- anisms used by Acyl Carrier Proteins able groups increases and the energy of their Coulomb interaction decreases, Terra Sztain-Pedone, Michael D. Burkart, James A. McCammon. the balance of these energies will favor burial of the groups in the neutral Chemistry & Biochemistry, University of California, San Diego, La Jolla, state, eliminating any direct electrostatic interaction between them. To CA, USA. examine the magnitude of electrostatic crosstalk between buried ionizable Engineering carrier protein dependent pathways relies upon a thorough un- groups experimentally, many variants were engineered on a highly stable derstanding of the molecular basis of carrier protein-partner enzyme interac- form of staph nuclease. All variants have His66 in the hydrophobic core tions. AcpP, the acyl carrier protein involved in Escherichia coli fatty acid and either Asp or Glu residues buried at another internal position. Many biosynthesis, delivers a vast array of substrates to the appropriate partner His-Asp/Glu pairs were studied, and in no case were medium- or long- enzyme, efficiently distinguishing between at least 21 different enzymes range Coulomb interactions observed. When the groups were within H-bond in both primary and secondary metabolic pathways. Fatty acyl substrates distance, strong, favorable coupling energies were measured, but only when are covalently tethered to the AcpP and at the same time sequestered inside the side chains of the buried pair could achieve a geometry favorable for a hydrophobic cavity of the protein, leading to an allosteric modification of an H-bond interaction. These data suggest that at short range the buried the AcpP surface that participates in a transient binding event. Here, the sub- pair behaved as a strong hydrogen bond, but no evidence was found that strate is flipped out of the AcpP pocket and into the active site of the they ever behave as titratable point charges interacting at a distance through enzyme. Due to the complexity of this interaction, it has been difficult to Coulomb forces enhanced by the low dielectric protein medium. delineate the mechanisms used by AcpP to convey substrate identity to part- ner enzymes. Herein, we provide a detailed description of how AcpP struc- 926-Pos ture is effected by sequestration of various substrates by combining nuclear Polymorphisms Modulate Sheep Prion Protein Susceptibility to Misfolding magnetic resonance spectroscopy with molecular dynamics simulations. by Altering the Residue Network of Interactions Additionally, we investigate the interaction of AcpP with enzymes from India Claflin, Noah Yoshida, Patricia Soto. fatty acid biosynthesis and secondary metabolic pathways. These findings Creighton Univ, Omaha, NE, USA. elucidate information on carrier protein substrate shuttling, which, taken The autocatalytic misfolding of the cellular form of the prion protein, PrPC,to with previous data will facilitate rational design of carrier protein-partner the infectious form, PrPSc, is critical to the development of prion diseases. enzyme complexes. Though the mechanism for this misfolding is not well understood, previous research recognizes the role of genetic mutations in varying susceptibilities 924-Pos to prion diseases in sheep. Using structural bioinformatics techniques, our study Dissecting the Allosteric Effects of pVHL G123F Mutation in Type 2A examines the residue interactions in three different sheep PrPC structures. pVHL Disease These structures contain different point mutations and have different levels Hongsheng Qian, Yu Zou, Qingwen Zhang. of susceptibility to scrapie prion disease. We will discuss our results in relation Physical Education and Training, Shanghai University of Sport, Shanghai, to the role of mutations on networks of interaction, and propose a mechanism China. for the primary misfolding of the a-helical rich PrPC into the b-sheet rich PrPSc. The von Hippel-Lindau tumor suppressor protein (pVHL) plays an essential This work was made possible partly by grants from the National Institute for role in the regulation of the hypoxia response pathway in tumor growth and General Medical Science (NIGMS) (5P20GM103427), a component of the Na- hypoxia training. This regulation is mediated by the ubiquitin-mediated degra- tional Institutes of Health (NIH), and its contents are the sole responsibility of dation of hydroxylated HIF-1a at proline564 occurs by binding to pVHL. the authors and do not necessarily represent the official views of NIGMS or Experimentally, the mutation Y98N, which leads to the 2B phenotype disease, NIH. was shown to destabilize pVHL and substantially reduce its binding affinity to HIF-1a. Besides, the type 2A pVHL disease mutant Y98H in pVHL is more 927-Pos disruptive to microtubule stability in cells than is the Y98N mutation. Previous Towards the Inhibition of Calpain-Dependent Desmoplakin Cleavage in molecular dynamics (MD) simulation study reported that mutant G123F in 2B Arrhythmogenic Cardiomyopathies phenotype disease can stabilize unbound pVHL and lower the binding free en- Taylor Albertelli1, Kendahl Ott1, Heather R. Manring2, Stuart G. Campbell3, ergy of pVHL with HIF-1a. However, whether G123F in type 2A pVHL dis- Maegen Borzok2, Nathan T. Wright1. ease (Y98H) plays the similar essential role remains elusive. Herein, we 1Department of Chemistry and Biochemistry, James Madison University, performed all-atom MD simulations to investigate the structural effect and Harrisonburg, VA, USA, 2Department of Physiology & Cell Biology, Ohio interaction between pVHL and HIF-1a in status of Y98H mutant, and both State University, Columbus, OH, USA, 3Department of Biomedical Y98H and G123F mutants (Y98H_G123F). Our cumulative 5 ms MD simula- Engineering, Yale University, New Haven, CT, USA. tions reveal that the structure of pVHL/HIF-1a complex in the Y98H_G123F Desmoplakin (DSP) is a large (260 kD) protein found in the desmosome, a sub- system is more stable than that in Y98H system. The complex in the Y98H sys- cellular structure that links the cytoskeleton of one myocyte to that of its tem shows a large structural flexibility of HIF-1a, while it is decreased in the neighbor. In cardiomyocytes, desmoplakin’s main function is to maintain Y98H_G123F system, specifically for the HIF-1a and residues V181 to L201 cell-to-cell adhesion and synchronization during heart contractions. A mutation in pVHL a-domain. Binding energy calculations show that it is less favorable hot spot, centered around the SH3 domain of DSP, is associated with arrhyth- for the binding of HIF-1a to pVHL in the Y98H system, whereas both Y98H mogenic cardiomyopathy, but the underlying mechanism(s) of this association and G123F mutations enhance the interactions between HIF-1a and pVHL. isn’t well studied. Here, we show that many of these disease-causing mutations Our results reveal the effects of Y98H_G123F on the interaction between display increased calpain sensitivity. Additionally, structural and computa- HIF-1a and pVHL, which is helpful to provide a rescue strategy to restore tional studies on DSP variants show that this cleavage event is driven not pVHL function in type 2A pVHL disease. through a gross structural change, but instead through the discrete exposure of a normally occluded calpain cleavage site. Initial in silico and in vitro 925-Pos screens of small molecules suggest the feasibility of developing a pharmaceu- Ion Pairs Buried in Hydrophobic Environments within Proteins: Electro- tical solution to prevent calpain-mediated DSP cleavage. static Crosstalk between Buried Groups Aaron Robinson1, Andrea Theodoru1, Jamie L. Schlessman2, 928-Pos Bertrand Garcia-Moreno3. Cryo-EM Structural Analysis of Neuronal Nitric Oxide Synthase 1Johns Hopkins Univ, Baltimore, MD, USA, 2Chemistry Dept, US Naval Thomas H. Pospiech1,2, Yoshihiro Morishima3, Yoichi Osawa3, Acad, Annapolis, MD, USA, 3Dept Biophysics, Johns Hopkins University, Daniel R. Southworth2. Baltimore, MD, USA. 1Graduate Program in Chemical Biology, University of Michigan, Ann Internal ion pairs buried in the hydrophobic interior of proteins are essential Arbor, MI, USA, 2Biochemistry and Biophysics, Institute for for processes such as Hþ transport, e- transfer, and catalysis. Despite their Neurodegenerative Diseases, University of California - San Francisco, San importance, the properties of buried ion pairs remain poorly understood. It Francisco, CA, USA, 3Pharmacology, University of Michigan, Ann Arbor, has been suggested that medium- or long-range Coulomb interactions could MI, USA.

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Nitric oxide (NO) is an essential signaling molecule for neurotransmission, 931-Pos cardiovascular function, and cellular defense. However, NO overproduction Obscurin Acts as a Semi-Flexible Chain in Solution from neuronal nitric oxide synthase (nNOS) increases infarct size after Jake Whitley1, Aidan Ex-Willey2, Daniel Marzolf2, Oleksandr Kokhan1, ischemic stroke and has been linked to the pathogenesis of Alzheimer’s and Maegen Ackermann2, Anthony Tongen1, Nathan T. Wright1. Parkinson’s disease. Despite fundamental roles of NO in cellular signaling 1James Madison Univ, Harrisonburg, VA, USA, 2Ohio State Univ, and disease, intact nNOS holoenzyme structures have remained elusive, and Columbus, OH, USA. the structural basis for the electron transfer cycle across the reductase and Obscurin, a giant modular cytoskeletal protein, is comprised mostly of tandem oxygenase domains is unclear. In previous studies, we used negative-stain Ig-like domains. This architecture allows obscurin to connect distal targets electron microscopy (EM) to determine conformational states and the overall within the cell. The linkers connecting the Ig domains are usually short (3-4 res- domain organization of nNOS during its catalytic cycle. In current work, we idues). The physical effect arising from these short linkers is not known; such have interrogated the nNOS and nNOS:Calmodulin (nNOS:CaM) complexes linkers may lead to a stiff elongated molecule or, conversely, may lead to a by cryogenic-EM (cryo-EM). In these intact complexes, the oxygenase dimer more compact and dynamic structure. In an effort to better understand how structure was determined to 4A˚ , revealing its active-state architecture, while linker length affects obscurin flexibility, and to better understand the physical the nNOS reductase domains were identified to be flexibly linked in both underpinnings of this flexibility, here we study the structure and dynamics of CaM-free and CaM-bound states. Additional structural analysis of four representative sets of dual obscurin Ig domains using experimental and crosslinked-stabilized complexes, including 2D and 3D classification, will computational techniques. We find that in all cases tested, tandem obscurin be presented that reveals the overall architecture and highlights the use of Ig domains interact at the poles of each domain and tend to stay relatively cryo-EM methods in elucidating different conformations of the nNOS:CaM extended in solution. NMR, SAXS and MD simulations reveal that while tan- complex. dem domains are elongated, they also bend and flex significantly. By applying this behavior to a simplified model, it becomes apparent that obscurin can link 929-Pos targets up to 200 nm away. However, as targets get further than 200 nm apart, Using 4-Cyanophenylalanine to Probe the Degree of Water Exposure in a obscurin begins acting as a spring, and requires progressively more energy to Peptide Hydrogel elongate further. Benjamin F. Frost, William E. Fox, Fiona Berry, Katherine Chung, Karin Akerfeldt. 932-Pos Chemistry, Haverford College, Haverford, PA, USA. Large-Scale All-Atom Simulations of T4P Filaments Reveal Critical Inter- A peptide comprised of residues 37-49 of human semenogelin I (SgI 37-49), actions for T4P Stability sequence KGSFSIQYTYHVD, spontaneously aggregates in water in a pH Rebecca B. Goncalves1, Nicolas Biais2,3, Joseph L. Baker1. dependent manner to generate a hydrogel. Structural analysis by Fourier trans- 1Chemistry Department, The College of New Jersey, Ewing, NJ, USA, form infrared (FTIR) spectroscopy indicates that the aggregated peptide fibrils 2Biology Department, CUNY Brooklyn College, Brooklyn, NY, USA, 3The consist of extended b;-sheet. Three SgI 37-49 mutants were synthesized in Graduate Center of CUNY, New York, NY, USA. which the aromatic residues, and tyrosine, were each substituted Type IV pili (T4P) are protein filaments that function as molecular ‘‘grappling for 4-cyanophenylalanine, FCN, to generate F4FCN, Y8FCN, and Y10FCN. The hooks’’ for bacteria and archaea. They are found to support very large forces, frequency of the nitrile group in the hydrogel state was then assessed with and undergo a force-induced polymorphic transition that is poorly understood. FTIR. The cyano group, with a stretch vibration of 2237 cm1 in water and Most of what is known about T4P comes from cryo-electron microscopy 2228 cm1 in a non-polar solvent, such as tetrahydrofuran, has been used pre- based models, for example from the bacterial organisms Neisseria meningiti- viously to probe the degree of water exposure, inferred through extent of dis and Neisseria gonorrhoeae. However, these models are static structures, hydrogen bonding, in aggregating systems such as the islet amyloid peptide, and do not shed light on the underlying dynamics of T4P filaments. In this IAPP. Our studies are consistent with the core residue in position 8 of SgI study we have run more than 1.5 microseconds molecular dynamics simula- 37-49 being embedded in the hydrogel whereas the flanking positions four tion on the largest T4P systems that have been studied using all-atom models. and 10 appear more solvent exposed, in agreement with their positions in the Simulations have been carried out for both the N. meningitidis (Nm) and sequence. These results suggest that the N- and C-terminal ends of the self- N. gonorrhoeae (Ng) T4P, which exhibit very similar quaternary structure, assembled peptides within the aggregated fibrils might be at least partly frayed. with slightly different primary sequences for their pilin subunits. The simula- tions are run in explicit solvent using the AMBER molecular dynamics soft- 930-Pos ware, and in order to enhance the sampling in these simulations the hydrogen Evaluation of the HIV Genome to Promote Gag Hexamerization mass repartitioning method is implemented. All simulations are carried out on Noel Getachew. graphics processing units (GPUs). These large-scale simulations are used to University of Maryland Baltimore County, Woodbine, MD, USA. calculate various filament physical properties including the pilin subunit Human immunodeficiency virus (HIV-1) is a retrovirus that attacks CD4 T- rise, subunit twist around the helical axis, and the filament persistence length cells of the immune system. This can lead to the development of acquired im- and torsional rigidity. Additionally, the filaments from the Nm and Ng sys- munodeficiency syndrome (AIDS), resulting in the body becoming more sus- tems are compared to one another based on these metrics, as well as based ceptible to opportunistic diseases and infections. Current drug therapies target on the results of the clustering of conformations that the pilin subunits in the actions of proteins that are prone to mutation, but one aspect of the repli- each filament system explore over microsecond timescales. Pilin-pilin interac- cation cycle that does not have any drug therapies is that of genomic recog- tions are also studied in order to ascertain the most stable contacts in the sys- nition. During virus particle production, the dimeric viral genome has to be tem, including hydrogen-bonding, salt-bridge formation, and hydrophobic selectively packaged by the Gag-polyprotein. Previous studies have identified packing interactions within the filament core, and interactions between pilin the minimal packaging region, termed the Core Encapsidation Signal (CES) globular domains along the T4P surface. of the viral RNA necessary to promote selective packaging by Gag. It is hy- pothesized that the structure of the CES promotes the selective recognition 933-Pos and formation of Gag hexamers. This may result in a nucleation complex Effects of Local and Global Dynamics on the Supertertiary Organization that allows for the formation of the immature hexagonal lattice seen within of Postsynaptic Density Protein 95 new virions. Our research aims to characterize the interaction between the George L. Hamilton1, Jakub Kubiak2, Claus A.M. Seidel3, Hugo Sanabria4. CES and the Gag polyprotein through the use of crosslinking experiments 1Physics and Astronomy, Clemson University, Clemson, SC, USA, in order to determine if the CES can support the formation of Gag hexamers. 2Molecular Physical Chem, Univ Duesseldorf, Dusseldorf, Germany, The preliminary data we obtained was unable to provide any additional infor- 3Chemistry, Heinrich Heine University, Duesseldorf, Germany, 4Dept Phys/ mation for Gag hexamerization due to a lack of lysine residues available in Astro, Clemson University, Clemson, SC, USA. the area of interest within the protein. In order to alleviate this problem we Postsynaptic density protein 95 (PSD-95) is a critical scaffolding protein in are utilizing a new crosslinker known as succinimidyl-[4-(psoralen-8- the excitatory postsynaptic density (PSD) with key functions in synaptic orga- yloxy)]-butyrate (SPB) to study this interaction. SPB covalently links uracils nization and regulation of synaptic strength. PSD-95 is a canonical multido- within CES to lysines of the RNA binding domain of the Gag protein. Under- main scaffold protein comprised of three PDZ domains, an SH3 domain, standing whether the CES promotes hexamer formation during selective and a guanylate ‘‘pseudokinase’’ (GuK) domain connected in series by flex- packaging could potentially allow for the development of new antiretroviral ible polypeptide linkers. The domains of PSD-95 show varying degrees of therapy. interaction and were suggested to partition into two independent supramod- ules. The first two PDZ domains display weak interactions but the third

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PDZ domain preferentially interacted with the SH3 and GuK domains. to investigate the effects of formulation conditions in order to ensure quality Despite a trove of structural studies, we do not understand how the interac- consistency. Since structural changes in proteins and nucleic acids are re- tions of PSD-95 with its diverse set of synaptic binding partners are regulated. flected in their CD spectra, CD measurements are extremely effective for PSD-95 exchanges between supertertiary configurations as part of its func- structural analysis and stability evaluation of these biopolymers. Conse- tional cycle. Interactions and posttranslational modifications can change the quently, CD measurements have begun to be utilized during the development conformation with effects on function. This suggests that internal dynamics phase of biopharmaceuticals, and for identifying the original drug and bio- may regulate access to binding sites in PSD-95. In this work, we utilize a similars in order to ensure consistent quality. In response to this demand, FRET network designed to probe all pairwise combinations of the five do- JASCO has developed a system that can automatically measure the CD spec- mains. We used multiparameter fluorescence detection and filtered fluores- trum of multiple specimens formulated under different conditions, in addition cence correlation spectroscopy to identify interdomain interactions and to software that can quantitatively evaluate CD spectral changes associated resolve dynamics from picoseconds to milliseconds. The labeling sites were with structural changes in proteins. We used this high-throughput screening on a-helices or surface loops. We distinguished locally rigid or flexible sites system to measure CD spectra of VHH antibodies formulated under different based on their relative contribution to FRET efficiencies and correlation am- pHs and salt concentrations, and we compared the CD spectra of native and plitudes. Global analysis of the entire FRET network allowed comparison of denatured antibodies using a Z-test. A high correlation was obtained between the timescales associated with local and interdomain motions. Describing how the Z-score for each formulation condition and the denaturation temperature. local and interdomain dynamics drive the supertertiary organization of PSD- This suggests that a Z-test is a very useful primary screening method before 95 provides a basis for understanding how conformational changes regulate conducting a thermal denaturation evaluation, which generally requires a great multidomain scaffold proteins. deal of time.

936-Pos Posters: Protein Stability, Folding, and Probing (UN)Folding Transition Paths of Fast-Folding Proteins by Single- Molecule Fluorescence: Exploring the Role of Secondary Structure, Fold Chaperones I Topology and Sequence Nivin Mothi1, Mourad Sadqi2, Victor Munoz2. 934-Pos 1Department of Chemistry and Chemical Biology, University of California UV Peroxidation Reduces Poloxamine T1107 Capability to Disaggregate Merced, Merced, CA, USA, 2Department of Bioengineering, School of Lysozyme Engineering, University of California Merced, Merced, CA, USA. Michelle X. Ling, Colin A. Mcfaul, Raphael C. Lee. Protein folding involves formation of secondary structural elements and Plastic & Reconstructive Surgery, The University of Chicago Biological their collapse into well-defined 3-dimensional structure. Attempts to study Sciences Division, Chicago, IL, USA. how these processes cooperate to form the native structure and determine Poloxamers (poly-oxy-amers) and poloxamines (poly-oxy-amines) are amphi- folding mechanisms has been focus of research for decades. Addressing philic multi-block polymer surfactants. Because their backbones are made of these questions require looking at the distributions of behaviors of indivi- polyethylene oxide and polypropylene oxide blocks, they are susceptible to dual molecules, which has been successfully attained by theory and computer oxidative formation of hydroperoxide side groups that alter surfactant proper- simulations. Experimentally, the problem must be approached by single- ties. Several poloxamers and poloxamines are able to prevent protein aggrega- molecule spectroscopic methods, which have not had the required time- tion by binding to exposed hydrophobic domains. The purpose of this study was resolution. to determine the effect of oxidative degradation on T1107 catalysis of unfolded We are investigating protein folding at the single-molecule level combining lysozyme. advanced custom-built fluorescence-based SM-FRET techniques optimized To peroxidate Poloxamine T1107 [Maroon Biotech], 2 mL of 15 mg/mL in for high-count rates and a maximum likelihood analysis (MLA) of photon phosphate-buffer saline (PBS) was exposed to a range of UV (254 nm, arrival times that we developed for the detection of (un)folding transition @2000 Gy/hr) radiation for 2, 4, 6, 8, 24, 32, 48, or 72 hours. DTT (5mM) paths of microsecond folding proteins. To investigate the roles of secondary reduced and aggregated hen egg white lysozyme (HEWL, 0.5mg/ml) structure, fold topology, and amino acid sequence we are performing these [Sigma-Aldrich] was used to quantify T1107 (0.5mg/mL) catalyzed disaggre- experiments on three fast-folding protein models, all consisting of three sec- gation at relevant temperatures (37 C). Aggregation was quantified by ondary structure elements in an antiparallel arrangement: En-HD (three a-he- measuring absorption at 400 nm using a Beckman DU800 spectrophotometer lices), and Nedd4-WW3 and FBP11WW2, which fold onto homologous (n=10). A reducing agent-compatible bicinchoninic acid (BCA-RAC) assay 3-stranded b-sheets but with different amino acid sequence. We are investi- kit [Thermo Fisher] was used to measure the concentration of soluble native gating their folding transition paths by 2-color SM-FRET using variants protein (n=3). A PeroxiDetect Kit [Sigma-Aldrich] was used to quantify the labeled with FRET pairs at the two ends in free diffusion experiments. concentration of peroxides (n=6). P values <0.05 were considered statistically Because these proteins fold-unfold in microseconds, free diffusion offers significant. the opportunity to catch folding transitions during the diffusive trajectories Non-irradiated T1107 caused 6% disaggregation of DTT HEWL. T1107 irradi- without immobilization and thus without significantly perturbing folding ated for 72 hours was 99% as ineffective in disaggregating HEWL as no T1107. landscape. Our analysis of these data using MLA of photon trajectories Increasing UV exposure was inversely related to T1107 ability to prevent ag- (rather than binning) provides the free energy landscape as a function of gregation. The non-irradiated T1107 indicated <5% loss in protein, while the experimental order parameter (FRET-pair distance) and the intramolecu- the most irradiated T1107 indicated 47% loss. CD showed highly irradiated lar diffusion coefficient. Subsequent reconstruction of the molecular trajec- T1107 and samples without T1107 did not preserve any secondary structures, tories from the photon trajectories using stochastic methods provide a 1 whereas non-irradiated and less-irradiated T1107 did. H NMR (8-9 ppm), direct glimpse of barrier crossing events. FTIR (1723cm1), and PeroxiDetect showed the formation of peroxide groups. This study demonstrates that peroxidated Poloxamine 1107 is incapable of 937-Pos catalyzing proteins disaggregation. Monitoring Dynamics of Protein Nascent Chain on the Ribosome using PET-FCS 935-Pos Marija Liutkute1, Ekaterina Samatova1, Manisankar Maiti1, New Approach to Evaluating the Stability of Antibodies using Circular Wolf H. Holtkamp2,Jo¨rg Enderlein3, Marina V. Rodnina1. Dichroism Spectroscopy Screening 1Dept Phys Biochem, MPI Biophysical Chemistry, Goettingen, Germany, Satoko Suzuki1, Yasuo Horiguchi1, Leah Pandiscia2, Koushi Nagamori1, 2Paul-Ehrlich-Institut, Langen, Germany, 3Biophysics / Complex Systems, Hiroshi Nakayama3, Kouhei Tsumoto4. Georg-August-Universit€at Go¨ttingen III. Physical Institute, Goettingen, 1Jasco Corporation, Tokyo, Japan, 2Jasco Inc, Philadelphia, PA, USA, Germany. 3Panasonic Corporation, Osaka, Japan, 4School of Engineering and Institute Many cellular proteins begin folding while the peptide is still attached to the of Medical Science, The University of Tokyo, Tokyo, Japan. translating ribosome. Contrary to well-studied folding of isolated proteins Biopharmaceuticals have attracted a great deal of attention in recent years in vitro, co-translational folding is vectorial. The ribosome interacts with since antibody drugs and protein formulations can target specific regions of the emerging peptide, protects it from aggregation and misfolding, and may the body with few side effects. However, compared to conventional low mo- stabilize intermediates that do not exist in solution but are important for vecto- lecular weight drugs, biopharmaceuticals can more easily change their struc- rial folding. Currently, only few methods can address the dynamic properties ture depending on formulation conditions such as the solvent, pH and or the folding pathways of the emerging nascent chains. Here we investigate temperature, and may consequently lose their activity. It is therefore important co-translational folding and nascent chain dynamics of the N-terminal HemK

BPJ 9352_9354 190a Monday, March 4, 2019 domain (HemK NTD) in a minimal in vitro translation system consisting of 940-Pos pre-assembled initiation complexes, elongation factors, and aminoacylated A Structural, Dynamic, and Thermodynamic Explanation of Thermosta- tRNAs. It was previously reported that the HemK NTD folds on the ribosome bility in a De Novo Designed Three-Helix Bundle through the formation of compact intermediate states. We used a single mole- Natali Gonzalez, Emily Hamlin, Parwana Khazi, Catrina Nguyen, cule equilibrium technique combining photoinduced electron transfer-based Jennifer Young, Michelle E. McCully. quenching and fluorescence correlation spectroscopy (PET-FCS) to probe Biology, Santa Clara University, Santa Clara, CA, USA. the dynamics of these compact intermediate states on the ribosome. We devel- UVF is a de novo designed, three-helix bundle protein built based on the back- oped a PET reporter between a native tryptophan and fluorescently labeled bone template of the Engrailed homeodomain (EnHD). UVF was designed in N-terminal methionine of HemK. PET-FCS data of purified ribosome nascent the Mayo Lab by placing only hydrophobic residues at buried positions and chain complexes resulted in multi-exponential curves reporting on nascent only polar residues at surface positions. When expressed, the protein folded peptide dynamics in the nanosecond to microsecond timescale. By analyzing as expected, and it was also highly thermostable (Tm >99 C vs. 52 C for the data, we obtained rates of interactions between the N-terminal dye and the EnHD). Hybrid proteins combining the buried and surface residue sets from internal tryptophan or the quencher residues of the ribosome surface. By UVF and EnHD were built to assess independently the contributions of comparing the rates of different length and varied stability nascent chains UVF’s fully hydrophobic core and polar surface to its thermostability. A com- with those of peptides released from the ribosome, we could further charac- plement of molecular dynamics simulations and equilibrium unfolding exper- terize the folding pathway itself and the dynamics of the observed co- iments were performed to quantify the proteins’ dynamics and their translational folding intermediates of the HemK NTD while on the ribosome. thermodynamic parameters of folding.

941-Pos 938-Pos Long-Ranged Protein-Glycan Interactions Stabilize Von Willebrand Fac- Enhanced pH Dependent Modulation of Alpha Crystallin Chaperone tor A2 Domain from Mechanical Unfolding Function and Subunit Exchange in an N-Terminal Phosphorylation Mimic Chuqiao Dong1, Jumin Lee2, Seonghoon Kim2, Whitney Lai3, Kashmeera Baboolall, Belelot Birhanu, Natalie Braun, Yusrah Kaudeer, Edmund B. Webb1, Alparslan Oztekin1, Xiaohui Zhang4, Wonpil Im5. Patricia B. O’Hara. 1Department of Mechanical Engineering and Mechanics, Lehigh Univ, Dept Chemistry, Amherst Coll, Amherst, MA, USA. Bethlehem, PA, USA, 2Department of Biological Sciences, Lehigh Univ, It has been hypothesized that N-terminal phosphorylation can modulate chap- Bethlehem, PA, USA, 3Department of Bioengineering, Lehigh Univ, erone function of alpha crystallin in vitro. We used an alpha crystallin mutant Bethlehem, PA, USA, 4Dept Mechanical Engineering/Bioengineering, in which 3 serines at positions 19, 45 and 59 have been replaced by gluta- Lehigh Univ, Bethlehem, PA, USA, 5Biological Sciences and mates. Since the negative charges of the 3 glutamate residues at neutral pH Bioengineering, Lehigh Univ, Bethlehem, PA, USA. mimic the phosphorylation that can happen in vivo, the mutant is referred von Willebrand Factor (vWF) is a large multimeric protein that binds to plate- to as the 3E phosphorylation mimic. pH can play a role in the modulation lets and collagen in blood clotting. vWF A2 domain hosts a proteolytic site for of chaperone function of the phosphorylated protein the 3E phosphorylation ADAMTS13 (A Disintegrin and Metalloprotease with a ThromboSpondin type mimic. We measured the capacity of wild type and mutant protein to modu- 1 motif, member 13) to regulate the size of vWF multimers. This regulation late insulin aggregation at pH 5 (acidic), pH 7 (neutral), and pH 8 (basic). process is highly sensitive to force conditions and protein-glycan interactions Light scattering produced upon insulin denaturation can be followed as a as the process occurs in flowing blood. There are two sites on A2 domain function of time to determine the rate of denaturation and extent of its disrup- (N1515 and N1574) bearing various N-linked glycan structures. In this study, tion by both wild type and the 3E phosphorylation mimic. We see that aggre- we used molecular dynamics (MD) simulation to study the force-induced un- gation is dependent on pH for both wild type and the 3E phosphorylation folding of A2 domain with or without a single N-linked glycan type on each mimic, with chaperone function being lowest under acidic conditions and ris- site. The sequential pullout of b-strands was used to represent a characteristic ing at neutral and basic conditions. Above pH 7, chaperone behavior of the 3E unfolding sequence of A2. This unfolding sequence varied due to protein- phosphorylation mimic far exceeds wild type. We have also attached fluores- glycan interactions. The force-extension and total energy-extension profiles cent labels to both wild type and 3E mutant in order to explore the exchange also show differences in magnitude but similar characteristic shapes between of the alpha crystallin subunits within the protein multimer. By observing the systems with or without glycans. Systems with N-linked glycans encoun- changes in FRET when one solution labelled with FRET-donor is mixed tered higher energy barriers for full unfolding and even for unfolding up to with a solution labelled with FRET-acceptor, we can measure the extent of the point of ADAMTS13 cleavage site exposure. Interestingly, there is not the subunit exchange and determine how exchange depends upon pH for much difference observed for A2 domain structure itself with or without gly- both wild type and the 3E phosphorylation mimic. We can infer that cans from standard MD simulations, suggesting roles of N-glycans in A2 un- in vivo phosphorylation of alpha crystallin can be used to modulate chaperone folding through long-ranged protein-glycan interactions. function in stressed cells. 942-Pos 939-Pos Studying the Folding Behavior of a 3D Lattice Protein under Oscillatory H20Y Mutation Stabilizes Structure of the Primary DNA Recognition Conditions Domain of the Sleeping Beauty Qizhang Jia, Xuanye Zhu, Austin Cheng, Cory J. Kim, Amy Y. Wang, Chenbo Yan1, Gage O. Leighton1, Janna Lustig2, Zola´n Ivics2, Kateri H. DuBay. Irina V. Nesmelova1. Chemistry, University of Virginia, Charlottesville, VA, USA. 1Department of Physics and Optical Sciences, Center for Biomedical By spontaneously and reliably folding from a primary amino acid sequence Engineering and Science, University of North Carolina at Charlotte, into a highly specific native geometry with bioactive functionalities, the Charlotte, NC, USA, 2Division of Medical Biotechnology, Paul-Ehrlich- natively-folded protein plays a significant role in fundamental biological pro- Institut, Langen, Germany. cesses. Protein folding is governed by the interactions between residues and The ability of DNA transposons to jump from one DNA site or molecule to between residues and the environment. The structural stability and bio- another is exploited in functional genomics, genetic engineering, and human functionality of proteins depend on physiological environmental factors, gene therapy. Sleeping Beauty (SB) is the most widely used DNA trans- such as temperature, acidity, crowding, and ionic strength. In addition, the poson in genetic applications. The onset of SB transposition is the recogni- local cellular environment is highly dynamic – both spatially and temporally tion of the transposon DNA by the PAI subdomain of SB transposase. heterogeneous. In this work, we employed a 3D lattice protein model to inves- Previously, we have determined that the PAI subdomain remains mostly un- tigate the changes to the equilibrium folded state properties and to the kinetics structured at experimental conditions that mimic physiologic. We have of folding pathways for small proteins that fold amidst ongoing temperature further established that only the folded conformation of the PAI subdomain oscillations. is capable of binding to the transposon DNA. Here, we computationally identify and experimentally prove, using NMR and CD spectroscopy, 943-Pos several structure-stabilizing mutation in the PAI subdomain, including A Comprehensive Investigation of the Stabilization of Monomeric Hfgf1 H20Y. We also show that introducing H20Y mutation increases DNA bind- by Heparin Hexasaccharide using Microsecond-Level MD Simulations ing of the PAI subdomain. Our data suggest that introducing structure- and Enhanced Sampling Techniques stabilizing H20Y mutation into the SB transposase could improve the effi- Vivek Govind Kumar, Shilpi Agrawal, T.K.S. Kumar, Mahmoud Moradi. ciency of SB transposition. Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA.

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Acidic human fibroblast growth factor 1 (hFGF1) is a major signaling molecule time of the intermediate as the conformational space available for the protein that is heavily involved in cell proliferation, angiogenesis, tumor invasion and to rearrange is constrained due to the presence of the disulfide bonds. The metastatic progression. Previous experimental studies have demonstrated that kinetically trapped intermediates are not observed in the folding pathways hFGF1 is naturally unstable and that it has a near-physiological denaturation when the disulfide bonds are absent. temperature. Heparin (a linear sulfated ) is known to stabilize hFGF1 and protect it from thermal and proteolytic degradation. Our study 946-Pos used experimental data to set up a rigorous computational investigation of Probing the Chaperone Activity of Erythroid Spectrin the hFGF1-heparin hexasaccharide complex. Dipayan Bose, Abhijit Chakrabarti. Three models were simulated for 4.8 microseconds each. Our equilibrium-MD Crystallography and Molecular Biology Division, Saha Institute of Nuclear simulations confirmed that the heparin-free monomer is less stable than the Physics, Homi Bhaba National Institute, Kolkata, India. heparin-bound monomer. The decreased stability of the heparin-free monomer Spectrin, the major protein of the RBC membrane skeleton has canonically is due to a conformational change in the heparin-binding region. This confor- been thought to only serve a structural function. We have described a novel mational change was not observed in the heparin-bound systems. Important in- chaperone-like property of spectrin and have shown that it is able to prevent teractions that contribute to the stability of the complex were also the aggregation of other proteins such as alcohol dehydrogenase, insulin and characterized. K113 and S117 were identified as important residues of the free globin chains. Moreover this chaperone activity is pH linked, with spec- heparin-binding region that contribute to intramolecular hydrogen bonding. trin displaying higher oligomeric states and consequently increased chap- Strong intermolecular hydrogen bonding was also observed between R123 erone activity at lower pH, with a maxima at pH 4.0. Hemoglobin and and IdoA(4) of the heparin hexasaccharide. We then used a combination of other heme proteins are implicated as clients of the chaperone-like activity non-equilibrium pulling and bias exchange umbrella sampling simulations to of spectrin and we have demonstrated that as a consequence of spectrin- determine the binding free energy of heparin. heme protein interaction, there is an increase in the enzymatic activity of Thus far, all published computational studies of the hFGF-1 heparin complex these heme proteins. The peroxidase activities of hemoglobin, hemoglobin have been based on nanosecond-level simulations. For the very first time, we variants, free globin chains, cytochrome-c and hemin increase upon spectrin have used a combination of microsecond-level MD simulations and large- binding, as does the enzymatic activity of catalase. The Km values of these scale enhanced sampling techniques to carry out a more realistic and biochem- heme proteins for H2O2 decreases in presence of spectrin while the Vmax ically relevant assessment of the hFGF1-heparin complex. values are found to increase. Raman spectroscopy indicates spectrin binding induced conformational changes in the prosthetic heme groups of these pro- 944-Pos teins as the source of increased enzymatic activity. We have also found that Identifying Intermediate States in Prion Protein Folding Pathway: A the chaperone-like activity of spectrin competes with its phospholipid/hemo- Possible Precursor to the Misfolded State? globin binding ability and phospholipid/hemoglobin bound spectrin is a Balaka Mondal. comparatively weaker chaperone. Non-enzymatic glycation has also been Solid State and Structural Chemistry Unit, Indian Institute of Science, shown to decrease spectrin chaperone potential. We have tried to localize Bengaluru, India. the molecular origin of chaperone-like activity in multi domain spectrin Aggregation of prion proteins causes neurodegenerative disorders. The by cloning and investigating individual domains and our current understand- misfolded form of monomeric cellular prion protein leads to toxic protein ing points to the presence of hydrophobic patches on the surface of these do- aggregates rich in b-sheets. Pathological scrapie form of prion protein also mains as the source of the chaperone activity of spectrin, as notably seen in catalyzes the conversion of cellular protein to the infectious conformation. the self-association domain. Therefore it is very important to understand the mechanism of structural tran- sition of a prion protein from a cellular functional form to the scrapie form. 947-Pos To probe the structures of transient intermediates populated in the folding Moltenprot: A High-Throughput Analysis Platform to Assess Thermody- pathways of prion protein, which can be probable precursors to the patholog- namic Stability of Membrane Proteins and Complexes ical misfolded forms, we performed molecular dynamics simulations to probe Vadim Kotov1,2, Oliver Vesper1,2, Maria Garcia Alai3, Christian Loew4, the folding pathways of prion protein using coarse-grained protein models. Thomas C. Marlovits1,2. The protein populates an intermediate, which is globular and resembles a 1University Medical Center Hamburg-Eppendorf (UKE), Institute for b molten globule like structure where only the 1 strand detaches from the Structural and Systems Biology, CSSB-Centre for Structural Systems rest of the folded protein structure. This observation also supports the Biology, Hamburg, Germany, 2German Electron Synchrotron Centre b 3 NMR studies, which report the partial unfolding of the 1 strand. This confor- (DESY), Hamburg, Germany, Sample Preparation and Characterization mation might possibly be a precursor to the misfolded or the scrapie prion Facility, European Molecular Biology Laboratory (EMBL), Hamburg, form. Germany, 4European Molecular Biology Laboratory (EMBL), CSSB-Centre References for Structural Systems Biology, Hamburg, Germany. 1) Prusiner, S. B. (1998) Prions. Proc. Natl. Acad. Sci. U.S.A. 95, 13363-13383 Structural biology has entered a new era, when high resolution analysis of 2) Legname, G., Baskakov, I., Nguyen, H., Riesner, D., Cohen, F., DeArmond, increasingly complex samples was made possible. Still, spectacular technolog- S., and Prusiner, S. (2004) Synthetic mammalian prions. Science 305, 673-676. ical developments, such as lipidic cubic phase crystallization or direct electron 3) Julien O, Chatterjee S, Thiessen A, Graether SP, Sykes BD (2009) Differen- detectors, do not address the primary challenge that any structural biologist tial stability of the bovine prion protein upon urea unfolding. Protein Sci 18, faces, namely, how to determine optimal buffer conditions for maintaining sta- 2172–2182 ble proteins or protein complexes. To overcome this issue we developed a software package MoltenProt, which 945-Pos estimates thermodynamic characteristics of protein stability from thermal un- Disulfide Bonds Modulate Lysozyme Folding Pathways folding assays. The analysis integrates both melting temperature and slope of Aswathy Muttathukattil Narayanan. the unfolding curve to determine standard Gibbs free energy of unfolding. Solid State and Structural Chemistry Unit, Indian Institute of Science, This approach is applicable to proteins of any complexity and origin, con- Bangalore, India. sumes minimal amounts of sample and is compatible with most common Experiments on lysozyme folding [Dobson et al., 1994; Kiefhaber, 1995] buffer reagents. Data processing can be massively parallelized, and the whole show that it folds in parallel pathways: a slow kinetic pathway with well pipeline is typically done within one week. We demonstrate increased sensi- populated partially folded states and a faster pathway without any intermedi- tivity and selectivity of MoltenProt analysis compared to standard melting ates. We studied lysozyme folding using coarse-grained protein models and temperature based hit detection and illustrate the utility of the assay by testing molecular dynamics simulations. The simulations do show that lysozyme the stabilizing properties of 94 widely used detergents on a panel of mem- has slower and faster folding pathways as predicted by the experiments. brane proteins. The folding timescale in the fast pathway is on the order of tens of millisec- onds as expected for two-state folding globular proteins. In the slower 948-Pos pathway, the folding timescale is higher as the intermediate populated in Structural Dynamics of c-Myb DNA-Binding Domain Revealed by DXT this pathway is stable for hundreds of milliseconds. The intermediate popu- and Thermal Analysis lated is partially structured where the b domain is fully formed and the a Satomi Inaba1,2, Yuhi Hosoe2, Yuji C. Sasaki3, Hiroshi Sekiguchi1, domain is partially formed and there are no inter-domain contacts. These in- Masayuki Oda2. termediates are kinetically trapped and require rearrangement in order to fold 1JASRI/SPring-8, Sayo, Japan, 2Kyoto Prefectural Univ, Kyoto, Japan, 3Dept to its native state. The disulfide bonds play a major role in enhancing the life- Adv Matl Sci, Univ Tokyo, Kashiwa City, Japan.

BPJ 9352_9354 192a Monday, March 4, 2019

The transcriptional factor, c-Myb R2R3, is minimum unit for DNA-binding model protein, we demonstrate that we can extract the free energy of unfold- and shows largely flexible conformation in solution, which is important for ing (DGU) from a single HXMS experiment over 30 minutes with less than specific DNA-binding function. Here we investigated the structural dynamics 10mlof10mM protein in no denaturant. The HXMS-derived DGU matches of c-Myb R2R3 induced by the DNA-binding, using circular dichroism (CD), that determined from traditional equilibrium denaturation monitored by fluo- diffracted X-ray tracking (DXT), and isothermal titration calorimetry (ITC). rescence. We will discuss how we are modifying this approach to extract DXT is recently developed methods and can evaluate the protein structural other landscape parameters and access a broader range of experimental fluctuations by detecting the movement of a gold-nanocrystal attached to conditions. This will allow us to apply N-HXMS to map a more diverse the target protein. Thermal stability of R2R3 was increased in the presence collection of proteins, expanding our understanding of the interplay between of cognate DNA, suggesting that the structure was changed in more rigid protein landscape and function. upon the DNA-binding. The resultant curve of the mean square angular dis- placements (MSD) obtained from DXT clearly showed that the flexibility of R2R3 was decreased upon DNA binding, and the DNA-binding energies 951-Pos determined using the angular diffusion coefficients were in good agreement Investigating the Effects of Missense Mutations in MSH2 Gene Associated with those determined using ITC. The results of the MSD curves also indi- with Lynch Syndrome cate that the translational length reduces by approximately half upon DNA Bohua Wu, Yunhui Peng, Julia A. Eggert, Emil Alexov. binding. Healthcare Genetics, Clemson University, Clemson, SC, USA. MSH2-MSH6 is a DNA mismatch repair (MMR) protein complex. 36 mu- 949-Pos tations in MSH2 have been reported to cause Lynch Syndrome. At the same The Earliest Stages of a Protein’s Life Influences its Long-Term Solubility time, individual genetic testing results suggest that MMR deficiency is asso- and Structural Accuracy ciated with breast cancers in some individuals with Lynch Syndrome. Here Matthew D. Dalphin1, Angela Varela1, Andrew Stangl1, we demonstrate that the sequence position that have pathogenic mutations Robert Kirchdoerfer2, Rayna Addabbo1, Yoo Jin Song1, Yue Liu1, in MSH2 protein are highly conserved among species, while the harmless Silvia Cavagnero1. are not. We explore the effects of disease-causing and harmless mutations 1Univ Wisconsin Madison, Madison, WI, USA, 2Scripps Research Institute, on the conformational dynamics, stability and binding of MSH2 protein. La Jolla, CA, USA. Our analysis indicates that pathogenic mutations in MSH2 affect protein Despite its fundamental importance for life, many details regarding how the stability and dynamics and alter binding affinity and flexibility of the cell promotes the solubility and structural accuracy of proteins remains protein. poorly understood. This lack of knowledge poses serious challenges in basic science, biotechnology and medicine as the inability to prevent inclusion 952-Pos body formation limits the efficient overproduction of recombinant proteins Unsupervised Learning for Decoy Selection in Protein Structure Prediction and protein-based therapeutics. In this work, experimental and computational Nasrin Akhter1, Gopinath Chennupati2, Hristo Djidjev2, Amarda Shehu1. approaches were combined to explore factors that help discriminate folding 1Dept Compu Sci, George Mason Univ, Fairfax, VA, USA, 2Los Alamos and aggregation pathways at various stages of a protein’s life in the cell. If National Lab, Los Alamos, NM, USA. folded properly, the native states of many proteins, including apomyoglobin Despite rapid advances in template-free protein structure prediction, the funda- and the soluble E. coli proteome, were shown to be kinetically-trapped from mental question of what makes a tertiary structure of a protein native remains stable aggregate states under physiologically-relevant conditions. However, unanswered. This question, known as decoy selection in the context of thou- kinetic simulations suggest that this kinetic trapping can be circumvented sands of decoy structures generated by optimization algorithms, continues to at high protein concentrations and, importantly, in the presence of small motivate diverse computational approaches in protein structure modeling. pre-nucleated aggregate seeds. Attempts to understand how the cell correctly Some approaches focus on designing energy/scoring functions to score struc- biases nascent proteins into their kinetically trapped native states highlight tures and proceed to carry out decoy selection in a ranking-based manner. that the ribosome and molecular chaperones play distinct yet complementary Others ignore energetic scoring altogether and cluster structures based on their roles in this process. The ribosome confers solubility to aggregation-prone similarity. These approaches show varied performance and currently cannot be nascent proteins early during biosynthesis, but requires additional help reliably adopted for decoy selection. Moreover, these approaches ignore the from molecular chaperones to prevent cotranslational nascent protein aggre- knowledge that the decoys sampled by a decoy generation protocol constitute gation during the late stages of translation. Ongoing studies are further char- points in an unknown energy landscape whose underlying organization can be acterizing the structural aspects of cotranslationally generated aggregates and extracted and leveraged to reveal regions housing native and near-native de- investigating their effects on the long-term solubility and structural accuracy coys. In this work we showcase a machine learning approach that leverages of nascent proteins upon release from the ribosome. Taken together, this discovered components of a decoy-sampled energy landscape and outperforms work sheds light on the unique mechanism by which the cell shapes, and ul- both clustering and energy ranking-based methods for decoy selection. Our timately traps, proteins into their native state during the earliest stages of pro- approach also demonstrates a consistently better performance on varied decoy tein life. data sets, suggesting the promise of machine learning-based approaches for decoy selection. 950-Pos N-HXMS: A Method to Directly Measure Protein Folding and Stability un- der Native Conditions 953-Pos Nejc Nagelj1, Minjee Kim2, Kaeli Mathias3, Sheila Jaswal1. Biophysical Characterization of Differences in Domain-Domain Interac- 1Biochemistry and Biophysics, Amherst College, Amherst, MA, USA, tions between the Apolipoprotein E4 and E3 2Harvard Medical School, Boston, MA, USA, 3St. Jude Children’s Research Subhrajyoti Dolai, Kanchan Garai. Hospital, Memphis, TN, USA. Biophysics, Tata Institute of Fundamental Research, Hyderabad, India. Protein function is intimately linked to the dynamics and energetics of the Apolipoprotein E4 is the strongest risk factor for Alzheimer’s disease while native state. Most proteins need to fold into a specific conformation and the ApoE3 is considered normal and the ApoE2 is protective. ApoE3(112C) maintain that structure in order to function properly. The conformations and ApoE4(112R) differ by one amino acid change at 112th position. Struc- sampled, their thermodynamic stability differences, and the transition rates ture of ApoE consists of two domains, a 22 kDa N-terminal domain (1-167) between them are defining features of the protein’s landscape. Traditionally, and a 10 kDa C-terminal domain (238-299), connected by a hinge region. these landscape parameters are extracted from equilibrium denaturation ex- Here we investigate the role of the amino acid at position 112 on the stabil- periments coupled with spectroscopic methods. The aforementioned ity and the domain-domain interactions by performing substitution of C112 approach requires numerous measurements as a function of denaturant or to different amino acids of varying hydrophobicity. Our results indicate that temperature, only to then empirically extrapolate results to native conditions. stability of the N-terminal domain increases dramatically with increasing Furthermore, this approach is limited to proteins which can unfold and refold hydrophobicity of the residue at 112. Intramolecular Fosters Resonance En- reversibly without misfolding or aggregating under these harsh conditions. ergy Transfer (FRET) between N- and the C-terminal domains indicate that Many biologically interesting and disease-related proteins are hence not the stability of the domain-domain interactions is correlated with the stabil- accessible to these methods. Hydrogen exchange mass spectrometry ity of the N-terminal domain. Furthermore, presence of the C-terminal (HXMS) detects the sampling of unfolded states from the native state even domain destabilizes the N-terminal domains of ApoE4 but not ApoE3. under non-denaturing conditions. Using protein L, a well-characterized Taken together our data indicate that nature of the residue at position 112

BPJ 9352_9354 Monday, March 4, 2019 193a plays strong role on both the stability and the domain-domain interactions in 957-Pos ApoE. Site-Specific Characterization of Intermediates in Folding-Tetramerization of Melittin by the Rapid Mix/Freeze Method and Magic Angle Spinning 954-Pos Dynamic Nuclear Polarization (MAS-DNP) NMR at Low Temperature Native Quantitative Determination of Antibody-Drug Conjugate Affinity (25K) and Stability Jaekyun Jeon, Kent Thurber, Wai-Ming Yau, Robert Tycko. Colette Quinn1, Shawn Owen2, Keith Arlotta2. 1 2 Laboratory of Chemical Physics, National Institute of Diabetes and Digestive TA Instruments, Lindon, UT, USA, University of Utah, Salt Lake City, UT, and Diseases, National Institutes of Health, Bethesda, MD, USA. USA. Protein folding study has been regarded very important for fundamental under- Protein stability, particularly antibody stability, can be determined by calorim- standing of protein structures and functions. Although significant progresses etry without restrictions on buffers, excipients, and detergents. This native have been made, the characterization of the kinetic transition pathways and in- assay works equally well for novel biotherapeutics some of which are bispecific termediate states has been challenged and limited by either time or spatial res- antibodies, biosimilars, and modified antibodies including antibody drug con- olutions. In this presentation, I will introduce a newly developed rapid mixing/ jugates (ADC). For this ADC example, maytansine bound to trastuzumab freezing method for the sensitivity-enhanced solid-state NMR study at low tem- was prepared using two different methods: on-bead and off-bead. The DSC perature (25 K) by using the dynamic nuclear polarization (DNP) method. Our thermogram was able to capture and quantify the potential differences with re- rapid mixer/freezer device can mix two solutions to introduce the folding gards to stability while ITC dissected the binding to the HER2 protein into its’ within a couple of milliseconds and rapidly freeze at liquid nitrogen tempera- enthalpic and entropic contributions all while providing stoichiometry, which ture (80K) within few microseconds to trap the intermediate states during the could have relevance to bioavailability. folding transition. In this study, the folding and tetramerization of a small bee venom peptide, melittin was investigated. Melittin, selectively isotopically 955-Pos 13 15 Using Computational Modeling to Understand the Binding Mechanism of C and N labeled to monitor the folding and the possible dimerization, un- Designed Cyclic b-Hairpin to MDM2 derwent rapid pH change and was freeze-trapped at different time points (2, Yunhui Ge, Vincent Voelz. 4, 9, 30 ms). The low temperature MAS-DNP NMR spectra of frozen samples Chemistry Dept, Temple University, Philadelphia, PA, USA. captured at varying transition time points revealed the site-specific changes of Understanding mechanisms of protein folding and binding is crucial to the simultaneous folding and oligomeric transitions (monomer-dimer- designing their molecular function. Molecular dynamics (MD) simulations tetramer). The detail information of the mixer/freezer design, and the character- have become a powerful tool to understand the atomic details underlying com- ization of melittin intermediates and its pathway will be discussed in the plex processes. Together with Markov State Models (MSMs), these ap- presentation. proaches have shown great success in understanding conformational change 958-Pos that occurs over long timescales, which may be important in drug design. Radioligand Thermostability Assessment of Agonist-Bound Human Type 2 The MDM2-p53 protein-peptide interaction is a well-studied model system Cannabinoid Receptor that is both therapeutically relevant and used frequently in methodology Ryan L. Beckner, Klaus Gawrisch, Alexei Yeliseev. development. Cyclic peptide binders of MDM2 have become attractive as LMBB, NIAAA-NIH, Rockville, MD, USA. therapeutics due to their high stability and preorganization in solution. In The human type-2 cannabinoid receptor (CB2) is a 7-helix transmembrane G- this work, we performed massively parallel explicit-solvent molecular dy- protein coupled receptor (GPCR) implicated in anti-inflammatory and anal- b namics simulations of designed cyclic -hairpins on the Folding@home gesic processes of considerable therapeutic interest. However, biophysical distributed computing platform. MSM analysis of the over 3-ms aggregate tra- characterization of CB2 has proven challenging due to its low thermostability jectory data enabled us to build a detailed mechanistic model of coupled and high structural lability in detergent micelles and liposomes. We hope to folding and binding of cyclic peptides that can be compared to experimental surmount this issue by identifying thermostabilizing CB2 point mutations, a binding affinities and rates. The MSMs allows us to assess the relative stabil- method that has produced thermostabilized and conformationally-locked con- ity of each ligand in solution and capture a number of non-native interactions structs for several other GPCRs. To evaluate the thermostability of GPCR which lead to misfolded states. Our MSM models imply a conformational se- point mutants, a radioligand-binding assay is often employed wherein b lection mechanism of cyclic -hairpin binding, a result which helps inform GPCR-containing extracts are incubated with radioligand at elevated temper- future drug design studies targeting MDM2. atures and subsequently purified using size-exclusion chromatography. Ratios 956-Pos between radioactivity measured before and after heat treatment and purifica- Single-Molecule Force Spectroscopy Shows that the Anti-Prion Compound tion relay information on the impact of a given mutation on receptor stability, Pentosan Polysulfate Binds Heterogeneously to Folded and Unfolded Prion with higher ratios indicating a larger fraction of correctly-folded receptor and Protein increased thermostability. Melting curves can also be determined by incu- Rafayel Petrosyan, Patra Shubhadeep, Negar Rezajooei, Craig Garen, bating radioligand at several temperatures. Utilization of such radioligand- Michael T. Woodside. based thermostability assessments of CB2 has previously been impeded due Department of Physics, University of Alberta, Edmonton, AB, Canada. to high background values resulting from nonspecific association of lipophilic The misfolding of the prion protein (PrP) causes fatal neurodegeneration in cannabinoid ligands with detergent micelles. We report that a novel usage of a mammals. Some ligands have been reported that bind to PrP and inhibit the facial steroid-based detergent, Fac¸ade-TEG, enables radioligand thermosta- progression of the disease, but the underlying molecular mechanisms of this bility assessment of CB2 using a tritiated cannabinoid agonist CP-55,490 inhibition are unclear, in part because it is difficult to observe directly the and hypothesize that the small micelle size of Fac¸ade-TEG enables the effects of anti-prion ligands on PrP folding. We studied the interaction of observed dramatic reductions in background over conventional detergents. PrP with pentosan polysulfate (PPS), a polymer that was shown to be an We also demonstrate that detergent exchange into Fac¸ade-TEG from large effective anti-prion agent and is known to directly interact with PrP. Using scale solubilization of recombinant CB2 is feasible with nickel-affinity immo- optical tweezers to measure unfolding and refolding of single PrP molecules bilization. The assay is used to demonstrate the impact of selected mutations via force spectroscopy, we examined the effects of PPS by comparing the as well as a maltose-binding-protein fusion on CB2 thermostability. Assay re- folding with and without PPS present. For these studies, we used PrP from sults are compared against alternate measurements of thermostability using Syrian hamsters and bank voles; both are disease-prone rodents, but bank intrinsic tryptophan fluorescence and G-protein activation. Ongoing work as- vole PrP is more likely to misfold. We found that PPS bound to single sessing thermostability of members of an alanine-scanning CB2 library is also PrP molecules heterogeneously. The unfolding force was generally much described. higher than without PPS, but was very broadly distributed. PPS binding also introduced a variety of new unfolding intermediates, especially in the Posters: Protein Assemblies II unfolding and refolding of hamster PrP (which is normally two-state), and led to a broad distribution of unfolding contour length changes, indicating 959-Pos heterogeneous binding modes. Interestingly, PPS was also able to bind to Towards Understanding Amyloid Formation Mechanism of Beta2- unfolded or partially folded PrP, delaying or preventing refolding into the Microglobulin Induced by Copper Ions native state, similar to what was seen previously for a very different type Chungwen Liang. of anti-prion agent, a tetrapyrrole. The effects were similar for both hamster IALS, UMass Amherst, Amherst, MA, USA. and bank vole PrP, despite the differences in their native folding mecha- Beta2-microglobulin (b2m) is one of the well-known amyloid forming proteins nisms. that cause human degenerative diseases. The mechanism of b2m amyloid

BPJ 9352_9354 194a Monday, March 4, 2019 formation has been extensively studied for more than few decades, however, Cells actively maintain a vast array of complex, tightly regulated, chemical the detailed oligomer structures and underlying driving force are still unclear reactions, which occur on a multitude of time scales. The condensation of pro- and under intense debate. Remarkably, recent experiments suggest that copper teins, nucleic acids and other biomolecules by liquid-liquid phase separation ions play a crucial role in promoting b2m oligomers and facilitating aggrega- (LLPS) into membraneless organelles likely plays an important role in orga- tion process. Here, we combine ion mobility mass spectrometry (MS) and mo- nizing the myriad processes that sustain life. While this hypothesis has gained lecular dynamics (MD) simulation to investigate possible oligomer structures traction via macroscopic studies of cellular organization, atomic-level under- along the assembly pathway. We found that b2m dimers contain coppers, standing of the interactions that drive condensation remains elusive. Protein and two intermolecular salt bridges nearby the copper-binding site play a regions of low sequence complexity (LCRs) are particularly well poised to crucial role in stabilizing b2m dimers. Heterogeneous tetramers, which adopt serve as a model system for studying the requisite transient intermolecular in- different sizes and copper-binding states, are the key elements for initiating teractions in atomic detail given that specific subset of LCRs are prone to further amyloid formation. Upon losing coppers, one type of tetramers trans- demix at physiologically relevant solution conditions. Using a combination forms to a pre-polymer state that is assumed to be the building block of b2m of small angle x-ray scattering (SAXS) and nuclear magnetic resonance polymer. The polymer shows a worm-like structure consisting of native-like (NMR) coupled to all atom Monte Carlo and coarse-grained simulations, it monomers, which is consistent with previous NMR and MS studies. In addi- is possible to characterize both residue-specific interactions and the global tion, two distinct binding interfaces between native-like monomers play an properties of individual proteins and protein solutions. Using this multi- important role in maintaining both the rigidity and flexibility of the polymer. length scaled approach the single chain conformational landscape of the Based on the structural information of b2m oligomer states that we observed, hnRNPA1 LCR per-residue and globally can be coupled to the critical condi- a copper-induced assembly pathway is then proposed. This suggests that, by tions for phase separation. The favorable enthalpy of interaction between ar- designing a proper salt bridge blocker to prevent b2m dimer formation, a prom- omatic sidechains is observed to both stabilize the compact, disordered protein ising treatment for b2m related amyloidosis would be established in a near ensemble and drive phase separation. Further, SAXS measurements on phase future. separated solutions suggests that single chain behavior is conserved system- wide. This hierarchical experimental and computational approach suggests 960-Pos that a defined amino acid composition is capable of recapitulating the symme- Studying Multi-Protein Interactions by Fluorescence Detected Sedimenta- try between disperse protein conformation and phase separation theorized for tion Velocity Combining Hydrodynamic Separation of Complexes with homopolymers and suggests possibilities for symmetry breaking. Such ana- Fluorescence Quenching Analysis lytic rules could allow for prediction of phase behavior based on amino 1 2 1 1 Huaying Zhao , Siddhartha A. Datta , Sung Kim , Sumit K. Chaturvedi , acid sequence. Alan Rein2, Peter Schuck1. 1Dynamics of Macromolecular Assembly, Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging 963-Pos and Bioengineering, National Institutes of Health, Bethesda, MD, USA, 2HIV Multi-Step 2D Protein Crystallization via Structural Changes within an Dynamics and Replication Program, Center for Cancer Research, National Ordered Lattice Cancer Institute, Frederick, MD, USA. Jonathan Herrmann1, Colin J. Comerci2, Fatemeh Jabbarpour1, Sedimentation velocity analytical ultracentrifugation is a classical biophysical Lucy Shapiro3, William E. Moerner4, Soichi Wakatsuki1. 1 2 method to study protein interactions. It has the capability of hydrodynamically Structural Biology, Stanford University, Stanford, CA, USA, Biophysics 3 resolving co-existing complexes free in solution. Combined with fluorescence Program, Stanford University, Stanford, CA, USA, Developmental Biology, 4 detection it is possible to study high-affinity interactions even in the low pM Stanford University, Stanford, CA, USA, Chemistry, Stanford University, range, which can be enhanced to multi-component discrimination through Stanford, CA, USA. the use of photoswitchable proteins. This allows the determination of size, Macromolecular phase transitions govern a wide variety of biological and shape, and composition of reversibly formed complexes. Here we introduce industrial processes. Previous studies have identified single- and multi- the combined analysis of total fluorescence intensity and sedimentation profiles step protein crystallization pathways that can control the final crystal as a function of protein concentration. This adds microscopic structural infor- polymorph. We examined RsaA, the 98 kDa exported protein from Caulo- mation on the different hydrodynamically resolved complexes through the bacter crescentus that forms the surface layer, a 2D crystalline layer around identification of changes in the proximity of the fluorophore upon complex for- the entire outer surface of the cell. RsaA exhibits calcium-mediated 2D mation. We illustrate the potential of this method in the application to the early crystallization in vivo and in vitro, forming a 22 nm lattice of hexameric re- stages of HIV-1 Gag assembly initiated by nucleic acid. peats. Using time-resolved electron cryo-microscopy, x-ray scattering, and FRET to observe RsaA’s nucleation and crystallization pathway in vitro, 961-Pos we find first a calcium-induced conformational change followed by Non-Classical Nucleation of Tumor Suppressor P53 Fibrils Hosted by clustering into an initial hexameric nucleation lattice with an observed Mesoscopic Protein-Rich Clusters spacing of 6 nm. We then observe a proto-crystal lattice that exhibits the Peter G. Vekilov. expected 22 nm spacing, but still requires an additional conformational Dept Chem/Biomolec Eng, Univ Houston, Houston, TX, USA. change involving the N-terminus to reach the physiologically observed About half of human cancers are associated with mutations of the tumor sup- crystal form. Therefore, RsaA crystallizes via ordered, structurally discrete pressor p53. Gained oncogenic functions of the mutants have been related to intermediates driven by changes in the protein’s tertiary and quaternary aggregation behaviors of wild-type and mutant p53. The thermodynamic and structures. kinetic mechanisms of p53 aggregation are poorly understood. Here we find that wild-type p53 forms an abnormal phase, mesoscopic clusters. The clusters exhibit several behaviors beyond the scope of classical phase transition the- 964-Pos ories: their size, ca. 100 nm, is independent of the p53 and crowder concentra- Modeling the Assembly Order of Multimeric Heteroprotein Complexes tions and decoupled from the protein mass held in the cluster phase. Lenna Peterson1, Yoichiro Togawa1, Juan Esquivel-Rodriguez2, Thermodynamic analyses elucidate another unusual cluster property: the lack Genki Terashi1, Charles Christoffer2, Amitava Roy3, Woong-Hee Shin1, of constant solubility. The nucleation of p53 fibrils deviates from the accepted Daisuke Kihara1. 1Dept Biol Sci, Purdue Univ, West Lafayette, IN, USA, 2Computer Science, mechanism of sequential association of single solute molecules. We find the 3 mesoscopic clusters serve as pre-assembled precursors of high p53 concentra- Purdue Univ, West Lafayette, IN, USA, Rocky Mountain Laboratories, NIH, tion that facilitate fibril assembly. Fibril nucleation hosted by precursors repre- Hamilton, MT, USA. sents a novel biological pathway, which opens avenues to suppress protein Protein-protein interactions are the cornerstone of numerous biological pro- fibrillation in aggregation diseases. cesses. Although an increasing number of protein complex structures have been determined using experimental methods, relatively fewer studies have 962-Pos been performed to determine the assembly order of complexes. In addition Correlating Amino Acid Sequence and Solvation to Disordered Protein to the insights into the molecular mechanisms of biological function provided Collapse Transitions and Phase Separation by the structure of a complex, knowing the assembly order is important for Erik W. Martin1, Alex S. Holehouse2, Ivan Peran1, Anne Bremer1, understanding the process of complex formation. Assembly order is also prac- Rohit V. Pappu2, Tanja Mittag1. tically useful for constructing subcomplexes as a step toward solving the 1Structural Biology, St Jude Childrens Res Hosp, Memphis, TN, USA, entire complex experimentally, designing artificial protein complexes, and 2Center for Biological Systems Engineering, Washington University, St developing drugs that interrupt a critical step in the complex assembly. There Louis, MO, USA. are several experimental methods for determining the assembly order of

BPJ 9352_9354 Monday, March 4, 2019 195a complexes; however, these techniques are -intensive. Here, we pre- 967-Pos sent a computational method that predicts the assembly order of protein com- Is the Sickle Hemoglobin Polymer Structure a Frustrated Spin-Glass? plexes by building the complex structure (PLoS Comp. Biol., 2018). The Emily Harkness1, Mark Davis1, Marilyn F. Bishop1, Kimberly C. Grasty2, method, named Path-LzerD, uses a multimeric protein docking algorithm Patrick J. Loll2, Frank A. Ferrone1. that assembles a protein complex structure from individual subunit structures 1Dept Physics, Drexel Univ, Philadelphia, PA, USA, 2Biochemistry, Drexel and predicts assembly order by observing the simulated assembly process of Univ, Philadelphia, PA, USA. the complex. Benchmarked on a dataset of complexes with experimental ev- Sickle hemoglobin differs from normal adult hemoglobin by a val in place of idence of assembly order, Path-LZerD was successful in predicting the assem- glu at the b6 position of the beta chain. This causes deoxygenated HbS to bly pathway for the majority of the cases. Moreover, when compared with a form 14 strand polymers with virtually no change to the structure of the hemo- simple approach that infers the assembly path from the buried surface area of globin molecule itself. The b6 site comprises one class of intermolecular con- subunits in the native complex, Path-LZerD has the strong advantage that it tacts known as lateral. A second class of contacts known as axial contacts can be used for cases where the complex structure is not known. As the first comprises connections that run roughly along the axis of the fiber. The axial method of its kind, Path-LZerD opens a new area of computational protein contacts present in the linear double strands found in crytallized HbS have structure modeling and will be an indispensable approach for studying protein been customarily thought to constitute the contacts present in the twisted fiber. complexes. However, it was recently observed that an alternative configuration of the same contacts might also be possible, interchanging donor and acceptor regions of the axial contact, but retaining the lateral contact. In preliminary simulations 965-Pos we have discovered that these alternatives spontaneously generate twisted dou- Variable Binding of Thioflavin T to Amyloid Fibrils ble strands instead of linear ones. We are exploring the possibility that neither Hiroaki Komatsu, Claire Meurice, Paul H. Axelsen. form is exclusively present, but that instead the polymer contains a non- Dept Pharmacology, Univ Pennsylvania Sch Med, Philadelphia, PA, USA. stoichiometric mixture of both types of axial contacts, in a structure similar Thioflavin T (ThioT) fluorescence is commonly used to quantify amyloid for- to a frustrated spin glass. To test this notion, we are constructing more exact mation in vivo and in vitro, but its binding mode and the basis for binding models, as well as creating site direct mutants. The mutants are being created selectively to fibrillar material is not understood. Moreover, there is no with a plasmid-based prokaryotic expression system that has previously been consensus on the relationship between fluorescence intensity and fibril used for high-level protein production (Shen, et al., PNAS 90:8108-8112). mass. In our previous study, to examine the quantitative relationship between The effects of the variation in contact partners will be tested by measurements ThioT-binding and conditions of fibril formation, the fluorescence of ThioT of the nucleation kinetics of molecular hybrids, which is highly sensitive bound to fibrils formed by 40-residue amyloid beta (Ab40) proteins was method to their presence and ability to participate in the growing structure. measured under conditions where the rate of fibril formation was varied by We will report on the current theoretical and experimental status of this project; changing the protein monomer concentration. It was demonstrated that the in- if correct, this could have implications for all patholoigal polymerizing tensity of ThioT fluorescence increased with the concentration of monomeric systems. protein during fibril formation. A similar dependence on monomer concentra- tion was observed for insulin fibrils. In the present study, the binding of ThioT 968-Pos to Ab40 protein fibrils formed at different rates was characterized by The Role of Charge Interactions in Liquid-Liquid Phase Transitions isothermal titration calorimetry (ITC). The binding isotherm indicated that Iuliia A. Antifeeva1, Alexander V. Fonin1, Olesya G. Shpironok1, the binding ratio was Ab40 protein:ThioT = 30:1 when the fibrils were formed Irina M. Kuznetsova1, Konstantin K. Turoverov1,2. at the low protein concentration (25mM). When fibrils were formed at higher 1Laboratory of Structural Dynamics Stability and Folding of Proteins, protein concentration, the binding ratio decreased, but the affinity remained Institute of Cytology of the Russian Academy of Sciences, St Petersburg, constant. These results suggest that ThioT binds to irregular features on the Russian Federation, 2Department of Biophysics, Peter the Great Saint- fibril, and that these irregular features increase in number when fibrils form Petersburg Polytechnic University, St. Petersburg, Russian Federation. rapidly. Recently, it became apparent that liquid - liquid phase transitions have a vital role in the cell life. Charge interactions between residues of intrinsically disor- 966-Pos dered proteins (IDPs) are important driver of phase separation in cell. In this P-Nitrophenylalanine as a Novel Probe of Hydrogen Bonding in Peptide work we studied phase separations of negatively charged IDP, prothymosin Hydrogels and Proteins alpha (ProTa), in dilute solution, highly concentrated PEG solutions simulating Nicholas R. John, Casey H. Londergan, Karin Akerfeldt, macromolecular crowding conditions within cell and in the presence of posi- Eliana V. von Krusenstiern. tively charged linker histone H1. ProTa status was monitored by fluorescence Chemistry, Haverford College, Haverford, PA, USA. of mCherry fluorescent protein fused with ProTa. It was shown that ProTa is Using vibrational probes to report on the local environment of specific protein unable to form droplets both in diluted and crowded solutions. Injection of posi- residues has became very popular in recent years. Aromatic nitro groups are tively charged histone H1 in diluted solutions of ProTa (low PEG concentra- easily incorporated into peptides and proteins, and the vibrational modes of tion) did not resulted in formation of any supramolecular structures. the nitro groups have been proposed as IR (or Raman) probes. Currently, However, injection of histone H1 in ProTa crowded solution (high concentra- quantitative data on how the nitro group’s frequencies respond to the local tion of PEG) lead to the formation of large droplets of ProTa. The obtained re- environment is unclear. Previous IR work led to focus on a mode near sults allow concluding that charged IDPs which are not capable to form 1600 cm-1. Now, we see a very strong Raman scattering peak at about coacervates individually and together in diluted solutions, may interfuse into 1350 cm-1: calculations indicate that this comes from a normal mode that liquid droplets in macromolecular crowding conditions through charge contains symmetric nitro and ring motions. We used the compound 4-nitro- interaction. benzyl alcohol and a home built Raman spectrometer to determine the solva- The work was supported by a grant from Russian Science Foundation RSCF tochromism of this band in a wide range of solvents. Our results showed that 18-75-10115 (AVF) and Russian Foundation for Basic Research RFBR 18- strong hydrogen bond donor solvents led to a blue shift in the frequency of 34-00975 (IAA) this Raman band, and a linear regression analysis using empirical solvent scales indicated that H-bond donors are the main factor that determines the 969-Pos nitro stretching frequency. Using this new information, we placed p-nitrophe- Mechanisms of Protein Fibril Formation in Amyloid Beta and Lysozyme nylalanine as a Raman probe in a hydrogel-forming peptide derived from se- Proteins menogelin I. Our results showed that the peptides stayed intact and the probe Carlos M. Perez, Ghanim Ullah, Tatiana Miti, Martin Muschol. was clearly visible across different concentrations in both H2O and D2O, with Dept Physics, Univ South Florida, Tampa, FL, USA. different frequencies in three different positions where p-nitrophenylalanine Rigid fibril (RFs) formation of amyloid proteins is a precursor to many degen- replaced either phenylalanine or tyrosine residues along what appears to be erative disorders, thus understanding the kinetics behind their generation could a hydrophobic cluster that holds together the hydrogel’s beta sheet superstruc- yield important information with respect to disease pathology. These amyloid ture. P-nitrophenylalanine was also incorporated into the E. Coli acyl carrier proteins exhibit what is known as the ‘‘critical oligomer concentration’’ (COC), protein at key sites using the suppression technique. In all peptide exhibiting similar behavior as that of micelle formation by surfactants. At low and protein samples, this band is visible despite its spectral overlap with other concentrations these proteins generate only smaller oligomeric species and vibrations. RFs, upon crossing the COC the growth of globular oligomers (gOs) and

BPJ 9352_9354 196a Monday, March 4, 2019 curvilinear fibrils (CFs) begin, aggregate species that have been shown to be 4) M. Kouza, N. Co, M. S. Li, S. Kmiecik, A. Kolinski, A. Kloczkowski, I.A. strongly correlated with disease pathology. Below the COC, RFs result in a Buhimschi, The Journal of Chemical Physics 148, 215106 (2018) sigmoidal rise with an initial lag period generated by delayed polymer nucle- ation. Above the COC, the rapid generation of gOs/CFs result in the elimination 972-Pos of the initial lag period, followed by a secondary rise due to RF growth, produc- Integrating Reaction-Diffusion Dynamics with a Biophysically Driven ing a biphasic onset. Here, we simulate the growth kinetics of amyloid beta and Deformable Membrane Model lysozyme proteins utilizing the theory of nucleated polymerization with off- Yiben Fu, Margaret Johnson. pathway aggregation, with the assumption that on-pathway nucleation and Biophysics, Johns Hopkins University, Baltimore, MD, USA. off-pathway aggregation occur so rapidly that they can be represented as a sin- The self-assembly of cytosolic proteins on membranes plays a critical role in gle step. We show that in order to simulate experimental fibril formation, the driving membrane remodeling in cell division, vesicle trafficking and viral primary nucleation rate must be significantly decreased at initial monomer con- bud formation. Simulating these processes at the cell-scale is challenging due centration where there is a significant amount of off-pathway aggregates, re- to the relatively slow time-scales and the coupling to nonequilibrium processes. sulting in a corresponding increase in initial lag periods. In addition, Reaction-diffusion is a powerful technique for these types of simulations, but attempting similar fitting with models of the same type produce comparable capturing the biophysics of membrane remodeling in combination with binding trends, further supporting our initial findings, and hinting at a possibly inhibi- reactions on surfaces has not been done. We present progress on implementing tory effect on RF growth by gOs/CFs. a dynamic membrane that is integrated with an accurate propagator for reaction-diffusion dynamics in solution, on surfaces, and transitioning between 970-Pos them. These tools will help characterize pathways of self-assembly of retroviral The Effect of Solution pH on the Structure and Stability of Lysozyme Gag protein and viral RNA in virion assembly, as it couples to membrane Amyloid Fibrils budding. Anna I. Sulatskaya1, Olga I. Povarova1, Maksim I. Sulatsky1, Irina M. Kuznetsova1, Konstantin K. Turoverov1,2. 973-Pos 1Inst Cytology, Russian Acad Sci, Saint Petersburg, Russian Federation, Study of Self-Assembly in Protein-Protein Interactions in Ultrafast Endo- 2Peter the Great Saint-Petersburg Polytechnic University, Saint Petersburg, cytosis Russian Federation. Yasaman Moghadamnia. Amyloid fibrils are ordered protein aggregates, enriched with beta-structures, T. C. Jenkins Department of Biophysics, Johns Hopkins University, formation of which is a marker of numerous serious human diseases. One of Baltimore, MD, USA. them is hereditary systemic lysozyme amyloidosis, in which aggregated forms Endocytosis is an essential process for regulating the intake of extra-cellular of lysozyme accumulate in the tissues and organs of patients. Earlier it was macromolecules such as metabolites, hormones, and proteins in to the cells. shown that the conditions of fibrillogenesis (heating, denaturing agents, Neural synapses use ultrafast endocytosis to recycle vesicles 1000 times extreme pH of the solution, etc.) significantly affect the growth kinetics of lyso- faster than typical clathrin-mediated endocytosis (60s). It has been sug- zyme amyloid fibrils in vitro. In this work, we demonstrated that the solution gested that in contrast to other endocytic methods, ultrafast endocytosis oc- pH can also influence on the structure of the mature lysozyme amyloid fibrils curs through preformation of protein complexes in solution prior to binding (in particular, the degree of their clusterization), as well as on the stability of to the cell membrane. To investigate possible mechanisms of rapid protein these aggregates. The obtained results can be used to develop the approaches assembly on the membrane in ultrafast endocytosis, we study the depen- to the degradation of amyloid fibrils or reduce their cytotoxicity by changing dence of self-assembly kinetics on the protein and lipid concentration, cell the fibrils structure. size, effective diffusion constants (both in the cytosol and on the mem- This work was supported by grant 18-74-10100 from Russian Scientific Foun- brane), rate constants, and spatial heterogeneity. Our approach is to use dation and the RF President Fellowship (number SP-841.2018.4). reaction-diffusion simulations through recently developed algorithms that provide full spatial and temporal resolution of protein self-assembly as 971-Pos parameterized by protein-protein binding constants. Our study will help bet- On the Relationship between Aggregation Rate and Mechanical Stability ter understand the underlying principles of self-assembly in ultrafast in Protein Aggregation endocytosis. Maksim Kouza1,2, Andrzej Kolinski2, Andrzej Kloczkowski1, Irina Buhimschi3. 974-Pos 1Battelle Center for Mathematical Medicine, Nationwide Childrens Hospital, User-Friendly Software for Simulating Non-Equilibrium Self-Assembly Columbus, OH, USA, 2Dept of Chemistry, University of Warsaw, Warsaw, Using Reaction-Diffusion Poland, 3The Research Institute, Nationwide Children’s Hospital, Columbus, Matthew J. Varga, Margaret E. Johnson. OH, USA. Biophysics, Johns Hopkins University, Baltimore, MD, USA. Preeclampsia, a pregnancy-specific disorder has been shown to share typical Protein self-assembly is a critical step in many biological processes, pathophysiological features with protein misfolding disorders including Alz- including endocytosis and virion formation. Standard computational heimer’s disease1. All-atom explicit solvent model employed previously for methods to study self-assembly are limited in their ability to reach the protein unfolding and folding studies2, is used to study oligomerization of long, biologically-relevant timescales required to observe the formation KLVFF and FVFLM peptides which are linked to Alzheimer’s disease these assemblies within the cell, or to couple self-assembly with critical and preeclampsia. We report that FVFLM peptides from SERPINA1 aggre- nonequilibrium events such as chemical reactions. Methods for studying gate more rapidly and form more mechanically stable oligomers than cell-scale dynamics, such as single-particle reaction-diffusion, are typically KLVFF peptides3,4. The extent of superior mechanical stability of FVFLM not applied to self-assembly, as they neglect the geometry of the species be- as compared to KLVFF was quantified by subjecting both to steered molec- ing simulated. We detail here generalized software for performing reaction- ular dynamics. Our studies provide insight into the relationship between diffusion simulations of self-assembly through recently developed algo- mechanical stability and oligomer formation times, the faster the aggrega- rithms that include interface resolution and rigid-body structure while tion rate of peptide the stronger has noted to be its mechanical stability. simplifying position updates of species, to accurately reproduce kinetics, These results open a new way for predicting fibril formation rates based and equilibrium, of association in an efficient manner. Each protein is rep- on mechanical stability which can be easily estimated by steered molecular resented as a set of center of mass and interface coordinates and are prop- dynamics simulations. agated individually to provide spatiotemporal resolution at both single- 1) I. A. Buhimschi et al., Science translational medicine 6 (245), 245ra292 particle and interface levels. Interface interactions are provided by the (2014) user in a simple, rule-based format along with predefined angles for rigid- 2) M. Kouza et al., Molecules 23 (8), 1995 (2018); M. Kouza et al., The Journal body association, enabling formation of multi-protein structures and defini- of Chemical Physics 146, 135101 (2017); M. Kouza, S. Gotham, M. Seel, U. tions of chemical reactions. This software represents a powerful tool for Hansmann, Phys Chem Chem Phys 12 (37), 11390-11397 (2010) studying nonequilibrium self-assembly at biologically-relevant timescales, 3) M. Kouza, A. Banerji, A. Kolinski, I. A. Buhimschi and A. Kloczkowski, which is demonstrated here through simulations of clathrin-coated structure Phys Chem Chem Phys 19 (4), 2990-2999 (2017) formation on membranes.

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Posters: Intrinsically Disordered Proteins (IDP) and cell death. IDPs are also involved in various diseases, such as cancer and Alzheimer’s disease. Therefore, it is important to understand the mech- and Aggregates I anisms of association of IDPs with their targets. Colicin E3 is a ribosomal RNase toxin released by Escherichia coli. Although the colicin E3 rRNase 975-Pos domain is folded, alanine mutant of Tyr507 within the hydrophobic core The Intrinsic Folding Mechanism Affects the Coupled Folding-Binding unfolded the protein. Interestingly, the unfolded E3 rRNaseY507A is still Process of Unfolded Proteins able to bind to Im3 and forms a complex identical to the wild-type protein. Meng Gao, Zhengding Su, Yongqi Huang. In this work, pre-steady-state kinetic analysis, thermodynamic analysis and Department of Biological Engineering, Hubei Univ Tech, Wuhan, China. F value analysis were performed to investigate the coupled folding-binding Dynamic cellular signaling processes proceed via specific and rapid binding/ mechanism between E3 rRNaseY507A and Im3, and the binding mechanism unbinding events which require a high association rate constant and a high of the E3 rRNaseY507A was compared with the WT folded protein. We found dissociation rate constant for rapid kinetics and at the same time a sufficient that the encounter process was steered by complementary electrostatic inter- high association constant for specificity. It has been found that intrinsically actions between the two molecules. However, the effect of electrostatic disordered proteins (IDPs) are extensively involved in cellular signaling pro- steering is much weaker for the unfolded E3 rRNaseY507A than that for cesses and possess kinetic advantages over their ordered counter parts. the WT E3 rRNase. F value analysis revealed that the E3 rRNaseY507A is Although the coupled folding-binding processes of IDPs have been extensively folded and forms many native intermolecular contacts in the transition state. studied, it remains impossible to predict whether an unfolded protein is suitable Our results provide clues to explain why introducing disorder affects the for molecular signaling via coupled folding-binding. In this work, we studied association rate but not the dissociation rate for the E3 rRNase/Im3 the interplay between intrinsic folding mechanism and the coupled folding- interaction. binding process during target binding for unfolded proteins through coarse- grained molecular dynamics simulations. We first compared the folding free 978-Pos energy landscapes of three representative IDPs with different folded structures. The Rational Discovery and Design of Disordered Protein Ligands Simulations suggest that the free energy landscapes of IDPs involved in David W. Baggett, Abhinav Nath. coupled folding-binding are downhill or with low energy barriers. To further Dept Medicinal Chem, Univ Washington, Seattle, WA, USA. study the influence of intrinsic folding mechanism on the coupled folding- Intrinsically disordered proteins (IDPs) are core components of many biolog- binding process, we tried to modulate the folding mechanism of an unfolded ical processes and are central players in several pathologies. Despite being protein and simulated the coupled folding-binding process. We used the important drug targets, attempts to design small molecule ligands that would barnase-barstar complex as a model and used circular permutation to modulate help understand and attenuate their behavior are frustrated by the structural di- the folding mechanism of barnase. Although barnase is not an IDP, it become versity exhibited by these flexible proteins. To accommodate the dynamic na- unfolded under denatured conditions, thus we could simulate the binding pro- ture of IDPs, we have developed a procedure that efficiently identifies active cess between unfolded barnase and folded barstar. We found that the intrinsic small-molecule ligands for disordered proteins. This method utilizes enhanced folding free energy barrier is a key factor affecting the coupled folding-binding sampling molecular dynamics and adapts conformational clustering algorithms kinetics, where reducing the folding free energy barrier enhances binding rate to identify persistent local structures. Ligands are then screened using a com- up to two orders of magnitude. Our results suggest that IDPs may be evolved to bination of computational docking and machine learning to identify promising minimize the folding free energy barrier to achieve high specificity and rapid lead compounds. binding/unbinding kinetics simultaneously. We tested our strategy by targeting the microtubule binding region of the disor- dered protein tau, and successfully identified novel tau ligands. One of these 976-Pos compounds alters the kinetics of the pathological aggregation involved in Recognition Mechanism of the Intrinsically Disordered dsRNA Binding neurodegenerative disease. By exploring the chemical space around this ligand, Domain DCL1-A with its Substrate from Molecular Dynamics Simulations we have been able to refine the effect on aggregation and identify factors crit- Yuwen Chen, Meng Gao, Yongqi Huang. ical for activity and affinity. Hubei University of Technology, Wuhan, China. With this method we identified novel ligands that expand our toolkit of protein Double stranded RNA (dsRNA) is involved in many biological processes and is aggregation inhibitors into new areas of chemical space. Notably, the discovery recognized by the dsRNA binding domain (dsRBD). DCL1 protein from Ara- of this new family of disordered protein ligands was achieved more quickly and bidopsis thaliana contains two dsRBDs chich are characterized by a conserved with less expense than conventional high-throughput screening or docking topology of alpha-beta-beta-beta-alpha. In contrast to other studied dsRBDs, alone would have allowed. the N-terminal dsRBD of DCL1 (DCL1-A) is intrinsically disordered in isola- tion and only folds into the canonical structure upon binding with dsRNA. In 979-Pos this work, we studied the conformation ensemble of free DCL1-A and the bind- Polymer Theory for Sequence-Specific Phase Separation Behaviors of ing mechanism of DCL1-A with dsRNA through extensive all-atom and Charged Intrinsically Disordered Proteins Yi-Hsuan Lin1, Julie D. Forman-Kay2, Hue Sun Chan1. coarse-grained molecular dynamics simulations. Through all-atom molecular 1 2 dynamics simulations, we found that the alpha-helical structure elements and Dept Biochem, Univ Toronto, Toronto, ON, Canada, Molecular Med, The the antiparallel beta-sheet were transiently populated in the free state of Hosp for Sick Children, Toronto, ON, Canada. DCL1-A. We also confirmed that the dsRNA binding competent conformations Liquid-liquid phase separation in biology underpins the formation of meso- was present in the conformation ensemble of free DCL1-A, suggesting that scopic, liquid-like assemblies of DNA, RNA, and proteins, termed ‘‘mem- DCL1-A recognizes its dsRNA substrate through a conformational selection braneless organelles’’ or ‘‘biomolecular condensates’’. Without a membrane, mechanism. To further understand the binding mechanism, we performed bind- these assemblies can respond to environmental stimuli rapidly and play a crit- ing simulations using the coarse-grained Go-model. After calibrating the pop- ical role in many cellular regulation processes. Intrinsically disordered proteins ulation of preformed structure elements and stability of the DCL1-A/dsRNA (IDPs), proteins that do not fold when isolation because of the depletion of hy- complex, our simulations revealed that the recognition process proceeded drophobicity and the abundance of polar, charged, and aromatic residues, have mainly through a conformational selection of partially preformed bound-like been observed to form membraneless organelles in vivo with other biomole- mechanism, although disordered conformations could also initiate the binding cules as well as undergo phase separation in vitro by themselves. The phase process. We further performed a flux calculation for the unbound-to-bound separation propensity of an IDP is determined by its amino acid sequence, transitions initiated by bound-like conformations and disordered conforma- not only the composition but also the sequence pattern of its amino acids. To tions, respectively. Our results suggest that the structure preference plays a crit- investigate this ‘‘sequence-specific’’ phenomenon, we develop a polymer phys- ical role in determining the coupled folding-binding mechanism of DCL1-A, ics theory, the ‘‘random-phase approximation (RPA)’’ theory, for the phase which be general for many IDPs. separation of charged IDPs. The theory predicts that the charge sequence pattern and the number of aromatic residues are critical for the phase separation 977-Pos propensity of IDP. We have applied this theory to the RNA IDP Ddx4 Mechanism of Coupled Folding of Disordered Colicin E3 rRNase Domain and its two mutants with scrambled charge sequence and mutations reducing its Upon Binding with Im3 aromaticity. Our predictions are consistent with experiments. We also applied Xingyu Chen, Yongqi Huang, Zhengding Su, Meng Gao. the theory to 30 model IDP sequences with the same positively and negatively Hubei University of Technology, Wuhan, China. charged residues but different sequence patterns, and discovered a connection Intrinsically disordered proteins (IDPs) are universally observed in all do- between the sequence-specific single-chain compactness and multi-chain mains of life. They play critial roles in many cellular processes, including phase separation propensity. Aqueous solutions with two IDP species with signal transduction, transcription and translation regulation, cell division, different charge sequences have also been addressed by our formulation,

BPJ 9355_9357 198a Monday, March 4, 2019 whereby a ‘‘fuzzy’’ molecular recognition mechanism supported by the simi- While this is true for the most part, recently this paradigm was put into question larity/dissimilarity of the two IDP sequences is indicated. Taken together, as more and more proteins are being discovered that seem to lack any well our RPA theory establishes a framework for systematically investigating and defined structure per se, but still were proved to play important roles in variety rationalizing sequence-specific phase separation behaviors of charged of important biological processes, such as cell regulation and signalling. Such biomolecules. proteins are called Intrinsically Disordered Proteins (IDP) if they are fully disordered or proteins with Intrinsically Disordered Regions (IDR) if only 980-Pos part of the protein lacks any structure. Despite large number of studies, until The Coupled Folding-Binding Mechanisms of Intrinsically Disordered now all the advantages of disordered state of proteins are still not fully under- Proteins with Different Folded Structures stood. In this work we study potential capability of IDRs to accelerate protein- Jing Yang, Meng Gao, Zhengding Su, Yongqi Huang. protein association. We hypothesize that protein-receptor pairs containing Hubei University of Technology, Wuhan, China. multiple independent binding sites might associate faster if binding sites are Many cellular functions, including signal transduction and gene regulation, are connected by IDR or are located on IDR. Using theoretical methods we study carried out by interactions between intrinsically disordered proteins (IDPs) and possible range of accelerations and their properties. Using molecular dynamics their biological targets. Although the coupled folding-binding processes of we measured possible binding acceleration for ERK2-EtsD138 system and find IDPs have been extensively studied, previous works mainly focused on IDPs that the addition of IDR-located binding site might increase acceleration rate by folding into simple alpha-helical structures, it is not clear how folding of a factor of 3 to 4. beta-strands and more complex structures are coupled with target binding. In this work, we studied the coupled folding-binding process of three IDPs with 983-Pos different folded structures, i.e., c-Myb, AF9, and E3 rRNase Y507A mutant us- Biophysical Characterization of the Translational Isoforms of the Human ing topology-based coarse-grained molecular dynamics simulations. Although Glucocorticoid Receptor the three IDPs initiated the binding process via unfolded conformations, the Emily M. Grasso1, Ananya Majumdar2, Dominique P. Frueh3, coupling mechanism between folding and binding was different. C-Myb folded Vincent J. Hilser4. into an alpha-helix upon binding with KIX and the folding free energy land- 1Dept Biophysics, Johns Hopkins Univ, Baltimore, MD, USA, 2Biomolecular scape was downhill. When the binding free energy barrier was crossed, folding NMR Facility, Johns Hopkins Univ, Baltimore, MD, USA, 3Dept Biophys/ of c-Myb processed synchronously with binding. AF9 folded into an alpha-beta Bioph, Johns Hopkins Sch Med, Baltimore, MD, USA, 4Dept Biology, Johns structure upon binding with AF4. However, AF9 remained largely unfolded Hopkins Univ, Baltimore, MD, USA. even when 70% of intermolecular contacts were formed. Further binding Structural studies have driven the analysis of the biological function of pro- induced folding and packing of the N- and C-terminal helices of AF9. In teins for much of the era of modern biology. It has become increasingly contrast to AF9, E3 rRNase folded into native-like all-beta conformations obvious, however, that a more nuanced approach to the study of proteins upon encountering Im3 and further binding proceeded subsequently. Our that addresses the roles of intrinsic disorder, allosteric action, and excited state results suggest that the coupled folding-binding mechanisms of IDPs may conformations in effecting biological processes is required to truly understand vary significantly depending on their folded structures and intermolecular biological activity. The human glucocorticoid receptor (GR) is one such interactions. biological macromolecule for which these phenomena are of critical impor- tance. Eight translational isoforms of GR exist and involve truncations of 981-Pos the intrinsically disordered N-terminal domain (NTD) of the protein. Much Characterizing Time-of-Day Conformational Changes in the IDP Fre- is still unknown about the role the disordered NTD plays in modulating quency at the Heart of the Circadian Clock in N. Crassa using the Crafty GR activity. Additional detail regarding residual structural features of the Protocol NTD and its interactions with other domains of the protein would greatly Jacqueline Pelham, Alexander E. Mosier, Jennifer M. Hurley. benefit our understanding of this protein. NMR spectroscopy has emerged Biology, Rensselaer Polytechnic Institue, Troy, NY, USA. as a powerful tool for the study of structure, disorder and dynamics in proteins Continuous oscillation of environmental conditions has led to the ubiquitous at atomic-level detail. We use NMR spectroscopy and fluorescence anisotropy evolution of anticipatory mechanisms known as circadian rhythms. From the to study GR and its binding to DNA, focusing first on the N-terminal and cellular to the organism level, these rhythms coordinate a plethora of physio- DNA-binding domains (DBD) of the protein. Slight, though significant, chem- logical and behavioral responses. They provide organisms with elevated sur- ical shift perturbations exist in the NMR spectra of different NTD-DBD iso- vival fitness, evident by the increased survival and reproductive rates of forms, indicating that NTD truncations have some effect on the structural organisms maintaining clocks vs. those without a mechanism to time daily features of the protein in the absence of DNA. DNA binding affinities rhythms. Circadian rhythms are maintained by a conserved molecular clock measured by fluorescence anisotropy indicate that each NTD-DBD isoform orchestrated by a transcriptional/translational negative feedback loop differs in binding affinity for DNA, with all isoforms showing significantly (TTFL). Many of the proteins that organize this feedback loop are Intrinsically stronger binding than the isolated DBD. NMR spectra reveal critical differ- Disordered Proteins (IDPs), which lack a fixed or ordered three-dimensional ences in the local chemical environments of different NTD-DBD constructs structure. This includes the conserved disorder of the clock proteins in fungi, in the presence of DNA. We are pursuing further NMR experiments to eluci- plants, and . Little is known about the impact of intrinsic disorder on date the structural and dynamical underpinnings of these differences in bind- clock proteins and this lack of comprehension is compounded by the fact ing affinities. that efficient techniques to understand the inherent nature of IDPs are only now developing. To address this gap in knowledge we developed a novel 984-Pos protocol to track the conformational dynamics of a core clock protein Coarse-Grained Simulations of Disordered Proteins: Effect of Interaction (FREQUENCY) in a fundamental clock (Neurospora crassa). Potentials and Charge Pattern Parameters FREQUENCY (FRQ) is at the heart of the repressing arm of the TTFL and Suman Das. drives the length of the circadian period in N. crassa. Our protocol, circadian Biochemistry, University of Toronto, Toronto, ON, Canada. native fast parallel proteolysis (CRAFTY), utilizes a parallel proteolysis Recent experimental studies have revealed that membraneless organelles are method in native conditions to determine the conformational shifts in FRQ multi-component viscous liquid droplets, assembled through phase separations over circadian time. CRAFTY yields biologically relevant information, of proteins and RNAs. These organelles are involved in many crucial biological enhancing our understanding of the importance of disorder in the circadian functions. Understanding their formation and genetic basis is challenging due clock. to the intrinsic complexity of cellular environment and heteropolymeric nature of the biomolecules. Extensive Langevin dynamic simulations have been per- 982-Pos formed with coarse-grain model using three different interaction potentials to Study of Potential Kinetic Advantages of Intrinsically Disordered Regions understand how phase separation propensity depends on the charge pattern of for Protein Association the amino acid sequences. Charge patterns are characterised by two well- Mikita Misiura, Anatoly B. Kolomeisky. known parameters, namely the ‘‘blockiness measure’’ k and the ‘‘sequence Chemistry, Rice University, Houston, TX, USA. charge decoration’’ (SCD) parameter. Our new simulations indicate that Protein-protein interactions are critical for the most biological processes inside liquid-liquid phase separation propensity for a previously-studied set of se- living organisms and are being actively studied. Until recently it was thought quences correlates positively with k and also with –SCD. Moreover, quantita- that in order to function properly proteins need to have a rigid, well defined tive analysis suggests that overall phase separation propensity is enhanced 3D structure and that if a protein fails to fold, it will be unable to function. by background residue-residue attraction. However, for a novel set of

BPJ 9355_9357 Monday, March 4, 2019 199a sequences we designed to exhibit an anti-correlation between k and -SCD, the 987-Pos simulated ’s are quite insensitive to k or SCD. Additionally, we find that the Theoretical Saxs Signatures of Conformational Heterogeneity and Homo- phase separation propensity of a strictly alternating charge sequence crucially geneity of Disordered Protein Ensembles depend on spatial alignments that are difficult to achieve in real space. In gen- Jianhui Song1, Hue Sun Chan2. eral, our results reveal both the utility and limitations of analytical theory as 1School of Polymer Science and Engineering, Qingdao University of Science well as the charge pattern parameters, and point to several fruitful future direc- and Technology, Qingdao, Shandong, China, 2Department of Biochemistry tions in the development of theory and simulation for the phase behaviors of and Department of , University of Toronto, Toronto, ON, disordered proteins. Canada. The biological function of an intrinsically disordered protein (IDP) is based upon 985-Pos its conformational ensemble which is in turn governed by the IDP’s amino acid Modeling the Effects of Ligand Binding on the Phase Behavior of sequence. In view of the different functions served by different IDPs, this Aggregation-Prone Proteins ensemble-function view reinforces the physical expectation that the disordered Kiersten M. Ruff, Ammon E. Posey, Rohit V. Pappu. chains of non-homopolymeric amino acid sequences—IDPs and unfolded chains Department of Biomedical Engineering, Washington Univ St Louis, Saint of globular proteins—are heterogeneous, i.e., their ensemble distributions of con- Louis, MO, USA. formations are different from those of homopolymers that habor a uniform intra- Many diseases are associated with aberrant protein aggregation. Recently, chain interaction. Indeed, recent analyses indicate that conformational there has been resurgent interest in co-opting the framework of phase tran- heterogeneityis key toreconciling the apparent inconsistency between IDP dimen- sitions to understand the distinct concentration thresholds separating sions determined by SAXS and FRET. This development notwithstanding, it was different phases of aggregation-prone molecules. This analogy, which was stated recently that SAXS data on disordered proteins can be adequately accounted purely conceptual, has now been shown to have quantitative significance. for by homopolymer chain models. It is important, therefore, to address the infor- Importantly, instead of searching for elusive ‘‘toxic conformations’’, dealing mation content of SAXS data, to assess to what extent SAXS data can discriminate with the deleterious consequences of protein aggregation and phase separa- between heterogeneous and homogeneous conformational ensembles. We have tion can be approached via methods that alter or modulate the sequence- conducted extensive simulations of subensembles of chain conformations with specific phase behavior. One route to accomplishing this is by shifting phase narrow ranges of radius of gyration Rg and end-to-end distance REE, and compared boundaries via ligand binding through a phenomenon known as polyphasic the molecular form factors (MFFs) and dimensionless Kratky plots of these highly linkage. Disease-causing mutations, such as polyglutamine expansions in heterogeneous (sub)ensembles with those computed for homogeneous ensembles. the N-terminal region of huntingtin, lead to aggregation and phase separation We find that the MFFs of heterogeneous and homogeneous ensembles are quite at lower, physiologically relevant concentration thresholds. Ligand binding similar when the chain conformations are relatively open, suggesting that SAXS can shift these phase boundaries based on the phase that is preferentially may not be capable of clearly distinguishing heterogeneous from homogeneous bound. Here, we show that a ligand can reduce the driving force for protein conformational ensembles of well-solvated or slightly compact chains. In contrast, aggregation in vitro and may help to explain the reduced cellular aggregation MFFs for more compact ensembles are more sensitive to conformational hetero- and toxicity observed when overexpressed in the context of Huntington’s geneity. We have also developed an analytical theory for sequence-dependent disease. Through a combined biophysical and computational approach, we MFFs. The theory provides insight into how different conformational ensembles further show that multi-valency in binding appears to be important for shift- that share the same average REE can have different average Rg’s. Ramifications ing phase boundaries in therapeutically relevant directions. However, in the of our findings for the interpretation of experimental data are discussed. context of the cell, a protein such as huntingtin has a large interactome. Each 988-Pos of the interaction partners will have a different effect on the intrinsic phase Investigating the Role of Charge-Altering Post-Translational Modifica- boundaries and therefore, designing a therapeutic approach that leverages the tions on Tau Peptide Conformational Ensembles using Polarizable Molec- tenets of polyphasic linkage requires a general framework that considers the ular Dynamics Simulations competing effects within a network of interactions. Using the huntingtin Darcy S. Davidson, Justin A. Lemkul. example, we develop a framework for designing modulators of phase Biochemistry, Virginia Tech, Blacksburg, VA, USA. behavior via polyphasic linkage that accounts for competing interactions Aggregation of amyloidogenic proteins, such as the amyloid b-peptide, within an interactome. microtubule-binding protein tau, and a-synuclein, is linked to a number of human diseases. Amyloidogenic proteins unfold, aggregate, and self-assemble into 986-Pos highly stable, insoluble fibrils characterized by cross-b structure. Despite having Redox of Cysteines and Protein Folding of Snap-25 unique primary sequences, these amyloidogenic proteins all undergo similar 1 2 2 1 Aidan H. Mourik , Matt Pettit , Robert E. Coffman , Graham M. Pingree , conformational changes, from disordered and only partially structured, to highly 2 1 Chandler B. McSpadden , Dixon J. Woodbury . ordered in the fibril form. The forces driving such changes in structure are not 1Physiology & Dev Biol, Brigham Young University, Provo, UT, USA, 2 completely characterized and a better understanding is needed to potentially Neuroscience Center, Brigham Young University, Provo, UT, USA. combat a wide range of diseases that are currently incurable. In this study, we per- The SNARE proteins SNAP-25, syntaxin, and VAMP play critical roles in formed molecular dynamics simulations with the Drude polarizable protein force neuronal exocytosis by providing the four helical regions (SNARE domains) field to gain a better understanding of protein aggregation. We previously found that form a coiled-coil complex required for neurotransmitter release. This that the explicit representation of electronic polarization was required to delin- complex is continually formed and unwound as exocytotic vesicles fuse eate mutation-specific unfolding pathways of the amyloid b-peptide. Building and are recycled. Using multiple techniques, we observe clear structural upon those findings, here we explore the impact of charge-altering post-transla- changes in SNAP-25B due to temperature, pH, ionic strength, methanol, tional modifications (PTMs) on the dynamics of tau, the protein that forms intra- and oxidation state. Here we focus on redox state and structural changes cellular, neurofibrillary tangles in Alzheimer’s disease. PTMs like acetylation of due to temperature and methanol. Using Circular Dichroism to measure lysine and phosphorylation of serine and threonine are known to induce tau ag- the secondary structure of SNAP-25B in low ionic strength solutions, we gregation, but the underlying molecular mechanism remains unknown. By study- observe an increase in alpha-helical structure when methanol is added, or ing fragments of tau harboring these PTMs, we sought to gain novel insights into the temperature is lowered, similar to the helical shift observed when how changes in charge caused by PTMs shift the conformational ensembles of SNAP-25 forms a complex with syntaxin and VAMP. Methanol (30%) or fragments of the tau protein. Polarizable simulations of these charge-altering heating (34C) also breaks up large aggregates of SNAP-25B, as observed PTMs shed light on how electrostatic interactions, including those between per- by decreased opacity in the CD signal and a loss of high molecular weight manent and induced dipoles, affect side-chain properties, salt bridges, water and bands in non-denaturing gels. The four cysteines in SNAP-25B are thought ion interactions, and changes in secondary structure. to be palmitoylated in vivo and can be modified by addition of iodoacetic acid (IAA). Using non-denaturing gels, we observe a shift of mobility of 989-Pos SNAP-25B depending on the redox potential of the reaction solution. We Protein Unfolded States are Characterized by the Duality of Sequence- þ previously showed that these cysteines are oxidized by micromolar Cu2 Specific Conformational Preferences and Ensemble-Averaged Features by measuring the number of cysteines accessible for labeling with a of Canonical Random Coils Biotin-maleimide tag technique developed in our lab (Woodbury et al., Alex S. Holehouse1, Ivan Peran2, Natalie E. Stenzoski3, Junjie Zou3, 2011. Anal. Biochem. 417:165-173). These data support the hypothesis Andrea Piserchio4, Ranajeet Ghose4, Isaac S. Carrico3, Osman Bilsel5, that SNAP-25B structure and cysteine accessibility are sensitive to environ- Daniel P. Raleigh3, Rohit V. Pappu1. mental changes, which may modulate SNAP-25 palmitoylation and neurose- 1Biomedical Engineering, Washington University in St. Louis, St. Louis, cretion in vivo. MO, USA, 2Department of Structural Biology, St. Jude Children’s Research

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Hospital, Memphis, TN, USA, 3Dept Chemistry, Stony Brook Univ, Port structural information from vibrational spectra, we used molecular dynamics Jefferson, NY, USA, 4Department of Chemistry and Biochemistry, The City simulations to simulate dynamic proteins with probe groups, which can then be College of New York, New York, NY, USA, 5Dept Biochem Mol Pharm, used to calculate infrared absorption lineshapes. By comparing simulated and UMass Medical Sch, Worcester, MA, USA. experimental lineshapes, an experimentally validated structural ensemble can A consensus regarding rigorous, quantitative descriptions of unfolded states of be generated. This approach is was first tested on Calmodulin, which has a rela- autonomously foldable proteins under folding conditions has remained elusive tively narrow structural ensemble according to simulations from several groups. despite its fundamental importance for a variety of processes in vivo. Here, we To use this approach to study intrinsically disordered proteins, the proteins combine time-resolved Fo¨rster Resonance Energy Transfer (FRET) using multi- NTAIL and XD from Nipah virus were chosen because their interaction is crucial ple non-perturbing, amino-acid-sized dye pairs, paramagnetic relaxation enhance- to viral replication and NTAIL is highly disordered. The goal of this project is to ment (PRE) experiments with multiple labels, equilibrium and time-resolved generate a structural ensemble for the NTAIL-XD complex by using computa- small angle X-ray scattering (SAXS), extensive all-atom simulations, and poly- tional simulations to interpret previously completed spectroscopic experiments mer theory to construct a rigorous, atomistic descriptions for unfolded states of on these proteins. Docking algorithms and replica exchange molecular dynamics different proteins under their respective folding conditions. Our descriptions sum- were used to sample many possible structures for the NTAIL-XD complex, which marize sequence-specific contact distributions while provide a framework for were then separated into clusters based on a set of geometrical classifiers for the quantifying the amplitudes of conformational fluctuations and correlations among complex. Vibrational probes were inserted into various sites of representative these fluctuations. The picture that emerges suggests that unfolded states are con- structures from each cluster; then on these structures, molecular dynamics simu- formationally heterogeneous, and this heterogeneity is sequence-specific both in lations and IR lineshape calculations were performed. Results and conclusions terms of the biases for native as well as non-native contacts. Despite significant from both Calmodulin and the NTAIL-XD system will be discussed. conformational heterogeneity, quantifiable biases toward the sequence-specific 992-Pos folded state topologies result from conformational fluctuations in unfolded states. Measures Adapted from Information Theory and Energy Landscape Importantly, we find that converging on a coherent picture for heterogeneous en- Theory for Quantifying Sequence-To-Conformation Relationships of sembles such as unfolded states under folding conditions requires a combination Intrinsically Disordered Regions of readouts from multiple type of experiments that are then integrated with theory Megan Cohan, Alex S. Holehouse, Rohit V. Pappu. and simulation. These methods are also directly applicable to studies of sequence- Dept Biomedical Engineering, Washington Univ in St. Louis, St. Louis, to-conformation relationships of intrinsically disordered proteins. MO, USA. 990-Pos The biological relevance of intrinsically disordered regions (IDRs) is coming A High Throughput Method for Exploring the Sequence Space of Polypep- into sharp focus through a combination of methods that connect sequence- tides That Exhibit Thermoresponsive Phase Behavior encoded information to conformational ensembles and the functions / pheno- Xiangze Zeng1, Martin J. Fossat1, Nicholas Tang2, Ashutosh Chilkoti2, types they support. Many IDRs show evidence for convergent evolution. Rohit V. Pappu1. This implies that a set of IDRs make up a sequence family that support a spe- 1Department of Biomedical Engineering, Washington University in St. Louis, cific set of functions. Examples of this include the disordered C-terminal tails St. Louis, MO, USA, 2Department of Biomedical Engineering, Duke (CTTs) of FtsZ, the bacterial protein that is involved in the formation of con- University, Durham, NC, USA. tractile rings. Across roughly 103 orthologs, the sequences of the folded Intrinsically disordered protein polymers (IDPPs) are built up of repeating units GTPase domain are highly conserved whereas the sequences of the CTTs are and they can demonstrate two types of thermoresponsive phase behavior. Sys- hypervariable. And yet, the CTTs are essential for regulation of GTPase activ- tems characterized by lower critical solution temperatures (LCST) undergo ity, for FtsZ polymerization, for Z-ring formation, and for growth in rod shaped phase separation above the LCST whereas systems characterized by upper crit- bacteria. Clearly, the information encoded in the sequences of IDRs is germane ical solution temperatures (UCST) undergo phase separation below the UCST. for function and the control of cellular phenotypes, but the relevant information The amino acid composition and sequence of the repeat as well as the number remains refractory for extraction by naı¨ve sequence comparisons. We pursue of repeats will determine the thermoresponsive phase behavior of IDPPs. There the ansatz that the encoding of information that yields sequence families de- is congruence between thermoresponsive coil-to-globule transitions and phase rives from the totality of pairwise sequence-to-conformation, sequence-to- behavior. The theta temperature above or below which the IDPPs transition function, conformation-to-function, and triplet relationships. To quantify the from coils to globules serve as useful proxies for the LCST/UCST values. This influence of sequence-to-conformation relationships as determinants of IDR implies that one can design sequences with desired values for the theta temper- sequence families, we have developed a series of information theoretic mea- ature and either increasing or decreasing radii of gyration (Rg) as a function of sures that enable comparative assessments of conformational distributions. increasing temperature (T). However, the vastness of sequence space makes it We have developed additional measures based on energy landscape theories impossible to combine conformational sampling with sequence design algo- to quantify sequence-specific breadths, depths, and ruggedness of free energy rithms. We recently discovered that the Monte Carlo simulations performed in landscapes. Together, the two sets of measures allow us to understand informa- the intrinsic solvation (IS) limit version of the temperature-dependent ABSINTH tion encoding within IDRs that give rise to sequence families. Our analyses model, a limit that includes all terms excepting the descreened electrostatic inter- highlight how and why key determinants of convergent evolution become actions, affords highly efficient estimates of the temperature-dependence of the masked by the hyper-variability of IDR sequences and how these can be un- sequence-specific free energies of solvation. For sequences that demonstrate masked by consideration of sequence-to-conformational relationships. LCST behavior, the Rg vs. T plots have negative slopes whereas sequences 993-Pos with UCST behavior have Rg vs. T plots with near zero or positive slopes. Taking Characteristics of the Binding Interaction between Pdx1 and SPOP advantage of the efficiency of IS limit simulations and the robust classifications Grace A. Usher1, Roman Rohac2, Amie K. Boal1,2, Scott A. Showalter1,2. they afford, we have developed a high-throughput sequence design engine based 1Biochemistry and Molecular Biology, Penn State University, University on evolutionary algorithms to design novel sequences with LCST vs. UCST Park, PA, USA, 2Chemistry, Penn State University, University Park, PA, phase behavior. We are closing the loop with experiments to expand the libraries USA. of known thermoresponsive IDPPs for biomedical applications. Pancreatic and duodenal homeobox 1 (Pdx1) is a homeodomain transcription 991-Pos factor required for pancreatic development and activation of insulin expression Computational Simulations of the NTAIL-XD Complex from the Nipah in response to increased blood- levels. In addition to a DNA binding Virus for Constructing Experimentally-Validated Structural Ensemble domain, Pdx1 contains two intrinsically disordered regions (IDRs) that mediate ChuHui Fu1, Casey H. Londergan2, Rosalind J. Xu3. protein-protein interactions to regulate transcription factor activity. In partic- 1Haverford College, Haverford, PA, USA, 2Dept Chemistry, Haverford Coll, ular, speckle-type POZ protein (SPOP) binds to the disordered Pdx1 C-termi- Haverford, PA, USA, 3Chemistry and Chemical Biology, Harvard University, nus (Pdx1C) under low glucose conditions to facilitate ubiquitin-mediated Cambridge, MA, USA. degradation of Pdx1. Additionally, SPOP binds other disordered substrates Understanding the ensemble of the intrinsically disordered proteins’ structures, and is implicated in certain adenocarcinomas. Pulldown experiments identified rather than any one structure, is the most important thing for understanding these a short linear motif (SLiM) within Pdx1C that interacts with SPOP; deletion of proteins’ behaviors. Site-specific vibrational spectroscopy is a technique with a this region prohibited binding of SPOP to Pdx1. Using 13C direct-detect NMR fast timescale that can in principle capture dynamical information (and thus the experiments optimized for analysis of intrinsically disordered proteins (IDPs), entire ensemble) in its spectra. The technique involves using vibrational probe we identified a second SLiM within Pdx1C for SPOP interaction. I hypothesize groups that are inserted into proteins and which report on their local environ- that a single SLiM within Pdx1C is necessary, but not sufficient, to mediate a ments. The challenge is that spectra from vibrational probe groups are often over- functional interaction with SPOP. I probed the binding interactions between determined and hard to interpret quantitatively. To extract quantitative and SPOP and Pdx1C using structural biology and thermodynamic assays.

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Co-crystal structures of SPOP and Pdx1 peptides showed that Pdx1 occupies a California, San Francisco, San Francisco, CA, USA, 3Department of Cell and canonical groove on SPOP, despite deviations in the Pdx1 sequence from other Molecular Biology, University of Hawaii, Honolulu, HI, USA. SPOP binding partners. Multivalency may also play a role in the binding affin- Selenoprotein K (SELENOK) is an intrinsically disordered membrane protein ity and, thus, regulation of Pdx1 stability by SPOP. Fluorescence titration ex- that assists palmitoylation of proteins using its selenocysteine at position 92 periments showed that, absent the other, the SLiMs within Pdx1C bind to (Sec92) and also contributes to the degradation of misfolded proteins in SPOP with near millimolar dissociation constants; however, in the context of response to cellular stress. While only a small group of intrinsically disordered the full Pdx1 C-terminus, binding affinity increases dramatically. We demon- proteins are enzymes, the presence of Sec suggests that SELENOK may func- strate that, in cells, both SPOP binding sites within Pdx1 must be present to tion as an enzyme.We discovered that SELENOK undergoes autoproteolysis, allow proteins to co-localize into nuclear speckles. An enhanced understanding releasing its C terminal disordered fragment that contains Sec92, the first of SPOP binding mechanisms with various substrates will yield valuable such observation for a selenoprotein. We demonstrate that SELENOK has mul- insight into the roles of SPOP and Pdx1 in etiologies of disease. tiple cleavage sites located around its Src homology 3 (SH3) binding segment. The cleavage shifts to adjacent sites when its conformation is altered by muta- 994-Pos tions and the use of different detergents. Mutation of potential catalytic residues Dynamic Interactions between an Intrinsically Disordered Protein and its such as Ser, His, Asp and Thr to Ala led to slower cleavage rates. The presence Binding Partners Probed by Multiparameter Single-Molecule Fluores- of Sec92 accelerated autoproteolysis but, interestingly, cleavage was retained in cence the absence of this amino acid. This indicates that Sec92 is not the nucleophile Taehyung Chris Lee1,2, Gregory-Neal Gomes1,2, for cleavage but rather might be influencing the protein conformation. We sug- John Darvy M. Castroverde1,2, Claudiu C. Gradinaru1,2. gest that SELENOK autoproteolyis regulates its association with different pro- 1Chemical and Physical Sciences, University of Toronto Mississauga, tein complexes in response to ER stress and other cellular signals. Mississauga, ON, Canada, 2Physics, University of Toronto, Toronto, ON, Canada. 997-Pos The intrinsically disordered protein (IDP) Sic1 is a cell cycle regulator in S. cer- Local Chain Dynamics of Intrinsically Disordered Sic1 Protein Inferred evisiae. It functions by inhibiting cyclin dependent kinases (CDKs), and is sub- from Fluorescence Anisotropy Decay Measurements 1,2 1,2 jected to degradation by ubiquitination. The degradation pathway is tightly John Darvy M. Castroverde , Gregory-Neal W. Gomes , 1 3,4 1,2 controlled by phosphorylation of Sic1, where phosphorylation of 6 or more Taehyung C. Lee , Julie Forman-Kay , Claudiu C. Gradinaru . 1Chemical and Physical Sciences, University of Toronto Mississauga, among its 9 phosphorylation sites dramatically increases its binding affinity 2 to SCF complex, an ubiquitin ligase in eukaryotes. Such modulation of binding Mississauga, ON, Canada, Physics, University of Toronto, Toronto, ON, Canada, 3Molecular Structure and Function Program, Hospital for Sick affinity has been topic of interest as Sic1 remains disordered regardless of phos- 4 phorylation state. Here, we investigated this dynamic interaction between Sic1 Children, Toronto, ON, Canada, Biochemistry, University of Toronto, and its binding partners, CDK and WD40 domain of SCF complex, using multi- Toronto, ON, Canada. parameter single-molecule FRET measurements. To investigate the structural As part of the cell cycle of the budding yeast Saccharomyces cerevisiae, the dynamics of Sic1 in multiple angles, we developed and applied a novel chem- cyclin-dependent kinase inhibitor Sic1 is an intrinsically disordered protein ical fluorophore labeling method for the C-terminus of protein, orthogonal to (IDP) that is degraded by the Cell division control protein 4 (Cdc4) substrate the commonly used thiol-maleimide reaction. Our results were interpreted in of the SCF ubiquitin ligase complex upon multi-site phosphorylation. More combination with the previous structural and computational studies of Sic1. specifically, the binding affinity of Sic1 to the WD40 binding domain of This study provides new insights towards a physical mechanism for the Cdc4 increases nonlinearly with the number of phosphate groups added to ‘‘fuzzy’’ interaction between this IDP and its binding partners, while pioneer- the Sic1 sequence. Previous studies have shown that the overall compaction ing a widely applicable novel protein labeling method. of Sic1 is not altered significantly upon phosphorylation and/or binding. In fact, the Sic1 protein remains disordered upon binding and it is thought to 995-Pos form a ‘‘fuzzy’’ complex with Cdc4. The Importance of Sequence Order Versus Composition in the Cryopro- The internal chain dynamics of the disordered region of Sic1 were examined by tective Function of an Intrinsically Disordered Protein fluorescence anisotropy decay (FAD) measurements using site-specific label- Steffen P. Graether1, Sharall Palmer1, Ray De Villa1, Andrew Harris2, ling with Alexa Fluor-488 at six different positions along the chain. Anisotropy 2 decays for each labelled construct were fit with two rotational correlation times Leonid S. Brown . 1Department of Molecular and Cellular Biology, University of Guelph, ( 0.3 ns and 2 ns), much faster than the tumbling rate of the whole protein. Guelph, ON, Canada, 2Department of Physics, University of Guelph, Guelph, Conversely, these data provide a map of the dye/linker rotational freedom and ON, Canada. the intrinsic backbone torsional mobility of separate regions in the Sic1 Dehydrins (dehydration proteins) are a class of intrinsically disordered proteins sequence. Differential quenching as measured by the fluorescence lifetime that are able to protect plants from damage caused by drought, cold and salt stress. points to local/distant contacts between some of the labelling sites and the Dehydrin sequences are modular, consisting of three conserved motifs separated only aromatic residue in Sic1 (Y14). Additionally, the backbone segmental dy- by large, poorly conserved regions. To examine the importance of sequence order namics are due to dihedral angle fluctuations, and they were compared with a compared to sequence composition, we created several dehydrin constructs where Ramachandran plot obtained from the (p)Sic1 conformations available in the the sequences were scrambled and where key charged residues were mutated to ENSEMBLE database. Going forward, chain dynamics data obtained from their opposite charge or neutralized. One of the proposed roles of dehydrins is fluorescence anisotropy decays can be incorporated as an additional (local) to protect protein denaturation during cold stress. We examine the effect of the constraint in ENSEMBLE to complement existing constraints from NMR dehydrin constructs on protecting (LDH), an enzyme (local) and SAXS (global). that is susceptible to aggregation during freezing, and protecting yeast frataxin 998-Pos homolog-1 (Yfh1), a protein that is susceptible to secondary structure loss during Exploring the Structural Properties of Synaptotagmin’s Intrinsically freezing. The results show that charge is important for protecting from damage Disordered Region caused by aggregation, while disorder is important when protecting from damage Michael E. Fealey1, Anne Hinderliter2, David D. Thomas1. caused by secondary structure loss. We also examined the secondary structure of 1Univ Minnesota, Minneapolis, MN, USA, 2University of Minnesota Duluth, the dehydrin constructs both in the solution state (using circular dichroism) and in Duluth, MN, USA. the dry state (using infrared spectroscopy). Most of the constructs remained fully We used time-resolved FRET, circular dichroism, and all-atom simulation to disordered in solution, though scrambling one construct resulted in less disorder, investigate the structural impact of phosphorylation and dielectric constant despite online prediction tools saying otherwise, while drying the constructs re- on synaptotagmin 1’s intrinsically disordered region (IDR). We found that sulted in a diversity of secondary structure content. Our results suggest that the full-length IDR sequence, a 60 residue strong polyampholyte that when even the poorly conserved regions are under some selective pressure. studied in the form of a peptide, undergoes structural collapse consistent with its k-predicted behavior. Furthermore, we found that the exocytosis- 996-Pos modulating phosphorylation of Thr112, a residue located in the IDR’s more A Membrane-Bound Selenoprotein Regulates its Activity by Autopro- sequence diverse central core region, disrupts a local disorder-to-order transi- teolysis tion that occurs when solution dielectric constant is lowered and helical struc- Rujin Cheng1, Marina Grossi1, Jun Liu2, Peter R. Hoffmann3, ture is stabilized by addition of trifluoroethanol. Implicit solvent simulations Sharon Rozovsky1. testing the impact of dielectric constant alone converge on a similar result, 1Department of Chemistry and Biochemistry, University of Delaware, showing that helical structure is formed with higher probability at a reduced Newark, DE, USA, 2Department of Pharmaceutical Chemistry, University of dielectric, where several lysine-aspartic acid salt bridges stabilize transient

BPJ 9355_9357 202a Monday, March 4, 2019 secondary structure. Phosphorylation, however, results in formation of salt solution and solid-state NMR. 2D 1H-13C INEPT and PRE experiments suggest bridges unsuitable for helix formation. These results suggest a model where that the N-terminal IDR of ChiZ remains dynamic in the presence of liposomes, compaction of the IDR sequence and phosphorylation may regulate structural and interactions with the lipid membrane are mediated by arginine residues. To transitions that in turn modulate neuronal exocytosis. help interpret these data with atomistic details, we have carried out multiple molecular dynamics (MD) simulations in water using several force fields 999-Pos tailored for disordered proteins. The best agreement with SAXS, chemical shift, Intrinsically Disordered HAX-1 Regulates Phospholamban in Membranes and NMR relaxation data was obtained by AMBER14SB/Tip4pD, followed 1 1 1 2 Erik K. Larsen , Daniel Weber , Songlin Wang , Seth Robia , closely by AMBER03ws/Tip4p2005s. In the MD simulations, the IDR tran- 3 1 Evangelia Kranias , Gianluigi Veglia . siently populated helices, especially in the N-terminal half, including residues 1Chemistry, Univ Minnesota, Minneapolis, MN, USA, 2Physiology, Loyola 3 11-15, 19-22, and 28-32. This result explains the lower amide proton exchange Univ, Maywood, IL, USA, Department of Pharmacology & Cell Biophysics, rates in the N-terminal half of the IDR observed by solution NMR. Simulations University of Cincinnati, Cincinnati, OH, USA. in the presence of a lipid bilayer, in line with the solid-state NMR data, showed Hematopoietic-subtrate-1 associated protein X-1 (HAX-1) is a 279 aa protein multiple arginine residues interacting with the membrane, where any one argi- expressed ubiquitously. In the cardiac muscle, HAX-1 was found to inhibit nine may engage 2 or 3 lipid headgroups at once. Ongoing work aims to deter- the sarco-endoplasmic reticulum calcium ATPase (SERCA) by shifting the 2þ mine the dominant conformations of the full-length ChiZ in the membrane apparent Ca affinity (pCa). Co-immunoprecipitation assays suggest that environment. HAX-1 binds to phospholamban (PLN), enhancing its inhibitory action. HAX-1 function is reversed upon PLN phosphorylation by cAMP-dependent 1002-Pos protein kinase A. Using a combination of NMR, CD spectroscopy, and Extracting Sequence-Dependent Intra-Protein Interaction Parameters SAXS, we found that HAX-1 is intrinsically disordered in the absence of a from Photo-Induced Electron Transfer Measurements of IDPs binding partner, and adopts secondary structure upon interaction with mem- Felicia Gibson1,2, Andrea Soranno3, Wenwei Zheng4, Sara M. Vaiana1,2. brane mimetics. Interaction with the lipid membranes was confirmed via 1Dept Physics, Arizona State Univ, Tempe, AZ, USA, 2Center for Biological Isothermal Titration Calorimetry measurements. Finally, preliminary solid- Physics, Arizona State University, Tempe, AZ, USA, 3Department of state NMR experiments show that HAX-1 interacts with the cytoplasmic region Biochemistry and Molecular Biophysics, Washington Univ. in St. Louis, St. of PLN in the presence and absence of SERCA. We propose that HAX-1 reg- Louis, WA, USA, 4College of Integrative Sciences and Arts, Arizona State ulates the SERCA/PLN complex in a Ca2þ-dependent manner that may be Univ, Mesa, AZ, USA. modulated through its interaction with the lipid membrane. This would add A fundamental understanding of structure/dynamics and function of intrinsi- another layer of control in Ca2þ homeostatic balance in the heart muscle. cally disordered proteins (IDPs), analogous to that reached for protein folding, requires experimental data that can be directly linked to physics based models. 1000-Pos Photo-induced electron transfer (PET) measurements between tryptophan and Solvent Effects on the Self-Assembly and Mechanical Properties of cysteine offer a convenient method to measure end-to-end contact formation Elastin-Like Peptides probabilities and rates in IDPs. This data can be directly linked to models Ananya Srivastava1,2, Zhuyi Xue1,2, Lisa D. Muiznieks1, Fred W. Keeley1,2, describing IDP structure and dynamics in terms of intra-protein interactions. Regis Pomes1,2. The information provided is similar to that of FRET and FCS, without the 1 Molecular Medicine, Hospital for Sick Children, Toronto, ON, Canada, need to label the IDP with prosthetic dyes. To understand sequence-specific 2 Department of Biochemistry, University of Toronto, Toronto, ON, Canada. intra-protein interactions, we have generated a wide set of PET data for Elastin belongs to an important but poorly-understood class of structural pro- different IDP variants under a range of solvent conditions. However, the in- teins that impart extensibility and elastic recoil to major arteries, lungs, skin, ferred average properties strongly depend on the distribution function chosen and other elastic human tissues. Its monomeric precursor, tropoelastin, is intrin- to describe the end-to-end distance. Moreover, IDPs with different sequences sically disordered. Although it is highly hydrophobic, it is soluble at room tem- or under different physico-chemical conditions might require different distribu- perature and undergoes liquid-liquid phase separation, or coacervation, upon tion functions. We therefore analyze the data set by applying a polymer model temperature increase. In addition, some elastin-derived peptides are able to that allows variations of the distribution function. We are able to explain all the coacervate while others form amyloid-like fibrils containing a large amount data by separating position-dependent electrostatic contributions from non- of b-sheets. However, fibril formation is impeded by depositing the amyloid- electrostatic contributions, including solvent effects. The results shed light on like peptides in methanol. In this study, we use all-atom molecular dynamics the amino acid based interaction parameters that can be used for both future simulations to uncover the structural and physico-chemical basis for the self- experimental and theoretical studies of IDPs. assembly of elastin-derived peptides into liquid-like or amyloid-like aggre- gates. To this end, we examine the structural ensemble of a set of model 1003-Pos elastin-derived peptides successively in explicit water and methanol. Results The Amino Acid Sequence Features of the FG Nucleoporins Affect the obtained with three different force fields generally suggest that compared to Movement of Cargo Complex Inside the NPC water, methanol reduces the burial of nonpolar groups in all peptides even as Mohaddeseh Peyro, Mohammad Mofrad. it promotes b-sheet formation in the amyloid-like peptides and lowers the Univ Calif Berkeley, Berkeley, CA, USA. elastic modulus of the elastin-like peptides. The latter result is confirmed by Nuclear Pore Complex (NPC) is the largest inside the cell. tensile mechanical measurements of cross-linked elastin materials. Together, Nucleocytoplasmic transport has been an active area of research for the past these findings provide an explanation as to why methanol prevents fibril forma- few decades. The transport process is facilitated by the interactions among tion in amyloid-like peptides and indicate that the hydrophobic effect is a sig- FG Nucleoporins (FG Nups) and transport factors (proteins that bind to cargo nificant driving force for the elastic recoil of elastin. to facilitate transport). One of the exceptional features of the nucleocytoplas- mic traffic is that cargo complexes are transported in a very fast yet selective 1001-Pos manner. This feature is known as the ‘‘transport paradox’’, since usually selec- Experimental and Computational Characterization of the Conformational tivity is correlated with long interaction times and high affinities. However, in Ensemble and Interaction Motifs of Chiz N-Terminal Intrinsically Disor- the NPC, the interactions are specific yet transient. Despite extensive research, dered Region the underlying molecular mechanisms that enable this process are not yet Alan Hicks1, Cristian Escobar2, Timothy A. Cross3, Huan-Xiang Zhou4. known. FG Nucleoporins are one of the major role players in the transport pro- 1Department of Physics, Florida State University, Tallahassee, FL, USA, cess. In this study, we examined the role of amino acid sequences of FG Nups in 2Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, the transport process and whether they have a role in formation of transient USA, 3Department of Chemistry, Florida State University, National High interactions. Magnetic Field Laboratory, Tallahassee, FL, USA, 4Department of Using our coarse-grained model of the NPC we studied the effect of specific Chemistry and Department of Physics, University of Illinois at Chicago, features in FG Nups sequences, known as Like Charge Regions (LCRs), on Chicago, IL, USA. the movement of cargo complex in the ring cross sections of yeast NPC. Cell division of Mycobacterium tuberculosis is crucial for the pathogenesis of Our analysis of all of the yeast FG Nups that contain positively charged resi- tuberculosis. This process is mediated by the divisome, a multi-component, dy- dues showed that mutation of charged residues in the LCRs leads to long inter- namic complex anchored by transmembrane proteins. One such protein is ChiZ, actions between FG Nups and transport factors. However, in the wildtype which contains a single transmembrane helix preceded by a 64-residue N-ter- system, the cargo complex makes more transient interactions with FG Nups, minal intrinsically disordered region (IDR). We have characterized the confor- moves between and engages with multiple FG Nups. Therefore, LCRs may mational ensemble and membrane interactions of the ChiZ IDR by a variety of be one of the reasons that transient interactions are enabled between FG experimental techniques, including small-angle X-ray scattering (SAXS) and Nups and transport factors.

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1004-Pos been explored through both in vitro and in vivo experiments These studies iden- Sensitivity-Enhanced Dnp NMR for In Situ Structural Biology tified a hydrophobic patch within the Nab2 N-terminal domain, centered on F73, Kendra K. Frederick. which was suggested as being critical for the interaction between the two pro- Biophysics, UT Southwestern, Dallas, TX, USA. teins. However, the underlying molecular mechanism of Mlp1-Nab2 complex Structural investigations of biomolecules are typically confined to in vitro sys- formation and the role of F73 in this process are still elusive. Here, we employed tems under extremely limited conditions. These investigations yield invaluable an integrated computational approach to study the pivotal interaction between insights, but such experiments cannot capture important structural features Mlp1 and Nab2. First, we conducted a modeling to predict imposed by cellular environments. We recently demonstrated that structural the structure of Mlp1 and then used it for a docking analysis on Nab2 and Mlp1 to studies of proteins in their native contexts are not only possible using state-of- identify most promising orientations of the molecules for complex formation. the-art sensitivity-enhanced (dynamic nuclear polarization, DNP) solid-state nu- Subsequently, we cut the disordered region of Mlp1 into small fragments clear magnetic resonance (NMR) techniques, but also that the native context can randomly distributed at the proposed site of binging to Nab2-N. Finally, molec- have a dramatic influence on protein structure. We aim to visualize such struc- ular dynamics simulations were carrid out to explore the detailed binding mech- tural changes with atomic level resolution and understand how genetic back- anism of Mlp1 and Nab2. We also investigated the effect of experimentally ground can influence protein folding. To do so, we have developed new tested mutations on the F73 residue in order to both validate the model and biotechnology for sample preparation for DNP NMR to investigate the structure further elucidate the role of F73 in Mlp1-Nab2 binding. of a protein containing both an environmentally sensitive folding pathway and an intrinsically disordered region. We have 1) optimized experimental sensitivity of Posters: Membrane Protein Dynamics I DNP NMR through modifications of the sample composition, 2) optimized experimental specificity of complex biological samples through careful use of 1007-Pos isotopically enriched and depleted carbon, nitrogen and proton sources and 3) Spatial Confinement Effects on Lipid Kinase and Phosphatase Reactions improved spectral resolution of DNP NMR through the reduction of chemical on Membrane Surfaces shift degeneracy via segmental and amino acid-specific isotopic labeling. Using Albert A. Lee. a self-polymerizing yeast prion protein, Sup35, we are determining how native UC Berkeley, Berkeley, CA, USA. and mutant cellular environments affect amyloid structure for proteins at endog- Phosphatidyl inositol phosphorylation and dephosphorylation reactions, cata- enous levels in biological contexts with atomic-level precision. lyzed by corresponding kinase and phosphatase enzymes, are a core element of many cellular signal transduction pathways. In cells, Phosphatidyl inositol 1005-Pos reactions span a range of spatial dimensions, from the plasma membrane The Functional Significance of Intrinsically Disordered Protein Regions with a scale in microns to vesicles on the nanometer scale. Moreover, the Encoded by the Diabetes Gene Clec16A cellular cytoskeleton, membrane topographical features, and molecular crowd- Morgan Gingerich1, Xueying Liu1, Michael Vincent2, Bioxian Chai3, ing can all create dynamic physical barriers that can locally confine reactions on Tracy Vozheiko1, Gemma Pearson3, Daniel Klionsky2, Santiago Schenll2, cellular membranes. However, little is known about how or if enzymatic reac- Soleimanpour Scott3,4. tion properties can be altered as a function of spatial confinement. Here, we 1Cellular and Molecular Biology, University of Michigan, Ann Arbor, MI, 2 3 employ micropatterned supported lipid bilayers to study phosphatidyl inositol USA, University of Michigan, Ann Arbor, MI, USA, Metabolism and enzymatic under defined spatial confinement. Two distinct and seemingly very Endocrinology Department, University of Michigan, Ann Arbor, MI, USA, 4 fundamental themes emerge from these studies. First, the corall-to-corral reac- VA Ann Arbor Healthcare System, Ann Arbor, MI, USA. tion rate variation exhibits strong dependence on details of the enzymatic Recent advances in structural biology implicate intrinsically disordered proteins mechanism of interaction with the membrane. Second, we find that the average (IDPs) and/or intrinsically disordered protein regions (IDPRs), which lack a observed kinetic rate for a reaction in an array of micron size corrals can differ fixed 3-dimensional structure, in several human diseases including diabetes. dramatically from the bulk average, and this effect is also strongly dependent However, comprehensive structure-to-function characterizations of IDPs or on the mechanism of enzyme recruitment to the membrane. Using a combina- IDPRs in the context of diabetes is lacking. Using in silico predictive algorithms, tion of stochastic kinetic modeling and biochemical engineering, we map out we observe that the diabetes-associated gene encoding the E3 ligase and mitoph- design rules to control these behaviors in real membrane systems. agy regulator Clec16a, contains two comparatively large putative IDPRs. Mitochondrial function and mass, governed by a balance of biogenesis and se- 1008-Pos lective mitochondrial turnover (mitophagy), is crucial to maintain insulin secre- Dynamin pH Domain Interactions with Lipid Membrane tion and glucose homeostasis. We previously identified Clec16a to regulate Joseph A. Marte, Dalia Hassan, Frank X. Vazquez. pancreatic beta-cell function, insulin release, and glucose homeostasis through Chemistry, St. Johns University, Jamaica, NY, USA. mitophagy. Little is known regarding the structure-to-function implications of During the final stages of endocytosis, large scale dynamin protein oligomers the putative IDPRs within Clec16a. We hypothesized that diabetes-associated are formed around lipid vesicle necks to cause membrane fission. The interac- Clec16a IDPRs control structure-function relationships of consequence for tion of dynamin with the lipid membrane of mammals is controlled by the PH beta-cell competence. Utilizing a mouse model lacking a putative C-terminal domain of the protein. PH domains are found in many proteins and tend to bind Clec16a IDPR, we observed that IDPR-deficientmice had impaired glucose ho- with phosphatidylinositol lipids in membranes. In order to understand the role meostasis, deficient insulin secretion, abnormal b-cell mitochondrial of the dynamin PH domain during membrane fission, we have used molecular morphology/respiration, and impaired mitophagic flux. Disorder-to-order (D- dynamics simulations to study the interaction between the PH domain and the O) mutagenesis revealed that Clec16a IDPRs are vital for binding, stabilization, lipid membrane. In these studies, we have varied the composition of the lipid and ubiquitination of its principal mitophagy effector Nrdp1. Taken together, membrane to understand the effect of phosphatidylinositol concentration and we identify novel roles for IDPRs in the function of a key diabetes- negative lipid charge. These simulations will help shed light on the role of associated protein. Extension of these structure-function relationships within the PH domain in modulating the binding of dynamin to the lipid membrane, other diabetes-associated proteins may provide a unique structural insight a key step in dynamin induced fission. into diabetes pathogenesis and elucidate novel approaches to protect beta-cells. 1009-Pos 1006-Pos Binding from Both Sides: TolR and Full-Length OmpA Bind and Maintain Molecular Mechanisms of the Interaction between the RNA-Binding Pro- the Local Structure of the E. Coli Cell Wall tein Nab2 and the Nuclear Basket Protein Mlp1 in mRNA Quality Control Alister T. Boags. Mohammad Soheilypour, Mohaddeseh Peyro, Hengameh Shams, Chemistry, University of Southampton, Southampton, United Kingdom. Stephanie Rider, Ali R. Kaazempur-Mofrad, Mohammad Mofrad. We present a molecular modeling and simulation study of the of the E. coli cell Dept Appl Sci & Tech, Univ Calif Berkeley, Albany, CA, USA. envelope, with a particular focus on the role of TolR, a native protein of the Transcription of mRNA from DNA is followed by several processing and pack- E. coli inner membrane in interactions with the cell wall. TolR has been pro- aging steps, eventually forming an export-competent messenger ribonucleopro- posed to bind to peptidoglycan, but the only structure of this protein thus far tein (mRNP) to be exported to the cytoplasm. The steps preceding mRNA export is in a conformation in which the putative peptidoglycan binding domain is are quality controlled at different stages of RNA biogenesis via various sophis- not accessible. We show that a model of the extended conformation of the pro- ticated, yet efficient, mechanisms in eukaryotic cells. Mlp1, a nuclear basket pro- tein in which this domain is exposed, binds peptidoglycan largely through elec- tein, plays a substantial role in mRNA quality control by interacting with other trostatic interactions. We show that non-covalent interactions of TolR and proteins involved in that process, specifically RNA-binding proteins (RBPs). OmpA with the cell wall, from the inner membrane and outer membrane sides Yet, the mechanism of the interaction between Mlp1 and RBPs is still elusive. respectively, maintain the position of the cell wall even in the absence of Braun’s The interaction between Nab2, one of the yeast RBPs, and Mlp1 has previously lipoprotein. When OmpA is truncated to remove the peptidoglycan binding

BPJ 9355_9357 204a Monday, March 4, 2019 domain, TolR is able to pull the cell wall down towards the inner membrane. The 4Department of Physics, University of Arizona, Tucson, AZ, USA, charged residues that mediate the cell-wall interactions of TolR in our simula- 5Department of Biomedical and Chemical Engineering and Sciences, Florida tions, are conserved across a number of species of Gram-negative bacteria. Institute of Technology, Melbourne, FL, USA. Rhodopsin is the archetype for the Family A of G-protein-coupled receptors 1010-Pos (GPCRs). Several putative X-ray crystal structures are available for rhodopsin The Open State of the Bam Complex is Stabilized by its Accessory Proteins in the active Meta-II state, yet the conformation and orientation of the retinal Zijian Zhang, David Ryoo, Karl Lundquist, James Gumbart. chromophore remain uncertain at present. Notably, the flip of all-trans retinal Georgia Institute of Technology, Atlanta, GA, USA. in the active state due to rotation of the central part of the polyene chain about Outer membrane proteins (OMPs) in Gram-negative bacteria are transmembrane its axis has been hotly debated [1]. Here we applied solid-state 2H NMR spectros- b -barrel proteins that are involved in transport, signal transduction and copy and QM/MM simulations to study the structure and orientation of the ret- export of virulence factors. The complex responsible for insertion and assembly inylidene ligand in the active state of rhodopsin in aligned membranes [2, 3]. b of OMPs is the -barrel assembly machinery (BAM). BAM structures have now Rhodopsin was regenerated with retinal containing selectively 2H-labeled been solved for all member proteins individually, and in complex, revealing two methyl groups at the C5, C9, or C13 positions, and was recombined into phos- conformational states. However, a detailed mechanism for OMP insertion by the pholipid membranes, then trapped in the light-activated state [4]. We determined BAM complex is still not known. Currently, there are two leading models for this the orientations of the 2H-labeled methyl groups versus the local membrane insertion process based on features proximal to the seam between the N-terminal normal from the 2H NMR lineshape. The solid-state 2H NMR spectra together b b b ( 1) and C-terminal ( 16) strands of the BamA -barrel: the assisted model and with linear dichroism data provided the orientational restraints for calculating the budding model. The assisted model claims that, due to the decreased hydro- retinal structures. In QM/MM simulations four models were tested with different phobic region near the lateral gate, the nascent OMP inserts itself into the outer orientations of the chromophore and different protonation states of Glu113 and membrane. In contrast, the budding model claims that the lateral gate opens and Glu181. We discovered that two structures (‘‘flipped’’ and ‘‘unflipped’’) are b forms a -sheet hybrid with the growing OMP. While both models are backed by possible without significant steric clashes in the binding pocket of the active experimental evidence, no conclusive study yet exists. To shed a light on this rhodopsin. We analyzed the energies of the calculated structures, rearrangement issue, we have performed molecular dynamics simulations to identify key inter- of water around the retinal, and determined the probable structures. Lastly we actions related to the insertion process. Using the previously identified crystal discuss the implications for understanding the rhodopsin activation mechanism structures, we have carried out microsecond-long equilibrium simulations of in a membrane environment. [1] J.Feng et al. (2015) Biophys.J. 108, 2754. [2] BAM complex systems with an open or a closed lateral gate and with or without A.V. Struts and M.F. Brown (2014) in Adv.Biol.Solid-State NMR, Royal Soc. the lipoprotein BamB. We discovered that the laterally open structure is unstable Chem., pp.320-352. [3] A.V. Struts et al. (2011) PNAS108,8263. [4] A.V. Struts in the absence of accessory proteins and the rotational movement of the acces- et al. (2015) Methods Mol.Biol.1271,133. sory proteins drives lateral gate dynamics. 1013-Pos 1011-Pos Ubiquitination of MHC II Changes Dynamics of its Recognition Structure Investigating the Activation Mechanism Alteration of Receptor Tyrosine Haruo Kozono1, Takashi Kawamoto1, Yuko Kozono1, Kinase Mutants Masahiro Kuramochi2, Yuji C. Sasaki2. Soyeon Kim1, Zhenfang Du2, Christine Lovly2,3, Adam W. Smith1. 1Res Inst Biomed Sci, Tokyo University of Science, Noda, Chiba, Japan, 1 Department of Chemistry, The University of Akron, Akron, OH, USA, 2Univ Tokyo, Kashiwa, Japan. 2 Division of Hematology/Oncology, Vanderbilt University Medical Center, Ubiquitination regulate the turnover of MHCII/peptide complex on antigen pre- 3 Nashville, TN, USA, Vanderbilt Ingram Cancer Center, Nashville, TN, senting cells. The rate of ubiquitination is tightly controlled by means of ubiq- USA. uitin ligase expression as well as ubiquitin ligase inhibitor protein CD83 Adequate cell to cell communication is vital for cell function and human health. expression. The malfunction of MHCII turnover results in failure of both pos- Most cell communication is initiated through receptor-ligand interactions. ErbB itive selection of CD4 T cell at thymus and induction of regulatory T cell in family receptors (ErbB1-4) belong to receptor tyrosine kinase (RTK), which is a peripheral. To find the mechanism of such outcomes, we used dendritic cells class of receptors sharing similar structural features: an extracellular domain that that lack MARCHI ubiquitin ligase, which showed distinct T cell activation dictates ligand binding, a single helical transmembrane domain, a juxtamem- pattern from wild type cells. Then, we deployed X-ray single molecule mea- brane domain, and a kinase domain with a C-terminal tail. The tyrosine residues surement method, Diffracted X-ray Blinking (DXB), which revealed real in kinase domain and C-terminal tail are trans-autophosphorylated upon ligand time motion of proteins on living cells. The motion of MHC II on the WT activation, leading to recruitment and activation of cytosolic downstream cell was faster than MARCHI KO mutant that may due to ubiquitination, signaling molecules. Lung carcinoma is the second common cancer in the United that explain the signals from ubiquitinated MHC II for TCR is different from States, and ErbB receptor family is the most common driver mutant that causes non-ubiquitinated MHC II. We suggest ubiquitinated MHC II may not inter- tumorigenesis. Numerous antibodies targeting the ErbB1 ectodomain or tyrosine nalize promptly but also present antigens to T cell to induce various critical kinase inhibitors targeting the kinase domain have been developed and approved outcome such as regulatory T cell induction. by FDA. However, these treatments are often ineffective towards the newly found ErbB1 transmembrane or kinase domain duplication mutants. To develop 1014-Pos specific treatments targeting those mutants, their effects on the activation mech- Exploring the Interhelical Landscape of the b-2 Adrenergic Receptor to anism and the downstream signaling pathway must be carefully and closely Identify Druggable Intermediate States using Enhanced-Sampling Molec- examined. We use pulse interleaved excitation fluorescence cross correlation ular Dynamics and Site Identification by Ligand Competitive Saturation spectroscopy (PIE-FCCS) to identify alterations to the activation mechanism (SILCS) of the ErbB receptor, the ligand-induced dimer/oligomerization, in live cell sys- Christoffer Lind1, Deepak Deshpande2, Alexander D. MacKerell1. 1 tem. PIE-FCCS is a two-color fluorescence spectroscopy that measures the fluo- Pharmaceutical Sciences, University of Maryland Baltimore, Baltimore, 2 rescence intensity fluctuation in a defined volume with respect to time. Collected MD, USA, Thomas Jefferson University, Philadelphia, PA, USA. data are used to calculate dynamic parameters such as diffusion coefficient, den- G-protein-coupled receptors (GPCRs) transmit information into a large number sity, binding affinity, and degree of protein co-localization. The sensitivity of our of signaling pathways based on various extracellular stimuli making GPCRs instrument system allows us to study molecular-level interactions between pro- one of the most sought-after pharmaceutical targets. Lately, structural studies teins of interest. Using PIE-FCCS, we investigate the homo- and heterodimeri- have generated essential understanding of how distinctive receptor conforma- zation of both wild type and mutated ErbB family members and their relationship tions can be of importance in triggering different signaling pathways. Hence, to oncogenic activities of the cells. one can potentially identify and specifically target these different conforma- tions in order to design biased agonists, directing an exact intracellular 1012-Pos response. The b -Adrenergic receptor (b AR) is of special interest as it is a 2 2 2 Retinal Flipping During Rhodopsin Activation Revealed by Solid State H GPCR target used for treatment of asthma. Many of the drugs used currently NMR and QM/MM Simulations have shown an increase in amplified mucus-production and tachyphylaxis. Andrey V. Struts1,2, Mikhail N. Ryazantsev3, Xiaolin Xu4, These side-effects ultimately worsen the symptoms of asthma. Therefore, it Trivikram R. Molugu1, Suchithranga M.D.C. Perera1, Charitha Guruge5, is of great importance to expand the knowledge of how intermediate conforma- Samira Faylough5, Carolina Nascimento5, Nasri Nesnas5, Michael F. Brown1. tions of b AR can be exploited in order to develop new drugs. To gain a deeper 1 2 Department of Chemistry and Biochemistry, University of Arizona, Tucson, understanding of the dynamics of intermediary functional conformations of the 2 AZ, USA, Laboratory of Biomolecular NMR, St. Petersburg State b AR, and how this is associated with the interhelical motions of activation, we 3 2 University, St. Petersburg, Russian Federation, Institute of Chemistry, have applied conventional long time-scale molecular dynamics simulations to St. Petersburg State University, St. Petersburg, Russian Federation, both agonist and antagonist bound b2AR at different known conformations.

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Additionally, enhanced sampling methods, such as targeted molecular dy- 1017-Pos namics, have been used where external forces were employed to transition Raf-1 Cysteine-Rich Domain (CRD) Promotes Active Orientation and the inactive conformation to the fully active state. Subsequently, all external Dimerization of KRas4B at the Membrane forces are released, and we can monitor the relaxation path of returning to an Hyunbum Jang1, Ruth Nussinov2. ‘inactive-like’ conformation. From this relaxation path we identify novel and 1Frederick Natl Lab, Leidos Biomed Res Inc, Frederick, MD, USA, 2Cancer potentially druggable intermediate states that are being characterized with and Inflammation Program, National Cancer Institute at Frederick, Leidos the Site Identification by Ligand Competitive Saturation (SILCS) approach. Biomedical Research, Inc., Frederick, MD, USA. Ras, a small GTPase, consists of highly homologous catalytic domains and flex- 1015-Pos ible C-terminal hypervariable regions (HVRs) that differ significantly across Ras Study of Ultra-Fast Rhodopsin Activation Dynamics with Molecular Dy- isoforms. Ras proteins control signal transduction pathways, promoting cell pro- namics Simulations liferation and survival. In cancer, KRas4B is frequently mutated. In the Raf/MEK/ 1 2  2 Letty Salas , Derek Mendez , Jose Domingo Meza-Aguilar , ERK (MAPK) pathway of tumor proliferation, KRas4B preferentially recruits 3 2 3 Suchithranga M.D.C. Perera , Abhishek Singharoy , Andrey V. Struts , Raf-1 and activates it. Raf kinases contain Ras binding domain (RBD) and 2 2 4 2 Nadia A. Zatsepin , Richard A. Kirian , Thomas D. Grant , Petra Fromme , cysteine-rich domain (CRD) at conserved region 1 (CR1), and kinase domain at 3 1 Michael F. Brown , Alan Grossfield . CR3. The high-affinity interaction of Raf RBD with Ras was solved in the absence 1University of Rochester, Rochester, NY, USA, 2Arizona State University, 3 4 of CRD. Recently, pivotal roles of CRD in the membrane interactions of KRas4B– Tempe, AZ, USA, University of Arizona, Tucson, AZ, USA, State Raf-1 RBD-CRD complex were reported. Here, we employ all-atom MD simula- University of New York at Buffalo, Buffalo, NY, USA. tions to investigate dimeric KRas4B–Raf-1 complex at the anionic lipid bilayer Rhodopsin is a photo-sensitive membrane protein that, when excited by light, composed of DOPC and DOPS (4:1). Active KRas4B dimer modulates Raf-1 acti- can activate the signaling pathway that leads to scotopic vision in mammals. vation, promoting Raf-1 kinase domain dimerization. In the dimeric complex, Although this receptor is well-characterized, there is limited structural informa- Raf-1 CRD stably binds anionic lipid bilayers inserting a positively-charged tion about the events that occur immediately after light-excitation, as they take loop into the amphipathic interface. The loop contains key basic residues, respon- place in notably short time regimes (fs-ns) and they are not easily captured exper- cis trans 0 sible for the membrane association, suggesting that CRD is an intrinsic membrane imentally. These events comprise the -to- isomerization of the protein s binding domain of Raf kinase. Our simulations suggest that the Raf-1 CRD not ligand after photon absorption, and a subsequent relaxation process that drives only offers an additional anchor for the KRas4B–Raf-1 complex, but also reduces the receptor through a series of spectroscopically distinguishable intermediates. the fluctuations of Ras–RBD, enhancing the high affinity interaction between Ras All-atom molecular dynamics simulations can be a powerful tool for the study of in silico and Raf. Raf-1 CRD supports the active-state Ras orientation, promoting Ras these ultra-fast dynamic events and aid the interpretation of emerging dimerization through the allosteric lobe helical interface at the membrane. The experimental techniques, such as time-resolved small- and wide-angle X-ray enhanced stability of the complex at the membrane rendered by CRD promotes scattering with free-electron lasers. The temporal resolution of simulations is Raf dimerization in the MAPK signaling, the key Ras proliferative pathway. only limited by storage space and, because the times of interest are quite short Here we suggest these significant roles of CRD at the membrane in the Raf acti- (fs-ps), it is feasible to run a large number of trajectories, improving the conver- vation. Funded by Frederick National Laboratory for Cancer Research, National gence of the results and facilitating the assessment of functionally relevant Institutes of Health, under contract HHSN261200800001E. phenomena. Starting from well-equilibrated microsecond-scale dark-state rhodopsin simulations, we run and analyze 3,000 pairs of 100 ps trajectories in 1018-Pos two conditions (dark and light-excited) to model the process of energy dispersion Dynamic ‘‘Molecular Portraits’’ of Proteins and Cell Membranes: A across the protein after light-excitation. Because these pairs of simulations start Computational View out with identical coordinates, cancellation of signals not related to photon ab- Roman G. Efremov1,2, Anton A. Polyansky3,4, Anton Chugunov1,2, sorption or retinal isomerization is expected to be optimal. Following this strat- Nikolay A. Krylov2,5, Dmitry Nolde1, Pavel E. Volynsky4, egy, we observe a small, but statistically significant, increase in the radius of Andrey Kuznetsov1,2, Pascal Maurice6. gyration of the receptor after light-excitation and a propagation of the light- 1Dept Struct Biol, Shemyakin-Ovchinnikov Institute of Bioorganic induced perturbation across the protein that occurs at speeds close to those found Chemistry, Russian Academy of Sciences, Moscow, Russian Federation, for other light-reactive proteins, like myoglobin and bacteriorhodopsin. 2National Research University Higher School of Economics, Moscow, 3 4 1016-Pos Russian Federation, Univ Vienna, Vienna, Austria, Shemyakin- Molecular Simulations Give Insights into the NDM-1/Membrane Interac- Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of 5 tion that Causes Rise of a Super-Bacterium Sciences, Moscow, Russian Federation, Joint Supercomputer Ctr Russian 6 Alessio Prunotto1, Guillermo Bahr2, Lisandro Gonza´lez2, Alejandro Vila2, Acad of Sci, Moscow, Russian Federation, Dept Struct Biol, Universitede Matteo Dal Peraro1. Reims Champagne Ardenne, Reims, France. 1E´ cole Polytechnique Federale de Lausanne, Lausanne, Switzerland, Molecular surfaces are the key players in processes of bimolecular recognition, 2Instituto de Biologia Molecular y Celular de Rosario, Rosario, Argentina. interaction, and signaling. Nowadays, state of the art methods exist for visual- Antibiotic resistance is widely seen as one of the biggest threats to global health izing molecule surface and surface distributed properties in three-dimensional for the next decades. WHO estimates that in 2050, up to 10 millions deaths per space. However, such a visual information could only be analyzed by human year will be caused by multi-resistant bacteria, unless action is taken. There- eye and therefore prompt to be biased and onerous in case of bio- fore, it is of vital importance to analyse and elucidate the mechanisms that bring macromolecules with a complex structural organization or for large sets of ob- resistance to microorganisms, in order to plan strategies that will lead, eventu- jects. This is especially true for such mesoscopic systems like cell membranes. ally, to the development of new generations of antibiotics. Metallo-b-lacta- Therefore, alternative (mostly computational) techniques operating with such mases (MBLs) are bacterial enzymes that generate resistance towards a wide ‘‘molecular portraits’’ are indispensable. Here, we present a number of original variety of antibiotics. The response of the immune system consists in supplying in silico methods to map and process multivariate and dynamic surface proper- metal chelators, which retrieve zinc ions, causing the degradation of MBLs. ties of biomolecules and their assemblies - proteins, membranes, New Delhi MBL 1 (NDM-1) is a B1 subclass MBL that came to the fore in and their complexes. These techniques were shown to improve protein-ligand the last years because of its resistance to metal chelators. It was demonstrated [1] and protein-protein [2] docking results. A new protein surface topography that a lipidated version of NDM-1 is able to anchor to the outer bacterial mem- (PST) method [3] is described. It permits pictorial visualization of the whole brane, hence providing resistance towards zinc starvation. However, the molec- protein surface in terms of 2D ‘‘earth maps’’. PST can be used to delineate ular mechanisms that lie beyond this phenomenon remain elusive. Molecular conformational changes, perform group analysis of molecules. It is a useful simulations show that, although lipidation is essential for stabilizing membrane tool to add to docking experiments. The proposed original tools have been anchoring, the first interaction is driven by simple electrostatics. The natural af- shown to be very efficient in analysis of atomistic details of the recognition pro- finity of NDM-1 for the membrane could explain why, among MBLs, this pecu- cesses in cell membranes - they allow easy monitoring of dynamic amphiphilic liar mechanism evolved within NDM-1 only, eventually helping to predict properties of lipid membranes, proteins, peptides, , etc. Imple- which other MBLs are likely to develop similar features. Indeed, the affinity mentation of the ‘‘molecular portrait’’ technologies is capable of rational for the membrane presents ample variability across different MBLs, as we design of new physiologically important compounds with predefined activities highlighted by comparing NDM-1 to other enzymes of the same class, such [4]. References. 1. Pyrkov T.V. et al. (2009) Bioinformatics. 25:1201. 2. Poly- as VIM-2. The simulations also show that a specific lipid patch is necessary ansky A.A. et al. (2014) Bioinformatics 30:889. 3. Koromyslova A.D. et al. to drive the protein/membrane recognition, and that removing charged lipids (2014) J. Chem. Inf. Mod. 54:1189. 4. Kasheverov I.E. et al. (2016) Scientific can strongly affect the binding. Experimental tests were conducted to support Reports, 6:36848. Acknowledgements Russian Foundation for Basic Research and confirm these findings. (18-54-15007), Russian Science Foundation (18-14-00375).

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1019-Pos mass spectrometry (DHX-MS) coupled with electron transfer dissociation Structural Insights into the Subtype-Selective Antagonist Binding to the (ETD) and a novel software-based evaluation approach. M2 Muscarinic Receptor We focus on the TMD of C99, a fragment of the amyloid precursor protein Sangbae Lee1, Suno Ryoji2, Maeda Shoji3, Takuya Kobayashi4, (APP) and a substrate of g-secretase. Due to its relation to Alzheimer’s disease, Brian K. Kobilka3, Nagarajan Vaidehi1. this fragment is one of the most extensively studied substrates in intramem- 1Dept Immunology, City Hope Med Ctr, Duarte, CA, USA, 2Department of brane proteolysis. The TMD consists of the N-terminal dimerization region, Medical Chemistry and Cell Biology, Kyoto University, Kyoto, Japan, a connecting double-glycine hinge, and the C-terminal cleavage region, where 3Department of Molecular and Cellular Physiology, Stanford University, the substrate is processed step-wisely after initial cleavage at the e-site. We Stanford, CA, USA, 4Kyoto University, Kyoto, Japan. observe a high flexibility in the dimerization region and a rigid cleavage region. G protein-coupled receptors (GPCRs) regulate diverse pathways in human In our studies, we focus on two areas: physiology, and therefore represent major pharmaceutical drug targets. A First, the double glycine hinge, which strongly affects the flexibility of the com- growing number of high-resolution GPCR structures have provided detailed in- plete TMD. Here, we introduce helix-promoting and helix-disturbing muta- formation about the mode of ligand binding, facilitating the development of tions, monitoring the impact on the overall backbone flexibility, including better drugs. However, due to the dynamic nature and low abundance of these changes in the cleavage domain. proteins, it remains challenging to obtain large quantities of stable GPCRs for Second, the ε-site, which is expected to unfold during substrate docking and structural studies. Therefore, we achieved thermostabilization of the human subsequent proteolysis. We investigate the impact of double glycine and double muscarinic M2 receptor by replacing the serine residue at position 3.39 with leucine substitutions, which are known to enhance or decrease cleavage effi- arginine (S110R) to obtain the thermostabilized M2 receptor in dramatically ciency, respectively. increased yields. We determined the structures of the thermostabilized M2 re- Furthermore, we analyzed the familial Alzheimer’s disease related mutation ceptor bound to the non-selective muscarinic antagonist NMS and the subtype- I45T. We observe altered dynamics in the cleavage region, suggesting a reason selective antagonist AF-DX 384 at resolutions of 2.3 A˚ and 3.0 A˚ , respectively. for the distorted cleavage-pathway which results in the pathogenic Ab42. Crystals of the mutant receptor bound to NMS diffracted to a higher resolution than those of the wild type, presumably because the mutation rigidified the 1022-Pos receptor to the antagonist-bound conformation. Comparison of the crystal Drug Permeation Across the Bacterial Membrane: Combining Theoretical structures and pharmacological analyses suggested that the side chain of and Experimental Approaches the arginine at 3.39 in the S110R mutant mimics the role of the allosteric so- Paula Gameiro, Carla F. Sousa, Joa˜o T.S. Coimbra, Pedro A. Fernandes, dium ion. In addition to increasing thermostability, the S110R mutation also Maria J. Ramos. enhanced the affinity for AF-DX384, enabling co-crystallization of the protein Quimica e Bioquimica, Faculdade de Ciencias, UP, Requimte, FCUP, with the ligand. MD simulations using this structure revealed that tightening Universidade do Porto, Porto, Portugal. Since many antibacterial drugs have intracellular targets, permeation studies are of the AF-DX 384-residue contacts in M2 receptor compared to M3 receptor caused to the ligand selectivity of AF-DX 384. crucial in the study of bacterial resistance and development of alternative drugs. Ciprofloxacin (Cpx) is an antimicrobial drug from the group of fluoroquinolones 1020-Pos that is widely used in the clinic. Cpx is mainly in its zwitterionic form at physi- Interplay between the Conformational Dynamics of a Bacterial ABC- ologically pH and its permeation in the bacterial cell is dependent on the transport Transporter and Surrounding Membrane Mechanical Properties through channel porins. Mutation of porin’s channel is an important mechanism Alicia Damm, Su-Jin Paik, Ajay Kumar Mahalka, John Manzi, Daniel Levy, of bacterial resistance to this drug. Complexation with a copper atom and a phe- Patricia M. Bassereau, Maxime Dahan. nantroline (CuCpxPhen) is an approach developed to counteract bacterial resis- UMR 168, Institut Curie, Paris, France. tance, since it is expected to improve permeation through the membrane. ABC (ATP Binding Cassette) transporters represent one of the largest families of In the present study, the permeation of Cpx was compared with the one of transmembrane protein. They use ATP hydrolysis to transport a wide range of CuCpxPhen. Two different models were used: one composed only by lipids, substrates such as lipids, antibiotics and drugs. Some ABCs, such as glycoprotein mimicking the negatively charged bacterial membrane, and one composed by P (PgP), a human ABC transporter, export anti-cancer agents out of the cell, and the OmpF porin embedded in a lipid matrix. contribute to a cellular phenotype of multi-drug resistance. ABC transporters Spectroscopic methods were used to determine drugs’ partition and interaction consist of two transmembrane domains (TMDs) that contain drug binding sites with the porin. To further explore structural and dynamical aspects of the and two nucleotide binding domains (NBDs) that hydrolyze ATP. Substrate permeation, a theoretical approach was considered, using molecular dynamics transport is associated with both rearrangements of NBDs and TMDs. We study simulations. BmrA, a bacterial homologue of PgP from B. Subtillis, for which a large confor- The results indicate that the Copper complex has higher partition for the bacte- mational change is a priori expected. However, the BmrA structure not being rial model membrane when compared to Cpx. The energy profiles also show that solved yet, this change has to be scrutinized. We aim at investigating the inter- CuCpxPhen’s permeation across the membrane is more favorable but Cpx’s play between the conformational dynamics of the protein and the mechanical transport across the porin OmpF is energetically more advantageous. Overall, properties of its surrounding membrane, e.g. curvature and tension. We have it is shown that CuCpxPhen permeation in the bacterial membrane should be shown that the purified protein is highly active when incorporated into different porin independent. Copper complexes are then expected to be good candidates membrane model systems. In WT both NBDs contain a single cysteine, labelled to bypass at least one type of bacterial resistance, related with membrane perme- with fluorescent dyes. We have used single molecule FRET method (Forster ability. The findings of this work are important to proceed with the study of Resonance Energy Transfer) to probe the transporter conformation in immobi- CuFQPhen complexes as an alternative to free FQs in resistant bacteria. lized liposomes. Our results show little difference in the FRET efficiency histo- Acknowledgments:UID/QUI/50006/2013-POCI/01/0145/FEDER/007265 grams between the apo and the post-hydrolysis conformations, suggesting that (LAQV/REQUIMTE) the distance between the two NBDs does not change significantly, in contrast 1023-Pos with initial expectations. Based on Electron Microscopy observations and other Dynamic Actin Mediated Nanoclustering of Cd44 Regulates its Meso-Scale biophysical results from our group, we suggest that a significant conformational Organization at the Plasma Membrane change occurs in protein TMDs, in line with recent models proposing a transport Parijat Sil1, Sangeeta Nath2, Nicolas Mateos3, Takahiro Fujiwara4, mechanism with constant contact between NBDs. Maria F. Garcia-Parajo3, Akihiro Kusumi4, Satyajit Mayor1. 1021-Pos 1NCBS, TIFR, Bangalore, India, 2INSTEM, Bangalore, India, 3ICFO, Intramembrane Proteolysis by Gamma-Secretase: Efficiency as a Matter Barcelona, Spain, 4Dept iCeMS, Kyoto Univ, Kyoto, Japan. of Substrate Flexibility Trans-membrane proteins at the plasma membrane of the cell are equipped with Philipp A. Ho¨gel, Dieter Langosch. modules to interact with the extra-cellular and the intra-cellular space. CD44 is Chair of Chemistry, Technical University of Munich, Freising, a type I trans-membrane cell adhesion receptor that has been recently shown to Germany. compartmentalize the membrane into domains by acting as a membrane picket. Intramembrane proteolysis is a crucial step in numerous processes in the cell. This compartmentalization has been show to be necessary for membrane func- Yet, the criteria for substrate/non-substrate discrimination, cleavage efficiency tion of signaling receptors. While the extra-cellular domain of CD44 interacts and site-specific cleavage are still unknown today. Backbone dynamics with extracellular matrix (ECM) ligands hyaluronic acid and galectins, the of transmembrane domains (TMDs) of intramembrane cleavage protease intra-cellular domain interacts with a range of cytoskeletal adaptors, including (i-CLIPs) substrates are expected to determine the binding to the proteases as ezrin and ankyrin at the cytoplasmic side. High spatial and temporal resolution well as cleavage efficiency. We investigate the backbone dynamics of several imaging of living cells revealed a meso-scale architecture comprising of a substrate TMDs on a residue-specific level with deuterium/hydrogen exchange meshwork pattern that is enriched with nano-scale clusters of CD44 along its

BPJ 9355_9357 Monday, March 4, 2019 207a perimeter. The nano-scale clustering in turn is orchestrated by engaging with gating MscL through well-tempered metadynamics. The free energy barrier the underlying actin cytoskeletal machinery. The cytoskeletal interaction medi- separating the open and closed state of MscL is 10 kcal/mol. Further, the ated by specific sites on the intra-cellular domain influences the organization mutation-induced perturbations (quadruple Lys mutant (K3T/K6T/K99T/ and dynamics of the protein both at the nano-scale and meso-scale providing K100T) and gain-of-function mutant (V21D)) on the characteristic features a selective engagement with actin filaments nucleated by formin-based actin of free energy surface in MscL are investigated in detail. nucleation machinery over Arp2/3. Additionally, the extra-cellular domain and its interaction with elements of the extra-cellular matrix also influence 1026-Pos the dynamics of the protein and its nano as well as meso-scale organization Selectivity and Substrate Translocation Mechanism in Eukaryotic Sweet at the plasma membrane. Taken together, our data captures a hierarchical na- Proteins: Bioinformatics and Molecular Dynamics Studies ture of organization of CD44 at the cell surface with cytoskeleton-templated Ankita Gupta, Ramasubbu Sankararamakrishnan. nano-scale clusters populating the meso-scale domains. Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur, India. 1024-Pos Sugar transport across the cell membrane is essential for all the life forms. G-Protein-Coupled Receptor Activation Mediated by Internal Hydration Amongst the known sugar transporters, SWEET proteins are the recently identi- Steven D.E. Fried1, Anna R. Eitel1, Nipuna Weerasinghe1, fied ones. These proteins play crucial roles in plants and their significance in other Carolanne E. Norris1, Johnathan D. Somers1, Gabrielle I. Fitzwater1, organisms is ambiguous. In , two triple-helix-bundles (SemiSWEETs) Michael C. Pitman1, Andrey V. Struts1,2, Suchithranga M.D.C. Perera1, dimerize to form a functional pore, whereas a linker-helix connects them in eu- Michael F. Brown1,3. karyotic SWEET proteins. Biochemical and structural analysis along with molec- 1Department of Chemistry and Biochemistry, University of Arizona, Tucson, ular dynamic studies have suggested an alternating access transport mechanism AZ, USA, 2Laboratory of Biomolecular NMR, St. Petersburg State for SemiSWEETs. SWEET homologs in plants are found to be selective for their University, St. Petersburg, Russian Federation, 3Department of Physics, monosaccharide or disaccharide substrates. Residues interacting with the sub- University of Arizona, Tucson, AZ, USA. strate and responsible for selectivity have been identified in experimental studies. Rhodopsin is a canonical member of the Class A (rhodopsin-like) G-protein- However, the molecular basis of selectivity remains to be elucidated. To under- coupled receptors and is responsible for scotopic vision under dim light in stand the diversity of SWEET family members, we have identified more than retinal rod cells. Utilizing rhodopsin as a model GPCR, we explored the 2000 SWEET homologs and performed exhaustive structure-based sequence role of water in the structural activation mechanism of GPCRs using osmotic analysis. With the help of homology modeling using multiple templates, models stress techniques. We hypothesized that rhodopsin activation is hydration were generated for all homologs in the three conformations. Along with phyloge- mediated as shown by nanosecond molecular dynamics simulations [1], which netic studies, our analysis elucidated positions crucial for transport and substrate reveal a bulk influx of water into the protein core following photoactivation. interactions in the SWEET subfamilies. With all the curated annotations of To experimentally validate these results, we subjected the protein within SWEET homologs from different kingdoms of life, we have developed a database native lipid membranes to environments of varying osmotic pressures, using dbSWEET, an integrated resource for the SWEET family members (bio- different molecular-weight polyethylene glycols as osmolytes. By photoacti- info.iitk.ac.in/bioinfo/dbSWEET/Home). We are further investigating the role vating the protein while recording time-dependent UV-visible spectra, we of sub-family specific features along with the interactions with the substrates measured the fraction of protein in the active metarhodopsin-II (MII) confor- with the help of Molecular Dynamics (MD) studies. MD simulations of a wild- mation, the receptor state capable of activating the G-protein. High-molecular type plant SWEET protein and it’s in silico mutants in the substrate-bound form weight osmolytes comprehensively favored the more closed, inactive reveal the substrate transport mechanism and the importance of subfamily- metarhodopsin-I (MI) conformation by withdrawal of water from the protein specific conserved positions in facilitating selective transport. core. By contrast, small osmolytes penetrated into the protein core, and stabi- lized the active MII conformation until a quantifiable saturation point was 1027-Pos reached. A universal osmotic response was discovered for the limit of Topographic Modulation of Acetylcholine Receptors Diffusion Dynamics increasing osmolyte size and maximal polymer exclusion from the protein. on Live Cell Membrane By measuring the thermodynamic dependence of the MI-MII equilibrium on Yusheng Shen, Chengjie Luo, Penger Tong. osmotic pressure, we determined that rhodopsin activation entails a bulk influx Department of Physics, The Hong Kong University of Science & of 80-100 water molecules into the protein core with a substantial increase in Technology, Hong Kong, China. compressibility. A new model is proposed for the functional role of water in In a recent single-molecule tracking experiment [1], the acetylcholine receptors GPCR signal transduction, enabling a wet-dry cycling mechanism that am- (AChRs) on muscle cell membranes were found to display non-Gaussian statis- plifies the activation of G-proteins. Our results necessitate a new understand- tics due to dynamic heterogeneity resulting from the complex cellular environ- ing of GPCR activation, in which the influx of water plays a critical role in ment of the AChRs. In this study, we vary the cellular environment of AChRs establishing the active receptor conformation. [1] N. Leioatts et al. (2014) by using the microgroove patterning techniques to control the spatial distribution Biochemistry 53, 376-385. of adhesion complexes on the basal membrane of live cells and study its effect on the diffusion dynamics of AChRs. Cultured Xenopus muscle cells are found to 1025-Pos align along the microgroove patterns with their adhesion complexes, such as Dynamics and Energetics of Gating Mechanism in Mechanosensitive b1-integrins, predominantly anchored on the ridge region of the microgrooves, Channel of Large Conductance (MscL) forming a stripe-like pattern. Interestingly, we find the movement of AChRs is Rajitha R. Tatikonda, Juan M. Vanegas. largely restricted within the microgroove-aligned integrin stripes. Detailed sta- Physics, University of Vermont, Burlington, VT, USA. tistical analysis of the AChR trajectories reveals that the AChRs are slowed Molecular-level insights into the key factors controlling the gating mechanism down by the patterned integrins, and immobilization and clustering of the AChRs in the tension-activated Mycobacterium tuberculosis mechanosensitive channel are correlated with the spatial distribution of integrins. The experiment demon- of large conductance (MscL) are unravelled using molecular dynamics (MD) strates that the spatial distribution of integrins plays a crucial role in modulating simulations. The number of hydrogen bonds between protein and lipids on the diffusion dynamics of AChRs. *The work was supported by the Research the cytoplasmic side is more than 50% larger compared to the periplasmic Grants Council of Hong Kong SAR. [1] W. He, H. Song, Y. Su, L. Geng, B. J. side. This is due to a cluster of positively charged Lys residues in the cyto- Ackerson, H. B. Peng, and P. Tong, Nature Communications, 7:11701 (2016). plasmic side of MscL, which contribute roughly half the total protein-lipid interaction energy. In our previous study, these strong electrostatic interactions 1028-Pos were used to gate the channel by the application of forces directly on the bound Electromigration of Cell Surface Macromolecules during Galvanotaxis lipids. Here, we have developed a new approach where a collective variable Anyesha Sarkar1, Brian M. Kobylkevich1, David M. Graham2, (CV) mimicking the lateral tension can be used to gate the channel in more pro- Mark A. Messerli1. nounced manner. This new CV method allows fast opening of MscL (<1 ns) in 1Biology and Microbiology, South Dakota State University, Brookings, SD, the absence of tension and also allows asymmetric exploration of tension in USA, 2Cell Biology & Physiology, University of North Carolina, Chapel Hill, each leaflet independently. The transition from closed to open state of MscL NC, USA. involves large conformational change with large increase in its pore radius. Weak, DC electric fields (EFs) direct cellular polarity, migration and The opening of the channel is highly asymmetric with respect to steering forces outgrowth. They are used to induce polarity during tissue engineering and in the two bilayer leaflets and is observed to be significantly more sensitive to direct migration to promote epidermal wound repair. Fields as weak as 10 perturbations in the cytoplasmic side. Upon release of steering forces, the open mV/mm, are sufficient to direct migration, although the mechanisms used by state MscL return to the closed state spontaneously. This newly defined CV al- cells to sense such weak fields are still debated as weaknesses exist in the lows us to obtain the free energy surface and minimum energy pathway for competing models. We hypothesize that cellular polarization is achieved by

BPJ 9355_9357 208a Monday, March 4, 2019 the redistribution of a cell surface, electric field receptor (EFR) in the presence 1031-Pos of an applied EF. We are investigating the electromigration model that depends Dynamic Coupling of the Aromatic Rotamer Conformation with the Bacte- on electrically generated forces in the plane of the plasma membrane and par- riorhodopsin Photocycle as Revealed by the Chemical Shift Assisted QM/ allel to the electric field vector, specifically, electrophoretic and electro- MM Calculations osmotic forces. Electrophoretic force drives net negatively charged macromol- Sijin Chen1, Xiaoyan Ding1,2, Chao Sun1, Haolin Cui1, Anthony Watts2, ecules to the anode while electro-osmotic flow of water, in the boundary layer, Xiao He1, Xin Zhao1. drives macromolecules with large extracellular domains to the cathode. We 1East China Normal University, Shanghai, China, 2University of Oxford, have derived a model based on these opposing forces, to predict the relative Oxford, United Kingdom. concentration of surface proteins over space and time, allowing us to test the Aromatic residues are well known to be highly conserved in G-protein-coupled redistribution of known plasma membrane surface macromolecules in applied receptors (GPCRs) and play very important roles in activating the global toggle EFs and under controlled conditions. The model closely describes accumula- switch through a local rotamer switch mechanism. However, little is to know tion of a net negatively charged, GPI-anchored, fluorescent protein to the cath- about the mechanism of the highly conserved aromatic residues during the pho- ode under different field strengths, showing that redistribution reaches steady- tocycle of the microbial rhodopsin family proteins. In this work, tyrosine 185 state within minutes, significantly faster than cathodally migrating cells turn to- (Y185), one of the highly conserved aromatic residues within the retinal (Ret) ward the cathode. The model is useful for determining the most likely candi- binding pocket of bacteriorhodopsin (bR) was used as an example to calculate dates for the EFR on many different cell types. its rotamer conformation in the inactive and active states during the bR photo- cycle by the automated fragmentation quantum mechanics/molecular mechanics 1029-Pos (AF-QM/MM) method, assisted by the chemical shifts of the Ret chromophore Analysis of Ion Channel Dynamics by Single Molecule Tracking in Live obtained by 2D solid-state NMR correlation experiments. Our results showed Cells that Y185 not only underwent a rotamer configurational changes during the Yeonki Hong, Jiseong Park, Daeha Seo. bR photocycle, but also affected the protein dynamics and the proton-pumping Daegu Gyeongbuk Institute of Science and Technology, Daegu, Republic of efficiency through a rotamer switch mechanism. In the AF-QM/MM calcula- Korea. tions, the whole protein was divided into three regions, the core region, buffer Protein dynamics play an important role in signal transduction in association region and remaining region. The protonated Schiff base, including the Ret chro- with their activation mechanisms, functions, and so on. Single molecule mophore and K 216, is defined as the core region. The buffer region is defined by tracking technique have been widely used for investigating diffusion behavior the residues within the retinal binding pocket and some other residues within 2.5 of protein in live cells. Especially membrane proteins have been studied since A˚ of the core region where the contacting atoms are hydrogen, and the residues they are important to understand cell responding to the surroundings. However, within 4.0 A˚ where at least one of the contacting atoms is non-hydrogen. The re- not many ion channel proteins such as AMPAR, NMDAR, etc., have been maining part of the protein is defined as the rest region. Both the core region and monitored due to their complex system. Here, we observed ionotropic gluta- its buffer region were treated by QM calculations, and the remaining region was mate receptor in neuroblastoma SH-SY5Y cell using single-molecule imaging. calculated at the MM level as background charges. The diffusion coefficient of the receptor was significantly low because the re- 1032-Pos ceptor has four subunits (tetramers) and each subunit possesses a four trans- Exploring the Hydrophobic Barrier of Human K2P Channel TWIK1 with membrane domain. Furthermore, we also analyzed interaction between Steered MD Simulations subunits using single molecule tracking, further investigation of the protein dy- Bharat Poudel1, Rajitha R. Tatikonda2, Juan M. Vanegas2. namics of the membrane proteins such as ionotropic receptors, their structure 1University of Vermont, Winooski, VT, USA, 2Physics, University of and protein activation mechanism will be possible. Vermont, Burlington, VT, USA. We used steered molecular dynamics simulations to explore the hydrophobic 1030-Pos barrier within the two pore domain potassium channel TWIK-1. This hydro- Gating Mechanism of a Potassium Channel, Experimental and Theoretical phobic barrier is formed by Leucine 146 and Leucine 261 that lie below the Studies selectivity filter on the cytoplasmic side of the protein. Experimental studies 1,2 1 1 Charline Fagnen , Ludovic Bannwarth , Iman Oubella , have shown that mutations of Leucine 146 and 261 allow more water to 1,2 1,2 3 2 Yasmina Mhoumadi , Aline De Araujo , Eric Forest , David Parahia , permeate to the selectivity filter and increase ion conduction (Nature Com- Catherine Venien-Bryan1. mun., 1   5, 4377, 2014). We use a collective variable (CV) that mimics tension Institut de mineralogie et de physique des Materiaux et de Cosmochimie, by applying forces on lipids in the vicinity of the channel based on their prox- Sorbonne Universite, Paris, France, 2Laboratoire de Biologie et  3 imity to explore the role of membrane interactions in modulating the hydro- Pharmacologie Appliquee, ENS Paris-Saclay, Cachan, France, Institut de phobic barrier. Our group has studied the mechanosensitive channel of Biologie Structurale, Grenoble, France. large conductance using a similar approach. By applying a harmonic potential Inwardly-rectifying potassium (Kir) channels are transmembrane proteins that þ to this CV, we observed different solvent accessibility to the selectivity filter. regulate membrane electrical excitability and K transport in many cell types We found that TWIK-1 interacts strongly with lipids on the cytoplasmic side generating and controlling the membrane resting potential. The function of this of the membrane through hydrogen bonding, which is higher compared to the channel although simple is primordial for physiological processes such as the periplasmic side. Positive amino acid residues Lysine (176, 275, 278) and creation and the propagation of the neuronal action’s potential, the regulation Arginine (155, 162, 163, 171) alone contribute to one third of the hydrogen of cellular volume, the muscular contraction and the cardiac pulse. bond made by cytoplasmic side with protein. Furthermore, we explored the Their physiological importance is highlighted by the fact that genetically in- effects of mutating the hydrophobic L146 and L261 residues to Asparagine herited defects in Kir channels are responsible for a wide-range of channelopa- on solvent accessibility to the channel’s selectivity filter and compare to the thies including Andersen’s syndrome a pathology that can cause periodic steered CV model. paralysis or serious heart problems. To date unfortunately, this disease does not have any effective treatment. Posters: Transcription The goal of these studies is to understand how the channel gates and what are the conformational changes required. Our work focused on the KirBac3.1 chan- 1033-Pos nel, homologous to Kir2.1 human. We started with known crystallographic data Nuclear Myosin VI Stabilizes RNA Polymerase II in Transcription from the KirBac3.1WT and two mutants S129R and W46R. We explored Factories conformational changes using Molecular Dynamics using Excited Normal Yukti Hari Gupta1, Natalia Fili1, Alia dos Santos2, Teng-Leong Chew3, Modes (MDeNM), a new method which gives access to vibrational motions Jesse Aaron3, Lin Wang4, Christopher P. Toseland2. (Molecular Dynamics) and global motions (Normal Modes) at the same time. 1University of Kent, Canterbury, United Kingdom, 2School of Biosciences, MDeNM successfully opened the closed state of the protein giving the oppor- University of Kent, Canterbury, United Kingdom, 3Advanced Imaging tunity to identify the key motions involved in the gating such as the role of the Center, HHMI Janelia, Ashburn, VA, USA, 4Research Complex - Octopus, cytoplasmic domain, the slide-helix as well as the transmembrane helices dur- STFC, Harwell, United Kingdom. ing the opening of the channel. In parallel, HDX-MS Spectrometry studies had While it is widely accepted there is temporal regulation of , been performed and gave important information on the flexibility of the protein. more recently there has been increase studies of spatial regulation. Indeed, tran- Experimental and theoretical studies were compared for validation. scription is known to occur within discrete foci throughout the nucleus with In addition, human Kir2.1 was purified and imaged with a Titan Krios micro- RNA Polymerase II arranged in hubs or factories. The factories bring together cope. The structure will help to understand the gating mechanism of this human several genes and arrange the chromatin to enable simultaneous gene expres- channel. sion. The hubs are estimated to contain 4-30 RNAPII molecules in a region

BPJ 9355_9357 Monday, March 4, 2019 209a of 40-200 nm. Despite this quantitative knowledge, the mechanism of hub as- the fact that the sequence is shared. While these splicing variations can be iden- sembly, organisation and stability remains unknown. tified using ensemble methods such as RNA-seq, this does not preserve spatial To this end, we have used advanced single molecule techniques to visualise information. We demonstrate a new in situ fluorescence technique based on RNAPII hubs and track their dynamics in live cells. Following our previous sequential barcoding and hybridization chain reaction, which can uniquely work on nuclear myosin VI, we know myosin-actin interactions are critical identify RNA isoforms in both budding yeast and C. Elegans. We demonstrate for RNAPII transcription. Using STORM imaging, we found a decrease in this technique on the SUS1 gene in single yeast cells and on the NURF1 gene in RNAPII hubs when myosin VI or actin was perturbed. Moreover, using 3D sin- the C. Elegans germline. We report the spatial distribution of five classes of iso- gle molecule tracking we found an increase in RNAPII mobility when myosin form of the NURF1 gene. We demonstrate the specificity of the strand displace- VI was knocked-down or inhibited. We conclude that myosin VI is critical for ment based probe design and the ability to deconstruct the amplified DNA the RNAPII hubs to enable gene expression whereby the loss of myosin VI ac- polymer for repeated hybridizations using a shared labeled sequence. Our tivity leads to a disruption of RNAPII organisation. method represents a cost effective means to fully characterize splicing variation Lastly, we show how myosin VI uses a load-induced anchoring ability to stabilise of an RNA transcript at the single molecule and single cell level. RNAPII within the hubs as transcription occurs. In this manner, myosin VI uses its force-sensing properties to regulate gene expression. For the first time, we 1037-Pos reveal how the properties of myosin are specifically required for transcription. G-Quadruplex Forming Sequence Modulated Transcription Chun-Ying Lee1, Christina McNerney2, Kevin Ma1, Sua Myong1. 1034-Pos 1Biophysics, Johns Hopkins University, Baltimore, MD, USA, 2Biology, Nuclear Myosin VI Regulates Estrogen Receptor Driven Gene Expression Johns Hopkins University, Baltimore, MD, USA. Yukti Hari Gupta1, Natalia Fili1, Alia dos Santos2, Teng-Leong Chew3, G-quadruplex (G4) is a noncanonical secondary structure that forms in G-rich se- Jesse Aaron3, Lin Wang4, Christopher P. Toseland2. quences of DNA or RNA. G4 has been implicated in gene regulation and human 1University of Kent, Canterbury, United Kingdom, 2School of Biosciences, disease. Current evidences indicate that formation of G4 in DNA and RNA mod- University of Kent, Canterbury, United Kingdom, 3HHMI Janelia, Ashburn, ulates replication, transcription and translation. It’s hypothesized that G4 can act as VA, USA, 4STFC Research Complex, Harwell, United Kingdom. a barrier to repress DNA and RNA polymerase (RNAP),but itcan also facilitate the Myosins are actin-based molecular motor which are involved in various same process by keeping DNA unwound, for example. However, how G4 forming cellular processes including cell migration and endocytosis. More recently, sequenceimpacts enzymatic processof polymerase isstill unclear.Here,we sought both myosins and actin have been directly implicated in transcription through to investigate if and how G4 forming sequences at 5’-UTR alters transcription by interactions with the three mammalian RNA , yet the precise func- comparing G4 sequences inserted in template vs. non-template orientation. Using tion remains unknown. in vitro transcription assay coupled with molecular beacon (dequenching probe), Myosin VI is over-expressed in breast, ovarian and prostate cancers, and when we found that all tested G4 sequences positioned in template strand reduced, but depleted inhibits proliferation and cell migration. We propose this is related to a the same G4 in non-template strand enhanced mRNA production. To dissect direct link between Myosin VI and nuclear receptors, and its role in hormone- various stages involved in transcription, we devised four sequential single mole- dependent gene transcription. We have identified that myosin VI is not only cule assays which enablemeasurement ofT7 RNAP binding, initiation, elongation, trafficked to the nucleus upon hormone stimulation, but it is also bound to pro- mRNA product formation. Our result shows that while binding and initiation are motor regions of hormone-responsive genes. Depletion of myosin VI leads to a similar, the proportion of successful elongation and the final mRNA yield is lower loss of gene expression and subsequently an inhibition of proliferation. when G4 is on template and higher when G4 is on non-template. In addition, the Using advanced single molecule microscopy techniques we have found that duration of transcription bubble at the G4 sequence is shorter on template than myosin VI regulates the assembly of RNA Polymerase II transcription factories in non-template, suggesting a potential secondary structure such as G-loop or R- in response to stimulation. We propose a model whereby myosin VI uses a loop modulating the transcriptional output at the G4 site. Our study provides a load-induced anchoring ability to hold the complexes together as transcription detailed kinetic map of transcription of G4 bearing template in which G4 orienta- occurs. In this manner, myosin VI uses its force-sensing properties to regulate tion plays a major role in determining the transcriptional output. hormone-induced gene expression. 1038-Pos 1035-Pos Transcription Machine Studied in a Nutshell on T7 RNA Polymerase Determining Dynamics of RNA Polymerase Elongation and Pausing using Mechanochemistry, Fidelity Control, and Bursting Activity Nanopore Tweezers Jin Yu. Ian C. Nova1, Jonathan M. Craig2, Andrew H. Laszlo2, Complex System Research, Beijing Computational Science Research Center, Abhishek Mazumder3, Henry Brinkerhoff2, Ian M. Derrington2, Beijing, China. Matthew T. Noakes2, Jonathan W. Mount2, Jesse Huang2, Jasmine Bowman2, RNA polymerase (RNAP) is an essential enzyme that transcribes DNA into Richard H. Ebright3, Jens H. Gundlach2. messenger RNA to further express proteins. Bacteriophage T7 RNAP is widely uti- 1Molecular Engineering, Univ Washington, Seattle, WA, USA, 2Physics, lized in lab gene expression system and synthetic circuit as it transcribes self- Univ Washington, Seattle, WA, USA, 3Rutgers University, New Brunswick, sufficiently, without transcription factors, and with high . We NJ, USA. have investigated T7 RNAP on both its mechano-chemical couplings and fidelity Single-molecule picometer resolution nanopore tweezers (SPRNT) is a single- control during transcription, combining physical modeling with structural dy- molecule technique that enables observation of enzyme movement along nu- namics simulations. Inparticular, we have implementedextensive atomistic molec- cleic acids under an applied force. SPRNT measures enzyme position with ular dynamics (MD) simulations and constructed the Markov state model (MSM) to sub-Angstrom spatial and millisecond temporal resolution, while simulta- investigate product release and translocation process of T7 RNAP, as it couples neously providing DNA sequence within the enzyme. We use SPRNT to Brownian thermal motions with essential structural elements to assist PPi release, monitor many E. coli RNA Polymerase (RNAP) core complexes during tran- to facilitate the RNAP translocation, and to prevent backtracking. Meanwhile, we scription elongation and pausing with an assisting force. We determine that systematically quantified the nucleotide selectivity of this non-proofreading RNAP during elongation at low [NTP], RNAP primarily stalls in a post-translocated at multiple kinetic checkpoints prior to the full nucleotide incorporation. Essential state, with brief deviations forward to a hyper-translocated state and backwards selection against the non-cognate nucleotide species appears to happen at early to a pre-translocated state. The rates and frequencies of these transitions vary checkpoints at the pre-insertion and right after, with a critical amino acid serving with DNA sequence. During transcription pausing at a pre-determined pause as a paw for selective ratcheting RNAP along the DNA. In addition, we also model sequence, we observe RNAP entering a partially-translocated state, with the complete transcription process along with RNAP traffic, as positive supercoil- brief deviations to backtracked, pre-, post- and hyper-translocated states. We ing is accumulated in front of a leading RNAP to inhibit the transcription, and then develop a model for RNAP pausing by varying the applied force and moni- being resolved gradually to allow transcriptional bursting. toring RNAP mutants with SPRNT. 1039-Pos 1036-Pos Tfiih Generates a Six-Base-Pair Open Complex during Eukaryotic Tran- RNA Isoform Identification via Sequential Hybridization and Strand scription Initiation Displacement Based Amplication in the Caenorhabditis Elegans Germline Eric A. Galburt1, Eric J. Tomko1, James Fishburn2, Steven Hahn2. 1 Gable M. Wadsworth, Harold D. Kim. Biochemistry and Molecular Biophysics, Washington University in St 2 Dept Physics, Georgia Tech, Atlanta, GA, USA. Louis, St Louis, MO, USA, Fred Hutchinson Cancer Research Center, RNA splicing leads to the generation of multiple distinct isoforms which are Seattle, WA, USA. developmentally regulated and highly homologous. Current methods for iden- The initiation of messenger RNA transcription in Eukaryotes is directed by the for- tifying transcripts in situ are unable to uniquely identify most isoforms due to mation of a megaDalton-sized multi-protein complex known as the pre-initiation

BPJ 9355_9357 210a Monday, March 4, 2019 complex (PIC). After PIC formation, double-stranded DNA is unwound to form a RNAP mobile elements and promoter DNA the discriminator direct, are major single-stranded DNA bubble and the template strand is loaded into the polymerase determinants of the lifetime and the structure of the RNAP-promoter open com- active site. Initial DNA opening is ATP-dependent and is catalyzed by Ssl2/XPB, plex (OC). The OC lifetime and rate of formation can both vary by 4 orders of a dsDNA translocase subunit of the basal transcription factor TFIIH. In yeast, tran- magnitude for different promoter sequences, and together regulate the rate of scription initiation proceeds through a scanning phase where downstream DNA is initiation and escape of RNAP from these promoters. We are investigating a searched for optimal start-sites. Here, to test different models for initial DNA series of discriminators including those of the long-lived lPR OC (GGTTGC) opening and start-site scanning, we measure the size of the DNA bubble formed and the short-lived rrnBP1 OC (GCGCCACC), in the context of the upstream by Saccharomyces cerevisiae PICs in real time using a single-molecule magnetic lPR promoter and a modified lPR transcribed region. Fast kinetic methods with tweezers assay. We show that ATP hydrolysis by Ssl2 leads to the opening of a 6 filter binding detection are used to determine overall rate constants for open base-pair (bp) DNA bubble that is expanded to 13 bp in the presence of NTPs. complex formation and dissociation, and to dissect contributions to these rate These observations support a two-step DNA opening model wherein ATP- constants from initial equilibria involving closed or open complexes (CC or dependent Ssl2 translocation leads to a 6 bp open complex which RNA polymer- OC), and the rate-determining isomerizations step between them. Our results ase II expands via NTP-dependent RNA transcription. to date indicate that the discriminator affects primarily the steps that stabilize the initial OC, and has only limited effects on the DNA closing rate constant 1040-Pos or on the overall second order rate constant for OC formation. Nuclear Ndp52 - a Putative Transcription Regulator From previous studies, interactions of in-cleft (ss) DNA and downstream (ds) Alia dos Santos1, Lin Wang2, Christopher P. Toseland1. 1 DNA with RNAP change in the process of stabilization of the initial OC. Re- School of Biosciences, University of Kent, Canterbury, United Kingdom, s70 2 gions 1.1 and 1.2 of and downstream mobile elements of core RNAP are Research Complex, STFC, Harwell, United Kingdom. involved, and the orientation of the downstream duplex is changed. We are NDP52/CALCOCO2 has been well characterised as an autophagy receptor, investigating these interactions and conformational changes for different dis- however a population of the protein exists in the nucleus. Moreover, NDP52 criminators in more detail, and the relationship to initiation and promoter shares significant homology to a transcription co-activator CoCoA. We have escape of RNAP. recently described the activation of Myosin VI by NDP52 in RNA Polymerase II (RNAPII)-dependent transcription. Although depletion of NDP52 is known 1043-Pos to reduce the steady-state levels of mRNA in cells, the molecular role of Regulation of Mycobacterial RNA Polymerase Promoter Escape Kinetics NDP52 in the nucleus is not clear. by Transcription Factors Card and RbpA We are using cell biology combined with biophysical techniques to understand Drake Jensen1, Ana Ruiz Manzano1, Christina L. Stallings2, the functions of NDP52 in nucleus. Using SEC-MALS, Microscale Thermopho- Eric A. Galburt1. resis and Electron Microscopy, we can show that NDP52 is an elongated dimer 1Biochemistry and Molecular Biophysics, Washington University School of of approximately 110 kDa that is capable of binding double-stranded DNA Medicine, St. Louis, MO, USA, 2Molecular Microbiology, Washington in vitro with high affinity. This interaction can also be observed in cells through University School of Medicine, St. Louis, MO, USA. Chromatin Immunoprecipitation (ChIP). We observed NDP52 bound to pro- The process of bacterial RNA polymerase (RNAP) escaping the promoter re- knock-down moter regions of several oestrogen-responsive genes. When we gion during transcription initiation can often be rate limiting, making kinetic NDP52 in these cells, we see dysregulation of known gene-targets of the oestro- regulation of escape a common strategy employed to control the rates of tran- gen receptor, as well as of those relating to apoptosis, DNA repair and recruit- scription. Using a pre-steady state ensemble fluorescence assay, we present the ment of RNAPII. Our proteomics data now suggest that this regulation might first in-depth kinetic analysis of initial nucleotide incorporation and promoter not only be sustained through direct interactions with DNA, but also through escape for the Mycobacterium tuberculosis (Mtb) RNAP.We show that in rela- protein-protein interactions with binding partners of NDP52 in the nucleus. tion to E. coli RNAP, Mtb displays slower initial nucleotide incorporation but Overall, we propose that NDP52 may be functioning as a transcription regulator faster overall promoter escape kinetics on the MtbrrnAP3 promoter. Further- in human cells. To test this idea, we have started using super-resolution imag- more, in the context of the essential transcription factors CarD and RbpA, ing - PALM-STORM, combined with Cluster analysis software, to image Mtb RNAP promoter escape is delayed, albeit through different mechanisms. NDP52 at single-molecule level in combination with RNAPII and other candi- We also see that these factors increase the inactive fraction, i.e. complexes date binding partners. that are unable to escape the promoter. Given that CarD and RbpA affect mul- 1041-Pos tiple kinetic intermediates during transcription initiation, we modelled hypo- Characterizing Transient Intermediates in Productive RNAP Transcrip- thetical sets of rate constants derived from the initiation kinetics to suggest tion Initiation that CarD and RbpA can serve to both activate and repress transcription in a rrnA Claire E. Evensen, Kate Henderson, M. Thomas Record. promoter-dependent context. For instance, on the P3 promoter, where Dept Biochemistry, Univ Wisconsin Madison, Madison, WI, USA. DNA-opening is rate limiting, both CarD and RbpA activate transcription, Transcription initiation plays a key role in the regulation of gene expression, and a whereas on promoters that are rate-limited at escape, the prediction is that these biophysical characterization of its kinetics and mechanism is needed. To determine factors lead to transcriptional repression. the mechanism of promoter escape by E. coli RNA polymerase (RNAP), we deter- mine the kinetics of RNA-DNA hybrid extension in productive complexes during 1044-Pos Transcription Factor Regulation of RNA Polymerase’s Torque Generation single-round RNA synthesis by RNAP-lPR opencomplexes. Rapid-quenchmixing and phosphorimager analysis of gel separations determine amounts of each short Capacity 1,2 1,2 1,2 2 RNA over time is presented. At 19 oC, release of short RNAs from RNA-DNA hy- Jie Ma , Chuang Tan , Xiang Gao , Robert M. Fulbright , 3 1,2 brids is slow and short RNAs detected are either intermediates from full-length Jeffrey W. Roberts , Michelle D. Wang . 1Howard Hughes Medical Institute, Ithaca, NY, USA, 2Department of RNA synthesis or from stalled, non-productive complexes. Analysis of productive 3 initiation kinetic data yields second-order rate constants for each step in hybrid Physics-LASSP, Cornell University, Ithaca, NY, USA, Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA. extension up to the escape point (11 bp for lPR promoter). These rate constants are strikingly smaller than elongation rate constants and differ greatly for different During transcription, RNA polymerase (RNAP) supercoils DNA as it forward- steps. Rate constants for the synthesis of 3-mer, 6-mer, and 10-mer are much larger translocates. Accumulation of this torsional stress in DNA can become a road- thansubsequent steps, resulting in transient accumulation of these threeshortRNAs block for an elongating RNAP and thus should be subject to regulation during at all NTP concentrations investigated. We propose these reductions in rate con- transcription. Here we investigate whether and how a transcription factor may stants are due to a build-up of translocation stress (scrunching, steric) during hybrid regulate the torque generation capacity of RNAP and torque-induced RNAP extension and indicate stepwise breaking of in cleft, 10, and 35 DNA-promoter stalling. Using a real-time assay based on an angular optical trap, we found contacts to relieve translocation stress and allow promoter escape. that under a resisting torque, RNAP was highly prone to extensive backtracking. However, the presence of GreB, a transcription factor that facilitates the cleav- 1042-Pos age of the 3’ end of the extruded RNA transcript, greatly suppressed backtrack- Effects of Discriminator Changes on Open Complex Formation, Stabiliza- ing and remarkably increased the torque that RNAP was able to generate by 65%, tion, and Transcription Initiation from 11.2 to 18.5 pN,nm. Variance analysis of the real-time trajectories of Hao-Che Wang. RNAP position at a stall revealed the kinetic parameters of backtracking and Biophysics, University of Wisconsin Madison, Madison, WI, USA. GreB rescue. These results demonstrate that backtracking is the primary mech- The discriminator is the region of promoter DNA between the key 10 recog- anism that limits transcription against DNA supercoiling and the transcription nition region for RNA polymerase (RNAP) and the transcription start site factor GreB effectively enhances the torsional capacity of RNAP. These findings (TSS). The length, sequences of the discriminator, and the interactions between broaden the potential impact of transcription factors on RNAP functionality.

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Posters: Protein-Nucleic Acid Interactions I whether the protein first binds to unbent DNA and then deforms it, or if pre-bent DNA conformations are ‘‘captured’’ by protein binding. The former mecha- 1045-Pos nism would be supported by discovery of conditions where unbent DNA is Predicting Diffusion Coefficients of DNA-Protein Complexes by Convex bound by yNhp6A. Here, we examine the possibility of a ‘‘bind-then-bend’’ Hull Modelling mechanism for HMGB proteins. This key question touches on the fundamental Miles Lee, Quan Wang. mechanism by which DNA bending is accomplished by architectural proteins, Princeton University, Princeton, NJ, USA. an area illuminated by few experimental results. To explore conditions under The recent development of single-molecule diffusometry establishes the diffu- which DNA could be unbent within a stable yNhp6A complex, we combined sion coefficient as a new observable in single-molecule experiments to visu- an array of experimental probes: FRET on end-labeled DNA (to monitor alize biomolecular interactions. To decipher the rich information available DNA unbending), fluorescence anisotropy (to monitor complex dissociation), from these experiments, accurate modeling of the hydrodynamic properties and circular dichroism (to monitor protein stability), at various ionic and tem- of biomolecular complexes is needed. Currently available tools for predicting perature conditions. Our studies reveal that at 100 mM NaCl, an 18-base-pair diffusion coefficients of biomolecules, such as Hydropro and HullRad, rely on DNA oligomer remains bound to yNhp6A but starts to unbend, at temperatures the existence of atomic coordinates for the molecule of interest. Composite up to 45 C; above that temperature, dissociation of the complex and unfold- molecular complexes that lack detailed Protein Data Bank entries, but can ing of the protein is detected. In 200 mM NaCl, DNA straightening in the intact be modeled structurally from their constituent parts (i.e. a complex with yNhp6A complex is again detected up to 35 C. Microseconds-resolved laser multiple proteins bound to a single DNA) are frequently encountered in temperature-jump perturbation of the yNhp6A-DNA complex reveal single- exponential FRET loss kinetics that, under conditions dominated by DNA un- experiments but cannot yet be properly modeled for hydrodynamic properties. We present a simple program to predict the diffusion coefficients of bending within complexes (below 45 C at 100 mM NaCl), yield unimolecular composite DNA-protein complexes. Our algorithm extends the computational DNA bending/unbending rates on timescales of 500 us - 1 ms. These data pro- framework introduced by Fleming et al. in the HullRad software to construct a vide the first direct observation of bending/unbending dynamics of DNA bound convex hull of the molecular complex and then uses an ellipsoidal approxima- to yNhp6A, suggesting a bind-then-bend mechanism for this protein, and the tion to compute hydrodynamic parameters. In order to construct the convex first measurement of these rates for a nonspecific DNA-bending protein. hull of the whole complex, the algorithm incorporates a priori knowledge of the complex such as the crystal structures of the constituent biomolecules, 1048-Pos binding site positions, geometry and mechanics of DNA duplexes. The pro- Direct Observation of DNA Target Searching and Cleavage by CRISPR- gram is computationally efficient, integrates naturally with PyMOL, and out- Cas12a puts the 3D convex hull for visualization. We validate the program by Yongmoon Jeon1, You Hee Choi2, Yunsu Jang2, Jiyoung Gu1,3, Cherlhyun Jeong1,3, Sanghwa Lee2, Sangsu Bae4. accurately predicting hydrodynamic parameters of known crystal structures 1 2 of DNA-protein complexes. We then predict diffusion coefficients of the re- Center for Theragnosis, KIST, Seoul, Republic of Korea, Advanced 3 striction enzyme BamHI bound to double strand DNA of different lengths Photonics Research Institute, GIST, Gwangju, Republic of Korea, KHU- and compare these predictions with single-molecule diffusometry measure- KIST Department of Converging Science and Technology, Kyunghee 4 ments. We envision that this program will aid researchers in using diffusion University, Seoul, Republic of Korea, Department of Chemistry, Hanyang coefficient to identify and characterize relevant biological structures at the University, Seoul, Republic of Korea. nanometer scale. Cas12a (also called Cpf1) is a representative type V-A CRISPR effector RNA- guided DNA endonuclease, which provides an alternative to type II CRISPR- 1046-Pos Cas9 for genome editing. Previous studies have revealed that Cas12a has Loops Enhance Transcriptional Roadblocks unique features distinct from Cas9, but the detailed mechanisms of target Wenxuan Xu, Yan Yan, David D. Dunlap, Laura Finzi. searching and DNA cleavage by Cas12a are still unclear. Here, we directly Physics, Emory University, Atlanta, GA, USA. observe this entire process by using single-molecule fluorescence assays to Transcription factors bound to specific sites may represent roadblocks to elon- study Cas12a from Acidaminococcus sp. (AsCas12a). We determine that As- gating RNA polymerases (RNAPs). Also, the DNA loops formed by the long- Cas12a ribonucleoproteins search for their on-target site by a one- range bridging interactions of many transcription factors could be roadblocks. dimensional diffusion along elongated DNA molecules and induce cleavage LacI is a well-understood repressor of transcriptional initiation when bound to in the two DNA strands in a well-defined order, beginning with the non- a strong recognition site (O1) overlapping the promoter. However, the regula- target strand. Furthermore, the protospacer-adjacent motif (PAM) for As- tion of LacI binding involves a loop formed upon simultaneous binding to Cas12a makes only a limited contribution of DNA unwinding during R-loop O1 and a lower affinity O2 binding site 400 bp downstream. In this study, formation and shows a negligible role in the process of DNA cleavage, in either tethered particle microscopy (TPM) or magnetic tweezers (MTs) contrast to the Cas9 PAM. were used to monitor how such LacI binding or looping obstructed elongation. The linear DNA templates contained the T7A1 promoter, a ‘‘near’’ site 250bp 1049-Pos downstream of the promotor, and a ‘‘far’’, O1 binding site another 400bp Electric-Field-Driven Translocation of ssDNA through Hydrophobic further downstream. The ‘‘near’’ site contained either no recognition site or Nanopores O2 binding site. The duration of pauses by RNAP, at the ‘‘far’’ O1 site in Taylor Haynes1, Iain P.S. Smith1, Jayne Wallace2, Jemma Trick3, the template without O2, increased significantly under slight tension that Mark S. Sansom4, Syma Khalid1. opposed transcription and loop formation. At a low LacI concentration 1Dept Chemistry, Univ Southampton, Southampton, United Kingdom, 2Res, without any applied tension, LacI-mediated looping between the ‘‘near’’, Oxford Nanopore Technologies, Oxford, United Kingdom, 3Physics, Kings O2 and the ‘‘far’’, O1 operators slightly shortened pauses by RNAP at the Coll London, London, United Kingdom, 4Dept Biochemistry, Univ Oxford, O1 site. In other words, the vacant O2 operator may compete with the O1 Oxford, United Kingdom. operator for binding LacI, clearing the path for RNAP. However, the same The accurate sequencing of DNA using nanopores requires control over the loops lengthened the pauses by RNAP at O2, which validates similar studies speed of DNA translocation through the pores and also of the DNA conforma- using atomic force microscopy. Dogma holds that looping delivers LacI tion. Our studies show that ssDNA translocates through hourglass-shaped pores captured at O2 to O1 to enhance repression, but these loops attenuate elonga- with hydrophobic constriction regions when an electric field is applied. The tion through O2 as well. constriction provides a barrier to translocation and thereby slows down DNA movement through the pore compared with pores without the constriction. 1047-Pos We show that ssDNA moves through these hydrophobic pores in an extended DNA Bending/Unbending Rates Revealed for Nonspecific Architectural conformation and therefore does not form undesirable secondary structures that DNA-Binding Protein yNhp6A may affect the accuracy of partial current blockages for DNA sequencing. Viktoriya Zvoda1, Manas K. Sarangi1, Molly Nelson Holte2, Nicole A. Becker2, Justin P. Peters2, Louis J. Maher III,2, Anjum Ansari1. 1050-Pos 1Department of Physics, University of Illinois at Chicago, Chicago, IL, USA, TIN2 is an Architectural Protein Stabilizing TRF1 at Telomere 2Department of Biochemistry and Molecular Biology, Mayo Clinic College Hai Pan1, Saroj Dangi1, Parminder Kaur1, Pengyu Hao1, Keith Weninger1, of Medicine and Science,, Rochester, MN, USA. Robert Riehn1, Patricia Opresko2, Hong Wang1. The yeast Nhp6A protein (yNhp6A) is a nonspecific DNA-binding member of 1Physics Department, NC State University, Raleigh, NC, USA, 2Department the eukaryotic HMGB family of chromatin factors that promote apparent DNA of Environmental and Occupational Health, University of Pittsburgh, flexibility. yNhp6A sharply bends DNA by >60. However, it remains unclear Pittsburgh, PA, USA.

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Telomeres, consisting of duplex TTAGGG repeats and associating with pro- 1053-Pos tein complexes at chromosome ends, play a crucial role in maintaining the sta- Bridge Helix of Cas9 Impacts Target DNA Cleavage bility of chromosomes. The protein complex - shelterin contains six subunits Rakhi Rajan. (TRF1, TRF2, RAP1, TIN2, TPP1 and POT1), which bind to telomeres and Chemistry and Biochemistry, University of Oklahoma, Norman, OK, USA. protect the chromosome ends from DNA repairing, and recruit telomerase CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune when chromosome gets shortened abnormally. Among these six subunits, protection for bacteria and archaea against intruding genomic materials. Cas9, a TIN2 has no affinity to either double-strand or single-strand DNA. However, type II CRISPR effector protein, is widely used for gene editing applications it is a core component bridging the double-strand DNA binding proteins since a single guide RNA (sgRNA) can direct Cas9 to cleave DNA targets of (TRF1 and TRF2) to single-strand DNA binding protein complex (TPP1- interest. The complementary base pairing between the guide region of the POT1). Loss of TRF1 or TRF2 binding domain in TIN2 can trigger DNA sgRNA and the target DNA is essential to activate the endonuclease sites of damage response. Moreover, TIN2 without the TPP1 binding domain is Cas9. In addition, a three to five nucleotide long region, termed protospacer capable of fully supporting the stabilization of TRF1 and TRF2/RAP1. adjacent motif (PAM), adjacent to the RNA-DNA complementary region is Despite the significance of TIN2 in telomere maintenance, the mechanism un- essential for Cas9 function. The mechanism of RNA-mediated conformational derlying TIN2 remains elusive. To understand how TIN2 affects TRF1 bind- changes in Cas9 and how they contribute to faithful DNA targeting is being ing dynamics and how TIN2 influences with TRF1-mediated telomeric DNA pursued actively for developing Cas9 variants with minimal off-targeting pairing, we conducted Atomic Force Microscopy (AFM) and used nanochan- effects. nels confined DNA to study the telomeric DNA conformation upon TRF1 and It has been previously established that an arginine-rich bridge helix (BH) pre- TIN2 binding. We observed high-order protein-DNA complexes formation sent in Cas9 is critical for its activity. In the present study, we show that sub- indicating TIN2 facilitates TRF1 accumulation on telomeric DNA. We also stitutions within the BH of Streptococcus pyogenes (Spy) Cas9 impair DNA carried out single molecule fluorescence technique to investigate how TIN2 cleavage activity compared to the wild-type protein. The DNA cleavage defi- affects TRF1 binding dynamics on telomeric DNA sequences. Our results ciency is more pronounced in DNA targets that are mismatched with the guide show that TIN2 can accelerate DNA-DNA pairing and stabilize TRF1 on te- region of sgRNA at the PAM proximal side. BH substitutions cause an accumu- lomeric DNA. lation nicked products, leading to a reduction of target DNA linearization. Gene editing experiments performed on human cells (HEK293T) showed that even 1051-Pos though the BH-variant is not as efficient as the wild-type protein in editing Solution Dynamics in Histone-Based Archaeal Chromatin all the tested sites, the off-target effect on edited sites is considerably reduced Samuel Bowerman1,2, Karolin Luger1, Jeff Wereszczynski2. when compared to the wild-type protein. Mechanistic analyses show that the 1Department of Biochemistry and Howard Hughes Medical Institute, 2 BH-substitution does not reduce sgRNA-binding affinity, but substantially re- University of Colorado Boulder, Boulder, CO, USA, Department of Physics duces the stability of the protein-RNA complex. We propose BH-substitution and Center for Molecular Study of Condensed Soft Matter, Illinois Institute of as a mechanism to fine-tune CRISPR-based gene editing applications since Technology, Chicago, IL, USA. BH is conserved in several CRISPR systems. Over the last two decades, histone-fold containing proteins have been identi- fied in most known Archaea, and their similarities in sequence and structure 1054-Pos strongly suggest that they share a common evolutionary ancestor to the eu- Molecular Dynamics Simulations of RNA-Recognition Motif Complexed karyotic histones that form the nucleosome core particle. Recently, crystal with CAC-Containing RNA structures of archaeal histones in complex with DNA have shown that they Shan Chang, Hang Shi, Ren Kong. are capable of forming oligomers that bind DNA in superhelical turns that Institute of Bioinformatics and Medical, Jiangsu University of Technology, are identical to the nucleosome; however, archaeal chromatin can stack as Changzhou, China. an extended superhelix, in contrast to the defined particulate character of nu- The RNA-recognition motifs (RRMs) are the most abundant RNA-binding do- cleosomes. Here, we have conducted molecular dynamics simulations on mains in higher vertebrates, which play diverse roles in post-transcriptional archaeal histone chromatin oligomers of varying sizes, and we compare these gene expression processes. In this work, the free RRM domain and its RNA- systems with one another and with nucleosomes in order to elucidate the binding complex structure are studied by molecular dynamics (MD) simula- structure-dynamics-function relationship of this superhelical stacking. Our tions. Principal component analysis (PCA) and Molecular mechanics models provide further proof that the interaction between L1 loops in neigh- Poisson-Boltzmann surface area (MM/PBSA) methods were used to explore boring archaeal histone dimers is paramount to chromatin stack formation, as the dynamical and recognition mechanisms of RRM domain and CAC- its absence results in a highly dynamic complex. Furthermore, we find that containing RNA. Through the motion mode comparison between free RRM eukaryotic histones are tighter binders of DNA, even when reduced to the domain and complex systems, it’s found that the RNA-binding interface of hexasome complex and in the absence of the highly-charged histone tails. free RRM domain is not stable, especially the C-terminal loop. The C-terminal Together, these data provide further insight into the structural origin of chro- loop in free RRM domain adopts the conformation similar to that in the com- matin, and display an energetic rationale for the evolution of the nucleosome plex system. The energy decomposition strategy of MM/PBSA was used to complex. analyze the contribution of each residue or nucleotide. In complex system, 20 residues make favorable contributions for RNA binding, which are posi- 1052-Pos tioned on the homodimerization interface (such as Arg38, Glu39 and Lys36) Mutation of the Dead-Box ATPase Prp5 Impacts Dynamics of the Reca- or the RNA-binding interface of RRM domain (such as Phe27, Phe65, Like Domains and Branch Site Usage During Pre-mRNA Splicing Lys100, Thr103 and Lys104). These residues are also reported by the previous David H. Beier, Tucker J. Carrocci, Aaron A. Hoskins. experiments, which may form hydrogen bonds or intermolecular stacking inter- Dept Biochemistry, Univ Wisconsin Madison, Madison, WI, USA. actions with RNA. Meanwhile, 11 residues in RRM domain contribute unfavor- In the first ATP-dependent step in splicing, the U2 snRNP becomes paired able energies for RNA binding. These residues are mainly located on the loop to the intron branch site (BS), identifying the nucleophile that will be used structure or the regions far away to the RNA binding interface. In the energy for 5’ splice site cleavage by the spliceosome. This process requires the decomposition of RNA, all of 4 nucleotides make favorable energy contribu- DEAD-box ATPase Prp5. Mutations in Prp5 that impact ATPase activity tions. The three nucleotides CAC contribute most of the binding energies, so also affect usage of weak BS with limited complementarity to the U2 snRNA. these 3 nucleotides are more stable than U1 in the representative structures. In addition, retention of Prp5 on the pre-spliceosome inhibits tri-snRNP addi- This study provides some new insights into the RNA recognition mechanisms tion, resulting in a block in spliceosome assembly and potential checkpoint. of RRM domain. How changes in Prp5 ATPase connect to its retention or release from the pre-spliceosome is unclear. We have used smFRET to study conformational 1055-Pos changes between Prp5’s RecA-like domains. Our data support formation of Investigating the Effect of Various FMRP Isoforms on microRNA a stable, ‘‘twisted open’’ conformation of the domains in the absence of li- Biogenesis gands. By monitoring the opening and closure of the domains in response Joshua A. Imperatore, John Roth, Mihaela Rita Mihailescu. to ATP and RNA, we correlate Prp5’s conformational equilibria with BS us- Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA, USA. age in yeast. Together our data support a model in which retention or release Fragile X syndrome (FXS) is the most common form of inherited mental of Prp5 from the pre-spliceosome is dependent on conformational changes impairment and is a result of a trinucleotide repeat expansion in the 5’ untrans- of the protein. Selection of BS by the splicing machinery may rely on a lated region (UTR) of the fragile X mental retardation-1 (FMR1) gene. The coordinated set of ATP-driven conformational switches among U2 snRNP expression of the protein product of this gene, fragile X mental retardation pro- components. tein (FMRP), is subsequently lost in patients with FXS. FMRP functions as a

BPJ 9358_9361 Monday, March 4, 2019 213a transporter protein and translational regulator for specific neuronal messenger Our results suggest that the DNA/Brg1 binding mechanism is different in pres- RNA (mRNA) targets. Recent studies have additionally proposed the involve- ence and absence of the AT-hook segment in the Brg1 sequence. ment of FMRP in the microRNA biogenesis pathway. These short, non-coding microRNAs (miRNAs) associate with the RNA-induced silencing complex 1058-Pos (RISC) and bind to target mRNAs, regulating translation and degradation Studying Nucleosome Assembly via FRET 1 1 2 events. Previous studies have demonstrated that FMRP interacts with members Caitlin Aguirre , Loiselle Gonzalez Baez , Elizabeth Jamieson , 3 of the RISC, such as Argonaute 2 (AGO2) and Dicer, the endonuclease Megan E. Nunez . 1Biochemistry, Wellesley College, Wellesley, MA, USA, 2Chemistry, Smith responsible for the final cleavage step in the miRNA biogenesis pathway. In 3 this study, we hypothesize that the FMRP isoforms 1 and 7 (ISO1 and College, Northampton MA, MA, USA, Chemistry, Wellesley College, ISO7), as well as their phosphorylated mimics (ISO1-P and ISO7-P), regulate Wellesley, MA, USA. the maturation of miR-125a and miR-125b-2 through interactions with its pre- Oxidative damage to DNA by reactive oxygen species (ROS) can result in the mature (pre-miRNA) form and the enzyme Dicer. Here, we utilized native formation of the highly mutagenic Spiroiminodihydantoin (Sp) lesion. The Sp polyacrylamide gel electrophoresis (PAGE) in vitro Dicer assays to evaluate lesion disrupts the shape of the DNA double helix due to the introduction of a the maturation of miR-125a and miR-125b-2 in the presence of various propeller-like chiral center. If left unrepaired, oxidative stress is harmful to FMRP isoforms. DNA stability as it may lead to permanent mutations, cancer, cellular aging or apoptosis. Our lab has previously demonstrated that Sp lesions cause subtle 1056-Pos changes in nucleosome stability and positioning. Nucleosomes are formed Mismatch Recognition by Msh2-Msh6: Role of Structure and Dynamics when 146 base pairs of DNA are wrapped around octamers of histone pro- Zane Lombardo, Yan Li, Meera Joshi, Manju M. Hingorani, Ishita Mukerji. teins. These structures compact our DNA and control access by repair pro- Dept Molec Bio/Biochem, Wesleyan Univ, Middletown, CT, USA. teins, transcription factors, and polymerases. We are using Fluroescence The mismatch repair (MMR) pathway is responsible for maintaining the Resonance Energy Transfer (FRET) to explore in more detail how the Sp integrity of the genome by correcting errors such as insertions, deletions, or lesion influences nucleosome assembly and disassembly. We synthesized mismatched bases formed during DNA replication. MMR is initiated by and characterized 146-mer duplexes with Cy3 and Cy5 fluorphores and Sp le- Msh2-Msh6 (Msh26), a heterodimeric protein that recognizes single base mis- sions at various locations, allowing us to assemble a variety of nucleosome matches and small insertion/deletion loops in DNA. Much is known about the core particles containing fluorescent donor-acceptor pairs. In undamaged nu- structure of Msh26 and its role in MMR, but the precise mechanism for recog- cleosomes, FRET occurs between the donor and acceptor fluorophores consis- nition of mismatched DNA remains unclear. In this study, we use the fluores- tent with their proximity; gradual unfolding of the nucleosomes by titration cent DNA base analog 6-MI (6-methylisoxanthopterin) at or adjacent to a with NaCl leads to a loss in acceptor fluorescence, consistent with peeling mismatch site to observe changes in the dynamics of single base mismatches of the DNA ends from the histone octamer surface. We are measuring quan- upon interaction with Msh26. We observe a 10 nm spectral shift in 6-MI titatively the correlation between the stability of the nucleosome and presence peak emission upon binding, consistent with an increase in local hydrophobic- and location of the lesion and hope to elucidate whether the histone tetramer ity possibly reflecting the intercalation of a Phe residue at the mismatch as or dimer is more sensitive to DNA distortions. These experiments will observed in the Msh26 X-ray crystal structure. We also find that the amount advance our understanding of how the Sp lesion affects the process of of protein-induced bending is the same for all substrates, as measured by en- DNA compaction into nucleosomes. ergy transfer experiments and does not correlate with binding affinity. Msh26 1059-Pos K < exhibits a higher affinity for DNA substrates ( d 25 nM) when the 6-MI Wild-Type Fus Rescues Altered RNA Binding of ALS-Linked FUS Mutant < probe exhibits a fast rotational correlation time ( 0.5 ns) indicative of Kevin Rhine1, Jaya Sarkar2, Amirhossein Ghanbari Niaki2, Xinyi Cai2, greater mobility. Msh26 has a low affinity for poorly repaired mismatches Gabby Vidaurre1, Sua Myong2. such as T:T and T:C. These mismatches exhibit significantly slower 6-MI 1Department of Biology, Johns Hopkins University, Baltimore, MD, USA, rotational correlation times ( 1-2ns) signifying reduced local mobility that 2Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA. is comparable to that of homoduplex DNA. Thus, we attribute the relatively Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease low repair efficiency in vivo to poor recognition of the T:T and T:C mis- that affects more than 10,000 people in the United States. ALS is hallmarked matches associated with their reduced motion. Msh26 binding affinities by the progressive loss of neurons, leading to defects in motor function and for mismatched substrates linearly correlate with relative mobility at the eventually death. One molecular phenotype of ALS is the aggregation of pro- mismatch site and we suggest this mobility influences recognition and binding teins such as Fused in sarcoma (FUS), which causes loss of protein function affinity. and gain of toxicity. FUS is an RNA-binding protein involved in mRNA splicing and export. FUS bears prominent intrinsically-disordered regions 1057-Pos (IDRs) that drive liquid-liquid phase separation (LLPS). Aggregates of Molecular Simulations Discern the Cooperative Binding of Human mutated FUS proteins (ALS FUS) are a pathogenic signature found in neuro- Brahma-Related Gene 1 Bromodomain and At-Hook Regions in DNA degenerative diseases including ALS and frontotemporal lobar dementia Binding (FTLD). How an initial mutation in Fus eventually leads to ALS is unclear, Stefania Evoli, Jeffery M. Wereszczynski. especially in the context of RNA binding and aggregation. Our preliminary Dept Physics, Illinois Inst Tech, Chicago, IL, USA. data suggests that ALS FUS displays defective RNA binding. A heterozygous Human brahma-related gene 1 (Brg1) is a central ATPase in the human SWI/ patient has both wild-type FUS and ALS FUS, and it’s also unclear how these SNF chromatin remodeling complex. It contains a bromodomain, which asso- two versions of the same protein may interact. We investigated if and how ciates with acetylated nucleosomes and is critical in DNA damage response ALS FUS interacts with wild-type FUS and potentially alters FUS-RNA inter- along with a small AT-hook motif that recognizes DNA and enhances the actions. Using single-molecule fluorescence resonance energy transfer DNA-binding activity. NMR spectroscopy, CD spectroscopy and isothermal (smFRET), biochemical, and biophysical assays, we find that ALS FUS can calorimetry studies have been conducted on both the Brg1 bromodomain be rescued by wild-type FUS before aggregation occurs. However, pre- and the AT-hook interacting with the DNA separately. The results show formed ALS FUS aggregates exhibit a dominant-negative effect, trapping that the Brg1 bromodomain can bind the DNA through an interaction patch wild-type FUS into these aggregates. Together, our results reveal how the which includes the A helix and the AB and ZA loops while the AT-hook pre- defective ALS FUS-RNA interaction can be modulated by interacting with fers to bind in the minor groove. Information about the mechanism of the the wild-type FUS. simultaneous binding are still unknown and, since Brg1 mutations are involved in several human tumors, understanding the function of DNA- 1060-Pos Brg1 complex is of key importance to develop specific inhibitors. Here, Characterizing the Binding of the HIV-1 NC Protein to Hairpins Formed we’ve combined molecular docking, classic molecular dynamics, and metady- by CAG Repeats namics simulations to investigate how the Brg1 bromodomain identifies spe- Melanie Dillon, Yustinah Ndambakuwa, Henrietta Ehirim, cific binding sites within the DNA helix, both in the presence and absence of Catherine B. Volle. the AT-hook motif. Free energy surface discriminate the different binding Biology, Cottey College, Nevada, MO, USA. modes found for both complexes and show that the most likely configurations Trinucleotide repeats (TNRs) occur throughout the genome and their expansion adopted primarily involve the AT-hook binding in the minor groove while the affects varied cellular processes such as gene expression, mRNA processing, A helix is parallel to the DNA. Without the AT-hook, the DNA/Brg complex and protein folding. TNR expansion is facilitated by formation of stable non- binds to the DNA through the A helix interactions in a perpendicular orienta- canonical secondary structures and has been linked to several neurodegenera- tion with the major groove. tive diseases, such as Huntington’s Disease (HD). HD occurs when the CAG

BPJ 9358_9361 214a Monday, March 4, 2019 repeats in exon 1 of the huntingtin gene expands. When the repeats become sin- in understanding DNA recognition and cleavage by Cas9, consistent structural gle stranded, either during DNA replication or through long-patch base exci- and functional information about the catalytic state of the HNH nuclease sion repair, they form a hairpin containing a four base loop and a stem domain remains to be elucidated. On the basis of our recent work (Nature Sci- containing AA mismatches. While hairpin formation is the causative mecha- entific Reports, 7:17271), we here report a new Cas9 active state complex nism of HD, hairpins are also useful regulatory system. In the HIV-1 genome, structure discovered by molecular dynamics simulations and validated by hairpin formation by the trans-activation response (TAR) element is a key part site-directed mutagenesis experiments. In this structure, the HNH domain is of regulating reverse transcription of the genome. The nucleocapsid (NC) pro- poised for cleaving the target DNA strand using a canonical catalytic triad tein binds to the loop of the TAR hairpin and destabilizes the stem, allowing as seen in phage T4 Endonuclease VII. Guided by the derived new structural reverse transcription to proceed. Interestingly, there is a noticeable similarity information, we rationally designed and tested a library of new Cas9 variants between the structure of the TAR hairpin and the hairpin formed by the to improve Cas9 specificities. Our ultimate goal is to offer several high- CAG repeats in the huntingtin gene. Thus, we are interested in determining fidelity Cas9 enzymes that can be broadly used in basic research and medical if the NC protein can recognize the hairpin formed by CAG repeats. Using a therapy. crude purification of recombinant NC protein, we demonstrated putative bind- ing of a (CAG)10 hairpin by the NC protein, and now provide more detailed 1064-Pos characterization of the NC/CAG hairpin binding. A Versatile Method to Quantify DNA-Protein Interactions on Negatively Supercoiled DNA 1061-Pos Graeme A. King1, Federica Burla2, Erwin J.G. Peterman1, Gijs J.L. Wuite1. DNA Sequence and Histone Core Composition Control the Unwrapping of 1Department of Physics and Astronomy, Vrije Universiteit Amsterdam, DNA from Nucleosome Core Particles Amsterdam, Netherlands, 2FOM Institute AMOLF, Amsterdam, Netherlands. Alex Mauney, Joshua Tokuda, Yujie Chen, Lois Pollack. Many genomic processes are regulated by torsional stress, resulting in a range Cornell Univ, Ithaca, NY, USA. of supercoiled DNA structures. In order to understand the effect of such struc- As controlling access to DNA is a major component of transcriptional regula- tures on protein binding and activity, several single-molecule techniques are tion, understanding the effects of interactions between core proteins and the often employed. These include magnetic, micro-pipette and angular optical DNA is crucial to gaining an overall understanding of transcription. We report tweezers. However, two factors can limit the study of DNA-protein interactions on the use of small angle X-ray scattering with contrast variation (CV-SAXS) on supercoiled DNA. First, it is challenging to combine DNA-torque control to analyze DNA unwrapping from nucleosome core particles. Interactions be- with fluorescence microscopy. Second, the DNA substrate is typically tethered tween the histone core and the DNA are destabilized using increasing concen- to a surface, hindering rapid buffer exchange. trations of neutral salt, lowering the strength of electrostatic interactions that Here, we present a novel method to prepare negatively supercoiled DNA that bind the components together. As the complex is destabilized many unwrap- overcomes both of these limitations, using a dual-trap optical tweezers assay. ping states are accessible and the DNA explores possible unwrapping path- Our approach, which exploits the intrinsic mechanical properties of DNA, en- ways. The CV-SAXS data reports on full range of structures present in the ables stable and controlled generation of supercoiled DNA with between 5% sample. They are fit using sequence-dependent models of unwrapped structures and 70% lower linking number than that of unconstrained DNA. Showcasing with thermal motions, selected by an ensemble optimization method [1]. the power of this method, we first report combined force-manipulation and Different combinations of DNA sequence and histone composition also alter fluorescence imaging studies that reveal how the human single-stranded their interactions, and the unwrapped structures reflect biophysical differences DNA-binding protein RPA can modulate negatively supercoiled DNA struc- in the nucleosome compositions. ture as a function of both sequence and tension. Next, we apply the method 1. Mauney A., Tokuda J.M., Gloss L.M., Gonzalez O., Pollack L. Local DNA to unravel how negative supercoiling can influence the DNA-binding dy- sequence controls the cooperativity and asymmetry of DNA unwrapping from namics of the vital mitochondrial transcription factor TFAM. By imaging nucleosome core particles. Biophys J. 2018 Sep 4;115(5):773-781 fluorescently-labelled TFAM on supercoiled DNA (at 5 pN), we uniquely demonstrate that the protein’s mobility is hindered by local underwound struc- 1062-Pos tures, resulting in sub-diffusive behavior. This finding has important implica- Observation of Allosteric Signaling through DNA with Single-Molecule tions for the ability of TFAM to locate and interact with promoter sites in vivo FRET and Cryo-EM and suggests a mechanism by which supercoiling could regulate mitochon- Gabriel Rosenblum1, Nadav Elad2, Felix Wiggers1, Hagen Hofmann1. drial transcription. 1Structural Biology, Weizmann Institute of Science, Rehovot, Israel, Taken together, this work establishes that our approach can provide unprece- 2Chemical Research Support, Weizmann Institute of Science, Rehovot, dented insight into the role of DNA supercoiling in genomic transactions. The Israel. method thus represents a powerful addition to the single-molecule toolkit. Gene networks often obey complex dynamics such as pulsing and oscillations that require a nearly binary ‘‘on’’ and ‘‘off’’ switch in gene activity. At a mo- 1065-Pos lecular level, a high cooperativity in the interaction between transcription fac- Atomic-Level Characterization of an Allosteric Gene Regulatory System tors (TFs) and DNA is key for this switch. However, how this cooperativity is Michael V. LeVine, Stefano Piana, Maxwell Tucker, Jesus Izaguirre, achieved is unknown for many systems. We used a combination of cryo- David E. Shaw. electron microscopy (cryo-EM) and single-molecule fluorescence spectroscopy D. E. Shaw Research, New York, NY, USA. to unravel the emergence of cooperativity in the interaction between ComK, a Living systems must acquire, analyze, and adapt to information in their envi- factor from Bacillus subtilis, and its promoter sequence. ronment. The canonical Tryptophan (Trp) repressor/Trp (TrpR/TrpO) The promoter contains two binding sites (AT-boxes) that are separated by 18 system found in many bacteria converts a chemical signal, the concentration base pairs (6 nm). Single-molecule FRET experiments show a low cooperativ- of free L-Trp within the cell, into a gene regulatory action, the inhibition of ity for the binding to individual AT-boxes whereas the combination of two tryptophan synthesis. Here, we characterize the molecular mechanism by distant binding sites in the natural promoter boosts cooperativity. Importantly, which L-Trp binding to TrpR allosterically modulates sequence-specific the boost does not result from interactions between ComK-molecules at the two DNA binding to TrpO. To study the minute-timescale DNA binding process, sites, as revealed by a cryo-EM structure of the ComK-DNA complex. Instead, we have utilized two advances: i) a new, state-of-the-art DNA force field the results unravel a new allosteric mechanism. Cooperativity between the that can accurately model DNA/protein complexes, and ii) an enhanced sam- ComK-binding sites results from an axially transmitted signal through the pling method, namely, accelerated weighted ensemble (AWE) molecular dy- DNA-structure. Hence, ComK-binding to one site increases the affinity of namics (MD), implemented on the Anton2 supercomputer. Our computations the 18 base pair distant site. This communication forms a toggle in which com- reveal a multi-step, induced-fit-binding mechanism, in which DNA shape plete binding, i.e., the switch from ‘‘off’’ to ‘‘on’’, is achieved within a narrow adapts to changes in the conformation of TrpR in a sequence-specific manner. ComK concentration range. In addition, we find that the most significant contribution to DNA binding af- finity comes from the formation of a direct interaction between the ligand and 1063-Pos the phosphate backbone in the final stage of binding. We performed surface Structural and Functional Insights into CRISPR/Cas9 Catalytic Activation plasmon resonance (SPR) experiments and obtained binding free-energies and Specificity Enhancement consistent with the estimates from both AWE and free energy perturbation Zhicheng Zuo, Jin Liu. (FEP). These results indicate that a combination of accurate force fields and Univ North Texas Health Science Center, Fort Worth, TX, USA. MD simulations on Anton can enable the study of complex allosteric gene CRISPR/Cas9 has been repurposed as a powerful genome editing tool, with regulatory systems at biological timescales and at an atomistic level of immense potential toward therapeutic applications. Despite recent advances detail.

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1066-Pos dimeric forms, using a qualitative gel electrophoretic mobility shift assay Regulation of Rep Helicase Unwinding by an Auto-Inhibitory Subdomain (EMSA) and a radiolabeled, 20 bp hybrid, shows that the dimeric form has a Monika A. Makurath1, Kevin D. Whitley2, Binh Nguyen3, higher affinity than the monomeric HBD. We also show that an ‘‘unnatural’’ Timothy M. Lohman3, Yann R. Chemla4. hybrid, in which the DNA strand is replace by a locked nucleic acid (LNA) 1Center for Biophysics and Quantitative Biology, University of Illinois at oligonucleotide, is also recognized with high affinity by the dimeric HBD. Urbana-Champaign, Champaign, IL, USA, 2University of Illinois at Urbana- The dimeric as well as higher multimeric forms are being analyzed by SPR Champaign, Champaign, IL, USA, 3Dept Biochemistry and Molecular for sensitivity to base mismatches and backbone chemistries, using model Biophysics, Washington Univ St Louis, St Louis, MO, USA, 4Dept Physics, hybrid substrates. University of Illinois at Urbana-Champaign, Urbana, IL, USA. are biomolecular motors that unwind nucleic acids, and their regula- E. coli 1069-Pos tion is essential for proper maintenance of genomic integrity. Rep heli- Elucidating the Molecular Binding Mechanism of the Tata-Binding case, whose primary role is to help restart stalled replication, serves as a model Protein using Pie-Pife for Superfamily I helicases. Rep-like helicases contain a flexible subdomain Evelyn Ploetz, Anders Barth, Lena Voith von Voithenberg, Ganesh Agam, (2B) that regulates their activity; however, the mechanism of control is not Don C. Lamb. well understood. While a monomer of Rep cannot unwind duplex DNA, com- Chemistry and Pharmacy, CeNS, Ludwig-Maximilians-University Munich, plete removal of 2B activates the motor for unwinding, though not as proces- Munich, Germany. sively as locking 2B into one conformation. Here we investigate the D The TATA-box binding protein (TBP) plays an important regulatory role in behavior of a 2B-deletion variant (Rep 2B) in relation to wild-type Rep. Using regulating gene expression by mediating the formation of the pre-initiation a single-molecule optical tweezers assay, we show that DNA unwinding by complex on promotor DNA. When binding to DNA, TBP melts the double RepD2B monomer is more processive than by wtRep oligomer. Additionally, o D helix and forms a stable complex while bending the dsDNA by 90 along we find that Rep 2B can switch between strands and form a single-stranded the TATA sequence. In vitro single molecule experiments based on single- DNA loop, both of which limit unwinding. By exploring the effect of force pair Fo¨rster resonance energy transfer (FRET) have recently revealed a dy- and DNA geometry on helicase activity, we further show that the behaviors namic, step-wise binding process via different intermediate conformations mentioned above are highly influenced by duplex stability. In light of our re- of the dsDNA. In vivo, TBP exists in different forms, e.g. as dimerbound sults, we propose a new model for regulation of unwinding by Rep-like to the dsDNA, or as part of different co-activator/deactivator complexes helicases. in the subsequent steps of pre-initiation complex formation. These states however are only observed at cellular concentration of TBP in the micro- 1067-Pos molar range, which is too high for in vitro single molecule fluorescence Specific at One Side While Unspecific at the Other: the Interaction of a imaging. Blood Protein with Extracellular DNA In this contribution, we present a new strategy to visualizes unlabelled TBP Angelica Sandoval-Perez, Camilo A. Aponte-Santamaria. binding on DNA with changes in DNA conformation at high concentrations: Max Planck Tandem Group in Computational Biophysics, University of Los binding of TBP is monitored via protein-induced fluorescence enhancement Andes, Bogota, Colombia. (PIFE) while single-pair FRET reports on ongoing conformational changes DNA from neutrophil cells is released into the blood stream to forms neutro- in the DNA/TBP complex simultaneously. We combine this label-free PIFE- phil extracellular traps (NETs), as an immune response response to trap circu- FRET approach with pulsed interleaved excitation (PIE) spectroscopy, to lating pathogens. The mechanosensitive blood-coagulation adhesive protein, investigate conformational dynamics of freely diffusing protein-DNA com- von Willebrand Factor (VWF), has been recently identified to interact with plexes in solution at the focus of a confocal microscope. Without the need of DNA of NETs and suggested to play a key stabilization role of such traps dur- surface immobilization it can overcome concentration limitations of multi- ing inflammation. Nevertheless, the molecular nature, structural stability, and colour assays by monitoring two parameters, binding and conformation rear- biological implications of this interaction remained to be elucidated. Here, we rangement at the same. addressed this question by performing Brownian Dynamics and Molecular Dynamics simulations. Our simulations revealed an interesting mode of bind- ing in which a specific region in VWF, namely a helix 4 (H4) in its A1 domain, Posters: Membrane Dynamics I acts as the specific binding site for DNA. In turn, DNA offers multiple unspe- cific binding sites for the A1 domain. Three arginines of the H4 were identified 1070-Pos as key residues anchoring VWF to DNA. Furthermore, simulations varying Active Transport of Membrane Components by Dynamic Min Protein the ionic strength and extensive in silico mutational studies pointed to electro- Waves static attraction as the main driving force governing the VWF-DNA interac- Yu-Ling Shih1, Ling-Ting Huang2, Yu-Ming Tu2, Bo-Fan Lee2, tion. Our data predicts a naturally-occurring mutation of one of the Yu-Chiuan Bau3, Chia Yee Hong2, Hsiao-lin Lee1, Yan-Ping Shih1, arginines to significantly destabilize the VWF-DNA interaction, a prediction Min-Feng Hsu1, Jui-Szu Chen2, Zheng-Xin Lu1, Ling Chao2. that is currently under experimental validation. Moreover, our data attribute 1Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan, the partial diminish in binding of platelets to VWF, not to competition of 2Department of Chemical Engineering, National Taiwan University, Taipei, DNA and platelets for the same binding site at A1, but rather, to partial unspe- Taiwan, 3Institute of Cellular and Organismic Biology, Academia Sinica, cific steric preclusion of the platelet binding site upon binding of DNA Taipei, Taiwan. to VWF. All together, our study provides the molecular basis for the stabili- The Min proteins, which mediate placement of the cell division site in E. coli, zation of DNA-based NETs by VWF, with potential implications during possess robust ability to self-organize and generate patterns on the membrane inflammation. in vitro. Here we report that this self-organization process can serve as biome- chanical machinery to impose steric pressure on the membrane, generate cor- 1068-Pos responding membrane patterns, and transport the membrane-associating Expanding RNA-DNA Hybrid Affinity by Multimerization of a Conserved components. The consequence of these events is the creation of a concentra- Fold tion gradient of the components along the propagating Min protein waves. Alex Stopar1, Rhonda Nicholson1, Matteo Castronovo2, Hence, the Min protein dynamics can partition membrane-associating compo- Allen W. Nicholson1. nents to generate and maintain a heterogeneous distribution of the components 1Biology, Temple University, Philadelphia, PA, USA, 2University of Leeds, on or in the membrane. Such mechanical function of the Min system could Leeds, United Kingdom. influence the subcellular localization of membrane-associating components RNA-DNA hybrids are functionally essential structures involved in numerous in vivo. protein-dependent processes such as transcription and viral reverse transcrip- tion. The hybrid binding domain (HBD) is a conserved fold (60 a.a., 6 1071-Pos kDa) that selectively recognizes RNA-DNA heteroduplexes (hybrids) in a Dynamic Effects of Calcium on Membranes Containing Phospha- sequence-independent manner and with submicromolar affinity. To further un- tidylserine derstand the structural and environmental factors that could establish coopera- Mason L. Valentine1, Alfredo E. Cardenas2, Ron Elber2, Carlos R. Baiz1. tive behavior in hybrid recognition, starting with the cloned sequence encoding 1Chemistry, University of Texas at Austin, Austin, TX, USA, 2Inst Comp/ the HBD from RNase H1 of Thermotoga maritima, a multimeric form was Eng Sci, University of Texas at Austin, Austin, TX, USA. generated that contains two copies of the HBD connected by a 10-residue Lipid membranes are dotted with negatively charged phospholipids, which linker. Comparative assessment of the binding affinity of the monomeric and have been shown to interact with cationic species. Phosphatidylserine (PS)

BPJ 9358_9361 216a Monday, March 4, 2019 lipids in particular have attracted attention due to the dramatic effects that diva- 1074-Pos lent metal cations have on PS-containing membranes. When a membrane con- Properties of Neurotoxicant Antidote Transport Across the Blood-Brain taining PS is exposed to calcium ions, the effects range from changes in the Barrier lateral diffusion of the lipids, to a collapse of the membrane and formation Christian Jorgensen1, Martin B. Ulmschneider2, Peter C. Searson1. of ‘cochleate’ aggregates. Although these effects are well known, their exact 1Institute for , Department of Materials Science and molecular mechanism is not, and neither are their physiological implications. Engineering, Johns Hopkins University, Baltimore, MD, USA, 2Department Studying these effects is made more difficult by the fact that PS typically makes of Chemistry, King’s College London, London, United Kingdom. up less than 10% of biological membranes. The delivery of neurotoxicant antidotes and chemotherapeutic agents into the By attaching nonperturbative isotope labels to specific lipids, we have been brain is controlled by the blood-brain barrier (BBB). At present the physico- able to track the local effects of calcium ions on specific membrane species us- chemical details and mechanisms of molecular transport across the blood brain ing infrared spectroscopy. We have found using this approach that calcium barrier are not well understood, which prevents therapeutic optimization of slows down the sub-nanosecond fluctuations around the lipid-water interface. novel drugs. Here we employ multi-microsecond MD simulations of a library Additionally, we’ve demonstrated specificity in the effect of Ca2þ on binary of drugs permeating across in silico models of human brain microvascular mixtures of lipids; Ca2þ does not influence zwitterionic lipids in the mixture endothelial cell (hBMEC) membranes. This membrane is an atomic-detail the way it influences PS. Finally, we have used molecular dynamics simulations model of the BBB, built according to principles of in vitro hBMEC lines, to connect the observed changes in spectra to changes in the motions and inter- and these simulations allow us to calculate the permeability and free- actions of molecules. energies of drug permeation across a realistic model of the BBB. We find that the values obtained are directly comparable to in vitro measurements, 1072-Pos and reveal how the chemical space spanned by the individual group character- Optical Dyes to Monitor Tension and Growth in Model Membranes istics of permeating BBB drugs can be classified into discrete groups, thus Margrethe Boyd. bringing us closer to a discrete description of BBB transport. Biomedical Engineering, Northwestern University, Evanston, IL, USA. Cells have been shown to dynamically regulate physical properties of the mem- 1075-Pos brane in response to mechanical cues. In particular, membranes have been Spectroscopic and Microscopic Approach to Monitor the Changes in shown to undergo surface area changes in response to membrane tension, a pro- Bilayer Rigidity during Cell Penetrating Peptide Induced Self- cess which plays a role in cellular growth, migration and signaling. The ability of Phospholipid Vesicles to simultaneously monitor membrane strain and membrane surface area Pavel Banerjee, Siddhartha Pal, Niloy Kundu, Dipankar Mondal, changes would be beneficial in understanding this process, yet we lack robust Nilmoni Sarkar. methods to accomplish this. Here, we introduce an optical technique to monitor Chemistry, Indian Institute of Technology, Kharagpur, India. membrane tension and surface area changes in real time. Through the combi- Understanding peptide-membrane interaction in view of structural change of natorial use of two types of optical dyes, which localize to distinct regions of lipid bilayer is essential to develop new therapeutic strategies. Though different the bilayer membrane, we can monitor changes in lipid separation through mechanisms are proposed for cell penetrating peptides (CPPs) penetration in the processes of membrane stretch and lipid uptake. Laurdan, a polarity- lipid bilayer, the exact mechanism of cellular uptake of CPPs remains a signif- sensitive probe, is localized to the hydrophobic region of the membrane, while icant unanswered piece of puzzle. Here, we report a novel observation that phospholipid-conjugated FRET pairs localize to the hydrophilic head region. model phospholipid vesicles self-reproduce to generate unilamellar daughter These dyes act in concert to indicate changes in water permeation and phospho- vesicles in presence of a CPP, nonaarginine (R9). Fluorescence lifetime image lipid head separation, respectively, as membrane properties change. We analysis (FLIM) exquisitely captures the changes of L-a- observed linear shifts in fluorescence in response to applied tension, indicating (LAPC) bilayer in different times and also quantifies the changes through valu- the potential of this system as a membrane tension sensor. We then used these able lifetime distributions obtained. Mean square displacement (MSD) calcula- probes to observe the process of tension-induced lipid uptake, and discovered tions from FLIM images of single vesicle trajectory establish CPP induced self- that membranes under strain take up excess lipid faster and in greater amounts. reproduction of LAPC vesicles. Heterogeneous nature of lipid bilayer after 1 Ultimately, the combined use of these probes offers a more complete picture of hour of R9 addition is observed from fluorescence correlation spectroscopy membrane dynamics, and allows us to remotely track changes in tension and (FCS) which supposed to consider as the starting point of ‘‘mother vesicles’’ surface area in model membranes. reproduction. Solvation dynamic study delineates the change in dynamics of the encapsulated water inside vesicles which also states different rigidity of 1073-Pos lipid bilayer in different states of vesicle self-reproduction. Fluorescence Spatial Relationship and Functional Relevance of Three Lipid Domain anisotropy image analysis provides information regarding change in lipid Populations at the Erythrocyte Surface bilayer rigidity by quantifying the rotational motion of the fluorophore in pres-  Louise Conrard, Amaury Stommen, Hele`ne Pollet, Donatienne Tyteca. ence of perturbation of CPPs. From these experimental observations, we are Cellular Biology, Institut de Duve, Brussels, Belgium. able to establish phospholipid vesicles self-reproduction in presence of CPPs Red blood cells (RBC) have been shown to exhibit stable submicrometric which may be considered as a possible route of CPPs action to deliver cargos lipid domains enriched in cholesterol (chol), sphingomyelin (SM), phosphati- to cell interior through hydrophobic barriers. Overall, our results provide an dylcholine (PC) or ganglioside GM1, which represent the four main lipid clas- experimental underpinning of the role of lipid bilayer structural changes in ses of their outer plasma membrane leaflet. However, whether those lipid self-reproduction which can exhibit great potential to interpret the protein- domains co-exist at the RBC surface or are spatially related and whether lipid interaction in emergence of life. and how they are subjected to reorganization upon RBC deformation are not known. Using fluorescence and/or confocal microscopy and well- 1076-Pos validated probes, we compared these four lipid-enriched domains for their Emergent Shape Sensing of Dynamic Membranes abundance, curvature association, lipid order, temperature dependence, spatial Brian A. Camley. dissociation and sensitivity to RBC mechanical stimulation. Our data suggest Dept. Physics & Astronomy, Johns Hopkins University, Baltimore, MD, that three populations of lipid domains with decreasing abundance coexist at USA. the RBC surface: (i) chol-enriched ones, associated with RBC high curvature For cells to divide or branch, they need to be able to sense their own shape, areas; (ii) GM1/PC/chol-enriched ones, present in low curvature areas; and localizing proteins to regions of high or low curvature. Curved proteins prefer (iii) SM/PC/chol-enriched ones, also found in low curvature areas. Whereas to bind to curved membranes, which can explain some of this shape sensitivity. chol-enriched domains gather in increased curvature areas upon RBC defor- However, when the membrane has curvature on the micron scale, and proteins mation, low curvature-associated lipid domains increase in abundance either are on the nanometer scale, single-protein sensing may be challenging. Shape upon calcium influx during RBC deformation (GM1/PC/chol-enriched do- sensing can also arise from the collective action of many proteins even if single mains) or upon secondary calcium efflux during RBC shape restoration proteins don’t sense curvature - i.e. the shape sensing is emergent. In particular, (SM/PC/chol-enriched domains). Hence, abrogation of these two domain pop- it is known that the motion of traveling waves arising from reaction-diffusion ulations is accompanied by a strong impairment of the intracellular calcium processes on a curved surface is altered by surface curvature. Common balance. Lipid domains could contribute to calcium influx and efflux by con- biochemical reactions that distinguish the cell front from rear (Rho GTPase po- trolling the membrane distribution and/or the activity of the mechano- larity, modeled by a ‘‘wave-pinning’’ mechanism) also sense shape, with the activated ion channel Piezo1 and the calcium pump PMCA. We currently cell front pointing toward the narrow end of an elongated cell. However, it is investigate whether this contribution results from lipid domain biophysical not completely clear what aspects of the cell geometry the wave-pinning reac- properties. tion senses. Does the cell front localize to high mean or Gaussian curvature, or a

BPJ 9358_9361 Monday, March 4, 2019 217a different shape feature altogether? Is this shape sensing disrupted by the mem- 1079-Pos brane fluctuations? We address these questions with a combination of detailed Effect of Chitosan on Mechanical Properties of Lipid Bilayers using simulation of reaction-diffusion on a curved membrane surface and a phenom- Micropipette Aspiration enological theory that allows us to construct an energy landscape to predict Honey Priya James, Sameer R. Jadhav. where these domains localize. This landscape theory allows us to develop a Department of Chemical Engineering, Indian Institute of Technology coarse-grained model of how regions of high Rho signaling will diffuse on a Bombay, Mumbai, India. fluctuating membrane. Liposomes composed of phospholipids have been widely used as drug deliv- ery vehicles for both hydrophilic and hydrophobic moieties due to several 1077-Pos characteristics such as reduced side effects, cell-specific and controlled deliv- Tuning of Membrane Sphingolipid Content Influences the Links of Outer- ery of drugs for better therapeutics. But the use of liposomes is limited due to Leaflet Membrane Lipid Dynamics to Cholesterol and Cytoskeleton short circulation half-life and higher susceptibility to oxidation and hydrolysis Anjali Gupta1, Federico Torta2, Markus Wenk3, Thorsten Wohland3. 1 leading to the leakage of drugs. To overcome these limitations liposomes are Department of Biological Sciences, National University of Singapore, modified with both natural and synthetic polymers. The surface modification Singapore, Singapore, 2Department of Biochemistry, National University of 3 of liposomes with polymers influences the membrane mechanical properties Singapore, Singapore, Singapore, Department of Biological Sciences and and water permeability, which can directly affect the cellular uptake, drug Biochemistry, National University of Singapore, Singapore, Singapore. release and encapsulation efficiency of the liposome. Among several poly- The plasma membrane (PM) compartmentalization is crucial for specific mers, chitosan - a biodegradable and biocompatible polymer has shown biochemical processes where lipid domains and actin cytoskeleton are re- greater potential in improving the mucoadhesive property and stability of garded as the fundamental compartmentalization factors. The PM composi- the liposomal formulation. The present study focuses on how the surface con- tion and organization vary across cell types. For instance, in some cell centration of chitosan modulates the mechanical properties (bending and area types, the connection of outer-leaflet lipid diffusion with cholesterol and compressibility moduli, water permeability and lysis tension) of SOPC vesicle cytoskeleton is stronger than in others. Transmembrane proteins interacting using micropipette aspiration technique. Chitosan-vesicles were prepared us- with the actin cytoskeleton are thought responsible for mediating these con- ing inverse phase emulsion method and the homogenous distribution of poly- nections. There is less evidence to show that lipids can also be responsible mer on the bilayer surface was confirmed using FITC-tagged chitosan by for establishing these connections. Previously, in an ITIR-FCS based confocal microscopy. The bending moduli of chitosan-vesicles were found comparative study between CHO-K1 and RBL-2H3 cell membranes, we to increase in a concentration-dependent manner compared to pure SOPC ves- have shown that in RBL-2H3 cell membranes, DiI-C18 (an outer-leaflet icles. However, area compressibility modulus does not show any significant liquid disordered phase marker) shows confined diffusion and is sensitive difference. In addition, the effect of solution pH on chitosan vesicle showed to cholesterol depletion and cytoskeleton disruption. In this study, we a decrease in bending modulus at pH 4.5 as compared to neutral pH 7. explore the differences in DiI-C18 diffusion in RBL-2H3 and CHO-K1 cells The water permeability of chitosan coated vesicles decreased significantly by comparing their PM lipid compositions using lipidomics. In addition, we as compared to SOPC vesicle. The lysis tension of chitosan-vesicles was ε a investigate the influence of Fc RI receptor (an abundant transmembrane not significantly different from that of SOPC vesicles with increasing chitosan protein in RBL-2H3 cells associated with the cytoskeleton) concentrations concentration. These findings may contribute to the design of chitosan- lipo- ε a on the DiI-C18 diffusion in RBL-2H3 cells. Results, after Fc RI knock- somal drug delivery system. down, show that the FcεRIa does not link DiI-C18 diffusion to cytoskeleton and cholesterol. Subsequently, lipidomics data reveal a higher percentage of 1080-Pos sphingomyelin and ceramides in PM of RBL-2H3 cells as compared to CHO- Destruction of Nematode Ova in Wastewater using Electroporation K1 cells. Therefore, we manipulated the sphingolipid content in RBL-2H3 Michael Dryzer1, Caitlin Niven2, Scott Wolter1, Christopher Arena3, cells and monitored the DiI-C diffusion. Results suggest that higher mem- 18 Edgard Ngaboyamahina4, Charles Parker4, Brian Stoner2. brane ceramide content is responsible for cholesterol and cytoskeleton sensi- 1Department of Physics, Elon University, Elon, NC, USA, 2Center for Water, tive DiI-C diffusion. Furthermore, exogenous treatment of CHO-K1 cells 18 Sanitation, Hygiene and Infectious Diseases, Duke University, Durham, NC, with structurally distinct ceramides show DiI-C diffusion similar to 18 USA, 3Department of Biomedical Engineering and Mechanics, Virginia RBL-2H3 cells. Finally, we conclude that a higher membrane ceramide con- Tech, Blacksburg, VA, USA, 4Department of Electrical and Computer tent is sufficient to induce coupling of outer-leaflet lipid diffusion with Engineering, Duke University, Durham, NC, USA. cholesterol and cytoskeleton. Posited as a public health risk by the World Health Assembly in 2001, hel- 1078-Pos minth worms are a virulent family of parasites prominent in the developing Calculating Ethanol Permeability of Membranes through Molecular world with various species together having infected approximately one Dynamic Simulations quarter of the global population. Helminth eggs are incredibly resilient— Mahdi Ghorbani, Eric Wang, Jeffery B. Klauda. possessing the ability to resist inactivation via treatment with most conven- Dept Chem/Biomolec Eng, Univ Maryland, College Park, MD, USA. tional sanitation methods. Determining a cost-effective and sustainable Permeation of small molecules into membranes is a fundamental biological sanitation approach will lead to increased sanitizing capabilities in regions process that is important for drug design, toxicology and production of bio- that otherwise do not have access to clean water and food. This research fuels. Molecular dynamics simulations have proven to be a promising tool first reports on the effectiveness of electroporation to inactivate Caenorhab- for studying permeability of membranes and can provide precise character- ditis elegans (C. elegans), a non-parasitic nematode genetically and morpho- ization of free energy, diffusivity and permeability coefficient of the mem- logically similar to most helminths, and discusses progress toward brane. In this study, permeation of ethanol into three different membranes implementation in off-grid toilets. Electroporation is an electrophysical tech- of POPS, POPE and POPC lipids are studied. Microsecond simulation of nique that utilizes electric pulses to increase cell membrane permeability in double bilayer membranes of these lipids provided direct measurements of its conventional application but herein is used to open pores in nematode permeability by calculating the number of ethanol molecules crossing the eggshells—the first report of such an application to the best knowledge of bilayer and going into the water phase between the two bilayers. These sim- the authors. A parametric evaluation of electric field strength and treatment ulations showed that POPC has the highest permeability followed by POPE duration of eggs and worms in phosphate buffer solution and polished black- and POPS lipids. In another approach, single bilayer membranes of these water was performed using a 1-Hz pulse train with a 0.01% duty cycle. Pore lipids are simulated at different concentrations of ethanol ranging from 1% formation extent was quantified using a fluorescent label, propidium iodide, to 10% and a Bayesian-based method is used to estimate position dependent that targets embryonic DNA. Post-treatment egg viability assays demon- diffusion and free energy profiles of these bilayers. The inhomogeneous strated that electroporation deactivated C. elegans eggs without supplemen- solubility-diffusion model and a compartmental model will allow calcula- tary methods (i.e., chlorination or oxidation). Following optimization, tions for the small-molecule permeability in these single bilayers. Perme- electroporation was ultimately applied to Ascaris suum to demonstrate ability values from single bilayer and double bilayer simulations will be compliance to the emerging PC 305 ISO standard for sustainable non- compared and to the empirical values to determine the more accurate model. sewered sanitation systems. This research reveals that electroporation in- To mimic a real , we expect that a single membrane model creases eggshell permeability through potential channel formation within will need to represent the varying concentration of ethanol exposed to each the shell in both saline and blackwater media. Based on our observations leaflet. Free energy and diffusion profiles for single bilayers with different thus far, we discuss current treatment conditions and associated energy concentrations of ethanol will be combined to represent natural gradients consumption requirements for the destruction of helminth parasites in felt by a cellular membrane. wastewater.

BPJ 9358_9361 218a Monday, March 4, 2019

1081-Pos and director fluctuations. We rely on trajectories by Pastor et al., who have Lipid Nanotubes: A Possible Route to Formation and Growth recently conducted such an analysis, but we analyze the results using a more Elif S. Koksal1, Susanne Liese2, Ilayda Kantarci1, Ragni Olsson1, recent tilt-curvature theory due to Terzi and Deserno. This in particular permits Andreas Carlson2, Irep Gozen1. to determine a new tilt-curvature coupling parameter that is related to the 1Norwegian Center for Molecular Medicine, Oslo, Norway, 2University of Gaussian curvature modulus. As a stringent test of the theory, we also attempt Oslo, Oslo, Norway. to fit height and directional fluctuation spectra simultaneously to a single set of Membrane-enclosed cellular compartments create spatially distinct microenvi- elastic parameters, but this is only moderately successful. To evaluate the ac- ronments which confine and protect biochemical reactions in the cell. On the curacy of the fits and the errors in the obtained parameters, we use both para- early Earth, the autonomous formation of compartments is presumed to have metric and non-parametric bootstrap data analysis methods. enabled encapsulation of nucleotides, satisfying a starting condition for the emergence of life. Recently, surfaces have become into focus as potential plat- 1084-Pos forms for the self-assembly of prebiotic compartments, as notably enhanced A New Computational Method for Membrane Compressibility: Bilayer vesicle formation was reported in the presence of solid interfaces. The detailed Mechanical Thickness Revisited mechanism of such formation at the mesoscale however is still under discus- Milka Doktorova1, Michael V. LeVine2, George Khelashvili2, sion. Here we report on the spontaneous transformation of lipid reservoirs on Harel Weinstein2. 1Integrative Biology and Pharmacology, University of Texas Health Science solid substrates to unilamellar membrane compartments through a sequence 2 of topological changes, proceeding via a network of interconnected lipid nano- Center, Houston, TX, USA, Dept Physiol/Bioph, Weill Cornell Med Coll, tubes. We show that this transformation is entirely driven by surface-free en- New York, NY, USA. ergy minimization and does not require hydrolysis of organic molecules, or Because lipid bilayers can bend and stretch in ways similar to thin elastic external stimuli such as electrical currents or mechanical agitation. The vesicles sheets, physical models of bilayer deformation have utilized mechanical con- grow by taking up the external fluid environment, and can subsequently sepa- stants such as the moduli for bending rigidity (Kc) and area compressibility rate and migrate upon exposure to hydrodynamic flow. This may explain, for (Ka). However, the use of these models to quantify the energetics of membrane the first time, the details of self-directed transition from weakly organized bio- deformation associated with protein-membrane interactions and the membrane amphiphile assemblies on solid surfaces to with secluded internal response to stress is often hampered by the shortage of experimental data suit- contents. able for the estimation of the mechanical constants of various lipid mixtures. While computational tools such as Molecular Dynamics (MD) simulations 1082-Pos can provide alternative means to estimate Ka values, current approaches suffer Malaria Parasites Break and Degrade Two Membranes to Egress from significant technical limitations. Here, we present a novel computational frame- Human Erythrocyte work that allows for a direct estimation of Ka values for individual bilayer Svetlana E. Glushakova1, Josh Beck2, Matthias Garten1, Brad Busse1, leaflets. The theory is based on the concept of elasticity and derives Ka from Armiyaw S. Nasamu3, Tatyana Tenkova-Heuser1, John E. Heuser1, real-space analysis of local thickness fluctuations sampled in MD simulations. Daniel E. Goldberg3, Joshua Zimmerberg1. We explore and validate the model on a large set of single and multicomponent 1NICHD, Section on Integrative Biophysics, NIH, Bethesda, MD, USA, bilayers of different lipid composition and sizes, simulated at different temper- 2Biomedical Sciences, Iowa State University, Ames, IA, USA, 3Dept of atures. The calculated bilayer compressibility moduli agree with values esti- Medicine, Washington Univ Sch Med, St Louis, MO, USA. mated previously from experiments and those obtained from a standard The malaria parasite Plasmodium falciparum replicates in a parasitophorous computational method based on a series of constrained tension simulations. vacuole within a human erythrocyte. To egress that erythrocyte and initiate a We further validate our framework in a comparison with an existing polymer new replicative cycle, the parasite must break two limiting membranes. Using brush model (PBM) and confirm the PBM’s predicted linear relationship live cell microscopy and parasites expressing vacuole-targeted fluorescent pro- with proportionality coefficient of 24 using elastic parameters calculated teins, we described the time frame and the sequence of egress steps, and linked from the simulation trajectories. The robustness of the results that emerge steps to the known intracellular compartment markers. Egress inhibitors were from the new method allows us to revisit the origins of the bilayer mechanical used to associate egress steps with the molecules affected by drugs. Natural (compressible) thickness and in particular, its dependence on acyl chain unsa- parasite egress, a tightly coordinated multistep process, lasts approximately 9 turation and the presence of cholesterol. minutes. The initiation of egress requires intracellular calcium and is expressed 1085-Pos as a short-lasting ( 2 min) step of vacuolar rounding, a newly described Differential Actin Binding Affinity Leads to Protein Sorting in a Reconsti- morphological form. Rounding is immediately followed by rupture and a piece- tuted Active Composite Layer meal pattern of vacuolar membrane degradation. The mechanism of vacuolar Abrar A. Bhat1, Amit Das1, Kabir Husain1, Madan Rao1, Darius Koester1,2, membrane transformation is unknown, but it does not involve vacuole swelling, Satyajit Mayor1. thus excluding membrane rupture due to membrane stretching. Vacuolar break- 1National Centre for Biological Sciences, Bangalore, India, 2Warwick down depends on the secretion of activated protease SUB1 into the vacuole. University, Coventry, United Kingdom. Released upon vacuole rupture, vacuolar content initiates the slow deterioration Various functional cell surface proteins bind to cortical actin and undergo tran- of erythrocyte membrane, a process requiring cysteine proteases. Lasting sient clustering driven by actomyosin flows. Examples include the GPI- approximately 7 min, simultaneous degradation of the vacuolar membrane anchored proteins, Ras-signalling proteins, T-cell receptors and many glyco- and modification of the erythrocyte cytoskeleton enables parasite egress. The proteins such as CD44. Given the density and diversity of protein and lipid spe- final step of erythrocyte membrane modification includes its perforation. The cies in the plasma membrane, the question arises how the local accumulation of weakened host cell membrane then ruptures, and dissociated parasites escape specific molecules is achieved in a timely manner. Here we explore the possi- the host cell, thus completing the replicative cycle. We have shown how the bility that the combination of differential binding of surface molecules to actin exploration of biophysical aspects of membrane transformation is important and the non-equilibrium acto-myosin dynamics leads to molecular patterning for understanding the biology of malaria parasite replication. and cluster formation of membrane proteins. Using reconstituted acto-myosin networks tethered to supported lipid bilayers, as well as theoretical simulations, 1083-Pos we provide evidence for this proposed active sorting mechanism. We show that Novel Method of Analyzing Lipid Bilayer Elastic Moduli using Membrane differential affinity for actin templates a variety of patterns of membrane teth- Fluctuations ered proteins, including complete segregation, bull’s eye and mixed pattern. Muhammed F. Erguder, Markus Deserno. Our results suggest that this mechanism could help in understanding the Physics, Carnegie Mellon University, Pittsburgh, PA, USA. spatio-temporal organization and regulation of various processes at the cell Many important cellular processes take place at membranes, for example surface. signaling and sensing, energy conversion, and metabolism. In order to under- stand how the physical properties of cell membranes enable these processes, 1086-Pos we focus on membrane shape changes. These arise in many circumstances, Pathways and Molecular Mechanisms of Microdomain-Dependent Mem- such as endocytosis, along the fusion pathway, and when accommodating trans- brane Trafficking membrane proteins. When these shape changes occur at a scale not much larger Barbara Diaz-Rohrer, Joseph Lorent, Ivan Castello-Serrano, than the membrane thickness, standard curvature elasticity needs to be Kandice Levental, Ilya Levental. amended by contributions coming from lipid tilt. In order to get information Univ Texas Houston, Houston, TX, USA. about the associated elastic moduli, we use atomistic model simulations of The plasma membrane is the physical barrier and communication interface be- 13 different lipid membranes and analyze the power spectra of their shape tween the cell and its environment. The lipid and protein composition of this

BPJ 9358_9361 Monday, March 4, 2019 219a membrane is tightly controlled by endocytic recycling; however, the sorting inserted into two bilayers: One hemichannel into a planar bilayer and the other and trafficking mechanisms mediating efficient recycling are not known. One hemichannel into a nanodisc. The structure was equilibrated using Gromacs, hypothesis implicates membrane microdomains known as lipid rafts. Such do- yielding a conductance of 770 pS. 5.6 works well as a correction factor for mains are inherently selective for certain proteins and serve to organize and many pores (Smart et al. 1999). The corrected estimate, 140 pS, is close to concentrate many distinct cargoes, including lipids. We have developed and the experimentally measured value of about 110 pS (Kanaporis, et al. Am. J. characterized a robust experimental system for direct, quantitative measure- Physiol. Cell Physiol, 2011. 300:C600-9). To gain a better understanding of ments of raft affinity in intact plasma membranes and used it to explore the de- this correction factor we will investigate the dependence of the mobility of terminants of protein recruitment into raft domains and its consequences on ions on a) the pore diameter, b) the distance from the pore wall, c) pore wall subcellular traffic. We identified several structural features associated with material, and d) the diffusion and drift of ions confined between two slabs of raft affinity, and established that raft association was fully sufficient for PM re- the pore wall material. The two slabs are more symmetric such that the motion cycling of certain proteins. Abrogation of raft partitioning for these proteins led of ions can be approximated by planar and normal diffusion and a normal drift to their degradation in lysosomes. These findings strongly support a model force. The diffusion coefficients and drift forces at different positions obtained wherein ordered membrane domains mediate PM recycling in the endosomal from molecular dynamics simulation will be compared to finite volume method system. Using a set of proteis probes for raft and non-raft domains, we devel- simulations. oped a high throughput screen to dissect the molecular machinery and pathways of raft-mediated sorting in endocytic and biosynthetic pathways. We identified 1089-Pos several known players of the early endocytic traffic, but also novel players that Interfacial Effects of Ion Channels in Lipid Membranes: Mean-Field define a distinct class of trafficking mediators specific for raft-associated pro- Computation from 3D Atomic Structures Versus Analytical Estimates teins. We implicate Rab3 as a central regulator of this pathway, and show Marcel Aguilella-Arzo1, Antonio Alcaraz1, Maria Lidon Lopez-Peris1, that it is essential for PM homeostasis, as abrogation of Rab3 or other pathway Maria Queralt-Martin2, Vicente M. Aguilella1. effectors dramatically disrupts PM lipidomes and proteomes. Thus, our findings 1Dept Physics, Univ Jaume I, Castello´n, Spain, 2Section on Molecular reveal a fundamental role for raft microdomains in endocytic sorting and recy- Transpor, NICHD NIH, Bethesda, MD, USA. cling and support a novel role for Rab3 as a central regulator of this previously Recent experimental studies have stressed the importance of interfacial effects unrecognized mechanism for PM and endosome homeostasis. in pores of nanometer dimensions, particularly in protein channels with low aspect ratio. On the one hand, the well known concept of Access Resistance 1087-Pos (AR) or Converge Resistance is essential in the description of ionic transport Hopanoids, the Big ‘Small Things’ in Oligomerization of Proteorhodopsin across these biological channels. In fact, AR may become a dominant contribu- Eric Sefah, Blake Mertz. tion to the total channel resistance, as demonstrated experimentally for a bac- Dept of Chemistry, West Virginia Univ, Morgantown, WV, USA. terial porin, under low ion concentration or macromolecule crowding in the Hopanoids are pentacyclic triterpenoid compounds that are bacterial ana- surrounding solutions, two conditions that are often met in the cell environ- logues of sterols. Unlike sterols, hopanoids have a high degree of variation ment. On the other hand, charged polar head groups of the lipid membrane in the size and chemical nature of the substituent attached to the ring moiety, may have a strong influence on the electric potential and the ionic concentration leading to different effects on the structure and dynamics of biological mem- in the vicinity of the channel-solution interface. Charged residues within the branes. However, the relationship to the of mem- protein located near the pore mouth can also play a role, although to a lesser brane proteins remains poorly understood. As integral membrane proteins extent than AR and membrane surface charges. These three factors are obvi- are sensitive to physical changes in the surrounding membrane [1], our hy- ously coupled and are also strongly dependent on the channel aperture size, pothesis is that hopanoids would alter the behavior and function of proteins 3D structure and channel-lipid assembling. We perform mean-field calculations residing in the membrane, including oligomerization. In this work we based on the channel atomic structure and compare them with estimations ob- have used coarse-grained molecular dynamics simulations to characterize tained from analytical expressions and AR experimental measurements. Our re- the effects of two hopanoids, diploptene (DPT) and bacteriohopanetetrol sults indicate that the inverse relationship between AR and both bulk (BHT), on the oligomerization of proteorhodopsin (PR) in a model mem- conductivity and pore radius should be modified by additional channel- sn brane composed of 1-palmitoyl-2-oleoyl- -glycero-3-phophoethanolamine dependent effects. Our study tests the limits of the available description of sn (POPE) and 1-palmitoyl-2-oleoyl- -3-phosphoglycerol (POPG). PR is a ion transport at the nanoscale and provides tools for the characterization of pro- bacterial membrane protein that functions as a light-activated proton pump. tein channel function in physiologically relevant confined environments. We chose PR based on its ability to adopt a distribution of oligomeric states in different membrane environments [2]. Furthermore, the efficiency of pro- 1090-Pos ton pumping in PR is intimately linked to its organization into oligomers [3]. Probing the Mechanosensing Features of Mammalian Piezo Channels and Our results reveal that both types of hopanoids increase packing density of Plant OSCA Channels via Molecular Dynamics Simulations phospholipids, resulting in a noticeably more condensed membrane in the Che Chun(Alex) Tsui1,2, Kei Saotome2, Sebastian Jojoa Cruz2, lateral plane. Furthermore, BHT decreases membrane fluidity, further Andrew B. Ward2, Mark S.P. Sansom1. depressing the clustering of PR. Overall, BHT has greater packing density 1Department of Biochemistry, University of Oxford, Oxford, United at the protein hydrophobic core, making protein-protein interactions less Kingdom, 2Department of Integrative Structural and Computational Biology, favorable. Our results show a direct relationship between hopanoid structure Scripps Research, La Jolla, CA, USA. and lateral diffusion of PR, thus providing a connection between hopanoids Mechanically activated (MA) ion channels mediate many crucial mechanosen- and protein oligomerization. [1] Philips et al. Nature (2009) 459:379. [2] Ma- sory processes in animals and plants. In mammals, Piezo channels have roles in ciejko et al. JACS (2015) 137:9032. [3] Hussain et al. J. Mol. Biol. (2015) touch sensation, proprioception, vascular development and much more. The 427:1278. cryo-electron microscopy structures of mouse Piezo1 revealed a large homotri- meric propeller-blade architecture weighing about 880 kDa. Each monomer of Posters: Protein-Lipid Interactions: Channels mPiezo1 has 26 of the predicted 38 transmembrane helices modelled. To extend our structural understanding of Piezo1 beyond detergent environments, 1088-Pos we use molecular dynamics simulations to explore protein-lipid interactions of Structure-Based Estimate of Connexin 26 Conductance Piezo1. In our coarse-grained (CG) simulations, phospholipid molecules self- Nathan H. Zimmerberg1, Satyan Sharma2, Manfred Lindau1,2. assemble into a curved bilayer around Piezo1. In subsequent microsecond- 1Applied & Engineering Physics, Cornell University, Ithaca, NY, USA, duration CG simulations, independent fluctuations of blade curvature were 2Research Group Nanoscale Cell Biology, Max Planck Institute for observed. In plants, OSCA channels are also MA channels which were previ- Biophysical Chemistry, Go¨ttingen, Germany. ously reported as osmosensors. Recently, the cryo-EM structures of three Connexins (Cx) form gap junction channels (GJCs) traversing 2 membranes OSCA channels from Arabidopsis thaliana have been determined. These enabling signal transmission between coupled cells. A GJC is formed by the OSCA channels all form dimers with a wide cleft, and intersubunit interactions docking of two hemichannels, each composed of a connexin hexamer. A struc- are only mediated via the cytosolic domains. Strikingly, OSCAs have structural tural model of the Cx26 homomeric GJC based on Cx26 crystal structures and a homology with the TMEM16 family that function as calcium-dependent lipid cryo electron microscopy map of Cx43, shows a large pore with a minimum scramblases. We have performed CG and all-atom simulations on AtOSCA1.2, diameter of about 1 nm. For such large pores, a simple estimate of its conduc- and identified several putative mechanosensing features of the channel. Specif- tance may be obtained from the pore structure and conductivity of the solution ically, amphipathic helices on each monomer causes a local perturbation of filling the pore (Smart, et al. Faraday Discuss, 1999. 111:185-99). The Cx26 lipids in the lower leaflet, with lipid species occupying the wider, cytoplasmic GJC structure (pdb 5ERA) was converted to a Martini coarse grain model, side of the pore on each monomer.

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1091-Pos valuable information on the membrane lipid-binding sites of this complex.To Drug Regulation of Ion Channel Function Involves Both Direct and interrogate the functional significance of these protein-lipid interfaces and to Bilayer-Mediated Mechanisms compare them with that of lipid-devoid DkTx-TRPV1 interfaces, we generated Radda Rusinova, Olaf Andersen. a series of DkTx variants and characterized their TRPV1-activating properties Weill Cornell Medicine, New York, NY, USA. electrophysiologically. The results of these experiments together with those The tight (hydrophobic) coupling between membrane proteins and their sur- from our membrane partitioning experiments demonstrate that these structur- rounding lipid bilayer makes the membrane lipid regulation of membrane ally extremely similar toxin-lipid interfaces play dominant yet non- protein function key for understanding protein function. This regulation overlapping roles in channel activation. The lipid interfaces formed by one ranges from specific lipid binding to membrane proteins more general regu- of the knots of toxin were observed to confer the toxin with its strong mem- lation by the lipid bilayer. The latter mechanism arises from energetic brane partitioning ability resulting in its slow dissociation from the complex. coupling between the lipid bilayer and membrane protein where protein In contrast, the lipid interfaces formed by the other knot was found to contribute conformational changes will perturb the adjacent bilayer. To tease apart minimally to toxin dissociation rates yet profoundly to the toxin’s TRPV1- the specific and general mechanism(s) of membrane protein regulation activating potency. These studies demonstrate that protein-lipid interfaces by the membrane lipids, we examine how the prototypical bacterial potas- can play key roles in the function of membrane embedded protein-protein sium channel, KcsA, is regulated by bilayer-modifying drugs using a complexes. fluorescence-based approach to quantify both changes in KcsA function and the lipid bilayer (using gramicidin channels as probes). All tested drugs 1094-Pos alter KcsA channel function at the concentrations where they alter bilayer Effects of Membrane Protein nAChRs on Phase Separated Model Mem- properties. Comparing the concentration ranges at which molecules alter branes KcsA function and lipid bilayer properties, enables us to determine whether Jigesh Patel. the underlying mechanism primarily involves specific interactions with Physics and Astronomy, Texas Tech Univ, Lubbock, TX, USA. KcsA or may be bilayer-mediated. Most compounds tested slowed activa- Effects of Membrane Protein nAChRs on Phase Separated Model Membranes tion and accelerated inactivation at concentrations below those where they In this study, the effects of adding 2 mol% of membrane protein nAChRs alter bilayer properties, suggesting that KcsA likely has some non-specific to DOPC/DSPC/cholesterol lipid bilayers containing coexisting phases site that allows hydrophobic molecules to bind and alter channel properties. are investigated. Previously, no 4-component phase diagram, with nAChRs Some compounds, however, slow recovery from inactivation at concentra- protein as one of the components, has been studied. This work is the first tions that alter bilayer properties, with the greater effects in the thicker bi- study of this kind that investigates the effect of ion-transmitter nAChRs layers, which suggests that conformational changes that occur during on LoþLd phase boundaries. The modification of phase boundary by recovery from inactivation are coupled to the bilayer. Changes in bilayer nAChRs is determined using fluorescence microscopy on Giant Unilamellar thickness changes alter KcsA function (Rusinova et al 2014), and we now Vesicles (GUVs). After phase boundaries are determined, thermodynamic show that drug effects on KcsA vary depending on bilayer thickness. We tie-lines and protein’s partition coefficients will be measured. Those data thus conclude that KcsA function may be modified by a combination of will allow us to precisely determine the exact concentrations of nAChRs direct and bilayer-mediated effects. in various cell membrane domains. Accurate measurement of the perturba- tions of the phase boundaries by the protein could serve as a means to 1092-Pos quantitatively understand the universal behavior of a range of membrane Predicting the Promiscuous Effect of Amphipathic Drugs on Gramicidin proteins. Channel Stability with Simulations and Experiments Delin Sun Sun. 1095-Pos Lawrence Livermore National Laboratory, Pleasanton, CA, USA. Boundary Lipids of the Nicotinic Acetylcholine Receptor in Quasi-Native The amphipathic characteristics of many drugs suggest that they readily Membranes partition into cell membrane/solution interfaces, where they can alter mem- Liam M. Sharp, Reza Salari, Grace Brannigan. brane mechanical properties. This may have adverse effects on the function CCIB, Rutgers Univ, Haddon Heights, NJ, USA. of many membrane proteins, which are sensitive to small perturbations in the The nicotinic acetylcholine receptor (nAChR) is a neurotransmitter receptor cell membrane environment. The gramicidin A (gA) channel has been devel- and pentameric ligand gated ion channel (pLGIC) critical for signaling across oped as a molecular force probe to monitor the perturbation of lipid bilayers synapses, including the neuromuscular junction. In reconstituted membranes, by external stimuli. In this work, we have employed molecular dynamics the nAChR function depends heavily on cholesterol, leading to the hypothesis (MD) simulations and fluorescence quenching experiments to investigate that nAChR will partition into cholesterol-enriched liquid-ordered domains the effect of six amphipathic compounds: capsaicin, resveratrol, octanol, ("rafts’’). Native nAChR membranes are rich in lipids with saturated fatty hexaethylene glycol monododecyl ether (C12E6), Triton X-100 and dodecyl acid (like palmitic acid) or polyunsaturated fatty acid (PUFA) acyl chains phosphocholine on the dynamics of gA embedded in lipid bilayer of different (like docosahexaenoic acid (DHA)). Using coarse-grained molecular dy- thickness, i.e. DC18:1PC, DC20:1PC and DC22:1PC. The MD simulations namics simulations we characterized preferential lipid interactions and parti- show that the six compounds can thin the lipid bilayers and they tend to tioning behavior of nAChR in binary membranes (cholesterol and lipids with reduce the size of the membrane defects induced by the gA dimer. The calcu- two palmitic acid acyl chains) ternary membranes (cholesterol, a lipid with lated potential of mean force for gA channel dissociation indicates that the two palmitic acid acyl chains, and lipids with either two chains composed stability of gA in the thick DC22:1PC bilayer is drastically influenced by of the long-chain omega-3 PUFA DHA or of the omega-6 PUFA linoleic the amphipathic compounds. In the DC18:1PC and DC20:1PC bilayers, acid). the compounds have much less effect on gA channel stability. The conclu- We quantify occupation of non-annular and annular regions by cholesterol and sions from the molecular simulation results are supported by the fluorescence saturated and polyunsaturated lipids. In the absence of PUFAs, cholesterol is quenching experiments, which suggests that it may be possible to develop a enriched in the nAChR annulus in a concentration-dependent manner. Choles- computational approach to test for the toxicity of drug candidates in a high- terol is also distributed throughout the non-annular (embedded) sites, while pal- throughput way. This work was performed under the auspices of the U.S. matic acyl chains persistently occupy only one interface between the beta and Department of Energy by Lawrence Livermore National Laboratory under alpha subunits. When lipids containing long-chain PUFA acyl chains are intro- Contract DE-AC52-07NA27344 and was supported by the LLNL-LDRD duced, they displace cholesterol from two additional interfaces. Contrary to ex- Program under Project No. 18-ER-035. Release number: LLNL-ABS- pectations, in domain-forming membranes containing PUFAs, the nAChR is 758962. observed to consistently partition into PUFA-rich, cholesterol-poor domains. Saturated lipids with either phosphatidylcholine and phosphatidylethanolamine 1093-Pos head groups are significantly depleted in such systems, although the extent of Protein-Lipid Interfaces Drive DkTx-Mediated TRPV1 Channel depletion is reduced for phosphatidylethanolamine. While nAChR consistently Activation partitions into the cholesterol poor domain, alpha-gamma and delta-beta Debayan Sarkar, Yashaswi Singh, Jeet Kalia. faces interact more than other faces with the cholesterol-rich domains. We Department of Biological Sciences, IISER Bhopal, Bhopal, India. extend this approach to more complex membranes of interest, including A recent cryo-EM structure and a molecular model of the TRPV1 ion channel more realistic synaptic membranes and the Xenopus oocyte membranes used in complex with its potent agonist, the double knot toxin (DkTx), have provided for electrophysiology.

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1096-Pos TMEM16A is a homodimeric, calcium-gated chloride channel with a hydro- Effect of Late Endosomal Dobmp Lipid and Traditional Model Lipids of philic cleft within each monomer that has been proposed to transport anions. Electrophysiology on the Anthrax Toxin Channel Activity Experimental point mutations to the TMEM16A cleft confers lipid scramblase Nnanya Kalu1, Yoav Atsmon-Raz2, Sanaz Momben Abolfath1, Laura Lucas1, activity to the protein. Additionally, a fungal homologue, nhTMEM16, scram- Clare Kenney1, Stephen H. Leppla3, D. Peter Tieleman2, bles lipids through its hydrophilic cleft. The structural similarity of the hydro- Ekaterina M. Nestorovich1. philic clefts suggests that TMEM16A mutants might follow a similar 1Biology, The Catholic University of America, Washington, DC, USA, scramblase mechanism. It remains unclear, however, how these point muta- 2Department of Biological Sciences, Centre for Molecular Simulation, tions enable this drastic functional shift. Notably, current crystal structures University of Calgary, Calgary, AB, Canada, 3NIAID, National Institutes of of TMEM16A are all in ‘‘closed’’ conformations. Here, we use computational Health, Rockville, MD, USA. methods to probe details of the functional shift as well as obtain open confor- Anthrax toxin action requires triggering of natural endocytic transport mecha- mations of the mutants. Equilibrium molecular dynamics simulations showed nisms whereby the binding component of the toxin forms channels (PA63) penetration of lipid phosphate head groups into the inner vestibule of the hy- within endosomal limiting and intraluminal vesicle membranes to deliver the drophilic cleft, which was mediated by positive residues lining this vestibule. toxin’s enzymatic components into the cytosol. Membrane lipid composition However, transport of lipid head groups was blocked at the entrance to a varies at different stages of anthrax toxin internalization, with intraluminal constriction in the cleft. Our simulations show that salt bridges between alpha vesicle membranes containing 70% of anionic bis(monoacylglycero)phos- helices in the upper cleft potentially help restrict lipid movement past this phate lipid. Using model bilayer measurements, we show that membrane lipids constriction region. We also used principal component analysis of the cleft can have a strong effect on the anthrax toxin channel properties, including the alpha helices to identify key backbone regions involved in the dynamics of channel-forming activity, voltage-gating, conductance, selectivity, and enzy- the channel. Steered MD of lipid head groups through the channel were matic factor binding. Interestingly, the highest PA63 insertion rate was observed then used to induce ‘‘opening’’ of the channel. Of particular interest, the pull- in bis(monoacylglycero)phosphate membranes. The molecular dynamics simu- ing simulation triggered significant changes in the bend angle of a transmem- lation data show that the conformational properties of the channel are different brane helix around a glycine hinge; experiments predict that this residue is in bis(monoacylglycero)phosphate compared to PC, PE, and PS lipids. The important in determining the open vs. closed probability of the channel. anthrax toxin protein/lipid bilayer system can be advanced as a novel robust Lastly, targeted MD was used to bias the mutant TMEM16A cleft to the model to directly investigate lipid influence on membrane protein properties nhTMEM16 structure, aiding in identification of the main structural differ- and protein/protein interactions. ences between the proteins. 1097-Pos 1100-Pos Noise Properties of Ion Channels Formed by Pestivirus Viroporin p7 Inducing Chemical Concentration Gradients to Investigate Gas Perme- Antonio Alcaraz1, Vicente M. Aguilella1, Eneko Largo2, Jose L. Nieva2. ability of Rh-Protein Containing Membranes 1Laboratory of Molecular Biophysics. Dept Physics, Univ Jaume I, Castello´n, Eric Shinn1,2, Emad Tajkhorshid2,3. Spain, 2Biochemistry and Molecular Biology, Biofisika Institute (CSIC, 1Center for Biophysics and Quantitative Biology, University of Illinois at UPV/EHU) and University of the Basque Country (UPV/EHU), Leioa, Spain. Urbana-Champaign, Urbana, IL, USA, 2NIH Center for Macromolecular Permeabilization of the endomembrane system by viroporins is instrumental in Modeling and Bioinformatics, Beckman Institute, University of Illinois at the progression of host-cell infection by many viral pathogens. Previous studies Urbana-Champaign, Urbana, IL, USA, 3Department of Biochemistry, showed that p7 viroporin assembles into channels of nanometric dimensions. University of Illinois at Urbana-Champaign, Urbana, IL, USA. Here we analyze the power spectrum of current traces. We focus on the low- Membranes form essential barriers between cells and their environment as frequency range where the power spectrum shows typically 1/fa behavior well as between their intracellular compartments, endowing cells with con- with 0.5< a < 2 (the so called ‘‘pink noise’’). We investigate several lipid mix- trol over transmembrane molecular shuttling. Molecular dynamics (MD) tures to show that lipid composition determines not only the absolute value of simulations offer a high-resolution approach to understanding the atomistic current fluctuations but also the a parameter in the power spectrum of the cur- mechanisms of membrane transport. However, they struggle to calculate rent traces. Our findings give support to previous studies hypothesizing that p7 membrane permeability as their periodic nature inhibit the creation of chem- viroporin forms channels with at least two different types of pore architecture. ical gradients necessary for mass flux calculations. Here we present a method Overall, our observations suggest that pink noise is caused by conductance fluc- to generate concentration gradients in order to explicitly calculate membrane tuations governed by equilibrium processes, in particular the conformational permeability for small molecules in a model system containing RhAG. dynamics involving the proteins and lipids that assemble together to form pro- RhAG is an integral membrane protein expressed in erythrocytes, suggested teolipidic pores. to play a role in membrane permeation of dioxygen and carbon dioxide. Because no structure currently exists for RhAG, the PDB entry of RhCG, 1098-Pos a nephral homolog (sequence identity=51%), was used as a threading tem- Regulation of KcsA by Anionic Phospholipids plate to generate a structural model via I-TASSER (Z-score=3.35). Grid- Carmen Domene1, Victoria Oakes1, Simone Furini2. steered MD (G-SMD) was used to generate a field confined to space near 1Dept Chemistry, Univ Bath, Bath, United Kingdom, 2Univ Siena, Siena, the periodic cell boundary that forces target molecules into the next periodic Italy. image. This chemically deprives one side of the membrane and enriches the Membrane lipids are known to modulate the structure and function of integral other without biasing molecules throughout the majority of the system, es- membrane proteins. This effect can be achieved directly, by specific binding tablishing a self-replenishing chemical gradient. Applying this technique to sites on the protein surface, or indirectly, by changing the physical properties systems containing either dioxygen or carbon dioxide can create transmem- of the membrane bilayer. In the former, binding sites at the interface between brane concentration gradients with differentials up to 250 mM. RhAG- two protein subunits, known as non-annular sites, have been implicated. embedded lipid bilayers were subject to such chemical gradients in Anionic lipids have been shown to modulate various functional properties of hundred-nanosecond scale MD simulations to investigate their effect on þ the KcsA K -channel in different manners, and are required for channel func- membrane gas permeability. Mass flux calculations reveal the impact of tion. Here, we present molecular dynamics simulations of KcsA with anionic the protein on the diffusive permeability of gas across the membrane. Addi- and zwitterionic lipids bound in non-annular sites, in order to deduce the mo- tionally, explicit ligand sampling analysis of these simulations reveal the en- þ lecular mechanism of action of membrane lipids in the prototypical K - ergetics of gas permeation through the monomeric and central pores, as well channel. at the interface between protein and lipid. 1099-Pos 1101-Pos Elucidating Conformational Changes Underlying the Conversion of Ion Transport Through Large-Diameter DNA Origami Nanotube Chan- TMEM16A Mutants from Anion Channels to Scramblases nels Across Synthetic Membranes Archit K. Vasan1,2, Tao Jiang2, H Criss Hartzell3, Emad Tajkhorshid2,4. Naresh N. Dhanasekar, Rebecca B. Schulman. 1Center for Biophysics and Quantitative Biology, University of Illinois at Chemial and Biomolecular Engineering, Johns Hopkins University, Urbana-Champaign, Urbana, IL, USA, 2NIH Center for Macromolecular Baltimore, MD, USA. Modeling and Bioinformatics, Beckman Institute, University of Illinois at Transport through nanoscale channels is a fundamental mechanism of ex- Urbana-Champaign, Urbana, IL, USA, 3Emory University School of change in living systems. The ability to direct such transport using Medicine, Atlanta, GA, USA, 4Department of Biochemistry, Biophysics, membrane-based synthetic devices could make it possible to systematically University of Illinois at Urbana-Champaign, Urbana, IL, USA. study or control these transport processes. Techniques from structural DNA

BPJ 9358_9361 222a Monday, March 4, 2019 have recently made it possible to emulate the structural and phosphorylation of the channel proteins. Through site-directed mutagenesis, functional aspects of naturally occurring membrane channels using easy-to- we found that serine 673 (S673) is a crucial phosphorylation position affecting design DNA nanostructures. We have designed a system for controlled trans- channel gating and PIP2 sensitivity of the channels. We further identified that port that begins with 10 nm-diameter DNA origami structures that forms PIP2 interacts with arginine 486 (R486) in the first intracellular loop as a puta- pores in synthetic membranes. These DNA origami structures also serves as tive binding site. Notably, our simulation data show that the phosphorylation nucleation sites for DNA tile nanotubes that can grow to many microns in region causes the change of structure of PIP2-binding site in the distance, length, making it possible to study nanofluidic transport within extended thereby altering PIP2 sensitivity. Together, our present study supports that self-assembled biomolecular channels. The DNA origami pores spontaneously PIP2 in the plasma membrane regulates the activity of TMEM16A channel insert into membranes formed using the droplet interface bilayer technique. and structure simulation allow us to understand mechanistic insight into allo- Single-channel electrophysiological measurements indicate that these pores steric modulation of TMEM16A channel by phosphorylation and membrane have ohmic conductances in the range of 1.0-1.2 nS. DNA nanotubes growing PIP2. from the origami pore extend channel length without changing channel archi- tecture produced channels with larger conductances. An artificial DNA 1104-Pos 2D origami shutter that when bound to the DNA origami pore closes off the in- Pip2 Potentiates the Ca -Activated Cl Channel TMEM16A in Xenopus ternal diameter halves the channel’s conductance. Our work thus demonstrates laevis Oocytes that DNA nanostructures several microns in diameter form channels in mem- Maiwase Tembo, Rachel E. Bainbridge, Anne E. Carlson. branes and further how the transport ions across DNA origami nanostructures Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA. can be modulated by a DNA origami structure that serves as a shutter The Ca2þ-activated Cl- channel, TransMEMbrane member 16A (TMEM16A), complex. regulates critical functions including smooth muscle contraction, mucosal secretion, and signal transduction. Despite its importance, we are just begin- ning to understand TMEM16A’s biochemical and biophysical properties. To 1102-Pos study TMEM16A regulation, here we recorded endogenous TMEM16A cur- Effects of Lipid and Detergent Environments on Cystic Fibrosis Trans- rents from Xenopus laevis oocytes. Using the inside-out configuration of the membrane Conductance Regulator (CFTR) Function and Structure patch clamp technique, we found that TMEM16A-conducted currents ran- Kerry M. Strickland1, Brandon B. Stauffer2, Yusuf M. Uddin3, down within seconds of patch excision despite the continued presence of Barry R. Imhoff2, Ingeborg Schmidt-Krey4, Nael A. McCarty5. þ Ca2 . Current rundown is common amongst channels regulated by phospha- 1Chem, Georgia Inst of Technology, Atlanta, GA, USA, 2Emory Univ, tidylinositol 4,5-bisphosphate (PIP ). Thus, we tested the hypothesis that Atlanta, GA, USA, 3Bio, Georgia Inst of Technology, Atlanta, GA, USA, 2 TMEM16A is potentiated by PIP by exposing excised inside-out patches 4Dept Biol/Chem/Biochem, Georgia Inst of Technology, Atlanta, GA, USA, 2 5 to agents that either increased or sequestered membrane PIP2. We found Dept Pediatrics/PACS, Emory Univ Sch Med, Atlanta, GA, USA. þ that following rundown, dioctanoyl-PIP (diC8- PIP ) applied with Ca2 Several recently-released structures of the Cystic Fibrosis Transmembrane 2 2 recovered TMEM16A-conducted currents by 3.5-fold, but not when applied Conductance Regulator (CFTR) protein in a detergent-micelle have enabled þ without Ca2 . Conversely, application of dioctanoyl-phosphatidyl inositol great progress in understanding CFTR; however, many labs report that (diC8-PI), comprised of the backbone of diC8- PIP but lacking the two phos- CFTR has significantly reduced ATPase activity in detergent-micelles and 2 phate groups, had only a nominal effect. We also found that PIP sequestering has reduced stability as exhibited by aggregation over short periods of time. 2 agents, neomycin and anti-PIP , sped TMEM16A current rundown by 2-fold. This begs the question, why is wildtype-CFTR (wt-CFTR) so unstable in deter- 2 Current rundown was slowed by at least 2-fold when we enabled rephosphor- gent, and would a lipid environment improve stability? Is the ATPase rate, or ylation of PI with Mg-ATP application, or by inhibiting PIP dephosphoryla- channel activity, itself, different when CFTR is solubilized in detergent as 2 tion by application of the phosphatase inhibitor b-glycerophosphate. In compared to when it is purified within the native lipid environment? And another series of experiments, we also tested our hypothesis that PIP poten- what lipids are natively surrounding and interacting with CFTR? To begin 2 tiates TMEM16A but within intact cells. We used the two-electrode voltage answering these questions, we are using a variety of biochemical and biophys- þ clamp to record the Ca2 -activated Cl- currents from X. laevis oocytes exog- ical techniques, including the following: detergent-solubilized protein purifica- enously expressing pseudojanin, a PIP -depleting enzyme. After the tion, size-exclusion chromatography, reconstitution (into proteoliposomes and 2 rapamycin-induced dimerization of pseudojanin enabling its translocation to nanodiscs), styrene maleic acid lipid particles (SMALP)-based protein purifica- the membrane, we observed that reducing membrane PIP also reduced tion, ATPase assay, mass-spectrometry lipidomics analysis, and transmission 2 TMEM16A conducted currents in Xenopus laevis oocytes. Taken together, electron microscopy. Results thus far indicate that CFTR function, at least as þ our data demonstrate that PIP and Ca2 are both necessary for TMEM16A measured by intrinsic ATPase activity, is much higher when CFTR is 2 to pass current. embedded in a lipid nanodisc than when surrounded by detergent. These results suggest that CFTR function is indeed dependent upon its environment. Studies 1105-Pos are underway to further characterize the lipid-dependence on the activity of Determining the Mechanism of SMase-Mediated Inhibition of CFTR Cur- CFTR via reconstitution in different lipid mixtures and SMALP purification. rent in Primary Bronchial Epithelial Cells Mass-spectrometry lipidomics analysis provides a highly detailed characteriza- Kirsten A. Cottrill1, Brandon B. Stauffer2, Nael A. McCarty3. tion that can be correlated to activity in the different lipid environments. (Sup- 1Pharmacology, Emory University, Atlanta, GA, USA, 2Emory University, ported by CF Fdn. MCCART17G0, CF Fdn. MCCART15R0, and Marcus Atlanta, GA, USA, 3Dept Pediatrics/PACS, Emory University School of Professorship). Key words: Cystic Fibrosis Transmembrane conductance Medicine, Atlanta, GA, USA. Regulator (CFTR), nanodiscs, Styrene Maleic Acid lipid particle (SMALP), Cystic Fibrosis (CF) is a genetic disease characterized by insufficient activity lipidomics. of the chloride channel Cystic Fibrosis Transmembrane conductance Regu- lator (CFTR). Lung failure is the predominant cause of death due to persistent 1103-Pos infection and inflammation. Interestingly, the two bacteria most common to D Allosteric Modulation of Ca2 -Activated Cl Channles TMEM16A by CF airways secrete the virulence factor sphingomyelinase (SMase), which de- PIP2 and CAMKII grades sphingomyelin into phosphocholine and ceramide. Our lab has shown Woori Ko1, Seung-Ryoung Jung2, Cheon-Gyu Park1, Joo Hyun Nam3, that bacterial SMase applied to the basolateral membrane inhibits CFTR cur- Bertil Hille2, Byung C. Suh1. rents on the apical membrane of bronchial epithelial cells from non-CF pa- 1Dept of Brain & Cognitive Sci, DGIST, Daegu, Republic of Korea, 2Dept of tients. However, two important questions remain: (1) what is the Physiology and Biophysics, Univ Washington, Seattle, WA, USA, 3Dept of mechanism of inhibition, and (2) can inhibition be rescued by the FDA- Physiology, Dongguk Univ, Gyeongju, Republic of Korea. approved CFTR potentiator (VX-770)? Preliminary data suggest that ceramide Transmembrane member 16A (TMEM16A), a member of 10 TMEM16 family, is involved in inhibition, as preventing its degradation resulted in similar in- þ has been shown to encode Ca2 -activated Cl- channels that generate chloride hibition of CFTR and also compounded the inhibitory effect of SMase. Still, þ conductance in an intracellular Ca2 - and voltage-dependent manner. Recently how does ceramide accumulation on the basolateral side of airway epithelial many studies showed that TMEM16A channel activity can be regulated by cells affect chloride secretion on the apical side? Preliminary Ussing Chamber PIP2, a phospholipid placed in the inner leaflet of plasma membrane. However, data following permeabilization of the basolateral membrane to monovalent it is not clearly established yet how the membrane PIP2 modulates TMEM16A ions suggest that inhibition is not due to reduced chloride uptake through gating activity in molecular level. Here we present that intracellular ATP the basolateral side. Furthermore, ceramide is a known signaling molecule, changes PIP2 sensitivity of TMEM16A channels through CAMKII-dependent activating enzymes that are known to inhibit CFTR. Thus, future experiments

BPJ 9358_9361 Monday, March 4, 2019 223a utilizing pharmacological agents, siRNA knockdown, and post-translational which can aid in the refinement of allosteric modulation and current structural modification analysis will determine the exact signaling mechanism underly- models. ing inhibition. Lastly, our preliminary data suggest that VX-770 cannot rescue CFTR from inhibition by SMase. This stresses the importance of understand- 1108-Pos ing this inhibitory mechanism, as it may explain why VX-770 does not rescue High Cholesterol Diet Up-Regulates Atrial and Neuronal GIRK Channel CFTR function in a subset of patients as well as predicted. Co-treatment with Activity a drug that decreases ceramide may be necessary to more fully recover CFTR Anna N. Bukiya1, Avia Rosenhouse-Dantsker2. function. [Support: CF Fdn. MCCART17G0, NIH T32 GM008602, NIH F31 1Dept Pharmacology, Univ Tennessee Hlth Sci Ctr, Memphis, TN, USA, HL143863-01]. 2Dept of Chemistry, Univ of Illinois at Chicago, Chicago, IL, USA. Cholesterol is a principal lipid component of the plasma membrane in mamma- 1106-Pos lian cells that plays essential roles in maintaining cell viability, growth, and Implication of Cholesterol in Regulating the Membrane-Interaction Mech- proliferation. Consequently, both excess and deficiency of cholesterol result anism of Vibrio cholerae Cytolysin, a Beta-Barrel Pore-Forming Toxin in a variety of pathological conditions including cardiovascular and neurode- Reema Kathuria1, Kausik Chattopadhyay2. generative diseases. In recent years, cholesterol has been implicated in the 1Department of Biological Sciences, Indian Institute of Science Education regulation of a growing number of ion channels. Here we focus on G and Research Mohali, Mohali, India, 2Dept Bio Sci, IISER Mohali, Mohali, protein-gated inwardly rectifying Kþ (GIRK or Kir3) channels that regulate India. membrane excitability in neuronal and cardiac cells. The most common effect Vibrio cholerae cytolysin, a b-barrel pore-forming toxin forms an important of cholesterol on ion channels is a decrease in channel activity. In contrast, we virulence factor of Vibrio cholerae, contributing towards it’s pathogenesis. have recently shown that unexpectedly, high cholesterol diet up-regulates In order to generate functional transmembrane pores, it binds to target cell GIRK activity in atrial myocytes. Here we show that also in freshly isolated membrane and has exploited the presence of membrane cholesterol which CA1 pyramidal neurons from the hippocampus, high cholesterol diet results is the distinguishing component of eukaryotic cells. However, there is no in an increase in neuronal GIRK whole-cell currents. Subjecting rats to a detailed evidence of cholesterol being involved in regulating pore-forming high cholesterol diet for 18-23 weeks resulted in a 2-fold increase in choles- activity of VCC. Hence, in this study, we demonstrate that the presence terol levels in both atrial and hippocampal tissues. This effect could be of cholesterol in target membrane is obligatory/necessary for functional mimicked by cholesterol enrichment in vitro. Thus, to obtain mechanistic pore-formation mechanism of VCC by employing an array of experiments insights into the up-regulation of GIRK channels by cholesterol, we first em- using artificial membrane system and biomembranes. In this study, we ployed planar lipid bilayers. Our data demonstrated that cholesterol up- have shown that cholesterol governs the efficient interaction of the toxin regulated GIRK function by increasing the open probability of the channel. to liposome and how the presence of cholesterol in membranes modulates Second, using mutagenesis, we traced the unique impact of cholesterol on subsequent oligomeric species assembly and functional pore-formation. To GIRK channels (up-regulation rather than down-regulation) to a transmem- study the physiological relevance of cholesterol in context of eukaryotic brane region. Point mutations within this region switched cholesterol-driven cells, erythrocytes were treated with methyl b-cyclodextrin to sequester up-regulation into down-regulation. Lastly, using a combined computational- cholesterol from the host cell membrane that established the compromised experimental approach, we identified a putative cholesterol binding site that activity of VCC in membranes devoid of cholesterol. Cholesterol appears overlaps with the gating hinge region in the transmembrane domains of both to govern the initial binding of the toxin to lipid vesicle membranes whereas cardiac and neuronal channels. Together, our studies provide novel insights in case of biomembranes, cholesterol plays a crucial role in formation of into the cholesterol-driven up-regulation of GIRK channels in the heart and functional pore by VCC. Further, in the same direction, we explored the the brain. interaction and segregation of VCC in membrane microdomains, known as lipid rafts, wherein VCC seems to sequester in these cholesterol-rich mem- 1109-Pos brane microdomains. In sum, our results suggest the presence of cholesterol Phospholipid Scrambling Activity by TMEM16E/Ano5: Opposite Effects in target membranes to be critical for pore-formation mechanism of VCC of Mutations Causing Bone Dysplasia and Muscular Dystrophy that appears to cause association of VCC with cholesterol-enriched lipid Eleonora Di Zanni, Antonella Gradogna, Cristiana Picco, rafts. Joachim Scholz-Starke, Anna Boccaccio. Institute of Biophysics, National Research Council (CNR), Genova, Italy. 1107-Pos TMEM16E is highly expressed in bone tissue and skeletal muscle and Differential State-Dependent Crosslinking of Azi-Cholesterol with Human different mutations in the human TMEM16E (ANO5 or GDD1) gene are A1 Glycine Receptor using Mass Spectrometry associated with the autosomal dominant bone disease gnathodiaphyseal Nicholas A. Ferraro, Michael Cascio. dysplasia (GDD) or with different recessive forms of muscular dystrophy Duquesne Univ, Pittsburgh, PA, USA. (LGMD2L, limb-girdle muscular dystrophy-2L and distal MMD3, Miyoshi The glycine receptor (GlyR) belongs to a superfamily of pentameric ligand- muscular dystrophy-3). At present, eight GDD-causing TMEM16E/Ano5 gated ion channels (pLGICs) that mediate fast neurotransmission. GlyR mutations have been identified leading to amino acid exchanges at six posi- typically modulates inhibitory transmission by antagonizing membrane depo- tions, while more than 70 frameshift, nonsense, insertion, deletion and aber- larization through anion influx. Allosteric interactions between the receptor rant splicing mutations have been reported in muscular dystrophy patients. and its lipid surroundings affect receptor function, and cholesterol is essential TMEM16E is mainly expressed in intracellular membranes and its physio- for pLGIC activity. Human a1 GlyR was purified from baculovirus infected in- logical function and role in the pathophysiology of these diseases are sect cells and reconstituted in unilamellar vesicles at cholesterol:lipid ratios currently unclear. Members of the TMEM16/Anoctamin family are function- above the cholesterol activity threshold with aliquots of azi-cholesterol. ally split into two categories: TMEM16A/Ano1 and TMEM16B/Ano2 func- State-dependent crosslinking studies of receptors primarily in its resting (no tion as ‘pure’ ion channels, while others, among which TMEM16F/Ano6, are glycine), desensitized (10mM glycine) and open (F207A/A288G, 30nM iver- Ca2þ-dependent phospholipid scramblases mediating the transfer of phos- mectin) states were performed. After photoactivation, covalently crosslinked pholipids between the leaflets of the membrane bilayer. We have recently cholesterol-GlyR were trypsinized, mass fingerprinted by tandem mass spec- shown that human TMEM16E overexpressed in mammalian cell lines dis- trometry (MS), and sites of cholesterol crosslinks in peptides were refined by plays partial plasma membrane localization and gives rise to phospholipid targeted MS/MS. Differential cholesterol crosslinking patterns between resting, scrambling as well as non-selective ionic currents in presence of high intra- desensitized, and open states were observed, highlighting state-dependent dif- cellular Ca2þ, similarly to TMEM16F/Ano6. We use patch-clamp experi- ferences in GlyR lipid accessibility. Distinct state-dependent crosslinking pat- ments and annexin-V binding assays to address the effects of mutations terns indicative of alterations in either the lipid environment and/or channel causing GDD and muscular dystrophy on the function of human TMEM16E. structure were observed throughout GlyR, most prominently observed in the GDD-causing mutations have a gain-of-function phenotype showing phos- M4 transmembrane helix, ECD loops and regions nearing the bilayer interface, pholipid scrambling activity even at basal cytosolic Ca2þ concentrations. and the large intracellular M3-M4 loop. Strikingly, crosslinking patterns within Contrarily, two mutations associated to LGMD2L, involving amino acids the M3-M4 loop offer insight into the generalized structure of this unresolved located in the putative scrambling domain, cause a loss of both ionic current region of all current pLGIC structural models, by suggesting the crosslinked re- and scramblase activity at high intracellular Ca2þ concentrations. Our data gions of this intracellular loop are intimately associated or buried within the suggest that a gain-of-function of TMEM16E activity contributes to the path- lipid bilayer. Taken together, crosslinking coupled with tandem MS has the ophysiology of GDD in bone tissue, while a loss-of function leads to capability to accurately probe and define physiological protein frameworks muscular dystrophy.

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Posters: Membrane Structure II to assess. We ask, given a cellular membrane structure, what could be the dis- tribution and concentration of curvature-inducing proteins necessary to main- 1110-Pos tain this shape? We propose a theoretical approach based on the Helfrich Single-Lipid Sorting and Dynamics at Membrane Curvature Sites: the Ef- model extended for lipid-protein interaction that allows us to compute the field fects of Fluorescence Labeling, Composition, Phase, and Temperature of spontaneous curvature that can sustain a given membrane structure at me- Xinxin Woodward, Christopher V. Kelly. chanical equilibrium. We demonstrate the applicability of this approach by Physics, Wayne State University, Detroit, MI, USA. investigating the role of spontaneous curvature in catenoid-shaped necks — Curvature on cell membranes assists in the spatial organization and domain for- relevant to vesicle trafficking, tubulation, and nuclear pores — and helicoidal mation that are critical for life of eukaryotic cells. Lipids and proteins can ramps — relevant to the structure of the spine apparatus in dendritic spines. sense, be sorted by, and generate both functional domains and membrane cur- We show that there exists energy barriers associated with geometrical varia- vature. Solving the relationship between membrane curvature, phase separa- tions of the membrane structures, highlighting the need for a coordinated action tion, and single-molecule behavior is a key to understanding fundamental of at least two distinct curvature-inducing mechanisms. These findings are in processes, such as phagocytosis initiation. We studied lipids sorting and diffu- agreement with experimental observations of necking processes. sion of single molecules on engineered membrane curvature to understand the effects of fluorescence labeling, composition, phase, and temperature. Five 1113-Pos lipids with varying head-group to tail sizes and eight types of fluorescent lipids Mechanistic Studies of Membrane Remodeling in Receptor Mediated were examined. Single-particle tracking was used to study lipid diffusion coef- Endocytosis ficients in each membrane composition versus membrane topology. Super- Samsuzzoha Mondal, Sankalp Shukla, Tobias Baumgart. resolution imaging was used to observe the sorting of the lipids at curvature Chemistry, University of Pennsylvania, Philadelphia, PA, USA. sites. Our recently published result shows that the fluorophore labeling strategy Receptor mediated endocytosis requires the generation of membrane curvature. on lipids effected both diffusion and sorting, but non-fluorescent membrane Followed by external stimulation, various G-protein coupled receptors and composition had no significant effects when a single lipid phase was present epidermal growth factor receptors are internalized and recycled by this crucial (Woodward et al, BBA Biomembr., 2018). The surrounding, non-fluorescent membrane trafficking pathway. Bin/Amphiphysin/RVS (BAR) superfamily lipids do affect the single-lipid diffusion and sorting when distinct lipid phase proteins have emerged as key effectors in membrane reshaping during the en- separation was present. Preliminary data suggest that the greatest effects of cur- docytic events. In addition to a crescent shaped BAR domain, many of these vature are found when the surrounding membrane is slightly order preferring. proteins contain a Src homology 3 (SH3) domain. BAR proteins sense and These studies will contribute to the greater biophysical understanding of mem- generate membrane curvature with their membrane binding domain whereas brane curvature, which could potentially improve the effectiveness of therapeu- the SH3 domain regulates their interaction with other protein binding partners. tic design and pathogen protection. Receptors containing proline rich domains (PRD) have been found to interact with different classes of SH3 domain containing proteins. While it has been hy- 1111-Pos pothesized that the SH3 domain-PRD interaction plays an important role in Predicting Spectral Properties of Polarity Sensitive Dyes with QM/MM BAR protein mediated receptor internalization, the exact mechanism has thus Simulation far remained elusive. We mimic SH3 domain-PRD interactions in artificial Swapnil Baral1, Lars Gundlach1,2, Bjorn Baumeier3, Edward R. Lyman1,2. lipid bilayers and investigate their effects on the characteristic membrane cur- 1Physics and Astronomy, University of Delaware, Newark, DE, USA, vature generation properties of BAR proteins. PRDs covalently linked to the 2Chemistry and Biochemistry, University of Delaware, Newark, DE, USA, lipid bilayer are designed to recruit the BAR proteins. The associated mem- 3Department of Mathematics and Computer Science & Institute for Complex brane shape changes are monitored by both optical and electron microscopy. Molecular Systems, Eindhoven University of Technology, Eindhoven, Insights into BAR protein mediated membrane remodeling in receptor internal- Netherlands. ization processes from these biophysical studies will be discussed in this Polarity sensitive, lipophilic dyes such as Laurdan report lipid packing in bio- contribution. membranes, as the emission spectrum is red shifted in more polar environ- ments. In simple membranes, the dye is more accessible to solvent in more 1114-Pos disordered membranes, and the spectral shift is well-explained by dipolar relax- Driving Spontaneous Membrane Curvature by Tuning Cardiolipin Con- ation of the solvent. However, in more complex systems other factors may centration and Spatial Distribution in Model Mitochondrial Membranes contribute, especially hydrogen bonding between the environment and the Moeen Meigooni1,2, Emad Tajkhorshid2,3. 1 chromophore. Disentangling the factors which control the spectral shift is Center for Biophysics and Quantitative Biology, University of Illinois at 2 complicated by the fact that polar environments likely stabilize a charge Urbana-Champaign, Urbana, IL, USA, NIH Center for Macromolecular transfer-like state, and so predicting the emission requires accurate modeling Modeling and Bioinformatics, Beckman Institute, University of Illinois at 3 of the relaxation of the environment around the excited state. An approach Urbana-Champaign, Urbana, IL, USA, Department of Biochemistry, Center has been developed in which (i) the local environment is sampled by classical for Biophysics and Quantitative Biology, University of Illinois at Urbana- molecular dynamics (MD) simulation of the dye; (ii) prediction of the absorp- Champaign, Urbana, IL, USA. tion and excited state by numerical quantum mechanics (QM); (iii) parameter- Cell membranes are complex systems with diverse lipid compositions that ization of an excited state MD model (iv) relaxation of the environment around contribute to overall cellular function. Necessary functions like respiration the excited state by MD; (v) prediction of the emission by QM. The QM steps and oxidative phosphorylation are highly dependent on lipid spatial distribu- are computed using GW-BSE (as implemented in Versatile Object-oriented tions and overall membrane morphology, particularly in the inner mitochon- Toolkit for Coarse-graining Applications: Exciton Transport Simulations drial membrane (IMM) where the complex interplay between membrane (VOTCA-XTP)) with the environment modeled as fixed point charges, sampled lipids and bioenergetic proteins generates and maintains its characteristic in the MD simulation steps. Comparison to time dependent DFT shows that curved cristae structures. The anionic diphosphatidylglycerol cardiolipin is GW-BSE yields much more quantitative predictions for both absorption and found most abundantly in the IMM, with the majority asymmetrically distrib- emission, as expected for charge transfer states, because GW more accurately uted to the outer leaflet. Cardiolipin has an inverted cone geometry due to its captures the effective electron-hole interactions following an excitation. The four fatty acyl tails, and has been proposed to play a role in regulating the high- results reveal the mechanisms responsible for the spectral shift of Laurdan, ly curved nature of the IMM. It is not well understood whether spontaneous cur- and map an approach for similar applications to other polarity sensitive probes. vature is elicited by cardiolipin alone, its interactions with IMM bioenergetic proteins and other lipids, or from a more complex molecular process. Using 1112-Pos coarse-grained molecular dynamics, we simulate membrane bilayers with Modeling the Interplay between Curvature-Inducing Proteins and Mem- asymmetric inter-leaflet cardiolipin concentrations, and find that cardiolipin brane Geometry in Organelle Structures: Catenoid-Like Necks and Heli- content and spatial arrangement largely determine the rate and magnitude of coidal Ramps membrane curvature formation. We find that by constraining the lateral diffu- Morgan Chabanon, Padmini Rangamani. sion of cardiolipin to certain regions of the membrane, negative membrane cur- Mechanical & Aerospace Engineering, UC San Diego, La Jolla, CA, USA. vature forms at those regions and remains stable over long timescales, showing An astonishing variety of membrane structures can be observed in the cellular that complex large-scale membrane morphologies can be robustly controlled by environment, both at the plasma membrane and at the organelles. These mor- tuning the spatial distribution of cardiolipin. Further, we characterize the ef- phologies are intricately related to cellular functions, enabling and regulating fects of other mitochondrial lipids and cardiolipin protonation state on curva- fundamental membrane processes. However, the membrane composition of ture generation and stabilization. Therefore, we find that cardiolipin alone curvature-inducing proteins that shape these structures remains challenging can drive the formation of negative membrane curvature at equilibrium.

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1115-Pos dividual lipid species (such as area per lipid), but also reproduced the major- Lipid Phase Asymmetry in Mammalian Bilayer Membranes ity of the correct experimental phase separation. Our previous studies to Joseph H. Lorent, Lakshmi Ganesan, Ilya Levental. iteratively improve and evaluate the lipid parameters was carried out using Integrative Biology and Pharmacology, UT Health Science Center at standard system sizes of 3,000 lipids that were restrained to maintain a Houston, Houston, TX, USA. planar lipid bilayer.Here we further expand on this work to investigate Cells invest significant energy to maintain a compositional asymmetry between how the size of the system, as well as membrane undulations influence the the two apposing leaflets of the plasma membrane (PM), emphasizing the phase separating behavior of the various lipid mixtures. This work performed importance of this distribution for cell homeostasis. Despite the central involve- under the auspices of the U.S. Department of Energy by Lawrence Liver- ment of PM asymmetry in various physiological processes, the distinct, more National Laboratory under Contract DE-AC52-07NA27344. Release detailed, comprehensive compositions of the PM leaflets are not known, nor numbers: LLNL-ABS-759357. is the influence of asymmetric lipid distribution on leaflet biophysical proper- ties. We therefore combined a detailed lipidomic analysis of PM leaflet compo- 1118-Pos sitions with an investigation of leaflet-specific biophysical properties through Ripple and Gel Phases of Saturated Phosphocholine Bilayers Investigated microinjection of an environment sensitive probe. We further explored if leaflet with Simulations Pouyan Khakbaz1, Jeffery B. Klauda2. properties are maintained in intracellular organelles, and the consequences of 1 2 these asymmetries on the structure of transmembrane proteins. In plasma mem- Univ Maryland, College Park, MD, USA, Dept Chem/Biomolec Eng, Univ branes of human red blood cells, we discovered a dramatic disparity in lipid Maryland, College Park, MD, USA. acyl chains, with the inner leaflet containing approximately two-fold more Lipid bilayers with saturated acyl chains typically are in a liquid crystalline acyl chain unsaturations compared to the outer leaflet. Membranes containing (La) phase, but can condense to a ripple (Pb)orgel(Lb) phase. We focused highly unsaturated lipids are typically characterized by reduced lipid packing on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmi- and we indeed observed that the inner leaflet was much more disordered than toyl-sn-glycero-phosphocholine (DPPC) lipid bilayers and their mixtures to the outer leaflet. This lipid phase asymmetry with the outer leaflet comparable characterize these lipid phases. MD simulations with the CHARMM36 lipid to a liquid ordered ‘‘raft’’ phase was largely maintained during endocytosis. An force field accurately predict the main transition temperature of DMPC and analysis of the human proteome revealed that transmembrane proteins have DPPC bilayers. Our MD simulations focused on the less-characterized Pb asymmetric shape in their transmembrane domains in organelles which partic- phase and compared to x-ray experiments. The Pb major arm has similar struc- ipate in exo- or endocytosis. This asymmetric shape produces an energetically ture to that of the Lb, while the thinner minor arm has interdigitated chains and favourable interaction with the asymmetrically packed membranes, and we the transition region between these two regions has large chain splay and dis- conclude that those shapes have co-evolved with the lipid phase asymmetries. order. The length of these major/minor arms are in reasonable agreement with m The function of the observed phase asymmetry remains to be definitively deter- x-ray experiments based on several sofsimulation.TheLb phase form a mined, but we speculate that this arrangement is a ‘‘best of both worlds’’, chain tilt and lipid packing in good agreement with experiment. This work combining low permeability and high mechanical robustness in the exoplasmic demonstrates that the CHARMM36 lipid force field can accurately represent leaflet with high mobility and diffusion in the cytoplasmic leaflet required for more condensed phases and provides insight into the complex structure of the dynamic intracellular signalling. ripple phase. 1119-Pos 1116-Pos Vitamin E Promotes the Inverse Hexagonal Phase Providing Insights on Structure of Gel Phase DPPC Determined by X-Ray Diffraction Lipid Packing Stresses: Studies by SAXS and 2H NMR 1 1 2 John F. Nagle , Pierre Cognet , Fernando G. Dupuy , Andres T. Cavazos1, Paul E. Harper2, Jacob J. Kinnun1, Horia I. Petrache1, 3 Stephanie A. Tristram-Nagle . 1 1 2 Stephen R. Wassall . Dept Phys, Carnegie Mellon Univ, Pittsburgh, PA, USA, Biochemistry, 1 2 3 Dept of Physics, IUPUI, Indianapolis, IN, USA, Dept Phys/Astron, Calvin Chemistry and Pharmacy, National University of Tucuman, Argentina, Dept Coll, Grand Rapids, MI, USA. Physics Biol, Carnegie Mellon Univ, Pittsburgh, PA, USA. Vitamin E protects membrane lipids from oxidation. How vitamin E partitions High resolution low angle x-ray data are reported for the gel phase of DPPC ˚ 1 ˚ 1 within a membrane and whether it preferentially interacts with polyunsaturated lipid bilayers, extending the previous q range of 1.0 A to 1.3 A , and em- lipids are long-standing questions. Polyunsaturated fatty acids (PUFA) prefer- ploying a new technique to obtain more accurate intensities and form factors entially incorporate into phosphatidylethanolamine (PE) lipids, which have a |F(q)| for the highest orders of diffraction. Combined with previous wide angle tendency to form the inverse hexagonal (HII) phase. We present small-angle x-ray and volumetric data, a space filling model is employed to obtain gel phase X-ray scattering (SAXS) and solid state 2H NMR data that illustrate vitamin structure at a mesoscopic level. A new conclusion from this analysis is that the E promotes the transition to H phase in PE lipids. It has been established hydrocarbon chains from opposing monolayers are mini-interdigitated; this II that hydrophobic molecules, such as dodecane, promote the HII phase in PE would help explain the previously well-established result that the opposing lipids by packing in the hydrophobic interior and so relieving the extensive monolayers are strongly coupled with respect to their chain tilt directions. stress. The hydrophilic hydroxyl head group on vitamin E, in contrast, is Even more detailed structural features are described that have not been ob- anchored near the aqueous surface while the phytanyl chain extends into the tained from experiment but that could, in principle, be obtained from simula- hydrophobic region. Based on analysis of our data we then reason that vitamin tions that would first be validated by agreement with the wide angle and the E promotes the HII phase by a novel mechanism, instead relieving the compres- new low angle |F(q)| x-ray data. sive stress. These observations provide a deeper understanding of lipid packing stress and potentially give insight into a structural role for vitamin E in its func- 1117-Pos tion to protect polyunsaturated lipids that are most vulnerable to oxidative The Influence of Periodic Size Effects and Membrane Undulation on Phase attack. Separation in a DPPC/DOPC/Chol Coarse Grain Martini System Timothy S. Carpenter1, Helgi I. Ingolfsson1, Cesar Lopez2, Chris Neale2, 1120-Pos Sandrasegaram Gnanakaran2, Felice C. Lightstone1. pH-Tunable Floating Lipid Bilayers 1Biosciences and Biotechnology Division, Lawrence Livermore National Dennis J. Michalak1, Mathias Lo¨sche1,2, David Hoogerheide2. Laboratory, Livermore, CA, USA, 2Los Alamos National Laboratory, Los 1Physics, Carnegie Mellon University, Pittsburgh, PA, USA, 2National Alamos, NM, USA. Institute of Standards and Technology, Gaithersburg, MD, USA. Ternary mixtures containing a high melting temperature lipid (such as di- Continuous solvent exchange has been shown to be a reliable and simple palmitoyl-phosphatidylcholine, DPPC), a low melting temperature lipid method to form supported planar lipid bilayers on solid substrates, particularly (such as di-oleoyl-phosphatidylcholine, DOPC), and cholesterol (CHOL) those that are not amenable to vesicle fusion. Here we show that bilayers form bilayers consisting of up to three different lipid phases. The potential formed in this way can be manipulated using the characteristic surface proper- lipid phases that can form are liquid-disordered (Ld), liquid-ordered (Lo), ties of these substrates. In addition, the mechanism of formation involves an a gel-like phase (Lb), or any combination of the three. The phase(s) present initial partial monolayer of lipid near the substrate that serves as a template within these membranes are dependent on the specific percentage composi- for bilayer assembly during the solvent exchange process. Using neutron reflec- tion of the three components within the membrane. The mixture-dependent tometry, we determine the structural features, especially the bilayer-substrate phases formed by these specific constituents have been well mapped exper- separation distance and the amplitude of bilayer fluctuations, of solid supported imentally to construct detailed phase diagrams. In our recent work, we pre- bilayers on TiOx and SiOx thin films. On TiOx films, the separation of a bilayer sented a modified version of the CG Martini DPPC and DOPC lipid from the substrate is tunable from 1 to 5 nm by adjusting the pH and ionic parameters. These parameters not only reproduced the characteristics of in- strength of the bulk solvent. On the other hand, the separation distance of

BPJ 9358_9361 226a Monday, March 4, 2019 bilayers from SiOx control films shows no variation with pH. The difference resolution microscopy of tubes confined to the interface between the two can be attributed to the strong and weak variations, respectively, of the TiOx polymer-rich solutions. At certain deflation levels, the nanotubes transform and SiOx surface charge over the pH range studied. A free energy model is into disk-like structures which we identify as double-membrane sheets developed to predict bilayer stability based on electrostatic contributions (DMSs). The DMSs can be connected to the mother vesicle via a short neck derived from double layer theory, van der Waals forces, steric repulsion, and or a nanotube of larger diameter. They nucleate from the nanotubes and short-range hydration forces. The model accurately predicts the observed grow at the expense of them, and can either consume all nanotubes upon for- bilayer-substrate separations and accounts for the increased bilayer fluctuations mation or coexist with them at the interface between the two aqueous polymer when the bilayer is far from the substrate. Implications for new measurement solutions. The DMSs do not close up into a double-membrane vesicle but are platforms and devices involving tunable floating bilayers are discussed. stabilized by the interface and bend with it. Theoretical considerations show that the tube-to-DMS transformation depends on the interplay between the 1121-Pos interfacial tension, the intrinsic contact angle (PRL 103:238103, 2009), and Model for Stability of Lipid Droplet Connection to the Membrane of the spontaneous tension of the membrane. (Faraday Disc. 161:305, 2013). Endoplasmic Reticulum When we increase the concentration of the encapsulated polymers (via further Gonen Golani1, Michael M. Kozlov2. 1 2 deflation), both the intrinsic contact angle and the interfacial tension increase Physics, Tel Aviv Univ, Tel Aviv, Israel, Physiology and Pharmacology, (ACS Nano 10:463, 2016), which favors DMSs over tubes. Our system mimics Tel-Aviv Univ, Tel-Aviv, Israel. the coexistence of tubes and cisternae in the endoplasmic reticulum and may Lipid Droplets (LDs) store fat and play a critical role in the lipid homeostasis of help to understand the origin of double-membrane, sheet-like phagophores cells. Hydrophobic molecules fill the bulk of a LD while its surface is covered observed during autophagy. This work is part of the MaxSynBio consortium, by a phospholipid monolayer. LDs form between two leaflets of endoplasmic jointly funded by the Federal Ministry of Education and Research of Germany reticulum (ER) membrane as a result of accumulation of enzymatically synthe- and the Max Planck Society. sized triacylglycerols and sterol esters. The ER is a continuous network of membrane tubules and sheets with small or vanishing lateral tension. LDs for- 1124-Pos mation occurs mostly in the tubular membranes of the peripheral ER. The ar- High Yield Assembly of Giant Unilamellar Vesicles using Cellulose Paper chitecture of the connections between the LDs and the ER membranes plays and Cotton Fabric an important role in LD function. Our aim is to analyze the stability of the Joseph Pazzi. ER-LD structures in dependence on the phospholipid compositions of ER UC Merced, Merced, CA, USA. membranes and, specifically, on the spontaneous curvatures of the membrane We recently reported a cellulose-abetted method, PAPYRUS for Paper- leaflets. We found that LDs are expected to separate from the ER if the overall Abbetted amPhiphile hYdRation in aqUeous Solutions, for producing giant lipid intrinsic curvature of the membrane monolayers is positive. These results unilamellar vesicles (GUVs). The cellulose-abetted method produces GUVs suggest a mechanism for the cell to control the connectivity of the LD to the ER from a wide variety of charged and uncharged lipids, in various ionic and by controlling the lipid content of the ER membranes. non-ionic buffers, at variable temperatures within 1 hour without requiring a 1122-Pos power source or specialized equipment. Cellulose does not contaminate the re- Dewetting-Induced Formation of Bacterial Model Membranes using Sub- sulting GUVs. In this work, we report the effect of the porosity and the ordering micron Shell Double Emulsions of the cellulose fibers on the sizes and yields of GUVs. Using high-throughput 1 2 3 1 single-plane confocal image analysis, we quantified the properties of popula- Sepehr Maktabi , Noah Malmstadt , Jeffrey Schertzer , Paul Chiarot . 5 1Mechanical Engineering, State University of New York at Binghamton, tions of O(10 ) GUVs harvested from three different papers and a cotton fabric. 5 Binghamton, NY, USA, 2Chemical Engineering and Materials Science, We find that the yields of GUVs were extremely high, 1.5 x 10 vesicles per Biomedical Engineering, and Chemistry, University of Southern California, microgram of lipid deposited. This number exceeds the yields reported for elec- Los Angeles, CA, USA, 3Biological Sciences, State University of New York troformation. The populations of GUVs harvested from the substrates showed a at Binghamton, Binghamton, NY, USA. unimodal-skewed distribution of sizes centered around four micrometers with a The double-membrane cellular envelope in Gram-negative bacteria enables prominent right tail that extended to 60 micrometers. Ninety-eight percent of < them to endure harsh environments and represents a barrier to many clinically the GUVs were 20 micrometers in diameter. Although the formation > available antibiotics. The outer membrane (OM), which is exposed to the of GUVs 20 micrometers was statistically rare, due to the high yield of environment, is believed to be the first point of contact involved in funda- GUVs, we obtained 1000 vesicles with diameters that ranged from 20 - 60 mental bacterial processes such as signaling, pathogenesis, and motility. As micrometers in one microliter of the harvested suspension. Thus, the in the cytoplasmic membrane, the OM in Gram-negative bacteria has a cellulose-abetted method produces GUVs of comparable sizes to electroforma- phospholipid-rich inner leaflet; however, the outer leaflet is predominantly tion, at a higher yield, in an overall simpler format. composed of lipopolysaccharide (LPS). In this work, we report on a microflui- dic technique for fabricating monodispersed giant unilamellar vesicles (GUVs) 1125-Pos possessing the Gram-negative bacterial OM lipid composition. Regulation of Horizontal Gene Transfer via Bacterial Extracellular Our continuous microfluidic fabrication technique generates monodisperse Vesicles 50 to 100 mm diameter water-in-oil-in-water double emulsions at high- James Boedicker, Frances Tran. throughput. The water-oil and oil-water interfaces facilitate the self-assembly Dept Phys/Astro, Univ Southern Calif, Los Angeles, CA, USA. of phospholipid and LPS molecules to create the inner- and outer-leaflet of Recent experimental work has revealed the ability of bacteria to produce and the lipid bilayer, respectively. The double emulsions have submicron oil shells; uptake extracellular vesicles. Such vesicle exchange enables cells to exchange this minimizes the amount of residual organic solvent that remains trapped biomolecules, including genetic material, which enables horizontal gene trans- within the GUV membrane. We show that micropipette aspiration of the sub- fer between bacteria. Many of the biophysical mechanisms and parameters that micron oil shell triggers an attractive interaction between the two lipid mono- control the production and uptake of such extracellular vesicles have not been layers assembled on water-oil and oil-water interfaces (i.e. dewetting explored. We have systematically characterized how multiple cellular parame- transition), forcing them to bond and form a lipid bilayer membrane. The effect ters influence gene exchange in extracellular vesicles. Gene transfer rates were of different lipid compositions on the stability and growth rate of dewetting measured for a library of plasmids with variable size, copy number, and origin transition is investigated. of replication, revealing that although DNA was randomly loaded, plasmid properties such as the origin of replication strongly influence the rate of gene 1123-Pos exchange. Cells can also regulate vesicle exchange rates through the release Nanotubes Transform into Double-Membrane Sheets at the Interface be- of exogenous molecules. In eukaryotic systems, vesicle exchange often re- tween Two Aqueous Polymer Solutions quires molecules that modulate the large energetic cost associated with mem- Ziliang Zhao, Roland Knorr, Jaime Agudo-Canalejo, Tom Robinson, brane restructuring during vesicle production and uptake. Several compounds Reinhard Lipowsky, Rumiana Dimova. were identified that increased both the rate of vesicle production and the rate Theory and Bio-Systems, Max Planck Institute of Colloids and Interfaces, of vesicle uptake. These compounds include antimicrobial peptides, demon- Potsdam, Germany. strating that membrane binding antibiotics may have a secondary role of regu- Giant vesicles encapsulating polymer solutions (PEG and dextran) can undergo lating vesicle exchange at low, sub-inhibitory concentrations. In the larger morphological transformations upon osmotic deflation resulting in aqueous context of horizontal gene transfer within diverse populations of bacteria, these phase separation of the encapsulated solution (Adv. Mater. Interfaces results suggest that gene transfer via extracellular vesicles is a ubiquitous mech- 4:1600451, 2017). The vesicle membrane can exhibit budding and internal anism of gene exchange and the rate of exchange depends on multiple factors nanotube formation. We resolved the nanotube diameter using super- set by both the donor and recipient cells.

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1126-Pos were synthesized by treating the cells with lysing enzymes from Trichoderma Fatty Acid Compositions of Ceramides and Sphingomyelins in Mamma- harzianum. Spheroplast formation was characterized by lack of their ability to lian Tissues and Cultured Cells survive osmotic shock by deionized water. Membrane fluidity of spheroplasts Felix M. Goni1, Marco M. Manni1, Jesus Sot2, Enara Arretxe1, of the three different strains, studied using diphenylhexatriene (DPH) fluores- Ruben Gil-Redondo3, Juan Falcon3, David Balgoma4, Cristina Alonso4, cence anisotropy, revealed increased order in the membrane of WT compared Alicia Alonso5. to the mutant strains. Further biophysical characterizations is focused on anal- 1Dept Biochemistry, Basque Country Univ, Bilbao, Spain, 2Bioquimi, ysis of ordered domain formation using fluorescence spectroscopy and Unidad Biofisica (CSIC-UPV/EHU), Leioa, Spain, 3CIC-Biogune, Derio, microscopy. Spain, 4Owl Metabolomics, Derio, Spain, 5Instituto Biofisika UPV/EHU, Leioa, Spain. 1129-Pos Sphingolipids consist of a sphingoid base N-linked to a fatty acyl chain. Among A Novel Nitrone-Trolox Conjugate Inhibits Membrane Lipid Oxidation them, sphingomyelins (SM) are major components of mammalian cells, while Through Synergistic Antioxidant Effects ceramide (Cer) plays an important role as a lipid second messenger. We have Larissa Socrier1,2, Marie Rosselin3, Ana Milena Gomez Giraldo2, performed a quantitative lipidomic study of Cer and SM species in different Benjamin Chantemargue4,5, Florent Di Meo4, Patrick Trouillas4,5, mammalian tissues (adipose tissue, liver, brain and blood serum of human, Gregory Durand3, Sandrine Morandat2. mice, rat and dog), as well as in cell cultures of mammalian origin (primary he- 1Lehigh University, Department of Physics, Bethlehem, PA, USA, patocytes, immortalized MDCK cells, mice melanoma b16 cells, and mice pri- 2Universite de Technologie de Compie`gne, Genie Enzymatique et Cellulaire, mary CD4þ T lymphocytes) using an ultra-high performance liquid FRE 3580 CNRS, Compie`gne, France, 3Universite d’Avignon, Institut des chromatography coupled to time-of-flight mass spectrometry (UHPLC-ToF- Biomolecules Max Mousseron, UMR 5247 CNRS, Avignon, France, MS)-based platform. The data have been compared with published, in general 4Universite de Limoges, INSERM, IPPRITT, U1248, Limoges, France, semi-quantitative, results from 20 other samples, with good agreement. The 5Palacky´ University, RCPTM, Olomouc, Czech Republic. sphingoid base was predominantly d18-1 sphingosine (2-amino-4-octade- Polyunsaturated fatty acids are particularly sensitive to the deleterious effects cene-1,3-diol) in all cases. The fatty acid composition of SM was clearly of reactive oxygen species (ROS) as ROS can trigger their degradation through different from that of Cer. In virtually all samples the most abundant Cer spe- a chemical reaction called lipid peroxidation. Lipid oxidation has been reported cies were those containing C24:0 and C24:1 in their N-acyl chains, while the to be involved in the early stages of several diseases as well as the deterioration main species contained in SM was C16:0. Brain was the most divergent tissue, of food supplies. To prevent the deleterious effects of ROS, water soluble free in which Cer and SM C18:0 were very abundant. radical scavengers like a-phenyl-N-tert-butylnitrone (PBN) and 6-hydroxy- 2,5,7,8-tetramethylchroman-2- (Trolox) have been widely 1127-Pos used as protective agents in various biomimetic and biological models. In order Imaging Organization in the Escherichia coli Outer Membrane to increase the hydrophobicity and the ability of these compounds to interact Sandip Kumar, Nicholas G. Housden, Patrick Inns, Colin Kleanthous. with membranes, three amphiphilic Trolox and PBN derivatives were recently Biochemistry, University of Oxford, Oxford, United Kingdom. designed by adding a perfluorinated chain to Trolox and PBN. A sugar moiety Antibiotic resistance in bacteria is on the rise for all classes of antibiotic. In the was added as well to ensure water solubility. case of pathogenic Gram-negative bacteria, many are intrinsically resistant to In the present work, we studied the physical-chemical properties of the de- some classes of antibiotics due to the outer-membrane (OM) acting as a diffu- rivatives and related their antioxidant properties to their ability to interact sion barrier for drug molecules. Hence understanding how the OM is built and with lipid membranes. We first investigated their ability to inhibit the maintained offers new routes for combatting antimicrobial resistance. The OM AAPH-induced oxidation of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine is asymmetric, with an outer leaflet of lipopolysaccharides (LPS) and an inner (DLPC) liposomes as well as their ability to interact with DLPC membranes. leaflet of phospholipids. The predominant proteins within this asymmetric Results evidenced that the three derivatives are efficient to prevent lipid bilayer are b-barrel outer membrane proteins (OMPs). While a great deal is oxidation and that the location in the hydrophobic core of the membrane known about the biogenesis of OMPs and LPS and how they are deposited is a key parameter in the antioxidant efficiency. The derivative bearing on the surface of Gram-negative bacteria, surprisingly little is known about both PBN and Trolox moieties on the same fluorinated carrier exhibited a how these molecules are organised relative to each other in a live bacterium. synergistic antioxidant effect by delaying the oxidation process and by scav- Using OMP-specific fluorescent probes, we recently discovered that OMPs enging free radicals. Molecular dynamics simulations supported the under- are organised into large, supramolecular assemblies (OMP islands) the forma- standing of the mechanism of action, highlighting various key physical- tion of which underpins the turnover of OMPs in bacteria. In the work to be chemical descriptors. presented, we combine diffraction limited fluorescence and super-resolution microscopy methods with specific labelling of OMPs and LPS to investigate their relative organisation in live bacteria and how these change during cell di- 1130-Pos vision and in response to environmental stress factors. A Role for Lipid-Lipid Interactions in Vitamin E’s Function as a Mem- brane Antioxidant 1128-Pos Samuel W. Canner1, Fangqiang Zhu1, Scott E. Feller2, Stephen R. Wassall1. Biophysical Characterization of the Plasma Membrane in Live Crypto- 1Physics Dept, IUPUI, Indianapolis, IN, USA, 2Dept Chemistry, Wabash coccus neoformans Coll, Crawfordsville, IN, USA. Amid Vahedi, Amir M. Farnoud. Vitamin E (a-tocopherol) is the principle lipid soluble antioxidant in cell mem- Chemical Engineering, Ohio University, Athens, OH, USA. branes. Its purpose is to protect membrane lipids from oxidative damage. Cryptococcus neoformans is an environmental fungal pathogen, capable of Whether unequal affinity for different lipids optimizes the proximity of vitamin causing neurological infections in immunocompromised individuals. It has E to polyunsaturated phospholipids, the lipid species most susceptible to oxida- been shown that plasma membrane lipids, in particular, the glycosphingolipid, tion, is the question that we address with MD simulations on lipid bilayers. Our glucosylceramide (GlcCer), plays a major role in virulence capabilities of this studies suggest cholesterol, ubiquitous in the cell membranes of animals, ex- fungus. GlcCer produced by the wild type (WT) C. neoformans is unsaturated cludes vitamin E from saturated raft-like domains enriched in the sterol. Pref- in position 8 and methylated in position 9 of the sphingoid base. An engi- erential affinity for polyunsaturated phospholipids is not indicated - vitamin E, neered fungal strain in which GlcCer lacks the methyl group in position 9 like polyunsaturated phospholipids, is pushed towards non-raft regions shows severe attenuation of virulence in the mouse model. Interestingly, depleted in cholesterol. The binding energy measured for vitamin E in umbrella loss of both the methyl group and the double bond in another strain, leads sampling AA (all-atom) simulations is greater for SM (sphingomyelin) than to complete loss of virulence in mice. Studies using purified GlcCer from PDPC (1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine). Add- these strains have shown that increased lipid saturation results in membrane ing cholesterol to SM eliminates the differential in binding energy. CG lipid order and domain formation in vesicle models. However, how different (coarse-grained) simulations run on PDPC/SM/cholesterol mixtures in the pres- chemical structures of GlcCer alter the biophysical properties of the plasma ence of vitamin E indicate the vitamin locates at the boundary between SM- membrane in each strain, thereby regulating pathogenicity, remains unclear. rich/sterol-rich (raft-like) and PDPC-rich/sterol-poor (non-raft) domains. CG In this study, the effects of alterations in the of GlcCer simulations run on mixtures replacing PDPC by DDPC (1,2-didocosahexa- on the biophysical properties of the plasma membrane of live C. neoformans noyl-sn-glycero-3-phosphocholine) will reveal details of the molecular interac- strains was investigated. Fungal spheroplasts, living microorganisms without a tions of vitamin E at the distinct boundary between raft-like and non-raft cell wall, were used for membrane biophysical characterization to allow for domains that forms with the di-polyunsaturated phospholipid. The results of efficient incorporation of fluorescent probes in the membrane. Spheroplasts these studies will be presented.

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Posters: Intracellular Transport depleted cells, suggesting that the observed phenotype is not due to a compro- mised kinesin that is unable to move cargoes to the cell periphery. To investi- 1131-Pos gate the mechanism by which depletion of GSK3 disrupts cargo localization we Anomalous Diffusion of Endoplasmic Reticulum Constituents identified a downstream phosphorylation target of GSK3, a microtubule asso- Konstantin Speckner, Lorenz Stadler, Matthias Weiss. ciated protein (MAP), which not only co-localizes with the cargo, but also be- Dept Exp Physics I, Univ Bayreuth, Bayreuth, Germany. comes centrally clustered in the absence of GSK3. Insight into the mechanism Diffusion of macromolecules and higher-order structures in the crowded inte- by which this GSK3/MAP pathway results in altered cargo distribution will be rior of cells frequently shows an anomalous behavior with the mean-square presented. displacement (MSD) increasing nonlinearly in time, MSDfta. Here we 1134-Pos have probed to which extent also larger organelle structures show such an Investigating the Interplay between PIKfyve/PI(3,5)P2 and CLC-7 in anomalous diffusion and how non-equilibrium contributions affect their diffu- Lysosomal Acidification and Traffic sional motion [Phys. Rev. E 98, 012406 (2018), in press: Biophys. J. 115 Xavier Leray, Anowarul Amin, Mary Weston, Joseph A. Mindell. (2018)]. In particular, we have employed single-particle tracking to monitor NINDS, NIH, Bethesda, MD, USA. the motion of tubular junctions in the endoplasmic reticulum (ER) network Through the endocytic pathway, internalized vacuoles mature, acquiring pro- and of long-lived membrane domains on the ER, called ER exit sites gressively an acidic pH and eventually ending-up to fuse with lysosomes to (ERES). Our results show that both, ER junctions and ERES show a distinct form endolysosomes. PIKfyve is a membrane-located lipid kinase found in anomalous diffusion with a significant anti-correlation of successive step in- the endocytic pathway that participates in the maturation of endosomes. It is crements that is reminiscent of fractional Brownian motion. Disrupting the the only enzyme responsible for the conversion of phosphatidylinositol-3- microtubule cytoskeleton significantly altered the subdiffusive characteristics phosphate PI(3)P to PI(3,5)P2, a low abundance phospholipid located in the of both entities, highlighting that even anomalous diffusion is an actively membrane of late endosomes and lysosomes. PI(3,5)P2 is a signaling lipid driven process in living cells. While the diffusion behavior of ER junctions which has been shown to directly interact with and modulate the activity of was seen to be directly dependent on the presence and activity of microtu- several lysosomal membrane proteins, including the Ca2þ channel TRPML1 bules, ERES were only indirectly affected. ERES therefore can be seen as mo- involved in endolysosomal trafficking and function. Pharmacological inhibition bile membrane domains that perform a quasi-one-dimensional random walk of PIKfyve leads to PI(3,5)P2 depletion, which quickly and drastically disrupts on the shivering ER backbone. the late endosomal/lysosomal trafficking, leading to the generation of oversized 1132-Pos vacuoles. Transport Modes of Viral in Live Cells A recent study shows that knocking out ClC-7, a lysosomal Cl-/Hþ exchanger, George M. Holzwarth1, Lucas Tommervik1, Arnav Bhandari2, provided substantial resistance against endocytic trafficking defects resulting David Ornelles3, Douglas Lyles4. from PIKfyve inhibition, such as vacuole formation (Gayle et al., Blood, 1Dept Physics, Wake Forest Univ, Winston-Salem, NC, USA, 2Dept 2017). Given that ClC-7 has been suggested to play a role in lysosomal acidi- Computer Science, Wake Forest Univ, Winston-Salem, NC, USA, fication, these results raise the possibility that either PIKfyve or PI(3,5)P2 may 3Microbiology and Immunology, Wake Forest University Health Sciences, regulate ClC-7 activity, in turn modulating the lysosomal pH, and that endoso- Winston-Salem, NC, USA, 4Biochemistry, Wake Forest University Health mal/lysosomal pH could be a critical parameter for the fine tuning of the endo- Sciences, Winston-Salem, NC, USA. cytic trafficking. Within a few hours after infection of a cell by vesicular stomatitis virus (VSV), Using ratiometric live measurements of lysosomal pH in intact cultured cells, newly assembled VSV particles are released from the surface of the infected we first observe that pharmacological inhibition of PIKfyve significantly cell. In that time, the viral RNA and 5 viral proteins have travelled to the hyperacidifies lysosomes. Repeating this experiment in cells in which edge of the cell from the sites of synthesis near the nucleus, a radial distance ClC7 is knocked out abolish the pharmacologically-induced lysosomal of 5-10 mm. The modes of transport from the sites of synthesis to the edge of hyperacidification, providing new evidence for the role of ClC7 as a key regu- the cell are key questions. The movement of VSV ribonucleoprotein particles lator of lysosomal pH, and suggesting that PIKfyve/PI(3,5)P2 may inhibit ClC7 (nucleocapsids) in live A549 cells was recorded by fluorescence video micro- activity. Finally, we analyze the relationship between lysosomal pH and vacu- scopy at 100fps at 3 to 4h postinfection. Each nucleocapsid contains approxi- ole formation and present results indicating that they are uncoupled. mately 400 molecules of GFP-tagged P protein. Nucleocapsids are tracked to subpixel precision. About 60% of the tracks show spatially constrained, 1135-Pos ATP-enhanced Brownian motion. These particles jiggle within an approxi- The Role of the Chloride Transporter CLC-7 in Acidification in Mouse mately circular area (‘‘trap’’) with 2s radii = 0.18 5 0.05 mm. Particles stay Liver Lysosomes in one trap for 1-5 s, then move abruptly to an adjacent trap. Motion within Anowarul Amin, Joseph A. Mindell. a trap is not directional. 1-3% of tracks show short bouts (< 1s) of directed mo- NINDS, National Institutes of Health, Bethesda, MD, USA. tion between traps. The velocity during directed motion is similar in magnitude The lysosome is a membrane-bound compartment that digests macromolecules to the frame-to-frame velocity in the traps. Bayesian analyses based on 2D loca- and cellular debris. To function properly, lysosomes must maintain an acidic tion, particle velocity, and directional change are being used to identify the environment (pH 4.5-5.0), generated by the proton-pumping action of a v- þ traps and the periods of directed motion. Simulations, biochemical perturbation type H ATPase. However, since the ATPase is highly electrogenic, the (motor protein inhibitors) and colocalization of capsids with cytoskeletal fibers voltage generated by its action must be dissipated by another ion movement, are being used to discern the physical basis for the observed modes of travel. known as the ‘counterion pathway.’ It has been recently revealed that acidifi- Supported by grant 1R01 AI120623-01A1 from the US National Institutes of cation in mouse liver lysosomes is dependent on the presence of Cl-, and is þ Health. minimally affected by K , indicating that Cl- serves as a counterion. Liver spe- cific knockout of ClC-7, a gene for the lysosomal Cl-/Hþ antiporter, abolishes 1133-Pos Hþ-coupled Cl- transport in isolated liver lysosomes, implying ClC-7 acting as The Role of Glycogen Synthase Kinase 3 (GSK3) in Regulating Intracel- the primary counterion pathway in this system, also abolishing ATP-driven lular Transport acidification in these organelles. However, when measuring lysosomal pH in Ibtissem Nabti1, George T. Shubeita2. primary hepatocytes, the knockout mice show no substantial lysosomal pH 1New York University Abu Dhabi, Abu Dhabi, United Arab Emirates, change compared wild type, revealing important differences between these 2Physics, New York Univ Abu Dhabi, Abu Dhabi, United Arab Emirates. experimental systems. It has been suggested that mammalian target of rapamy- Intracellular transport is indispensable for proper functioning of the cell, cin (mTOR) complex dictates amino acid homeostasis for cell growth and reg- and failure in regulating the molecular motors that transport cellular cargo ulates the lysosome’s membrane potential and pH stability. Loss of mTOR has been linked to various diseases.Numerous studies have identified Glycogen regulation by knocking out of Tuberous sclerosis complex (TSC) leads to an Synthase Kinase 3 (GSK3) as an important regulator of intracellular transport, increase in mouse liver size. Interestingly, liver specific ClC-7 KO mouse yet the mechanism by which GSK3 acts remains elusive. In this study we used shows 30% increases of liver and body weight comparing to WT, a striking GSK3-depleted cells as a model system to understand how GSK3 regulates correlation. Indeed, we find that mTOR in liver-specific KO ClC-7 mouse be- motor-based transport. We found that GSK3 depletion leads to perinuclear comes fasting-resistant and expresses at higher levels compared to that of WT. clustering of various cargoes as well as the microtubule motors -kinesin and These observations are similar with the phenotype of TSC KO mouse. Given dynein- that carry them. To understand how GSK3 depletion results in central the suggested effects of the mTOR pathway on lysosomal ion channels, it is clustering of motors and cargoes, we inhibited dynein function. Interestingly, possible that mTOR changes account for the compensatory changes observed dynein inhibition rescues the perinuclear clustering of cargoes in the GSK3- in living hepatocytes.

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1136-Pos Firing rate and rhythm of spontaneous action potentials (APs) in sinoatrial Snare Priming Inhibition via Phosphatidic Acid Induced Sec18 Conforma- nodal cells (SANC) are controlled via the fidelity of clock coupling: rhythmic, tional Changes and Competitive Small Molecule Binding to Sec18 spontaneous, local Ca2þ releases (LCRs) that emerges beneath the cell mem- Andres S. Arango1,2, Robert P. Sparks3, Matthew L. Starr3, Zhiyu Zhao1,2, brane (‘Ca2þ clock’) during diastole couples to sarcolemmal electrogenic Muyun Lihan1,2, Rutilio Fratti3, Emad Tajkhorshid2,4. mechanisms (‘membrane clock’). We hypothesized that dormant guinea pig 1Center for Biophysics and Quantitative Biology, University of Illinois at SANC, i.e. cells that don’t fire spontaneous APs, represent an extreme form Urbana-Champaign, Champaign, IL, USA, 2NIH Center for Macromolecular of clock uncoupling, and increased cAMP-mediated, PKA-dependent phos- Modeling and Bioinformatics, Beckman Institute, University of Illinois at phorylation of the coupled-clock system proteins ignite rhythmic AP firing in Urbana-Champaign, Champaign, IL, USA, 3Department of Biochemistry, dormant SANC. Membrane potential of dormant GP SANC averaged University of Illinois at Urbana-Champaign, Champaign, IL, USA, 3851mV(n=46), ICaL,If, and IK current densities were comparable to those 4Department of Biochemistry, Center for Biophysics and Quantitative that fire spontaneous APs, and LCRs were small and random. Isoproterenol or Biology, University of Illinois at Urbana-Champaign, Champaign, IL, USA. CPT-cAMP led to de novo spontaneous AP generation in 50% of dormant Membrane fusion is mediated by SNARE proteins, and is vital for a multitude SANC. The initial response involved increases in LCR size and rhythmicity of cellular transport phenomena. Post fusion, cis-SNAREs are recycled for and small amplitude, spontaneous APs and whole-cell Ca2þ transients. Average continued fusion, in an ATP dependent process called priming. The unique LCR size continued to increase, average LCR period shortened, partially syn- job of priming all cellular SNAREs is the responsibility of AAAþ ATPase chronizing LCRs to late diastole, paralleled by MDP hyperpolarization to -60 NSF or Sec18. In a joint computational and experimental effort, we show mV. AP cycle length shortened similar to LCR period. b-AR stimulation 2þ that Phosphatidic Acid (PA) inhibits the priming of cis-SNARE complexes increased availability of ICaL and IK, and If. Inhibition of Ca clock by CPA by inducing conformational changes in Sec18 protomer. We further studied precluded the isoproterenol-induced AP generation in 91% of dormant priming inhibition via a new small molecule inhibitor of Sec18 called IPA, SANC, but If block by ivabradine did not inhibit the activation. Ivabradine, which inhibits fusion, priming, and competitively inhibits Sec18 binding to however, prolonged the transition between dormant and steady firing state PA. We identify potential PA binding sites to Sec18 using computational flood- following the initiation of b-AR stimulation. When b-AR stimulation/CPT- ing of short tailed PA, as well as using an HMMM PA rich membrane to capture cAMP was removed, all changes reversed in order and SANC again became unbiased spontaneous membrane insertion of Sec18 monomers. In addition, to dormant. At least in some SANC, dormancy represents membrane & Ca2þ further sample potential binding sites of both PA as well as IPA on Sec18 we clock uncoupling that is rescued via cAMP-mediated, PKA-induced increased employed ensemble molecular docking using AutoDock Vina, with consecu- function of both membrane and Ca2þ clock proteins. tive molecular dynamics simulations performed for the top poses using NAMD for characterization of ligand stability. Finally, we performed Random 1139-Pos Accelerated Molecular dynamics simulations on the most stable resultant poses Adenosine Decreases Sinoatrial Node Cell Firing Rate by Uncoupling its to estimate dissociation constants, which were compared to results from lipo- Membrane and Calcium Clocks some binding experiments providing insight into the mechanism of priming Ashley Wirth. regulation via Sec18. Laboratory of Cardiovascular Science, National Institute on Aging, Baltimore, MD, USA. 1137-Pos Endogenous metabolite adenosine is known to slow down the firing rate of Evidence for ATP Interaction with Phosphatidylcholine Bilayers sinoatrial node pacemaker cells (SANC) through activation of the outward Alvaro Garcia1, Ronald J. Clarke2. KACh current, which hyperpolarizes SANC resting membrane potential and in- 1Sch of Life Sciences, University of Technology Sydney, Broadway, hibits of adenylyl cyclase activity. Adenosine has not been studied in the Australia, 2Sch Chemistry, Univ Sydney, Sydney, Australia. context of the contemporary view of cardiac pacemaking i.e. that a coupled ATP is a fundamental intracellular molecule and is thought to diffuse freely clock system drives normal pacemaker function. through the cytosol. However, evidence obtained from nucleoside-sensing Here, for the first time, we have investigated adenosine effect on the membrane sarcolemmal ion channels and red blood cells suggest that ATP is compartmen- and calcium clocks of isolated SANC. We measured calcium and electrophys- talised or buffered, especially beneath the sarcolemma. Macromolecular iological characteristics both separately and simultaneously. Considering aden- crowding of the cytosolic compartment, tortuosity of cytoskeletal structures osine’s inhibition of adenylyl cyclase activity, which is linked to cAMP- þ and non-hydrolysing binding sites on cytoskeletal proteins are some of the mediated PKA-dependent protein phosphorylation and intracellular Ca2 þ physical barriers proposed that could potentially reduce free diffusion of cycling, we hypothesize that adenosine will indirectly change the Ca2 regula- adenine nucleotides through the cytosol. In this study we compared ATP and tion in these cells and will therefore change the coupling of the membrane and adenosine using a spectroscopic study of RH421, a membrane bound electro- calcium clocks in so far as they work together as a system. chromic dye, impedance spectroscopy measurements with tethered phospho- Electrophysiological measurements confirmed that adenosine slows the firing lipid bilayers and differential scanning calorimetry to determine the of cardiac pacemaker cells. As SANC slowed down, beat-to-beat variability þ interaction with zwitterionic phospholipid bilayers. We report a concentration increased. Ca2 measurements showed that adenosine decreases intracellular dependent shift of the excitation spectrum of RH421 indicating an interaction calcium cycling. Local calcium release (LCRs) characteristics of size, duration, between ATP and DOPC unilammelar vesicles, with little effect observed from events, and period significantly changed in response to adenosine. In a subset of adenosine. This was supported by a concentration dependent change in the SANC, substantial slowing of AP firing rate coincided with extreme uncoupling þ calculated membrane conductance of tethered phospholipid bilayers with of the M and Ca2 clocks. Simultaneous measurements informed how the ATP and not adenosine. Concentration dependent alterations to the measured changes to one clock influence the other through clock crosstalk. enthalpy of the DMPC main phase transition with ATP was also observed, Adenosine not only slow SANC firing rate by hyperpolarizing the SANC mem- þ which weren’t replicated with adenosine. These observations provide strong brane, also reduced intracellular Ca2 cycling. Thus, by changing SANC firing evidence for the interaction of ATP with zwitterionic phospholipid bilayers rate, adenosine caused clock uncoupling to occur. These results show how at physiologically relevant concentrations. An interaction between ATP and adenosine creates dysfunction and desynchronization at the cellular level and the phospholipid bilayer could create a population of ATP molecules with a may contribute to the SAN tissue dysfunction observed in vivo. divergent diffusion rate with respect to ‘‘free’’ ATP and thus establish spatially 1140-Pos distinct areas of compartmentalised ATP beneath the sarcolemma. Mechanism for cAMP Overshoot in Ventricular Myocytes Following b1- Posters: Cardiac Smooth and Skeletal Muscle Adrenergic Stimulation Emily E. Meyer1, Timothy J. Lewis1, Colleen E. Clancy2. Electrophysiology II 1Mathematics, UC Davis, Davis, CA, USA, 2Dept Physiology and Medicine, Univ Calif Davis, Davis, CA, USA. 1138-Pos The sympathetic nervous system (SNS) plays a fundamental role in regulating Coupling of Calcium- and Membrane Clocks Ignites De Novo Spontaneous cardiac function through activation of b-adrenergic receptors (b-ARs) and the Action Potential in Dormant Guinea Pig Sinoatrial Nodal Cells via Camp- subsequent increase in cAMP production. However, SNS input to the heart can PKA Signaling sometimes lead to arrhythmias. In particular, certain motifs of SNS input have Kenta Tsutsui, Oliver Monfredi, Mary Kim, Ashley Wirth, Cristina Florio, been shown to trigger large transient increases (i.e., overshoots) of cAMP con- Annie Yang, Dongmei Yang, Bruce Ziman, Victor A. Maltsev, centration in ventricular cells, which have the potential to induce ectopic beats. Edward G. Lakatta. The specific conditions promoting such proarrhythmic activity are unclear. Laboratory of Cardiovascular Science, National Institute on Aging, Stimulation of b1-ARs leads to a cascade of intracellular reactions: adenylyl Baltimore, MD, USA. cyclase activated by stimulatory G-protein increases production of cAMP,

BPJ 9362_9366 230a Monday, March 4, 2019 which activates PKA; PKA then phosphorylates many targets, including cal- Results: Species with larger BM exhibited longer APCL and LCR period, and cium and potassium channels, ryanodine receptors, and troponin, as well as more LCR events per cycle versus that in species with smaller BM. The average b1-ARs. Detailed computational models have been used to describe the b1- APCL and LCR period linearly scaled with BM with scaling exponent close to ¼. adrenergic signaling process, but the complexity of these models makes it diffi- Conclusions: LCR period and APCL in single spontaneously beating SANC cult to uncover the mechanisms underlying subtle behavior, such as cAMP obey the universal law for allometric scaling for BM as does in vivo HR, there- overshoots. Here, we exploit multiple timescales to reduce the 16-variable fore species differences in LCR characteristics of subcellular LCRs predict spe- adrenergic signaling component of the Soltis-Saucerman model to a system cies differences in EKG parameters in vivo. of two differential equations for cAMP and active b1-AR concentrations. We then analyze the reduced model to reveal the mechanism underlying cAMP 1143-Pos overshoot in response to b1-AR activation and identify parameters that deter- Combining Systems Pharmacology Modeling with Machine Learning to mine the magnitude of the overshoot. Our results allow us to make predictions Identify Sub-Populations at Risk of Arrhythmia for how pharmacological methods can be used to deter the potential proarrhyth- Meera Varshneya, Xueyan Mei, Eric A. Sobie. mic effects of SNS input to the heart. Pharmacological Sciences, Icahn School of Medicine Mount Sinai, New York City, NY, USA. 1141-Pos When assessing whether an otherwise healthy individual is at risk of devel- Rhythm and Rate of Action Potential Firing of Single Cardiac Pacemaker oping an arrhythmia, the most common assessment is based on the electrocar- Cells Emerge from Concordant Beat to Beat Variability of Coupled Cal- diographic QT interval. Although this trait predicts susceptibility, there remains cium and Membrane Potential Functions 1 1 1 a percentage of patients who still succumb to an arrhythmia with a normal QT, Dongmei Yang , Alexey E. Lyashkov , Christopher H. Morrell , or at the cellular level a normal action potential duration (APD). Machine Ihor Zahanich1, Yael Yaniv2, Tatiana M. Vinogradova1, Bruce D. Ziman1, 1 learning (ML) can unmask hidden features and provide greater insight needed Edward G. Lakatta . to distinguish between healthy and arrhythmia-prone patients. We sought to use 1NIA/NIH, Baltimore, MD, USA, 2Biomedical Engineering Faculty, this technique along with systems pharmacology models that describe the elec- Technion-IIT, Haifa, Israel. trophysiology of ventricular myocytes to determine key ionic channel abnor- Bi-directional entrainment of surface membrane electrogenic proteins (‘‘M- 2þ 2þ malities and experimentally quantifiable metrics of the AP shape that are clock’’) and intracellular sarcoplasmic reticulum Ca cycling (Ca -clock) unique to highly susceptible cells. We generated heterogeneous populations proteins regulates the mean action potential (AP) firing interval (APFI) of adult of cells using three human ventricular myocyte models, and examined the ef- sinoatrial node cells (SANC). We hypothesized that not only the mean APFI but fect in each population of applying various arrhythmic triggers (increased L- also APFI variability (APFIV) in any ‘‘steady state’’ are determined by concor- þ type calcium current, hypokalemia, block of hERG channel, increased inward dant beat to beat variability of membrane potential and Ca2 regulatory func- current). We then trained ML algorithms (SVM, Random Forest, logistic tions during an AP cycle. We tested this hypothesis in single, isolated adult þ regression, artificial neural network) on features pertaining to the pre- rabbit SANC by measuring mean and beat to beat variability of Ca2 -clock func- 2þ 2þ perturbed AP and the outcome of the applied trigger. Our main goal was to tions (spontaneous diastolic local Ca releases, kinetics of AP induced Ca - find a set of features that could predict risk no matter which perturbation was transient decay) and of M-clock functions (the kinetics of diastolic membrane applied. We found that: (1) 11% of each population was susceptible to all depolarization and AP repolarization) across a broad range of ‘‘steady state’’ four perturbations, (2) APD was a strong predictor of susceptibility, confirming mean AP firing rates effected by autonomic receptor stimulation. A three-fold the utility of testing QT interval (3) incorporating additional features (besides range of the mean APFI was accompanied by a four-fold range of APFIV. In 2þ APD) describing the AP and the calcium transient improves prediction accu- 2 2 comparisons, membrane potential and Ca functional parameters during racy, and (4) the accuracy of the ML algorithms changes based on the pertur- AP cycle were significantly correlated with each other, and each was corelated bation applied. Overall, this computational pipeline can reveal patients who with the mean APFI and APFIV. In unbiased, principle component analyses of appear to be healthy but are highly susceptible to an arrhythmia, thus providing all mean and SD’s, the first 3 principal components accounted for 86.8% of the an automated approach to screen for patient risk. total variance. Distributions of coupled clock regulatory parameter mean values and their beat to beat variabilities over the entire range of APFI obeyed similar 1144-Pos power laws. We conclude that both APFIV and mean APFI, within and across 3-Week-Old Rabbit Cardiomyocytes (3wRbCM): A Novel Cellular Model ‘‘steady states’’, emerge from coherent beat to beat variability of M- and þ for Studying Cardiac Excitation Ca2 -clock regulatory functions that inform on self-similar organization of Anatoli Y. Kabakov, Karni Moshal, YiChun Lu, Karim Roder, availability of molecules that underlie these measured clock functions to become þ Turan Nilufer, Weiyan Li, Kevin Murphy, Dmitry Terentyev, Gideon Koren. activated in response to voltage or Ca2 cues during an AP cycle. Cardiovasc Res Ctr, Brown Univ Medical Sch, Providence, RI, USA. 1142-Pos Primary cultures of rabbit cardiac myocytes (CMs) are preferable model to The Periodicity of a Ca2D Clock Intrinsic to Individual Cardiac Sinoatrial study molecular mechanisms of human cardiac excitation. Here we show that Nodal Pacemaker Cells Universally Scales to Body Mass from Mice to 3wRbCMs contain developed Ttuble network and can be used to study cardiac Humans excitation and EC coupling. Syevda Tagirova1, Kenta Tsutsui1, Dongmei Yang1, Bruce Ziman1, Ventricular myocytes were isolated from left ventricle of 3 weeks old neonatal Yael Yaniv2, Edward G. Lakatta1. New Zealand White rabbits by standard enzymatic techniques. Then 3wRbCMs 1The Laboratory of Cardiovascular Science, The National Institute on Aging, were infected with adenovirus encoding GFP and cultured for 48h in presence Baltimore, MD, USA, 2Biomedical Engineering Faculty, Technion-IIT, of 0.5 mM cytochalasin D. Wheat Germ Agglutinin stain (Alex Fluor 594 con- Haifa, Israel. jugate) was used to determine T-tubule structure. Standard biochemical, optical Prior studies showed allometric scaling relationships between body mass (BM) and whole cell patch-clamp techniques were used to determine expression and and EKG of mammal’s heart, including heart rate (HR), atrioventricular con- function of ion channels. duction (PR interval),etc., as a ¼-power of BM. But can ¼-power scaling Staining of 3wRbCMs revealed clear T-tubule structure, which was mostly pre- that regulates HR in vivo relate to BM when downscaled to the subcellular served after 48h in culture. After 48h, 3wRbCMs had robust expression of the level? Our prior studies revealed that coupling of spontaneous local intracel- following membrane proteins: L-type voltage-dependent calcium channel 2þ lular calcium releases (LCRs), generated by a Ca clock (sarcoplasmic (alpha 1C subunit, Cav1.2), voltage-gated sodium channel (alpha subunit 5, reticulum-SR) to a membrane clock sarcolemmal electrogenic molecules regu- Nav1.5), sodium-calcium exchanger (NCX), and gap junction connexin 43 lates spontaneous sinoatrial nodal cell (SANC) action potential cycle length (Cx43). 3wRbCMs expressing GFP displayed appropriate electrophysiological (APCL) via activating NCX inward current, the larger amplitude and early phenotype including human like action potential duration (APD90) of occurrence (period) of the spontaneous LCR Ca2þ signal, the earlier and larger 224523 ms, L-type calcium channels current of 6.150.3 pA/pF at þ NCX, the faster AP (shorter APCL). Relationships among LCR periods and 20 mV, fast sodium current of 2457 pA/pF (20mV, [Na ]e = 5 mM), þ APCL conforms to a unique linear function. We hypothesized that the LCR pe- late sodium current of 1.150.1 pA/pF (20 mV, [Na ]e = 100 mM), rapid riods, generated within SANC from hearts of different species that vary in BM delayed rectifier current (IKr) of 0.650.1 pA/pF (þ30 mV), transient outward 2þ and HR scale non-linearly as ¼-power of BM of the donor species. potassium current (Ito)of852 pA/pF (at þ50 mV). Ca transients in cultured Methods: Confocal-line-scan microscopy was used to record AP-induced-Ca2þ 3wRbCM were similar to the transients in acutely isolated adult rabbit CM transients and spontaneous LCRs in spontaneously beating, single SANC, iso- stimulated at 1.0 Hz. lated from mouse-(M), guinea-pig-(GP), rabbit-(R) and human-(H) hearts. The 3wRbCMs model is a reliable, reproducible, and low cost alternative to adult APCL and other LCR characteristics and variations of BM across species were rabbit cardiomyocytes. These cells can be used to study the trafficking and analyzed via double-logarithmic plots. expression of cardiac ion channels and EC coupling.

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1145-Pos Magnetic resonance imaging (MRI) can reveal subtle microstructural changes A Comparative Analysis of Parameter Estimation Strategies for Mathe- in the heart muscle following infarction, a major cause of sudden cardiac death matical Modeling of Ion Channel Gating worldwide. We have developed preclinical models of chronic infarction and Chiara Campana, Eric A. Sobie. used diffusion tensor methods to efficiently probe biophysical MR signals. Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New Here our specific goal was to identify subtle characteristics of the peri-infarct York, NY, USA. zone, where the mixture of viable cells and collagen fibrils form arrhythmia Mathematical modeling in cardiac electrophysiology is well established; howev- substrate. We induced myocardial infarction in n=9 Yorkshire swine using er, the veracity of model predictions for pharmacological design and testing is un- an occlusion-reperfusion technique. We occluded the left circumflex artery clear. Ion channel gating is often modeled with Markov schemes that reflect (LCX) in 5 pigs and the left anterior descendent artery (LAD) in 4 pigs. After different occupancy states of the channel. Although experimental data are always 5-6 weeks healing time, high resolution diffusion tensor imaging used to constrain the parameters of these models, it oftenremains unclear whether (0.6x0.6x1.2mm spatial resolution) was performed ex vivo in explanted a set of parameter values is unique, or whether alternative parameter sets could fit hearts. We then calculated pixel-based fractional anisotropy (FA) in 3 zones: the data equally well. Here we have compared alternative parameter estimation infarct core (dense fibrosis), peri-infarct (arrhythmia substrate) and remote/ strategies to build a patient-specific approach to in-silico drug testing. Using a normal tissue. For analysis, we selected four regions of interests (ROIs) þ published Markov representation of the cardiac Na channel, we aimed to opti- from each tissue zone. A total of 36 ROIs selected from DT images were mize the value of rate constants regulating the channel’s transition between analyzed and further registered via anatomical markers with corresponding different states. Data derived from voltage-clamp experiments, performed by im- ROIs in histopathology slides. For , tissue samples were stained plementing a traditional voltage square steps protocol in HEK293 cells, were with collagen-sensitive stain to evaluate fibrosis under bright field and used to test the following approaches: (1) ‘‘broad fitting algorithm’’ using polarized light microscopy, as well as CD31 (microvascular integrity) and elec- population-based mathematical modeling; (2) optimization through genetic al- trical gap junctions (Cx43). Our results demonstrated a significant decrease in gorithm; and (3) optimization through direct search simplex algorithm. We FA (25%) in peri-infarct compared to normal myocardium (p<0.05), generated a population (n=10000) of channel variants by randomizing Markov in agreement with quantitative histopathological and immunohistochemistry model rate constants and used it to replicate the electrophysiology results. While findings which showed a reduced CD31 density (33%) and Cx43 density approaches (2) and (3) identify a single solution that matches the experimental (56%). These results provide clear evidence of altered microstructure (i.e., fi- findings, approach (1) provided a population (n=48) of channel variants that ber disarray due to collagen deposition) and function (i.e., reduced electrical reproduce distinctive features of experimental activation and inactivation curves. connections and perfusion) in peri-infarct, in large models relevant to We conclude that adopting a population-based strategy allows us to find several scar-related human pathophysiology. Future work will focus on using these parameter sets that are all compatible with experimental results and cannot be novel findings to parameterize MRI-based models to predict arrhythmia captured by other common optimization strategies. These solutions recreate inducibility. the level of experimental variability and account for the fact that different rate constants can compensate for one another. Evaluation of the three algorithms 1148-Pos on action potential clamp and dynamic clamp experiments is currently ongoing. Sinus Bradycardia Due to Electrolyte Changes as a Potential Pathome- chanism of Sudden Cardiac Death in Hemodialysis Patients 1146-Pos Axel Loewe1, Yannick Lutz1, Alan Fabbri2, Stefano Severi2. The Effects of Frequency of Voluntary Exercise on Cardiac Function in 1Institute of Biomedical Engineering (IBT), Karlsruhe Institute of Technolgy Dilated Cardiomyopathy Model Mice (KIT), Karlsruhe, Germany, 2DEI, University of Bologna, Cesena, Italy. Masami Sugihara1, Ryo Kakigi2, Takashi Murayama3, Takashi Miida1, Chronic kidney disease patients undergoing hemodialysis (HD) have a 14x Takashi Sakurai3, Sachio Morimoto4, Nagomi Kurebayashi3. higher risk to die from sudden cardiac death (SCD) compared to patients 1Department of Clinical Laboratory Medicine, Juntendo Univ, Tokyo, Japan, with a history of cardiovascular disease and normal kidney function. Tradi- 2Department of Physiology (II), Juntendo Univ, Tokyo, Japan, 3Department tional SCD risk factors cannot explain this high rate. Recent human studies sur- of Phamacology, Juntendo Univ, Tokyo, Japan, 4Department of Health prisingly point towards bradycardia and asystole as the prevailing arrhythmias Sciences at Fukuoka, International University of Health and Welfare, causing SCD in HD patients. This suggests a decisive role of the sinus node. We Fukuoka, Japan. sought to identify the effect of altered electrolyte levels as occurring in HD pa- Dilated cardiomyopathy (DCM) is one of major causes of heart failure (HF). tients on pacemaking in a computational model of human sinus node cells. We Recent clinical guidelines take exercises up as one of therapy of HF. However, enhanced the Fabbri et al. model of human sinus node cells to account for the effects of exercise on patients with inherited DCM have not been established dynamic intracellular balance of all considered electrolytes. The model was because DCM is associated with high risk of worsening HF and sudden death. exposed to clinically relevant extracellular electrolyte concentrations of potas- A knock-in mouse model of human inherited DCM, TNNT2 DK210, shows sium, sodium, and calcium to study their effect on spontaneous beating rate and similar characteristics to DCM patients and is useful model for evaluating ther- underlying pacemaking mechanisms. The level of sympathetic stimulation was apeutic effects. We aimed to examine how the influence on cardiac function kept constant. Beating rate showed a monotonic relationship with altered elec- differs depending on amount of voluntary exercise using this model mouse. Ho- trolyte concentrations starting from a baseline value of 72.5bpm. It increased þ mozygous DK210 (DCM) mice showed enlarged heart and frequent SD with with sodium (70.8-73.8bpm for [Na ]o from 120-150mM), with potassium þ t1/2 of 70 days. DCM mice were divided into 3 groups based on the frequency (70.7-81.9bpm for [K ]o from 3-9mM), and most pronouncedly with calcium 2þ of voluntary exercise: no exercise control (no-ex), every 2 days (2D) and daily (33.5-133.8bpm for [Ca ]o from 0.8-3mM). The severe bradycardia under hy- exercise (ED). The 2D and ED groups started running at 1 month of age. pocalcaemic conditions was due to hyperpolarized maximum diastolic poten- Following each dose of exercise for 1 month, cardiac function was assessed tial and slower diastolic depolarization driven by attenuation of ICaT and by echocardiography. Mice were sacrificed and their heart, lung, lower extrem- INCX, the latter due to depletion of intracellular calcium. Our human computa- ity muscles (soleus, plantaris and gastrocnemius) and body weights were tional study suggests that hypocalcaemia causes a pronounced decrease of measured. Gene expressions of HF- and arrhythmia-related genes in myocar- cellular sinus node pacing rate. While increased sympathetic tone will likely dium were quantified by qPCR analysis. The mortality rates up to 2 months compensate the lower basal beating rate, patients developing severe hypocal- of age decreased to 12% in 2D and ED groups from 39% in no-ex group caemia are at high risk to experience severe bradycardia and die from SCD dur- (n=18-25).The weights of soleus muscles were significantly and similarly ing a sudden loss of sympathetic tone. increased in 2D group and ED group. In the echocardiography, the cardiac contractility was more improved in ED group than 2D group. Expressions of 1149-Pos HF- and arrhythmia-related genes were significantly improved by the voluntary Effects of Varying Transverse and Axial Tubules in a Three-Dimensional exercise with dose dependent manner. The daily voluntary exercise appears to Model of Calcium Signaling in the Human Atrial Myocyte be most effective in preventing HF and SD in DCM mice. Xianwei Zhang, Haibo Ni, Stefano Morotti, Daisuke Sato, Eleonora Grandi. Department of Pharmacology, Univerisity of California Davis, Davis, CA, 1147-Pos USA. High Resolution Imaging and Histopathological Characterization of T-tubules are invaginations of the sarcolemma that play a key role in excitation- Myocardial Infarction contraction coupling in ventricular myocytes. Atrial myocytes are generally Peter Lin1, Jared Westreich1, Mengyuan Li1, Adam Gribble1, thought to possess sparse irregular transverse-tubular (TT) components, as Susan Newbigging2, Alex Vitkin1, Mihaela Pop1. opposed to the highly dense and regular ventricular TT system. Axial tubules 1Medical Biophysics, Univ Toronto, Toronto, ON, Canada, 2Centre for (ATs) with extensive junctions to the sarcoplasmic reticulum that include Phenogenomics, Univ Toronto, Toronto, ON, Canada. ryanodine receptor (RyR) clusters, characterized by rapid activation of

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Ca2þ-induced Ca2þ release, have also been identified in atrial myocytes. AT We characterized MET receptor activation with single-molecule fluorescence and TT remodeling and changes in distribution, composition, and phosphoryla- microscopy and spectroscopy directly in cells. First, we found that MET is tion status of Ca2þ release units are thought to underlie Ca2þ abnormalities in partially dimeric before ligand binding and that the dimer fraction increases atrial fibrillation (AF), the most common cardiac arrhythmia. Here, we per- when activated with InlB. Second, we developed an assay to measure binding formed a computational analysis to investigate how changes in the tubular affinities of ligands to receptors directly on the cell surface; we carefully evalu- network affect human atrial electrophysiology. We modified our well- ated the role of the fluorophore and excluded an impact on binding. Third, we established three-dimensional model of Ca2þ signaling in the rabbit ventricular used single-particle tracking and imaging fluorescence correlation spectroscopy myocyte to develop an analogous model in the human atrium. We also coupled to study MET dynamics in live cells, from which we derived a mechanistic the Ca2þ signaling model to our well-established model of membrane electro- model of MET receptor activation and its linkage with endocytosis pathways. physiology in the human atrial myocyte. We systematically varied TT and AT density and RyR distribution and assessed the effect on Ca2þ spark and wave 1152-Pos properties. When TT density is low, as shown in isolated atrial myocytes, the Spatiotemporal Dynamics of Ron and EGFR Crosstalk at the Plasma model recapitulates the typical U-shaped Ca2þ wave seen experimentally in Membrane transverse confocal line-scan images. Introduction of ATs resulted in W-shaped Justine Keth, Carolina Franco Nitta, Elton D. Jhamba, Ellen W. Hatch, Ca2þ waves, reflecting more synchronous Ca2þ release. The frequency and Mara P. Steinkamp, Bridget S. Wilson, Diane S. Lidke. amplitude of Ca2þ release events were affected by both density of TT and Department of Pathology, University of New Mexico, Albuquerque, NM, AT, as well as RyR distribution. Our newly developed three-dimensional model USA. of the human atrial myocyte will be employed to investigate whether and how Recepteur d’Origine Nantais (RON) and the Epidermal Growth Factor Recep- AF-induced cellular electrical and structural remodeling effects collectively tor (EGFR) are Receptor Tyrosine Kinases, each with important roles in normal contribute to the membrane potential and Ca2þ abnormalities seen in AF. and oncogenic signaling. Previous studies have shown that RON and EGFR can undergo crosstalk that alters cellular signaling outcomes, yet the molecular 1150-Pos mechanisms facilitating this are unknown. We hypothesized that crosstalk oc- Disruption of Caveolar Microdomains Creates ‘‘Hot Spots’’ for Atrial Ec- curs from direct RON/EGFR interactions. We integrated biophysical, biochem- topy and Arrythmogenesis in Heart Failure Mice ical and imaging techniques to define the spatiotemporal details of RON/EGFR Di Lang, Leonid Tyan, Aleah Warden, Zachary D. Piro, Rylie Lodin, interactions. Stable human cell lines containing both EGFR and RON were Evi Lim, Ashley Irwin, Alexey V. Glukhov. generated. Our results show that RON is phosphorylated in response to Department of Medicine, Univ Wisconsin Madison, Madison, WI, USA. EGFR activation after EGF addition. In contrast, RON activation by its own Atrial fibrillation (AF) often occurs during heart failure (HF). Ectopic foci that ligand MSP (Macrophage Stimulating Protein) does not result in EGFR phos- trigger AF, are linked to discrete atrial regions that experience the highest re- phorylation. To further elucidate this crosstalk, we used fluorescence micro- modeling and clinically used for AF ablation; however, mechanisms of their ar- scopy techniques to determine the location and dynamics of RON/EGFR rhythmogenic propensity remain elusive. HF is associated with alterations in interplay. High-resolution Single particle tracking (SPT) of quantum dot- cardiomyocyte microarchitecture, including degradation of transversal-axial labeled RON showed reduced mobility upon EGF stimulation, consistent tubule system (TATS) and caveolae structures, that might be differently man- with EGFR-driven RON activation. Using two color SPT, we observed corre- ifested throughout the atria forming vulnerable regions for ectopy. We em- lated motion of RON and EGFR in live cells, consistent with the formation of ployed an in vivo ECG telemetry, in vitro optical mapping and confocal heterodimers. Additionally, RON and EGFR were found colocalized at the imaging of Ca2þ transients (CaT) from myocytes isolated from the right atrial plasma membrane by confocal microscopy. As expected, EGF stimulation appendage (RAA) and inter-caval region (ICR) of wild type (WT, n=10) and 8- led to rapid EGFR endocytosis, however, RON was not found in the EGFR- weeks post-myocardial infarction HF (n=8) mice. HF mice showed an positive endosomes. These results suggest that crosstalk between RON and increased susceptibility to pacing-induced AF (4/4 in HF vs. 1/6 in WT) and EGFR is a result of heterodimerization that occurs transiently at the plasma enhanced ectopy originated from ICR. When compared with RAA, ICR membrane. demonstrated a significantly higher elevation of interstitial fibrosis (113% vs. 38% in RAA, P<0.001), decline in conduction velocity (47% vs. 35% in 1153-Pos RAA, P<0.01) and prolonged CaT rise-up time (51% vs. 41% in RAA, Probing the Interaction between Receptor Tyrosine Kinases and Trans- P<0.01). In WT mice, RAA consists of structured myocytes with a prominent membrane Adhesion Proteins 1 2 1 TATS while ICR myocytes don’t have TATS. Disruption of caveolae by Taylor P. Light , Deborah Leckband , Kalina Hristova . b 1Materials Science and Engineering, Johns Hopkins University, Baltimore, methyl- -cyclodextrin in ICR myocytes prolonged CaT rise-up time and CaT 2 duration promoting delayed afterdepolarization while methyl-b-cyclodextrin MD, USA, University of Illinois at Urbana-Champaign, Urbana, IL, USA. didn’t lead to such effects in RAA. A similar decrease in caveolae density Receptor Tyrosine Kinases (RTKs) are transmembrane proteins involved in observed throughout the atria, would results in more severe effects in ICR signal transduction cascades that result in various cellular responses. RTKs which correlated with the origin of ectopic activity in HF atria. Our findings are activated by associating in the plasma membrane, a process which is modu- demonstrate that in HF, caveolar disruption creates ‘‘hot spots’’ for arrhythmo- lated by ligand binding to their extracellular domains. Interactions between genic ectopic activity emanated from discrete vulnerable regions of the right RTKs within the same sub-family as well as more recent studies between atrium which are associated with desynchronized SR Ca2þ release and RTKs of different sub-families have demonstrated the promiscuity of RTK as- elevated fibrosis. sociation. The current work moves beyond RTK-RTK interactions and explores RTK interactions with other transmembrane proteins. Previous studies have Posters: Membrane Receptors and Signal demonstrated that RTKs can be directly activated by cell adhesion proteins, yet no evidence has been presented to confirm that these proteins directly asso- Transduction I ciate. To quantify the interaction between these families of proteins, we use the fully quantified spectral imaging FRET method whereby the association state 1151-Pos can be measured in the plasma membrane of live cells. Here, we demonstrate Met Receptor Tyrosine Kinase Activation Studied at the Single-Molecule that RTKs can interact directly with adhesion proteins at the plasma membrane. Level The effect of RTK ligand binding to this complex is also explored. Marina S. Dietz1, Marie-Lena I.E. Harwardt1, Thorsten Wohland2, Hartmut H. Niemann3, Mike Heilemann1. 1154-Pos 1Single Molecule Biophys/Phys and Theor Chemistry, Goethe University Response of FGFR1 to Different Ligands Frankfurt, Frankfurt, Germany, 2Dept Biol Sci/Chem, Natl Univ Singapore, Kelly Karl. Singapore, Singapore, 3Fac Chemistry, Univ Bielefeld, Bielefeld, Germany. Molecular Biohysics, Johns Hopkins Univ, Baltimore, MD, USA. Receptor tyrosine kinases (RTK) regulate essential processes in vertebrate Fibroblast growth factor receptor 1 (FGFR1) is a membrane protein which is a development, proliferation, and motility. Dysregulation of RTK signaling path- member of the Receptor Tyrosine Kinase (RTK) family and is a critical protein ways is often observed in cancer and other diseases. One receptor tyrosine ki- in transmembrane cell signaling. It is known that FGFR1 interacts with a wide nase that is increasingly the focus of current research is the MET receptor. variety of ligands from the 18 member family of fibroblast growth factors Beside its physiological ligand hepatocyte growth factor/scatter factor, MET (FGFs). Recent research has shown that exposure of FGFR1 to different ligands is also targeted by internalin B (InlB), which is expressed by the pathogenic in the extracellular matrix results in different cellular outcomes and eventually bacterium Listeria monocytogenes, as a pathway to invade epithelial cells. A different tissue phenotypes. Using FRET and biochemical methods, we inves- molecular understanding of the activation and signaling initiation of MET is tigate how the identity of the different ligands is transmitted through the plasma crucial for targeted therapy. membrane and to downstream signaling molecules.

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1155-Pos not to that of the inactive receptor. However, it does so with a significantly Mechanism of EphA2 Dimerization in Response to Monomeric Ligands weaker binding affinity compared to Naþ. While the orthosteric and sodium Elmer A. Zapata-Mercado1, Randall Rainwater2, Elena B. Pasquale3, allosteric sites could be simultaneously occupied by Naþ in the inactive m- Kalina Hristova4. OR, binding of Mg2þ at the orthosteric site of the active receptor was found 1Program in Molecular Biophysics, Johns Hopkins University, Baltimore, to drastically reduce the affinity of Naþ for its allosteric site. Notably, we iden- MD, USA, 2Department of Biology, University of Central Arkansas, tified a number of metastable states in which these ions are bound to the extra- Conway, AR, USA, 3Sanford-Burnham Prebys Medical Discovery Institute, cellular region of the inactive or activated receptor. This information, coupled La Jolla, CA, USA, 4Materials Science and Engineering, Johns Hopkins with thermodynamic and kinetic properties derived from our simulations, sug- University, Baltimore, MD, USA. gests testable hypotheses of molecular determinants responsible for the modu- The receptor tyrosine kinase (RTK) EphA2 is involved in cell homeostasis, latory effects of Mg2þ. tissue organization, epithelial cell differentiation, and angiogenesis, among others. EphA2 is also involved in diseases such as cancer, cataracts, and 1158-Pos atherosclerosis, making this receptor a promising drug target. Previous Detecting Intramolecular Dynamics of GPCRS using Diffracted X-Ray research from our lab has found that EphA2 can form two different types Blinking Technique of dimers, utilizing different interfaces of the extracellular region. One of Kazuhiro Mio1, Masaki Ishihara2, Shoko Fujimura1, Masahiro Kuramochi2, Yuji C. Sasaki2. these dimers is the unliganded dimer, which can be further stabilized by the 1 2 engineered peptide YSA. A second type of dimer forms upon binding of Operand OIL, AIST, Tokyo, Japan, Dept Adv Matl Sci, Univ Tokyo, the physiological monomeric ligand m-ephrinA1. Currently it is not known Kashiwa City, Japan. if the differences in the dimer architectures of the extracellular region are G protein-coupled receptors (GPCRs) are seven-transmembrane receptors, transmitted to the intracellular region. Two different scenarios have been pro- which transmit extracellular signals inside the cells via activation of G pro- posed. In the first scenario, the extracellular and intracellular regions are teins. GPCRs are involved in a wide variety of physiological functions, that coupled and therefore changes in the architecture of the extracellular region include diverse signal sensing, immune system, and neurotransmission. are transmitted to the intracellular region. In the second scenario, the linkers Indeed, about half of the drugs directly or indirectly target GPCRs. In contrast between these regions are flexible, not allowing propagation of the signal to the huge number of researches conducted on the structure and function from the extracellular to the intracellular region. We are investigating which analysis of GPCRs all over the world, however, little has been known about of these scenarios apply to EphA2. the internal molecular dynamics during signal transduction. In this study, we measured the dynamic intramolecular motion of GPCR expressed on the cell 1156-Pos surface at single molecular level using Diffracted X-ray Blinking (DXB) Investigating the Role of the Transmembrane Helix of EphA2 in Signal method. DXB is an X-ray based single-molecule imaging technique that eval- Transduction across the Plasma Membrane uates the internal motion of a biomolecule from the fluctuation of the diffrac- Daniel Wirth1, Kalina Hristova1, Elena Pasquale2. tion spots from the gold nanocrystal. DXB uses monochromatic laboratory X- 1Johns Hopkins Univ, Baltimore, MD, USA, 2Sanford-Burnham Prebys ray source. Serotonin receptor subtype 2A (5-HT2A) was transiently ex- Medical Discovery Institute, San Diego, CA, USA. pressed on HEK 293 cells, and the gold nanocrystals were attached to a Receptor tyrosine kinases (RTKs), the second largest class of membrane recep- methionine-rich tag inserted to the 5-HT2A extracellular sequence. X-ray tors, transduce biochemical signals across the cell membrane via lateral dimer- blinking intensity from the labeled and moving gold nanocrystals, correlated ization or oligomerization. RTKs control cell growth, differentiation and with the velocity of diffraction spots, were recorded at 100 millisecond / motility, and are involved in many diseases such as cancer. The single trans- frame. Autocorrelation function (ACF) of each pixel obtained from the inten- membrane domains of RTKs have been hypothesized to play important roles sity fluctuation and the Poisson distribution of the detected intensity, in signal transduction. First, sequence-specific interactions between the trans- confirmed increased fluctuation of the 5-HT2A in response to the a-methylser- membrane domains are believed to contribute to the overall stability of RTK otonin. We could also measure its dynamic fluctuation range. These results dimers and oligomers. Second, it has been proposed that the transmembrane indicate that in the event of ligand binding, the movement of the 5-HT2A domain dimer controls the spatial separation and orientation of the catalytic do- and the fluctuating motion velocity of the cell membrane is intensified. This mains in the RTK oligomers. In this study, we focus on the transmembrane is probably the first report of experimental determination of intramolecular domain of EphA2, an RTK involved in many critical physiological processes, GPCR dynamics in live cells. including tissue homeostasis, angiogenesis and tumorigenesis. Previous studies in our lab have shown that EphA2 can associate into two types of dimers, as 1159-Pos well as clusters, depending on the type of ligand (dimeric EphrinA1-Fc, Ephrin Inter-Domain Interactions and Allosteric Modulation of Metabotropic A1 and the engineered peptide YSA), with distinct interaction interfaces in the Glutamate Receptors extracellular region. By mutating key interaction motifs in the transmembrane Vanessa Gutzeit, Jordana Thibado, Josh T. Levitz. domain and utilizing a quantitative FRET methodology, we seek to determine if Biochemistry, Weill Cornell Medicine, New York, NY, USA. the interaction between the transmembrane domains is affected by the type of Metabotropic glutamate receptors (mGluRs) are class C G protein-coupled re- bound ligands. These experiments will give new insights into the role of the ceptors (GPCRs) which function in synapses throughout the central nervous transmembrane domain in EphA2 signaling. system where they respond to the excitatory neurotransmitter glutamate to initiate intracellular signaling cascades. A comprehensive understanding of 1157-Pos mGluR activation requires a mechanistic description of the effects of both Thermodynamics and Kinetics of the Divalent-Monovalent Cation extracellular ligand binding domain(LBD)-targeting ‘‘orthosteric’’ and trans- Competition for Binding Sites at the m-Opioid Receptor membrane domain (TMD)-targeting ‘‘allosteric’’ compounds. Here we use a Xiaohu Hu, Davide Provasi, Marta Filizola. combination of high resolution, time-resolved functional and conformational Department of Pharmacological Sciences, Icahn School of Medicine at assays to dissect the mechanisms by which allosteric compounds activate Mount Sinai, New York, NY, USA. and modulate mGluRs. First, using an electrophysiological assay we find that Monovalent and divalent cations have been known for years to differentially mGluR2 can be activated by both orthosteric or allosteric ligands and that allo- þ modulate opioid receptor (OR) signaling. While Na was shown to reduce steric activation does not require LBD closure. We then use a single molecule þ agonist binding, Mg2 had the opposite effect, particularly in the presence of fluorescence-based subunit counting assay to find that mGluR transmembrane þ sodium chloride. The observation of Na bound in an inactive, but not active, domains efficiently dimerize with variable efficiency to a degree that is consis- OR crystal structure, further supports the idea that this monovalent ion reduces tently higher than that observed for class A GPCRs and is not sensitive to allo- agonist binding by stabilizing the receptor inactive state. In contrast, little is steric drugs. Next, we develop an inter-TMD FRET assay that reveals that þ known about the preferred interaction site(s) of the divalent ion Mg2 at activation-associated inter-TMD rearrangement occurs independently of allo- ORs, as well as the molecular determinants responsible for its effect on drug steric input from the LBDs. Using this assay, we test a panel of allosteric com- affinity and potency. pounds to uncover diversity in apparent affinity, efficacy and kinetics between þ þ Here, we studied the binding of Mg2 and Na to m-OR in an explicit lipid/wa- these compounds. Strikingly, we find that negative allosteric modulators are ter environment. Using machine learning algorithms and Markov state models either neutral or induce TMD rearrangement which correlates with degree of applied to tens of microseconds all-atom molecular dynamics simulations, we inverse agonism. Together this data is consistent with a model where multiple þ þ investigated the competition of Na and Mg2 for binding sites at inactive or (>2) intersubunit reorientations are part of the activation process and motivates þ active conformations of m-OR. We found that Mg2 preferentially binds to the further work on the role of multimeric assembly and inter-domain coupling in orthosteric ligand binding site of the ligand-free active m-OR conformation, but the activation of class C GPCRs.

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1160-Pos the functional consequences of melanopsin-phosphatase interactions, we used Conformational Free Energies of Metabotropic Glutamate Receptor in vitro calcium imaging in HEK293 cells transiently transfected with mouse Ligand-Binding Domains melanopsin. Using these methodologies, we found robust expression of a variety Tyler J. Wied. of serine/threonine phosphatases, of the Protein Phosphatase family. In the Biophysics and Biophysical Chemistry, Johns Hopkins Univ Sch of Med, mouse retina, immunolabelling suggests robust expression of Protein Phospha- Baltimore, MD, USA. tase 2A (PP2A) in all cell layers, including the ipRGCs. In vitro calcium imaging Metabotropic glutamate receptors (mGluRs) are dimeric class C GPCRs that using a ‘‘serial-flash’’ paradigm (90-second recordings with 30-minute dark mediate cellular responses to the neurotransmitter glutamate in the central ner- adaptation between recordings) suggests that chemical inhibition of PP2A re- vous system. Crystal structures and single-molecule FRET results suggest that duces the intensity of light responses, while also increasing deactivation rates closure of the ligand-binding domains (LBDs) as well as reorientation of the of light- activated mouse melanopsin. Conversely, overexpression of PP2A in dimer interface are necessary conformational transitions for receptor activation. HEK293 cells dramatically increases the amplitude of light responses, while The energetic factors that drive these coupled processes, however, are poorly decreasing deactivation rates during the initial 90-second light stimulus. Alto- understood. Here, we report free energy landscapes, or potentials of mean force gether, this study represents an initial examination of re-sensitizing mechanisms (PMFs), for monomer mGluR3 LBDs in apo and glutamate-bound states as that might contribute to ipRGC sustained light responses in the retina. well as dimer mGluR3 LBDs the active and inactive poses. PMFs of LBD monomers suggest the glutamate-bound LBD is bistable: the closed and 1163-Pos open-cleft conformations are easily accessible. In apo LBDs, the closed-cleft Modulation of Mu-Opioid Receptor Signaling by Cannabinoid CB1 Recep- conformation is destabilized relative to the glutamate-bound. In contrast, tor through Heteromerization, a Novel Analgesic Target 1,2 2 2 2 PMFs for glutamate-bound dimers indicate a strong preference for the closed Guoqing Xiang , Lia Baki , Takeharu Kawano , Diomedes Logothetis . 1Dept Physiol/Biophys, Virginia Commonwealth Univ, Richmond, VA, state in both active and inactive poses. These results suggest cooperativity be- 2 tween dimer subunits. Specifically, network analysis suggests subunit coupling USA, Dept Pharmaceutical Sciences, Northeastern University, Boston, MA, alters the dynamics of an alpha helix in the LBD lower lobe. The energetics cor- USA. responding to the reorientation of the dimer interface are also examined. Opioids are the most common analgesics in the clinic despite severe side ef- fects, including respiratory depression, tolerance and dependence. A principal 1161-Pos target for opioids is the mu-opioid receptor (MOR), a 7 transmembrane G Mechanisms of G Protein-Selectivity in Muscarinic Acetylcholine Recep- protein-coupled receptor (GPCR). Signaling of MOR to Gi/o will lead to a tor Family decrease in cAMP level as well as activation of G protein sensitive inwardly Luis Santiago1, Ravinder Abrol2. rectifying potassium channels producing analgesic effects, whereas signaling 1Biology, California State University Northridge, Northridge, CA, USA, towards beta-arrestin will result in receptor desensitization and internalization 2Chemistry and Biochemistry, California State University Northridge, causing a variety of side effects. Cannabinoids can also produce analgesic ef- Northridge, CA, USA. fects targeting the cannabinoid 1 receptor (CB1R). Clinical and animal behav- Muscarinic acetylcholine receptors are integral membrane proteins that regulate ioral studies suggest a functional interaction between opioid and cannabinoid physiological activity. Acetylcholine is a neurotransmitter that binds to the receptors. MOR was found to co-localize with CB1R in striatum GABAergic extracellular side of these G protein coupled receptors (GPCRs) activating the neurons and bioluminescence resonance energy transfer (BRET) studies sug- Ga subunit of the trimeric G protein inside the cell. There are 5 different musca- gest a close proximity between MOR and CB1R in HEK 293 cells. We have rinic receptor subtypes (M1-M5), where the same ligand acetylcholine causes utilized a calcium mobilization assay in HEK 293 cells and an electrophysio- M2 and M4 receptors to preferentially couple to the Gai subunit and the receptors logical in Xenopus oocytes to detect Gi signaling and a luciferase complemen- M1, M3, and M5 to preferentially couple to the Gaq subunit. This selectivity of tation assay in HEK 293 cells to assess arrestin recruitment. We show that co- Ga subunits mediates different secondary messenger pathways in the cell: Gai expression of CB1R enhanced the Gi signaling of MOR and depressed arrestin being responsible for deactivating adenyl cyclase activity and Gaq being respon- recruitment to MOR. These effects could be further accentuated by specific sible for activating phospholipase C. We are using computational biophysical CB1R agonists. The signaling change is likely due to heteromerization of methods to probe the mechanisms of G protein selectivity of this muscarinic re- MOR and CB1R since it could be abolished by co-expression of a CB1R trans- ceptor family. We have performed molecular dynamics (MD) simulations of M1 membrane domain 5 (TM5) but not a TM1 mini-gene. Our results suggest that and M2 receptors coupled to both Gai and Gaq to obtain relaxed structures of targeting MOR-CB1R heteromer may provide a novel therapeutic approach, receptor:G-protein complexes and to carry out the thermodynamic analysis of which may yield improved analgesic effects with reduced side effects. the receptor G protein interactions. The studies show that thermodynamically, the wild-type M1 receptor prefers to bind to the G subunit and the wild-type 1164-Pos ai Cannabinoid Receptor CB2 Oligomerization in a Lipid Matrix M2 receptor prefers to bind to the Ga subunit, which is opposite of the receptors’ q 1 2 1 1 specificity observed in the cells. The same analysis for the M2 mutant Alexei Yeliseev , Jonathan D. Nickels , Kirk G. Hines , Lioudmila Zoubak , 1 3 3 4 (M2AALS) with the switched G protein selectivity, which favorably couples Walter E. Teague, Jr , Diane L. Lynch , Dow P. Hurst , Kevin L. Weiss , John Katsaras5, Patricia H. Reggio3, Klaus Gawrisch1. to the Ga subunit in the cells, showed that thermodynamically the M2 mutant q 1LMBB, NIAAA NIH, Bethesda, MD, USA, 2College of Eng. & Appl. Sci., also prefers to bind to the Gaq subunit. These data suggest that for the wild- 3 type receptors, the kinetics might be playing a dominant role over thermody- Univ. of Cincinnati, Cincinnati, OH, USA, Dept. Chem. Biochem., Univ. North Carolina, Greensboro, NC, USA, 4CSMB, Oak Ridge National namics in determining G protein selectivity. We are currently testing this hy- 5 pothesis. These studies are beginning to provide us with the missing Laboratory, Oak Ridge, TN, USA, BSMD, Oak Ridge National Laboratory, mechanistic insight into the G protein selectivity of muscarinic receptors. Oak Ridge, TN, USA. The human CB2 receptor belongs to the large family of rhodopsin-like G 1162-Pos protein-coupled membrane receptors (GPCR) which are critical for transmit- The Role of Protein Phosphatase 2A in the Re-Sensitization of Melanopsin ting external signals to the cell interior. Oligomerization of GPCR has been During Continued Light Stimulation recognized as important for modulating the function of signaling pathways. Juan C. Valdez-Lopez, Meheret Gebreeziabher, Jair Flores, Currently, there is a lack of effective methods that allow studying receptor olig- Olanike Awotunde, Thomas Burnett, Adam Byerly, Phyllis R. Robinson. omerization under physiologically relevant conditions, which include a fluid Biological Sciences, University of Maryland Baltimore County, Baltimore, lipid matrix of proper composition and higher receptor concentrations. We MD, USA. used small angle neutron scattering (SANS) in combination with neutron scat- Melanopsin is a light-sensitive G-protein-coupled receptor, or visual pigment, tering length density (NSLD) contrast matching of the lipid matrix into which expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs), which the protein is imbedded for measurement of GPCR oligomerization. In order to constitute a small subset of the total ganglion cell population. These cells are pri- obtain a SANS signal of a membrane protein of highest intensity, the difference marily implicated in non-image formation; specifically, they regulate behavior between neutron scattering length densities (NSLD) of protein and lipid was such as circadian entrainment, pupil size, mood, and sleep. Melanopsin cells maximized. Furthermore, the NSLD of the lipid matrix was matched to the also exhibit characteristically sustained light responses, in contrast to the exqui- NSLD of the buffer such that the SANS spectrum is dominated by the protein. sitely transient light responses of the image-forming rod and cone photoreceptor Overall proton content of the sample was minimized to reduce incoherent scat- cells. Molecular mechanisms of re-sensitization in melanopsin, much like in vi- tering of neutrons that obscures the data at higher angles of diffraction. Optimal sual pigments in general, are poorly understood. Thus, we aimed to describe conditions were achieved by conducting experiments on protonated protein im- these mechanisms; specifically, we hypothesize melanopsin interaction with bedded into an almost entirely perdeuterated lipid matrix in the presence of phosphatases contributes to its re-sensitization. To test this, we examined phos- close to 100 % perdeuterated buffer. Experimental results are compared to phatase expression in vivo using RT-PCR and immunohistochemistry. To model the structure of CB2 in a lipid matrix predicted by molecular simulations.

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1165-Pos from various caveats. Here we describe a novel quantitative imaging and The Interplay of Structural and Cellular Biophysics Controls the Clus- computational analysis strategy to derive an explicit relationship between tering of Multivalent Signaling Molecules: The Nephrin-Nck-Nwasp cell surface receptor mobility and clustering on the one hand, and AC turnover System kinetics and filament movement on the other. As a test model system, we are Aniruddha Chattaraj, Leslie M. Loew. applying this imaging and analysis strategy to study the cell surface receptor R.D. Berlin Center for Cell Analysis and Modeling, Univ Connecticut School CD36, the movement and clustering of which in the plasma membrane are of Medicine, Farmington, CT, USA. thought to depend on the AC. To monitor both CD36 and AC behavior at the Dynamic molecular clusters are assembled through weak binding interactions same time in the same cell, we are employing TIRF-based simultaneous single between multivalent molecules. They are highly plastic structures with a distri- molecule imaging of CD36 and fluorescent speckle microscopy of the AC, in bution of stoichiometries and sizes; they are becoming increasingly recognized live endothelial cells. The imaged CD36 molecules are tracked to quantify their as molecular platforms to drive key cellular functions, especially receptor- movement and clustering state, while the actin speckles are tracked and mediated signaling. A comprehensive understanding of the principles governing analyzed to quantify AC turnover kinetics and actin filament movement. These cluster formation at the molecular level is still lacking. Systematically varying CD36 and actin properties are then combined for neighboring CD36 molecules the molecular and cellular features in biophysical models of these systems can and actin speckles to derive receptor-AC inter-relationships. In parallel, to ac- help to elucidate these principles. In this work, we used SpringSaLaD, a particle count for cellular heterogeneity, various bins based on experimental conditions based spatial simulation platform, to examine a prototypical multivalent system and cellular phenotypes are created to further refine our observations. Our anal- with a membrane anchored protein Nephrin containing 3 phosphotyrosine ysis thus far suggests that CD36 clustering is inversely correlated with its diffu- (pTyr) sites, an adaptor protein Nck containing one SH2 domain and 3 SH3 do- sion coefficient and neighboring actin density. Additionally, for freely moving mains, and an effector protein NWASP consisting of 6 proline-rich motifs CD36 molecules, the density of neighboring actin is a better predictor of CD36 (PRMs). We performed computational experiments to determine how structural diffusion coefficient than the speckle speeds. and cellular features influence the size of molecular clusters composed of a small number of these multivalent molecules. Steady state cluster size distributions favored stoichiometries that optimized binding, but still were quite broad. A bal- Posters: Calcium Signaling ance of enthalpy and entropy limited the number of molecules per cluster, with 1168-Pos complete annealing into a single complex being exceedingly rare. Domains The Role of -M (Pfkm) in Oscillatory and close to binding sites sterically inhibited clustering much less than terminal do- Insulin Secretion in Pancreatic Beta Cells mains, because the latter effectively restrict access to the cluster interior. Vishal S. Parekh, Jim Ren, Leslie S. Satin. Increased flexibility of interacting molecules diminished clustering by shielding Dept Pharmacology, Univ Michigan, Ann Arbor, MI, USA. binding sites within compact conformations. Membrane association of Nephrin Beta cells secrete insulin in a pulsatile manner but the underlying mechanism is increased the cluster size in a density-dependent manner. Crowded cellular envi- unclear. We have shown by modeling that Pfkm is a feasible mechanism for ronment significantly modulated the cluster size and dynamics. These properties slow (e.g. 3-5 min.) oscillations in glycolysis, ATP/ADP, free Ca and insulin provide insights into how molecular clusters function to localize and amplify secretion. To investigate the functional role of Pfkm for glycolytic oscillations cell signaling. (supported by NIH grant P41-GM103313). and beta cell function, a beta-cell specific Pfkm knockout mouse (betapfkm / ) 1166-Pos was made by floxing exon 3 of the Pfkm gene and crossing these mice with Allosterism in Oligomeric Receptor Models: Cycle Bases of Reduced beta-cell specific Pdx1 or rip2 cre mice. Betapfkm / mice were Graph Powers Provide a Theoretical Framework for Conformational born at the expected Mendelian frequency, had normal weight, and normal fast- Coupling ing glucose. Pfkm protein was lost in the knockouts, whereas liver (Pfkl) and Greg Conradi Smith. platelet (Pfkp) PFKs were maintained. Betapfkm / mice exhibited mildly Department of Applied Science, William & Mary, Williamsburg, VA, USA. impaired glucose tolerance, reduced insulin content, and reduced insulin exocy- Pharmacologists use mathematical models of receptors to understand the action tosis in response to 16.8 mM glucose ex vivo. Glycolytic oscillations - as of natural ligands and drugs on cell responses. When receptors function as two or measured using a activity biosensor - as well as islet free Ca more similar protein subunits working in concert (i.e., homodimers or oligo- oscillations were preserved in islets from both control and betapfkm / mice. mers), receptor models must (1) account for symmetry, (2) satisfy thermody- Patch clamp revealed relatively normal glucose-induced electrical activity namic constraints, and (3) properly account for subunit interactions (allostery) but with enhanced compound bursting. Thus, while Pfkm is apparently not mediated by conformational coupling. (1) Assuming a monomer state- needed to generate slow glycolytic oscillations, it does have a role in beta transition diagram given by the connected, simple graph G, we establish that cell function. Our data show that the role of beta cell glycolytic oscillations re- the structure of state-transition diagram of a receptor k-mer is the reduced graph mains to be determined. Supported by NIH RO1 DK46409. power denoted G^(k). (2) Using a minimal cycle basis construction for reduced 1169-Pos graph powers, we provide a method of identifying the thermodynamic con- 2D straints in the receptor oligomer model without having to construct its state tran- Biphasic Ca Regulation of SK Channels in Ventricular Cardiomyocytes sition diagram G^(k). (3) Using a distinguished spanning tree construction for Maximizes Their Conductance during a Late Phase of the Action Potential 1 1,2 1 1 reduced graph powers, we show how (a) the allosteric interactions in a receptor Peter Bronk , Iuliia Polina , Radmila Terentyeva , Shanna Hamilton , Dmitry Terentyev1. oligomer model may be systematically enumerated, and (b) the equilibrium 1 binding curves of receptor oligomers can be expressed as rational polynomial Department of Medicine, The Warren Alpert Medical School of Brown University, Rhode Island Hospital, Cardiovascular Research Center, functions of the equilibrium binding curves of the constituent monomers, 2 without approximation and without assuming independence of receptor mono- Providence, RI, USA, Department of Medicine, Medical University of South mers. This intriguing and novel combination of graph theory and quantitative re- Carolina, Charleston, SC, USA. Small conductance Ca2þ-activated Kþ (SK) channels are dormant in ventricu- ceptor pharmacology provides a general theoretical framework for the study of b allosterism in oligomeric receptors composed of any number of identical mono- lar cardiomyocytes (VCMs) under basal conditions, but activated during - adrenergic stimulation. Previous work demonstrated that SK channels can be mers. The methodology may prove valuable for studies of pharmacological 2þ activated by Ca via constitutively bound calmodulin with EC50 1 mM, alteration of GPCR activity by allosteric modulators, whose action is modeled 2þ m as a modification of equilibrium constants of one or more receptor monomers. and are sensitive to block by intracellular Ca at IC50 20 M. Here we aimed to determine the contribution of ISK to the action potential waveform þ 1167-Pos in rat VCMs during the Ca2 transient, given the biphasic regulation of SK þ þ Quantification of Surface Receptor-Actin Cortex Interplay via Two-Color channels by intracellular Ca2 . Simultaneous recording of Ca2 signals using High Resolution Imaging confocal microscopy and voltage step activated whole-cell currents from Aparajita Dasgupta, Deryl Tschoerner, Bruno Da Rocha-Azevedo, VCMs treated with the b-adrenergic agonist isoproterenol (100 nM) revealed Khuloud Jaqaman. a rapidly activating apamin sensitive SK current peaking 10.7 5 1.7 ms þ Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, (20 mV) after the stimulus onset. Calculation of submembrane Ca2 2þ 2þ USA. ([Ca ]sm) revealed that [Ca ]sm at the ISK peak was 6 5 2 mM(20 mV). 2þ 2þ Mounting evidence suggests that the dynamic organization of receptors in the [Ca ]sm continued to rise peaking at 13 5 4 mMCa as ISK decayed from plasma membrane (PM) is crucial for ligand binding and downstream 1.7 5 0.6 pA/pF to 0.3 5 0.1 pA/pF. The current-voltage relationship for signaling. The actin cortex (AC) is thought to play an important role in regu- the ISK shows a peak at 10 mV that decreases robustly at more depolarized lating cell surface receptor organization, although its role has been almost potentials. Voltage clamping VCMs with an action potential waveform showed 2þ exclusively deduced via cell-wide actin perturbation experiments, which suffer that an initial peak SK conductance of 5.6 nS is rapidly inhibited as [Ca ]sm

BPJ 9362_9366 236a Monday, March 4, 2019 rises. The SK conductance increases again to a peak of 7.2 nS as the action po- However, important questions remain about the fundamental properties of car- 2þ tential drops below 0 mV and [Ca ]sm decreases below 10 mM. This is the re- diac SOCE including: subcellular localization, effector targets, interaction with gion of the action potential where the VCMs are most sensitive to early the classical excitation-contraction coupling (ECC) mechanism and role in afterdepolarizations due to reactivation of L-type Ca2þ channels and enhanced heart disease. Goal: We investigated the functional and molecular properties activity of the Naþ/Ca2þ exchanger, suggesting SK channels have a potential of cardiac SOCE, its subcellular distribution and the relationships between anti-arrhythmic effect. SOCE and arrhythmia in CPVT. Methods and results: We used confocal Ca im- aging, immunocytochemistry and protein in situ co-localization to examine 1170-Pos SOCE in ventricular cardiomyocytes isolated from WT and CPVT mice. We Computational Modeling of Purinergic Receptor Activation in Microglia found that in both WT and CPVT myocytes SOCE consists of local transient Peter M. Kekenes-Huskey, Byeongjae Chun, Darin Vaughan. Ca influx signals (LoCIS) that occur primarily at the myocyte periphery, partic- Dept Chemistry, Univ Kentucky, Lexington, KY, USA. ularly at intercalated disks (IDs). These signals, although relatively scarce and Microglia function is orchestrated through highly-coupled signaling pathways weak in WT myocytes, were significantly upregulated in occurrence and þ that depend on calcium (Ca2 ). In response to extracellular adenosine triphos- strength in CPVT myocytes. In both groups, the localization of these events þ phate (ATP), transient increases in intracellular Ca2 can be driven through matched that of immunofluorescence for STIM1, Orai1 and STIM1-Orai1 com- the activation of purinergic receptors, P2X and P2Y. Their activation is suffi- plexes. Correspondingly, the CPVT-linked upregulation of LoCIS was associ- cient to promote physiological responses in microglia including cytokine ated with a significant enhancement of these immuno-signals at myocyte release and morphological changes. Toward improving our understanding of lateral membranes and near IDs in CPVT myocytes. Conclusions: Local Ca how purinergic receptor activation drives cellular-scale responses, we devel- influx signals (LoCIS) and enriched immunostaining for STIM1-Orai1 com- oped a minimal computational model for P2X4, P2X7 and P2Y12 activation plexes detected in cardiomyocytes revealed that myocyte SOCE is highly þ þ with concomitant changes in intracellular Ca2 and K homeostasis. In addi- compartmentalized at lateral membrane and intercalated discs, areas divergent tion to handling the intracellular cation transients, the model captures the sub- from known sites of ECC localization in the myocyte interior. These SOCE- sequent modulation of intracellular signaling networks including G-protein enriched domains are markedly expanded in CPVT myocytes, thereby contrib- signaling, the phospholipase C pathway and store operated calcium entry.These uting to arrhythmogenesis by ‘‘spilling’’ Ca from the SOCE pool to the EC networks ultimately control the release of cytokines and changes in cellular coupling pool. morphology associated with microglial function. With this model, we probe the sensitivity of evoking these microglial functions with respect to purinergic 1173-Pos receptor expression and their subcellular distribution, which are known to vary Diurnal Properties of Voltage-Gated Calcium Currents in SCN across microglia phenotypes. Beth McNally, Andrea Meredith. Dept. of Physiology, University of Maryland School of Medicine, Baltimore, 1171-Pos MD, USA. The Role of Dopamine in Pancreatic a-Cells Calcium Heterogeneity and The suprachiasmatic nucleus (SCN) of the hypothalamus houses the mamma- Synchronization Measured by Light-Sheet Microscopy lian central clock, governing circadian rhythms in physiology and behavior. Zeno Lavagnino1,2, David W. Piston3. These rhythms are produced by 24-hr oscillations in spontaneous action poten- 1 Experimental Imaging Center, IRCCS Ospedale San Raffaele, Milano, Italy, tial firing, with higher frequency firing during the day compared to the night. 2 Cell Biology & Physiology, Washington University in St. Louis, Saint Several ionic currents have been identified that regulate SCN firing, including 3 Louis, MO, USA, Dept Cell Biol, Washington Univ St Louis, St Louis, MO, voltage-gated Ca2þ channel (VGCC) currents, but the circadian regulation of USA. distinct VGCC currents has not been comprehensively addressed. In this study, The role of calcium in the stimulation/inhibition of glucagon secretion by whole-cell L- (Nimodipine-sensitive), N- and P/Q- (u-agatoxinIVA, u-cono- glucose remains controversial. Even though elevated glucose causes an overall toxinGVIA, u-conotoxinMVIIC-sensitive), R- (Ni2þ-sensitive), and T-type increase in intracellular free calcium activity, it leads to reduced glucagon secre- (TTAP2-sensitive) currents were recorded from day and night acute SCN tion. Elevated calcium activity is required for secretion, but evidence suggests slices. Using standard voltage protocols, Ni2þ-sensitive currents comprised that paracrine signaling from other islet cells drives glucose inhibition of the largest proportion of total VGCC current (daytime, 45%; nighttime, glucagon secretion. We hypothesize that a-cell calcium controls glucagon secre- 41%). Other components were 33% and 19% (Nim-sensitive), 21% and tion at low glucose, while it is uncoupled from secretion at high glucose. To test 20% (agaIVA), 44% and 25% (conoGVIA), and 15% and 33% (TTAP2) of this idea, we measured calcium activity in islets from transgenic mice expressing the total day and night VGCC current, respectively. Only the Nim-sensitive an a-cell specific calcium biosensor (GCaMP6). Calcium is measured with dual- current exhibited a diurnal difference in magnitude, with daytime current view light-sheet fluorescence microscopy (diSPIM) in all a-cells in the islet. The (1652pA/pF) larger than night (851pA/pF; P=0.002). No diurnal differ- observed heterogeneities in a-cell calcium activity vary as a function of glucose ence in current magnitude was observed for agaIVA- (952 versus concentration. At high glucose concentrations, calcium oscillations tended to 751pA/pF), conoGVIA- (1552 versus 1252pA/pF), Ni2þ-(1552 synchronize among a subpopulation of a-cells, similarly to what happens in in- versus 1552pA/pF), or TTAP2-sensitive (652 versus 952pA/pF) cur- sulin secreting b-cells. It remains unknown if or how signals between islet cells rents. The diurnal difference in Nim-sensitive current was due to larger peak underlie the synchronicity. One candidate factor is dopamine, which is co- current activated during the day, and was eliminated by BayK (BayK-sensitive secreted with insulin, and has been shown to modulate calcium activity and in- current: day 551 versus night 651pA/pF). Inactivation did not differ b sulin secretion in -cells. We tested the hypothesis that dopamine could play a significantly between day and night (ISS/Ipeak: 0.4550.03, 0.4550.04). Nim- role in regulating calcium activity and/or glucagon secretion from a-cells. At sensitive current was evoked by both day and night AP voltage commands low glucose, both glucagon secretion and a-cell calcium activity are inhibited (851 and 751pA/pF). In current-clamp mode, spontaneous membrane po- by physiological levels of dopamine (10mM). At high glucose concentrations, tential oscillations exhibited a diurnal difference (20% of cells during the day however, dopamine additionally reduces glucagon secretion, but does not and 8% at night), which was inhibited by Nim. These data demonstrate a unique further inhibit average a-cell calcium activity, nor does it affect the number of diurnal regulation of L-type Ca2þ current among the major VGCC subtypes in synchronous a-cells observed. These data demonstrate uncoupling of calcium SCN neurons. from glucagon secretion from a-cells at elevated glucose concentrations, but do not support a role of dopamine in synchronizing a-cell calcium. 1174-Pos Role of Orai1 and Store Operated Calcium Entry (SOCE) in Liver: Effects 1172-Pos on Hormone-Induced Calcium Signaling and Glucose Metabolism Cardiac Store Operated Calcium Entry (SOCE) is Compartmentalized at Gary S. Bird, Diane D’Agostin, Pooja Desai, James W. Putney Jr. Intercalated Disks and Linked to Catecholaminergic Polymorphic Ven- Laboratory of Signal Transduction, NIEHS NIH, Research Triangle Park, tricular Tachycardia (CPVT) NC, USA. Ingrid M. Bonilla Mercado1, Andriy Belevych1, Stephen Baine1, In the liver, calcium ions play a pivotal role in a variety of cellular signaling Tom Bodnar1, Bin Liu1,2, Przemyslaw Radwanski1, pathways, and calcium-dependent processes play critical roles in liver health Rengansayee Veeraraghavan1, Pompeo Volpe3, Silvia Priori4, and disease. In this study, we were interested to understand how hepatocytes Noah Weisleder1, Sandor Gyorke1. respond to the glycogenolytic hormones such as epinephrine, and the role of 1OSU, Columbus, OH, USA, 2Mississippi State University, Missisippi State, calcium signaling in the control of hepatic glucose metabolism. At physiolog- MS, USA, 3University of Padova, Padova, Italy, 4University of Pavia, Pavia, ical levels, hepatocytes respond to these hormones with well characterized Italy. intracellular Ca2þ oscillations, mediated by receptor activation of phospholi- Background: SOCE the primary pathway of Ca entry in non-excitable cells, has pase C (PLC). Intracellular Ca2þ oscillations involve a coordinated and regen- recently emerged as a significant component of Ca handling in cardiac muscle. erative release of calcium ions from intracellular calcium stores, and require a

BPJ 9362_9366 Monday, March 4, 2019 237a sustained entry of extracellular calcium ions via store-operated Ca2þ-entry centrations, also failed to trigger significant Ca2þ release or the accompanying (SOCE). In hepatocytes, the mechanisms underlying PLC-dependent Ca2þ-os- Naþ/Ca2þ exchange current in most mutant patch-clamped cells, but showed cillations, and nature of the SOCE Ca2þ influx, have not been thoroughly significant Fluo4 quenching signals, indicative of rapid rise of cytosolic characterized. caffeine. In intact mutant cardiomyocytes, however, the higher 20mM caffeine This study was facilitated by generating animal models to specifically knockout concentrations triggered either irregular Ca2þ ‘‘puffs’’ or smaller and slower the SOCE subunit, Orai1, and characterize the impact on calcium signaling and rises of cytosolic Ca2þ in less than 50% of cells. Such signals were mostly sup- glucose metabolism activated by glycogenolytic hormones. In addition, to pressed by 10mmofIP3R blocker, 2-APB, suggestive of expression of IP3 stores improve the ability to define calcium signals in primary hepatocytes, we gener- in developing hiPSC-CMs. Spontaneous Ca2þ sparks were also absent in most ated a mouse expressing GCaMP6f globally in all tissues, including liver, or Q3925E hiPSC-CMs. Our data suggests that Q3925E-RyR2 mutation may just specifically in the liver using the Alb1-Cre-driver. Using this approach, decrease the sensitivity of CICR and suppress caffeine-triggered release either hormone-induced calcium responses could be measured in the intact liver, by depletion of SR Ca2þ stores or by impairing caffeine binding to RyR2, and within minutes of isolating primary hepatocytes. Primary hepatocytes consistent with the proximity and possible interaction of the Ca2þ and caffeine derived from GCaMP6f mice displayed robust and reproducible calcium binding sites. (NIH, RO1 HL16152, P20GM103499). signaling events in response to thapsigargin, phenylephrine and vasopressin. Preliminary data from both the blood glucose and calcium imaging studies sug- 1177-Pos gest that agonist activation of calcium entering via SOCE plays a role in regu- Myocardial Rad Deletion Modulates L-Type Calcium Channel Current lating the metabolic pathway of hepatic glucose production, and this sustained Brooke Ahern1, Mihir Shah1, Andrea Sebastian1, Douglas A. Andres2, calcium signaling process requires the expression of the SOCE subunit, Orai1. Jonathan Satin3. 1Physiology, University of Kentucky, Lexington, KY, USA, 2Dept Molec/ 1175-Pos Cell Biol, Univ Kentucky, Lexington, KY, USA, 3Dept Physiology, Univ Functional Connectome of the Mechanically Loaded Cardiomyocyte I: Kentucky Sch Med, Lexington, KY, USA. Identifying Involved Subsystems Rad inhibits L-type calcium channel current (ICa,L), and in vivo deletion of Rad Zana A. Coulibaly1, Leighton Izu1, Ye Chen-Izu1, Zhong Jian1, results in elevated ICa,L density. The aim of this study is to test the hypothesis Rafael Shimkunas2. that Rad-deletion modulates L-type calcium channel current (I ). 1 2 Ca,L Dept Pharmacology, Univ Calif Davis, Davis, CA, USA, MyoKardia, South We engineered a cardiac-restricted inducible Rad knockout mouse (cRadKO). San Francisco, CA, USA. We examined ICa,L using the whole cell configuration and the cell attached An isolated cardiomyocyte has the ability to adapt its force of contraction to configuration of the patch clamp technique. We assessed global and local counter an increase in mechanical load. We hypothesize that this adaptive cellular calcium handling using Fura-2 AM and Fluo-4 AM, respectively. response involves a number of cellular subsystems that regulate calcium. How- cRadKO displayed an increase in ICa,L compared to WT (maximal conduc- ever, we do not know a priori which subsystems are modulated. This work de- tance: cRadKO = 254 5 19 pS/pF, n=15; WT= 144 5 12 pS/pF, n=18; scribes our approach to identifying cellular subsystems that are part of the p<0.0001). ICa,L activated at more negative voltages (activation midpoint: functional connectome of the mechanically loaded cardiomyocyte. cRadKO = 18.3 5 1.0 mV, n=15; WT= 8.1 5 1.9 mV, n=18; p<104). We use a mathematical model to simulate the effects of a mechanical load on a WT cells treated with forskolin and IBMX demonstrated a ceiling effect of cardiomyocyte. Our model implements a feedback-control system that involves modulation in WT ICa,L.ICa,L kinetics are accelerated in cRadKO concordant the cardiomyocyte’s contractile and calcium systems. Since the mechanically- with the upstroke velocity of Ca2þ (transient velocity 48.7 5 2.4 AU/s, activated messenger can affect many cellular subsystems in the calcium system, n=49; WT=38.1 5 3.5 AU/s, n=37; p=.014). cRadKO ICa,L was no different we consider the possibility of modulations that either inhibit or promote the ac- when treated with isoproterenol, consistent with saturable modulation of the tivity of many subsystems. The subsystems we choose to consider are the LTCC. L-type Ca2þ current (LTCC), Naþ-Ca2þ exchanger (NCX), ryanodine recep- Myocardial Rad deletion increases ICa,L and activates ICa,L at more negative tors (RyRs), and SR pump subsystems. This multi-subsystem approach is com- potentials that is beyond PKA induced modulation of the L-type calcium chan- plemented with a filtering method that takes advantage of multi-dimensional nel; cRadKO ICa,L remains unchanged when PKA is activated. This unmasks aspect of the experimental data. This multi-subsystem/multi-dimensional data a new form of modulation of ICa,L, demonstrating that Rad plays a critical approach allows us to cover many possible pathways of the mechanism role in how the L-type calcium channel responds to b-Adrenergic Receptor involved in the cell’s response to mechanical load while retaining only the stimulation. modulations that conform to the experimental results. We found that there is no unique solution that conforms to all available 1178-Pos experimental data. Instead, it is possible for the mechanically-activated Modeling the Impact of Spine Apparatus on Signaling and Regulation in messenger to modulate all considered subsystems in different ways. This sug- Realistic Dendritic Spine Geometries gests that there exists multiple ‘‘good enough’’ modulations capable of Justin G. Laughlin1, Christopher T. Lee2, J. Andrew McCammon2, affecting subsystems related to the efflux and influx of calcium in a manner Rommie E. Amaro2, Michael Holst3, Padmini Rangamani1. that qualitatively matches the experimental data. In a separate abstract, we 1Mechanical and Aerospace Engineering, University of California, San discuss the connections between subsystems that enable the myocyte to respond Diego, La Jolla, CA, USA, 2Chemistry and Biochemistry, University of to mechanical load. California, San Diego, La Jolla, CA, USA, 3Mathematics, University of California, San Diego, La Jolla, CA, USA. 1176-Pos Cell shape and function have been shown to be closely related. This is partic- CRISPR/Cas9 Engineered Q3925E-RyR2 Mutation in Human Induced 2D ularly true in the case of synapses, where dynamic changes to dendritic spine Pluripotent Stem Cells Impairs Caffeine Triggered Ca Release size and shape are associated with synaptic plasticity, learning, and memory 1 1 2 1 Xiaohua Zhang , Hua Wei , Naohiro Yamaguchi , Martin Morad . formation. Here we describe a workflow, using open-source community tools 1Dept Regenerative Med, Cardiac Signaling Center of USC, MUSC AND 2 GAMer 2.0, Blender, and IMOD, to generate computable and realistic mesh ge- Clemson University, Charleston, SC, USA, Dept Regenerative Med, ometries from FIB-SEM images of dendritic spines. The generated meshes are Medical University of South Carolina, Charleston, SC, USA. high-quality and suitable for use in finite element (FE) simulations. Using CRISPR/Cas9 mediated gene editing of human induced pluripotent stem cells open-source FE solver FEniCS to model reaction-diffusion using realistic ge- (hiPSCs) provides a novel approach in human disease studies. Here we report ometries from our workflow, we show that microanatomy influences spine on a ryanodine receptor (RyR2) mutation, Q3925E, associated with catechol- 2þ spatio-temporal signaling dynamics of various messengers. Specifically, we aminergic polymorphic ventricular tachycardia (CPVT1), located in the Ca show that in realistic geometries, spine neck plays a critical role as a diffusion binding site of the near atomic level model of RyR structure. Simultaneous 2þ barrier and this effect is enhanced by the presence of a fully-developed spine Ca imaging in patch clamped hiPSC derived cardiomyocytes (hiPSC-CMs) apparatus. allowed quantifications of Ca2þ signaling and electrophysiological parameters associated with the mutant hiPSC-CMs. We found that ICa density by depolar- 1179-Pos izations to zero mV and its accompanying Ca-transients in Q3925E hiPSC- The Role of S-Acylation in Store Operated Calcium Entry þ 2 1 2 2 CMs were comparable to the WT hiPSC-CMs, but ICa induced Ca release Savannah J. West , Qiaochu Wang , Michael X. Zhu , 1 1 (CICR) was smaller, when measured by rapid and brief ICa tail currents acti- Askar M. Akimzhanov , Darren Boehning . vated by repolarizations from þ100mV to 50mVs, as compared to WT car- 1Biochemistry and Molecular Biology, UTHealth Sci Ctr, Houston, TX, þ diomyocytes. Unexpectedly SR Ca2 release, triggered by 5mM caffeine, USA, 2Integrative Biology and Pharmacology, UTHealth Sci Ctr, Houston, was fully suppressed in mutant cardiomyocytes. Higher, 20 mM caffeine con- TX, USA.

BPJ 9362_9366 238a Monday, March 4, 2019

S-acylation is the reversible post-translational lipidation of cysteine resi- 1182-Pos dues. It is known that S-acylation plays an important role in the modulation Mitochondrial Calcium Signaling in Heart of ion channel membrane localization, trafficking, and function. Orai1 is Andrew P. Wescott, Joseph P. Kao, W. Jonathan Lederer, Liron Boyman. a plasma membrane calcium channel activated by ER calcium store deple- Sch Med, Univ Maryland, Baltimore, MD, USA. 2þ 2þ tion. Orai1 is gated by STIM1, an ER-localized protein with a calcium A key modulator of mitochondrial biology is the level of matrix Ca , [Ca ]m. binding domain within the ER lumen. When ER calcium is released, Virtually all aspects of this topic remain controversial, however. These include 2þ 2þ STIM1 and Orai1 form complexes to facilitate store operated calcium the actual physiological Ca level of [Ca ]m, the beat-to-beat variation of 2þ entry. In an unbiased proteomic screen we have found that Orai1 and [Ca ]m in cardiac myocytes, the regulation of proteins controlling the amount 2þ 2þ STIM1 are S-acylated proteins. Cellular stimulation to deplete calcium of Ca from the cytosol that gets into the matrix, and the amount [Ca ]m and stores results in rapid and transient palmitoylation of both Orai1 and corresponding molecular signaling targets that regulate mitochondrial meta- STIM1. Palmitoylation-deficient Orai1 mutants disrupt channel function as bolic function and those underlying the mitochondrial permeability transition determined using calcium imaging and electrophysiological approaches. pore (mPTP) for cell death. Two examine the first two points single cardiomyo- þ This was associated with defective recruitment of Orai1/STIM1 into plasma cyte experiments are presented to reveal how Ca2 flux through the mitochon- 2þ membrane/ER junctions. Our results suggest that Orai1 S-acylation is a crit- drial calcium uniporter (MCU) sets [Ca ]m during beat-to-beat elevations of 2þ 2þ ical and previously unappreciated component of stimulus-dependent associ- cytosolic Ca ([Ca ]i). These cardiomyocytes are voltage-clamped so that 2þ 2þ ation with STIM1. both [Ca ]i and [Ca ]m could be measured simultaneously using high reso- 2þ lution confocal microscopy. We find that in a quiescent state, [Ca ]i is 100 2þ 1180-Pos nM, while [Ca ]m is about 150 nM. On the other hand, a series of heart beats 2D 2þ 2þ Inhibition of Ca Influx by SARAF and Pancreatitis (0.5 Hz) evoked [Ca ]i transients that lead to a gradual elevation of [Ca ]m to Aran Son1, Shmuel Muallem1, Malini Ahuja2. a steady-state of about 700 nM with unresolvable small (<10 nM) transient in- 1 2 2þ NIH, Rockville, MD, USA, NIDCR, Bethesda, MD, USA. creases of [Ca ]m with each beat. Additional quantitative measurements of the 2þ Pancreatitis, an inflammatory disease of the exocrine pancreas is associated Ca flux through the cardiac MCU complex reveal a surprising new under- with aberrant Ca2þ signaling increase, primarily due to excessive Ca2þ influx standing of the channel conductance and its gating mechanism. These findings 2þ 2þ by TRPC and Orai1 channels, both of which are regulated by the ER Ca set the quantitative stage to identify the molecular targets of [Ca ]m relevant to sensor STIM1. STIM1 interacts with and directly activates Orai1, is the the regulation of ATP production and to mPTP activation. 2þ pore forming unit of CRAC current. Store Operated Ca -Associated Regula- 1183-Pos tory Factor (SARAF), an ER membrane protein interacts with activated þ Identification and Characterization of an NAADP Receptor Essential for STIM1 to trigger the fast and slow forms of Ca2 dependent inactivation þ þ NAADP-Evoked Calcium Release from Endolysosomal Organelles (CDI) of Orai1 to inhibit Ca2 influx and prevent pathological Ca2 overload. 2þ Jiyuan Zhang, Xin Guan, Jiusheng Yan. Knockout and inhibition of TRPC channels and Orai1, although reduced Ca Dept Anesthesiol/PeriOp, Univ Texas MD Anderson Cancer Ctr, Houston, toxicity, caused serious damage that in the case of Orai1 resulted in death. An TX, USA. alternative approach is to facilitate the CDI by SARAF at pathological situa- Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca2þ- tions. Here, we first used CRISPR/cas9 to tag the native SARAF with HA in mobilizing second messenger identified to date; it uniquely mobilizes Ca2þ from mice. Cell toxicity by induction of acute pancreatitis resulted in transient in- acidic endolysosomal organelles. However, the molecular identity of the NAADP crease in SARAF mRNA and protein that lasted for 1-2 hours, with subse- receptor remains unknown. Given that the endolysosomal two-pore channels quent reduction in SARAF expression and its interaction with STIM1, 2þ 2þ 2þ (TPCs) are essential for NAADP-evoked Ca release and likely part of the causing increased Ca influx, mitochondrial Ca load and cell toxicity. NAADP signaling protein complexes, we performed affinity purification of the Accordingly, deletion of SARAF in mice exacerbated acute pancreatitis and / 2þ 2þ TPC- and NAADP receptor-containing complexes in HEK293 cells. With quan- SARAF acini showed increased Ca influx and mitochondrial Ca titative proteomic analysis, we identified an Lsm protein, designated as load. Notably, transgenic pancreas specific SARAF over-expression reduced 2þ NAADPR1, as a sole interacting partner of both TPCs and immobilized NAADP. Ca influx and profoundly reduced all symptoms of acute pancreatitis with We found that both endogenous (HEK293) and recombinant NAADPR1 (ex- no apparent effect on the mice. These findings suggest that SARAF should 2þ pressed in and purified from E. coli) can bind to NAADP with high affinity while be a preferred and safe target for treatment of cell toxicity due to Ca influx, TPCs lost their affinities to NAADP in the absence of NAADPR1. NAADPR1 is including acute pancreatitis. essential for NAADP-signaling as its genetic knockout abolished and its reappear- ance rescued NAADP-evoked Ca2þ release. Deletion and point mutation in Lsm 1181-Pos 2þ 2D domain nullified the NAADPR1 function in NAADP-evoked Ca release. Thus, Ca Diffusion in the Large Peptidergic Nerve Terminals of the Posterior for the first time, we identified an NAADP receptor that is essential for NAADP- Pituitary evoked Ca2þ release from acidic endolysosomal organelles. Shane M. McMahon, Meyer B. Jackson. Neuroscience Department, University of Wisconsin - Madison, Madison, WI, 1184-Pos USA. Null-Sarcolipin Equine Muscle Shows Enhanced SERCA Calcium Trans- þ Ca2 signals play important roles in synaptic transmission and plasticity, in port Which May Potentiate the Prevalence of Exertional Rhabdomyolysis addition to a variety of other processes such as egg fertilization and cell Joseph M. Autry1, Bengt Svensson1, Christine B. Karim1, death. These signals span several orders of magnitude in space and time in Sudeep Perumbakkam2, Zhenhui Chen3, Carrie J. Finno4, David D. Thomas1, different biological contexts. After entering the cytoplasm, the fate of a Stephanie J. Valberg2. þ Ca2 ion is decided by the interplay between diffusion, buffering, and seques- 1Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, tration and removal mechanisms. A large body of literature has developed Minneapolis, MN, USA, 2Large Animal Clinical Sciences, Michigan State þ around the fields of Ca2 buffering, sequestration and removal. In compari- University, East Lansing, MI, USA, 3Krannert Institute of Cardiology, þ son, the investigation of Ca2 diffusion remains relatively sparse. We have Indiana University, Indianpolis, IN, USA, 4Population Health and þ investigated Ca2 diffusion in the nerve terminals of acute slices prepared Reproduction, University of California Davis, Davis, CA, USA. from the posterior pituitary. The large size (10 mm) and nearly spherical ge- We have analyzed gene expression, protein expression, and enzymatic activity ometry of these nerve terminals render them a convenient preparation for of the sarcoplasmic reticulum calcium-transport ATPase (SERCA) in horse electrophysiological recording. These advantages have previously been ex- gluteus, a locomotive muscle adapted for speed. Horses have a high prevalence ploited by our lab and others to study various aspects of nerve terminal phys- of exertional rhabdomyolysis (3-10%) with correlative associations to stress þ iology including Ca2 . Here, we have used two photon imaging of the latest and perturbed Ca homeostasis. To assess SERCA activity and Ca regulation þ generation of chemical Ca2 indicators (Cal- and Calbryte 520 and 630) to in horse gluteus, we developed an improved protocol for isolating horse sarco- þ quantitatively investigate Ca2 diffusion in posterior pituitary nerve terminals. plasmic reticulum (SR) vesicles. Whole transcriptome shotgun sequencing Imaging revealed the formation and collapse of spatial gradients. From these (RNA-seq) and quantitative immunoblotting determined that the ATP2A1 dynamic processes we estimated apparent diffusion coefficients ranging from gene and its protein product SERCA1 are the predominant isoform expressed þ 10-80 mm2/s near resting Ca2 concentrations, and up to 200 mm2/s at in horse gluteus, as in rabbit fast-twitch muscle. Gene expression of four þ elevated Ca2 concentrations. These measurements allow us to investigate SERCA regulatory peptides was assessed, showing that sarcolipin (SLN)is þ þ the impact of fixed and mobile Ca2 buffers on the mobility of Ca2 in the the predominant SERCA regulator transcribed in horse gluteus, as in rabbit cytoplasm of a mammalian nerve terminal. This work was funded by NIH muscle. Surprisingly, the gene transcription ratio of SLN/ATP2A1 in horse grant NS103206. gluteus is an order of magnitude higher than in rabbit muscle, but conversely,

BPJ 9362_9366 Monday, March 4, 2019 239a the protein expression ratio of SLN/SERCA1 in horse gluteus is an order of EVP4593 can be associated with the reducing of huntingtin expression level. magnitude lower than in rabbit muscle. We conclude that the SLN gene is The study was supported by RSF grant No 17-74-20068. not translated to a stable protein in horse gluteus. The extremely high level of SLN transcription in horse muscle suggests a non-coding role, similar to 1187-Pos regulation of mouse muscle contractility by analogous functional RNAs A Computational Framework to Study the Kinetics and Evolution of 2D b LINC00948(MRLN) and ZFAS1. The Ca transport rate by SR vesicles is Ca -Permeable Amyloid Pores Associated with Alzheimer’s Disease Syed Islamuddin Shah1, Ian Parker2, Angelo Demuro2, Ghanim Ullah1. 75% greater in horse than in rabbit; thus, we propose that horse myocytes 1 2 have augmented SR Ca stores that enhance Ca release and contractility in Dept of Physics, University of South Florida, Tampa, FL, USA, Dept the athletically-bred horse species. We hypothesize that lack of SERCA regu- Neurobiol/Behavior, Univ Calif Irvine, Irvine, CA, USA. Total internal reflection fluorescence microscopy (TIRFM), a high-throughput lation by SLN increases equine susceptibility to exertional rhabdomyolysis by 2þ potentiating stress-induced Ca release from saturated SR. Funding: Morris An- imaging technique used to monitor Ca ions passing through pores formed b b imal Foundation grant D16EQ-004 (SJV, JMA, DDT) and NIH grants by Alzheimer’s disease-associated peptides amyloid (A ) in cell mem- GM027906 & HL129814 (DDT). branes, providing detailed information about their gating and evolution with resolution of < 2 ms. This massively parallel technique is capable of 1185-Pos providing simultaneous and independent recording from thousands of pores miR-200C Exhibits an Age-Dependent Increase in the Rat Heart and Mod- for extended time. Manual analyses of these several thousands of pores in ulates Cardiomyocyte Function tens of thousands of image frames is challenging. To circumvent this chal- Cristina Florio1, Alessandra Magenta2, Rostislav Byshkov1, Kenta Tsutsui1, lenge, we have developed a pipeline of computational tools to retrieve, Bruce Ziman1, Edward G. Lakatta1, Maurizio C. Capogrossi3. analyze, and predict the behavior of these pores at extended timescale to 1NIH/NIA, Baltimore, MD, USA, 2Istituto Dermopatico dell’Immacolata, shed light on their toxicity. IDI-IRCCS, Rome, Italy, 3Division of Cardiology, Johns Hopkins Bayview Analyzing imaging data includes detection of pores, generating location maps, Medical Center, Baltimore, MD, USA. retrieval of time series data, and collection of their kinetics. The obtained time Reactive oxygen species (ROS) play a pivotal role in aging and in some cardio- series data often contains multiple pores/conductance levels at a time in addi- vascular diseases, and markedly enhance miR-200c expression in numerous tion to noisy and drifting background. Our pipeline of computational tools in- cell types. However, it is still unknown whether miR-200c cardiac expression cludes a software called CellSpecks, which is capable of performing all these is age-dependent and whether it modulates action potential (AP) and calcium tasks. In order to separate signal from noisy and drifting background along dynamics in adult cardiomyocytes (CM). We evaluated miR-200c expression with quantification of multiple pores/conductance levels, we have developed in the left ventricle of young (3 months) and old (15 months) rats and found another software called TraceSpecks. TraceSpecks uses maximal likelihood that it is 2-fold higher in older rats (p=0.05). The effect of miR-200c overex- formalism for separating signal from a noisy and drifting background. Trace- pression was evaluated in adult left ventricular CM infected either with lenti- Specks is capable of processing patch-clamp data containing single and multi- miR-200c or lenti-scramble. Action potentials (APs) were recorded in ple channels per trace, fluorescence traces from TIRFM representing Ca2þ 2þ current-clamp mode. Cytosolic Ca transients (Cai) were evoked by field signals generated by several channels in a cluster of IP3Rs, and Ab pores stimulation (0.5 Hz) or caffeine pulse in another subset of cells loaded with with multiple conductance levels. We have used these tools to extract and Fluo4. Although AP amplitude was unchanged, time to AP peak, upstroke ve- idealize time traces of tens of thousands of Ab1-42 pores and developed Markov locity and AP duration, measured at 50% and 90% of the repolarization, were chain model to understand and predict their behavior and level of toxicity on significantly (p<0.05) prolonged in miR-200c overexpressing CMs (n=11) vs longer timescale. control (n=8). These effects on the AP were associated with reduced Cai tran- sient amplitude and prolonged duration (miR-200c n=25; control n=18; 1188-Pos p<0.01). Similar effects were observed in the caffeine-induced Ca2þ transient Probing the Mechanisms by which Septins Regulate ORAI1 Function (miR-200c n=28; control n=22; p<0.05). In summary, (a) miR-200c expression Zachary Katz1,2, Chen Zhang1, Ariel Quintana1, Bjorn Lillemeier2, increases in the old heart, (b) miR-200c prolongs the duration of the AP, prob- Patrick G. Hogan1. 1 2 ably affecting potassium channels, (c) miR-200c decreases the amplitude and La Jolla Inst Allergy, La Jolla, CA, USA, Salk Institute for Biological prolongs both the AP- and the caffeine-induced Cai transients thus indicating Studies, La Jolla, CA, USA. that sarcoplasmic Ca2þ release via RyRs, initiated either by the AP or caffeine, The STIM-ORAI-mediated calcium release-activated calcium channel, or 2þ and Ca2þ extrusion from the cytosol are affected by miR-200c. These results CRAC channel, is a key source of calcium influx for intracellular Ca suggest that miR-200c may be implicated in impaired contraction and/or relax- signaling and effector function in T lymphocytes. STIM in the ER membrane ation such as heart failure, i.e in cardiac dysfunction in the elderly and condi- senses depletion of ER calcium stores and moves to ER-plasma membrane tions associated with enhanced oxidative stress. (PM) junctions where it recruits PM ORAI channels and triggers store- operated Ca2þ entry. We have shown previously that septins 4 and 5 are crucial 1186-Pos for efficient STIM1-ORAI1 cluster formation following store depletion. Sep- Potential Neuroprotective Drug Evp4593 Reduces Excessive Expression of tins were previously reported to specify PM barriers at certain sites and to serve Huntingtin in iPSC-Based Juvenile Model of Huntington’s Disease as scaffolds that recruit signaling proteins, but their detailed role in calcium Dmitry Grekhnyov, Vladimir Vigont, Elena Kaznacheyeva. signaling remained elusive. We used live-cell super-resolution microscopy Institute of Cytology RAS, Saint-Petersburg, Russian Federation. and single-molecule tracking to map ORAI1, STIM1, ER-PM junctions, and Huntington’s disease (HD) is a hereditary neurodegenerative disorder character- membrane-localized septin 4 in resting cells and store-depleted cells. We ized by neuronal damage in various brain regions with the predominant vulner- further investigated the trajectories of ORAI1 channels in septin 4/5-deficient ability of striatal GABA-ergic medium spiny neurons (MSNs). The pathology is cells or in cells where the interaction between ORAI1 and STIM1 was abol- caused by mutation in gene encoding huntingtin protein. Nowadays, the key ished by mutagenesis. Our data show that septins 4/5 are needed to maintain mechanisms underlying HD pathogenesis remain unclear. Toxic actions of a normal complement of functional ER-PM junctions, and to support efficient mutant huntingtin lead to the dysfunction of multiple cellular processes STIM-ORAI interaction at the junctions. Septins neither promote ORAI local- including autophagy, calcium homeostasis, mitochondrial functioning etc... ization to junctions independent of STIM, nor do they specify corrals surround- Especially, accumulated evidences indicate that improper regulation of intracel- ing junctions to confine ORAI, but they do contribute to decreased ORAI1 lular neuronal calcium signaling play a role in HD progression. One of the most mobility across the entire cell footprint after store depletion. This additional adequate HD cellular models is induced pluripotent stem cells based (iPSC- factor could support calcium signalling by slowing the escape of ORAI1 chan- based) patient-specific model endogenously expressing the mutant huntingtin. nels from junctions after they have been recruited there by STIM. Thus live-cell In our study iPSCs were derived from patient with juvenile form of HD and single-molecule tracking has allowed us to document three primary mecha- differentiated into MSNs (HD76Q) using somatic cell reprogramming technol- nisms by which septins 4/5 enhance STIM-ORAI signaling. ogy. We supposed that toxic effect of the mutant huntingtin can be potentiated by increase in the expression level of huntingtin in HD-specific cells. Using western 1189-Pos blot analysis we found that huntingtin expression level was significantly higher Pade Approximation of Single-Channel Calcium Nanodomains in the Pres- in HD76Q cells compared to WT neurons. Moreover, we explored the effect of ence of Cooperative Calcium Buffers potential therapeutic drug EVP4593 on huntingtin expression. EVP4593 was Yinbo Chen, Victor Matveev. previously shown to have a neuroprotective effect on HD-specific cells and flies. Dept Math Sci, NJIT, Newark, NJ, USA. þ þ Our data demonstrated that 24h treatment with 3 mkM EVP4593 significantly Ca2 elevations produced in the vicinity of single open Ca2 channels are þ decreased the expression level of huntingtin in HD76Q cells, returning it to termed Ca2 nanodomains, and are known to trigger secretory vesicle exocy- the normal values. Thus, we suggest that the neuroprotective effect of tosis, myocyte contraction and other fundamental physiological processes.

BPJ 9362_9366 240a Monday, March 4, 2019

Ca2þ nanodomains are shaped by the interplay between Ca2þ influx, diffusion Calmodulin (CaM) is a multifunctional calcium (Ca2þ)-binding messenger and binding to Ca2þ buffers, and can be approximated with reasonable accu- that directly interacts with the cardiac ryanodine receptor 2 (RyR2), a large racy by analytic approximations of quasi-stationary solutions of the corre- transmembrane Ca2þ channel that mediates Ca2þ release from the sarco- sponding reaction-diffusion equations. Such closed-form approximations help plasmic reticulum to activate cardiac muscle contraction. Genetic studies to reveal the qualitative dependence of nanodomain characteristics on buffering have reported CaM missense mutations in individuals with history of life- and diffusion parameters without resorting to computationally expensive nu- threatening arrhythmogenic heart disorders. A recent clinical report identified merical simulations. Although several nanodomain approximations had been a novel, long QT syndrome (LQTS)-associated CaM mutation (E105A), in a developed for the case of buffers with a single Ca2þ binding site, most biolog- child, who experienced an aborted first-episode of cardiac arrest. Herein, to ical buffers have more complex Ca2þ-binding stoichiometry. Further, Ca2þ determine the functional consequences of the E105A mutation in vivo,we buffers such as calretinin and calmodulin consist of distinct EF-hand domains, introduced this mutation into human CaM sequence and we injected synthetic each possessing two Ca2þ binding sites exhibiting cooperativity in binding, mRNA encoding CaMWT and CaME105A into zebrafish embryos. Although whereby the affinity of the second Ca2þ binding is much higher than the first. expression of CaMWT and CaME105A proteins in zebrafish did not affect the While the Rapid Buffering Approximation (RBA) has been recently general- normal embryo development, a slight change in the heart morphology was ized to such cooperative buffers, its performance is limited by the complex observed, with 31.5% of the CaME105A-injected zebrafish exhibiting interplay between the condition of slow diffusion required for RBA accuracy, extended hearts. Furthermore, analysis of the cardiac activity of the zebrafish and the slow rate of the first Ca2þ binding reaction characterizing cooperative ventricle revealed that CaME105A mutant-injected larvae displayed irregular Ca2þ binding. To resolve this problem, we extend the recently developed alter- pattern of heart beating in comparison to the median of the CaMWT and con- native method, the Pade approximation method, to the case of 2-to-1 Ca2þ trol groups, resulting to an increased arrhythmic potential in these embryos. In buffers. The Pade approximation interpolates between the short-range and addition, the average heart rate was significantly increased in this group long-range distance-dependence of nanodomain concentration using a rational (160.5 beats per minute (bpm) vs 152.5 bpm of control group). In vitro function Ansatz. We examine in detail the parameter-dependence of the lowest- Ca2þ-binding studies revealed that the C-domain of CaME105A mutant ex- order Pade approximation accuracy, and show that this method is superior to hibited a 10-fold reduced Ca2þ-binding affinity compared to CaMWT. RBA for a wide ranges of buffering parameter values. The limitations of the Finally, the functional effect of E105A mutation on RyR2 activity was as- Pade method, in particular its algebraic complexity, are also discussed. Sup- sessed by a [3H]ryanodine binding assay and suggested that CaME105A mutant ported in part by NSF DMS-1517085 (V.M) shows a dramatically reduced inhibition of ryanodine binding to RyR2 compared to CaMWT. Our findings suggest that LQTS-associated E105A 1190-Pos CaM mutation dysregulates normal cardiac function in zebrafish by altering New Red Fluorescent Calcium Indicators for Functional Analysis of 2þ D both CaM-Ca and CaM-RyR2 interactions. GPCRs and Ca2 Channel Targets Qin Zhao, Haitao Guo, Peng Ruogu, Liu Jixiang, Jinfang Liao, 1192-Pos Zhenjun Diwu. Calcium Channels Contributing to Action Potential Firing and Rhythms in AAT Bioquest Inc., Sunnyvale, CA, USA. the Circadian Clock The intercellular calcium flux assay is a widely used in monitoring signal Amber E. Plante, Andrea L. Meredith. transduction pathways and characterizing GPCRs agonists and antagonists. Physiology, University of Maryland Baltimore, Baltimore, MD, USA. Most common calcium indicators are green fluorescent and difficult for using Mammalian circadian rhythms are timed by suprachiasmatic nucleus (SCN) with GFP and YFP transfected cells or tissues, so red fluorescent calcium in- neurons, which generate a 24-hour time signal by firing spontaneous action dicators are highly desirable. Although Rhod-2 is commonly used for multi- potentials (APs) at a higher frequency during the day versus the night. The roles plexing with GFP-transfected cells, the higher staining background and of Ca2þ channels in regulating AP firing in the SCN have not been extensively cellular localization (mostly in mitochondria) limits its applications and makes studied. To identify which Ca2þ channels contribute to the circadian regulation the rhodamine-based calcium dyes less sensitive. In this study, two new red of AP frequency, we tested the effects of inhibitors of VGCCs (Cd2þ), L-(ni- fluorescent calcium indicators, Calbryte 590 and Calbryte 630, were developed modipine, Nim), N-(u-conotoxinGVIA, GVIA), P/Q-(u-agatoxinIVA, Aga), þ for monitoring Ca2 flux assays in living cells with Ex/Em= 580/592 nm and R-(Ni2þ), and T-(TTAP2) type Ca2þ channels, ryanodine receptors (dantro- Ex/Em=608/624nm, which are red-shifted wavelength than Rhod-2 with no lene, Dan) and SERCA (cyclopiazonic acid, CPA) on extracellular day and overlap with FITC filters. In this study, the performance of Calbryte 590 night AP frequencies from acute SCN slices. Compared to control, daytime and Calbryte 630 was evaluated with different receptor signaling pathways us- AP frequency decreased in Cd2þ (-85%), Nim (28%), GVIA (65%), Aga ing HEK-293, CHO-M1 and CHO-K1 cell lines with fluorescence microscope, (69%), Ni2þ (-46%), Dan (31%), and CPA (27%). Nighttime AP fre- microplate reader and flow cytometer. The fluorescence imaging data showed quency also decreased in Cd2þ (-63%) but increased in Dan (þ113%) and that Calbryte 590 and Calbryte 630 uniformly bind to cytosolic calcium ion. CPA (þ125%). The chronic effect of these inhibitors on AP rhythmicity was The microplate assay results showed that unlike Rhod-2, which requires the determined using 3 days of baseline firing and 3 days post-drug application presence of organic anion transporter inhibitor (e.g. probenecid) to prevent from organotypic SCNs on multi-electrode arrays. Compared to baseline, the the leakage of the indicators during dye loading, Calbryte 590 and Calbryte percentage of rhythmic recordings decreased in Nim (1656%), GVIA 630 requires minimal amount of probenecid and still has around 5 folds of (1656%), Aga (2955%) and Ni2þ (32510%). Of the rhythmic record- response over background, which is more than twice higher compared to ings remaining, the c2 amplitude decreased with Nim (55%) and Aga Rhod-2 assay (S/B 2 folds). Calbryte 630 flow cytometry assay results (48%) and relative power (FFT) decreased with Nim (57%). Period þ also showed 4 folds Ca2 flux change with APC channel. In conclusion, Cal- increased in Dan (þ5%), although Dan and CPA did not affect other rhythmic bryte 590 and Calbryte 630 are robust red-shifted fluorescent indicators suit- parameters. Our data suggests L-, N-, P/Q- and R-TCCs all contribute to day- þ able for multiplexing Ca2 flux assay in GFP- and YFP-transfected cells or time AP frequency and circadian AP rhythms. The strongest influence is with other green fluorescent dyes with improved cellular retention, brightness contributed by LTCCs, consistent with evidence that LTCC currents are diur- 2þ and S/B ratio. nally modulated. In contrast, Ca i stores contribute to daytime and nighttime AP frequency but only a limited influence on AP rhythmicity is revealed in this 1191-Pos study. The Arrhythmogenic E105A CAM Mutation Dysregulates Normal Cardiac Function in Zebrafish by Altering CAM-Ca2D and CAM-RyR2 Interactions Posters: Other Channels Michail Nomikos1, Sahar I. Da’as2,3, Angelos Thanassoulas4, Rola Salem1, Brian L. Calver5, Alaaeldin Saleh1, Ali Al-Maraghi1, 1193-Pos Gheyath K. Nasrallah6,7, Bared Safieh-Garabedian1, Egon Toft1, Functionally Identifying Members of the MscS Superfamily of Ion Chan- George Nounesis4, F. Anthony Lai1,5. nels in Paraburkholderia Membranes 1College of Medicine, Member of QU Health, Qatar University, Doha, Qatar, Hannah M. Dickinson, Brittni L. Miller, Hannah R. Malcolm. 2Translational Medicine, Sidra Medicine, Doha, Qatar, 3College of Science & Dept Chemistry, Univ North Florida, Jacksonville, FL, USA. Engineering, Hamad Bin Khalifa University, Doha, Qatar, 4National Center Bacterial mechanosensitive channels gate in response to tension in the mem- for Scientific Research ‘‘Demokritos’’, Athens, Greece, 5College of brane which can be driven by drastic changes in osmolarity. The mechanosen- Biomedical & Life Sciences, Cardiff University, Cardiff, United Kingdom, sitive ion channel of small conductance (MscS) from Escherichia coli gates in 6Department of Biomedical Sciences, College of Health Sciences, Qatar response to mechanical force, and is the most well studied member of the MscS University, Doha, Qatar, 7Biomedical Research Center, Qatar University, superfamily of ion channels. Despite limited genome space, several MscS su- Doha, Qatar. perfamily members are predicted in each genome. Members of the bCNG

BPJ 9362_9366 Monday, March 4, 2019 241a subfamily are heteromultimers in bacterial membranes. This suggests that 1196-Pos MscS superfamily members may have varying roles within the cell. Patch Heterotypic Docking Compatibility of Human Cx37 with Other Vascular clamp electrophysiology on native membrane fragments gives insight into Connexins the functional role that members of the MscS superfamily play. To functionally Nicholas K. Kim1, Artur Santos-Miranda1, Honghong Chen2, identify MscS superfamily members, we have developed methods to create Hiroshi Aoyama3, Donglin Bai1. spheroplasts in two unique Paraburkholderia strains. Bioinformatic analysis 1Physiology and Pharmacology, Univ Western Ontario, Ontario, ON, of these strains shows that each genome is predicted to encode for at least eight Canada, 2Physiology and Pharmacology, Univ Western Ontario, London, unique MscS superfamily members. Some of these MscS superfamily channels ON, Canada, 3Graduate School of Pharmaceutical Sciences, Osaka Univ, contain additional binding domains that may act as additional stimuli. To func- Osaka, Japan. tionally identify these channels, we conducted patch clamp electrophysiology Human vascular connexins (Cx37, Cx40, Cx43, and Cx45) can form various on native membrane fragments, under varying conditions. types of gap junction (GJ) channels to synchronize vasodilation/constriction to control local circulation and blood pressure. Most of our knowledge on heterotyp- 1194-Pos ic gap junctions of these vascular connexins was from studies on rodent connex- Molecular Dynamics Simulations of TMC1 Homology Models ins. In human vasculature, the same four homolog connexins exist, but whether Sanket Walujkar, Lahiru N. Wimalasena, Jeffrey Lotthammer, these human connexins can form heterotypic GJs as those of rodents have not Marcos Sotomayor. been fully studied. Here we used in vitro expression system to study the coupling Dept Chem & Biochem, Ohio State Univ, Columbus, OH, USA. status and GJ properties of human heterotypic Cx37/Cx40, Cx37/Cx43, and Mechanical stimuli from sound and head movements are converted into elec- Cx37/Cx45 GJs. Our results showed that Cx37/Cx43 and Cx37/Cx45 GJs, but trical signals in the inner ear during a process known as mechanotransduction. not Cx37/Cx40 GJs, were functional and each with unique rectifying properties. Vibrations in the inner ear fluid - endolymph result in deflections of the hair Cx37/Cx43 GJs showed inward rectification for its current-voltage relationship at cell stereocilia. These deflections cause opening of cation channels at the top the beginning Vj induced junctional currents, but an outward rectification near the of the stereocilia producing an electric signal that is sent to the brain. The mo- end of the Vj induced junctional currents. The Cx37/Cx45 GJs showed extremely lecular identity of the cation channel involved in inner-ear mechanotransduc- low threshold (5 mV) for the Vj-gating when Cx45 cell with -Vj and reverse Vj- tion has been unclear for years, although four transmembrane proteins gating at the opposite Vj (at 10 mV or higher on Cx45 cell). The failure of docking (PCDH15, TMIE, TMHS, and TMC1) have been shown to be part of the me- between Cx37 and Cx40 could be rescued by designed Cx40 variants. Reveal the chantransduction complex. Recent studies suggest that TMC1 is the pore- heterotypic Cx37/Cx43 and Cx37/Cx45 GJ properties may help us in understand- forming subunit of the mechanotransduction complex and that it assembles ing the intercellular communication at the myoendothelial junction, a key site for as a dimer (Pan et al, Neuron 2018), yet the structure of TMC1 and the mech- propagated vasomotor responses. Supported by CIHR. anisms of ion conduction and activation remain to be elucidated. Bio- 1197-Pos informatic analyses suggest that TMC1 is related to the dimeric TMEM16 Charged Residues at the Pore Mouth Affect Single-File Water Flow family of membrane proteins that includes anion channels and lipid scram- Andreas Horner, Christof Hannesschl€ager, Florian Zocher, Pohl Peter. blases for which high-resolution structures have been recently reported. Institute of Biophysics, Johannes Kepler University, Linz, Austria. Here we present molecular dynamics simulations of TMC1 homology models þ Single-file water flow through narrow channels is determined by the number of based on the mouse Ca2 -activated chloride channel TMEM16A and the þ hydrogen bonds a water molecule may form with channel-lining residues(1). fungal Ca2 -activated lipid scramblase TMEM16. Unlike most ion channels, Entering such narrow channels, water molecules lose two of their four bulk these templates and consequently, the TMC1 homology models, do not show neighbours. The unitary water permeabilities (p ) of three different aquaporins a central ion conduction pore. Instead, the ion conduction pore and lipid f (AQP1, APQZ and AQP4) suggest that this process is sensitive to the presence scrambling region lie in the side facing the lipids, which are hydrated and sup- of positively charged residues at the pore entrance and exits(2). We saw a port cation permeation in simulations. Results from further simulations carried similar effect on p of reconstituted gramicidin channels in membranes with out in the absence and presence of applied membrane potentials and tension f different surface charges. A positive surface potential increased p several will be discussed. f fold whereas neutral membranes or a negative membrane surface potential did not affect pf. Osmotic water permeabilities of nanopores are calculated 1195-Pos from the concentration of reporter molecules within the stagnant water layers Functional Annotation of Ion Channel Structures: Predicting Pore Solva- adjacent to planar lipid bilayers(3),(4). We conclude that due to their lower tion States Based on Local Radius and Hydrophobicity dehydration penalty positively charged residues at the channel mouth boost 1 1,2 1 2 Shanlin Rao , Gianni Klesse , Phillip J. Stansfeld , Stephen J. Tucker , single-file water flow. However the variation in p caused by charges at the 1 f Mark S.P. Sansom . pore entrance and exits is two orders of magnitude smaller than the p changes 1Department of Biochemistry, University of Oxford, Oxford, United f 2 that may be induced by hydrogen bond donating and receiving residues along Kingdom, Clarendon Laboratory, Department of Physics, University of the channel wall. Hence, the design of artificial channels should aim at (i) mini- Oxford, Oxford, United Kingdom. mizing the number of H-bond forming pore-lining residues and (ii) maximizing Ion channel proteins function as water-filled, ion-conducting pores that the number of positively charged residues at the channel mouth. constitute crucial components of biological membranes. Ionic flow through 1. Horner A, Zocher F, Preiner J, Ollinger N, Siligan C, et al. 2015. Science Ad- these proteins in their resting or inactivated state may be interrupted at one vances 1:e1400083 or more positions (‘gates’) along the channel; conformational changes upon 2. Horner A, Siligan C, Cornean A, Pohl P. 2018. Faraday Discuss. 209:55-65 receipt of an activation signal typically lead to widening of the channel pore 3. Erokhova L, Horner A, Kugler P, Pohl P. 2011. J. Biol. Chem. 286:39926-32 at its gate region(s). Driven by pharmacological interests in elucidating 4. Pohl P, Saparov SM. 2000. Biophysical Journal. 78:2426-34 mechanisms of different ion channel families, and with advances in cryo- electron microscopy, an increasing number of ion channel structures are 1198-Pos being determined, often capturing alternative conformations of the transbi- Partial Characterization of the Inactivation Process of the Human Eryth- layer pore. Annotation of the conductive state represented by a newly rocyte Mechano-Activated KD Channel a (HEMKCA): Effect of Mem- resolved ion channel structure (i.e. whether its conformation would allow brane Potential, Ca2D and RbD permeation of water and ions) forms a key aspect towards understanding Diana Isturiz1, Alejandro Mata1,2, Jesus G. Romero1,3. its functional relevance and requires consideration of physicochemical prop- 1School of Biology, Univ Central Venezuela, Caracas, Bolivarian Republic erties of the pore lining. A hydrophobic region may disfavour liquid-phase of Venezuela, 2Health Science Center, The University of Tennessee, water and undergo spontaneous capillary evaporation, thereby presenting an Memphis, TN, USA, 3Strich School of Medicine, Loyola University Chicago, energetic barrier to the flow of water and ions without steric occlusion of the Maywood, IL, USA. pore. The critical radii above which a channel pore may become stably With a lifespan of 120 days, Human Erythrocytes are non-nucleated cells, so these hydrated is modulated by the degree of hydrophobicity of the pore lining, cells cannot undergo a process of classic apoptosis. In our laboratory, we have pro- as initially demonstrated by simulation studies on model nanopores. posed a hypothesis to explain how the process of aging in these cells occurs. This Here we apply molecular dynamics simulation-based calculations (www. hypothesis involves two novel mechanisms: a Kþ/Ca2þ exchanger, which is acti- channotation.org) to 190 ion channel structures. Through water density vated in response to changes in membrane potential; and a mechano-activated Kþ and free energy estimations, we examine the influence of local radius and channel (HEMKCA), which presents a sigmoid dependence of Po with the pres- hydrophobicity on channel pore hydration. The results of our analysis pro- sure applied to the membrane, an average unitary conductance of approximately vide a heuristic model for predicting the solvation state of new channel 20pS and is modulated by the internal concentration of Ca2þ.Herewepresenta structures as they emerge. partial characterization of the process of inactivation of HEMKCA. This channel

BPJ 9362_9366 242a Monday, March 4, 2019 presents an exponential drop of the open probability in time with a t=2.4 min at 1201-Pos 60 mV. We tested the effect of various parameters on this process in order to Gap Junction Mediated Cellular Delivery of miRNA Modulates Pace- characterize this inactivation. The development of the inactivation presented a maker Activity U-shape complex dependence with the membrane potential at T=15 min, thus Virgis Valiunas, Chris Clausen, Ira S. Cohen, Peter R. Brink. the Po decreased by 85 to 95% at Vm=þ/60mV, but no statistically significant Physiology and Biophysics, Stony Brook University, Stony Brook, NY, USA. difference was found at Vm=þ/80mV with respect to T=1min; this result is dis- Increasing evidence indicates the importance of microRNAs in the pathogen- cussed in terms of the voltage dependence of the inactivation recovery. On the esis and development of heart diseases. other hand, Rbþ delays the inactivation by a factor of 3.3 with respect to Kþ, We hypothesized that pacemaker activity can be modulated by connexin from a tK=2.4min to a tRb=7.99 min, but it has no effect on the level of inactivation permeability to microRNAs that in turn regulate gene expression of specific reached at the end of the record. Changes in cytoplasmic Ca2þ concentration did membrane channels, i.e. microRNA can be delivered via gap junctions and alter not affect the kinetics parameters of the inactivation. We conclude that these re- the expression of specific genes in recipient cells. sults are consistent with a C-type inactivation. Our chosen target gene, SCN5A encodes the voltage-gated cardiac sodium chan- nel Nav1.5. The loss-of-function of Nav1.5 is associated with many cardiac dis- 1199-Pos eases, including atrial fibrillation. Recently, Zhang et al, 2018 reported that miR- A Skeletal Muscle Conditional KCNJ2 Knock-Out Mouse Model for Peri- 24 suppresses SCN5A expression and is associated with heart failure mortality. odic Paralysis in Andersen-Tawil Syndrome The HEK293 target cells endogenously expressing Cx43 were stably transfected 1 2 1 Nathaniel Elia , Ekaterina Mokhonova , Marbella Quinonez , with SCN5A. HEK293 source cells were transfected with miR-24 targeting Stephen Cannon1. 1 2 SCN5A. Whole-cell patch-clamp was used to measure the INav1.5 and junctional Physiology, UCLA, Los Angeles, CA, USA, Neurology, UCLA, Los conductance. To induce pacing in a passive HEK293/SCN5A cell, the dynamic Angeles, CA, USA. clamp computed IK1 and If, which were injected via patch pipette under current- Dominant negative mutations in KCNJ2, coding for the strong inward rectifier clamp conditions into: 1) a single cell, 2) one cell of a gap junction coupled pair. Kir2.1, cause Andersen Tawil syndrome (ATS). The ATS clinical phenotype We found that miR-24 resulted in a significant (7 times) down regulation of includes varying degrees of periodic paralysis, cardiac arrhythmias, and dys- INav1.5 in HEK293/SCN5A cells transfected with miR-24. Data also showed morphia due to a suppressed Kir2.1 current. The global KCNJ2 knockout that APs generated in miR24-transfected cells lacked the fast upstroke and mouse is neonatal lethal, most likely from an inability to feed because of a se- overshoot components. Further, the AP amplitude was reduced 3.5 times in vere cleft palate defect. We created a conditional KCNJ2 knock-out in skeletal miR-24 containing cells. muscle to develop a mouse model for the periodic paralysis in ATS. þ To demonstrate miR-24 gap junction mediated transfer, the source HEK293 Floxed mice (KCNJ2fl/ ) were provided by Prof. M.T. Nelson (Univ VT). The cells were transfected with miR-24 and co-cultured with target HEK293/ floxed mice were crossed with a Cre expresser, driven by the Myl1 promoter SCN5A cells. After 24-48 hours in co-culture, the INav1.5 was significantly (myosin, light polypeptide 1) and for which we confirmed high activity in skel- (7 times) diminished in the coupled target cells, yielding 3 times smaller etal muscle in crosses with the rosa reporter mouse. Disruption of KCNJ2 in APs without fast upstroke and overshoot. These data demonstrate that miR- skeletal muscle was confirmed at the mRNA level by qRT-PCR and at the 24 can pass from cell-to-cell via Cx43 gap junctions and subsequently protein level by Western blot for Kir2.1. Both the heterozygous and homozy- silence/downregulate the SCN5A gene. gous KCNJ2 knockout mice were viable, fed independently, produced Supported by NIH R01GM 088181 (VV). offspring, and survived into adulthood. Voltage-clamp recordings from disso- ciated fibers (flexor digitorum brevis) showed a 70% reduction of Kir current 1202-Pos in homozygous ko mice, but essentially normal current density for heterozy- Calcium-Dependent Rearrangements of the N-Terminal Domain in Cx26 gous ko mice. Based on visual inspection and handling the mice, motor activity Hemichannels appears to be normal. No spontaneous attacks of immobility or weakness were Juan M. Valdez Capuccino, Luyu Liu, Andrew L. Harris, observed. Jorge E. Contreras. We have created a conditional skeletal muscle KCNJ2 ko mouse that is viable Dept of Pharmacology, Physiology & Neuroscience, Rutgers University, and may serve as a model for the periodic paralysis that occurs in ATS. Studies Newark, NJ, USA. are in progress to characterize the ex vivo contractile properties of muscle from Connexin hemichannels are hexamers that are sorted to the plasma membrane KCNJ2 ko mice. preceding the formation of gap junction channels (GJCs) between adjacent cells. 1200-Pos Typically, these hemichannels are closed, which is achieved by physiological 2þ 2þ Characterization of Gating of the Voltage-Gated Proton Channel (HV1) extracellular Ca concentrations ([Ca ]ex) that significantly reduced open 2þ During Activation Using Non-Canonical Amino Acids channel probability. Structural and functional data indicate that the Ca sensing Esteban Suarez Delgado, Gisela E. Rangel-Yescas, Leon D. Islas. domain is located at the extracellular entrance of the hemichannel pore. When 2þ Dept Physiology, Facultad de Medicina, UNAM, Mexico City, Mexico. Ca ions are bound to the pore, they form an electrostatic ring that restricts, The voltage-gated proton channel (HV1) is a membrane protein which is pre- but does not prevent, access of ions and molecules. This suggests that the confor- 2þ sent in several organisms and participates in a wide range of physiological mational changes accompanying hemichannel closing by [Ca ]ex must involve functions, such as pH regulation and membrane potential control in the im- a gate in another region of the pore. Because the first segment of the N-terminal mune, airway and cardiac cells, snail neurons and spermatozoa, to mention a domain (NTD) is folded into the intracellular pore entrance, it has been proposed few. Its structure is conformed by four transmembrane segments (S1-S4), to act as a physical gate. In this study, we aim to design a methodology that al- which have identity with the Voltage Sensor Domains (VSD) of canonical lows us to report conformational changes at the NTD and evaluate whether 2þ voltage-gated ion channels. Unlike the latter, which have a pore domain, the changes in [Ca ]ex can trigger movements in this region. By introducing a VSD in HV1 is sufficient to sustain a proton selective conductance when a de- cysteine on the second residue of the NTD, we measured the kinetics of disulfide polarizing stimulus occurs. Also, voltage activation is modulated by the differ- bridge formation between adjacent subunits in response to oxidizing reagents. ence of pH between intracellular and extracellular bulk (DpH), so that when We found that disulfide bridge formation between NTDs blocked hemichannel DpH < 0, the channel open to more negative potentials. Several aspects of currents. The kinetics of disulfide bridge formation was greatly accelerated by 2þ 2þ the activation mechanism and its modulation remain poorly understood. increasing [Ca ]ex. Similarly, we found that Cd binding to NTDs harboring 2þ Of particular interest is the mechanism of activation by voltage and its DpH cysteine mutations occludes the hemichannel pore in a [Ca ]ex dependent modulation. We have adapted a method to evaluate the conformational changes manner. Our data strongly suggest that: 1) The NTDs of adjacent subunits 2þ of HV1 during its activation, using an orthogonal modified tRNA and come into close proximity when the channel is closed by Ca , and 2) that there 2þ aminoacyl-tRNA synthetase, a no-canonical fluorescent amino acid (ANAP) is an allosteric coupling between the Ca sensing domain at the extracellular with sensitivity to the solvent polarity properties. ANAP was replaced at side and the NT at the intracellular side of the pore. various sites in the amino acid sequence of HV1. HV1-Anap channels were ex- pressed in HEK 293 cells in order to measure fluorescence changes simulta- 1203-Pos neously with proton current in whole-cell patch-clamp experiment. ANAP Synthesis of Romk1/2 Protein in E.Coli has been incorporated along the N-terminal portion of S4 without majorly Milena Krajewska, Piotr Koprowski, Adam Szewczyk. modifying channel voltage activation and pH sensitive properties. Fluores- Nencki Institute of Experimental Biology, Warsaw, Poland. cence recordings of HV1-Anap channels show fluorescence intensity changes Kir1.1a, a product of the KCNJ1 gene, is a plasmalemmal inwardly rectifying related to channel activation that suggests a quantifiable movement of the S4 potassium channel inhibited by ATP. Kir1.1b is a mitochondrial isoform of segment. Kir1.1 lacking first 19 amino acids. Kir1.1b is believed to be a part of mito- Supported by grants IN203318 DGAPA-PAPIIT and 252644 CONACYT. K(ATP) channel. However, the data directly linking the protein to this channel

BPJ 9362_9366 Monday, March 4, 2019 243a activity is limited. It is known that Kir1.1 protein forms homotetramers. Each gle carbon nanotube channel by visualizing the ion and water flux under a Total subunit is characterized by cytoplasmic N- and C-termini and by a transmem- Internal Reflection Fluorescence Microscopy (TIRF). Using a droplet interface brane part of two helices flanking a P-loop segment. bilayer (DIB) technique, we place an aqueous droplet ( 200 nanoliters) on an The small amount of Kir1.1 protein in mitochondria of H9c2 cells prompted us oil-agarose hydrogel interface, which brings together the lipid monolayers on to express the Kir1.1 protein in E. coli and purify Kir1.1 channels for further the droplet and the interface to form a bilayer that mimics a cell membrane. studies. Codon-optimized ORF encoding for Kir1.1 was fused to sequences We then incorporate the carbon nanotube channels in this bilayer. The ion of several expression tags, which could assist membrane insertion and folding and mass fluxes in each channel were characterized through the optical image of eukaryotic proteins in bacteria (SUMO, pOmpF and MISTIC). In addition, of ion-sensitive fluorescence, which made it available to trace the flux through a fusion proteins contained affinity tag for purification (6-His tag). The Kir1.1- large number of nanoscale channels individually instead of averaging them. 6xHis construct was chosen for further studies due to its low degradation and The evanescent wave from TIRF also made it possible by eliminating the noise the highest membrane expression level. Several detergents were screened in the background and confining the illuminated region. We envision that our and n-dodecyl b-D-maltoside was used for protein purification (based on solu- method will allow probing of fundamental transport properties through biomi- bilization efficiency and tetramer stability). metic carbon nanotube porins. This work was supported by Polish National Science Center, grant no. 2015/19/ B/NZ1/02794. 1207-Pos Regulation of Pannexin-1 Channel Gating by Nitric Oxide and Camp 1204-Pos Signaling A Method to Quantify Transport Numbers of Charged Molecules across Pablo S. Gaete1, Mauricio A. Lillo1, Nelson P. Barrera2, Xavier F. Figueroa2, Biological Channels Jorge E. Contreras1. Jayesh Arun Bafna, Mathias Winterhalter. 1Pharmacology, Physiology & Neuroscience, Rutgers, The State University Dept Biophysics, Jacobs University, Bremen, Germany. of New Jersey, Newark, NJ, USA, 2Department of Physiology, Pontificia Biological channels facilitate exchange of small molecules but surprisingly Universidad Cato´lica de Chile, Santiago, Chile. there is a lack of general tool for the quantification of transport. Here we Pannexin-1 channels are permeable to ions and small molecules, such as ATP. demonstrated a possible approach where we present a permeation assay based The mechanisms that control pannexin-1 channel gating are largely unknown. on conventional electrophysiology. The method mainly involves application of We recently showed that calcitonin gene-related peptide (CGRP) receptor acti- concentration gradients of charged molecules with different electrophoretic vation in endothelial cells leads to Pannexin-1 channel opening. Activation of mobilities across a membrane channel. Thus an unbalanced flux creates an elec- CGRP receptor induces NO production and turn on the cAMP pathway. There- trostatic potential which provides direct information on relative ion fluxes. The fore, we tested whether pannexin-1 channel is gated by different concentrations experimental approach applied here involves measuring zero-current- of NO and cAMP. We used primary cultures of endothelial cells obtained from potentials. Using a numerical solution of the Goldman-Hodkin-Katz allows mesenteric arterial bed of mice and Xenopus laevis oocytes injected with hu- from a fitting the experimental I/V curve to get approximate transport numbers. man pannexin-1 cRNA. Pannexin-1 expression was assessed by immunofluo- Using this approach, we quantified for a number of molecules (salt, antibiotics) rescence and western blot. Activation of pannexin-1 channels was evaluated the flux across a dozen of porins involved in antibiotic resistance mechanism. by ethidium uptake in endothelial cells and by the two-electrode voltage clamp 1205-Pos technique in Xenopus oocytes. In endothelial cells, pannexin-1 was found to be Haematological Characterisation of Mice with Piezo1 Gain-Of-Function expressed at the plasma membrane. SNAP, a NO donor, increased ethidium up- take only at low concentrations (< 1 mM). SNAP-induced ethidium uptake was Mutation 10 Elizabeth L. Evans, Jian Shi, Melanie Bettale, Laeticia Lichtenstein, fully prevented by the pannexin-1 mimetic peptide, panx. 8-Br-cAMP, a cell David J. Beech. permeable analog of cAMP, increased ethidium uptake, which was prevented University of Leeds, Leeds, United Kingdom. by the pannexin-1 blockers carbenoxolone (CBX) or probenecid, and by the m Piezo1 proteins assemble as trimers to form mechanically-activated non-selec- PKA inhibitor, KT-5720. In oocytes expressing pannexin-1, 0.1 M SNAP tive cationic channels. In physiology, their role as mechanosensors is critical to increased CBX-sensitive currents mediated by pannexin-1 activation. In m vascular development and red blood cell volume regulation. Mutations in contrast, SNAP concentrations higher than 1 M reduced CBX-sensitive cur- Piezo1 are associated with two diseases which share overlapping features, rents. 8-Br-cAMP also increased the magnitude of CBX-sensitive currents despite opposing effects on the protein. Loss of function mutations are linked and accelerated activation kinetics. Our results suggest that NO and cAMP to lymphatic dysplasia; whilst gain of function mutations are connected to de- pathways regulate activity of pannexin-1 channels. hydrated stomatocytosis (DHS), a form of anaemia. Gain-of-function mutations occurring in the extracellular cap domain of the protein have been linked to 1208-Pos DHS. To test whether such mutations are sufficient to cause DHS, we generated The Origin of the Voltage Clamp Fluorometry Signal in Ci-Hv1 Proton the equivalent of a human DHS mutation in mice using CRISPR/Cas-9 technol- Channel ogy, mirroring the genotype observed in DHS patients. Mice homozygous for Zoltan Petho, Adrienn Bagosi, Zoltan Varga, Gyorgy Panyi, Ferenc Papp. the mutation were viable with normal Mendelian inheritance and body weight. Department of Biophysics & Cell Biology, University of Debrecen, Haematological analysis revealed that the mice had characteristics of DHS, Debrecen, Hungary. with an anaemia phenotype and the presence of stomatocytes in blood smears. Site-specific voltage clamp fluorometry (VCF) is a powerful tool to investigate The data suggest that point mutation in the Piezo1 cap is sufficient to cause conformational changes as a response to voltage change in voltage-sensitive anaemia and that the mutation affects murine Piezo1 similarly to human proteins. Although a large amount of functional data has been acquired by Piezo1. Further studies will investigate if the mutation has wider implications VCF from Ci-Hv1 in the last decade, the origin of the specific fluorescent signal for other non-haematological systems because of the suggested broad impor- has not been elucidated. Our aim was to assess how the specific fluorescent tance of Piezo1 in mammalian physiology. Support by the Wellcome Trust signal is generated in VCF and to understand more accurately the correspond- and a British Heart Foundation PhD Studentship to EE. ing conformational changes. We performed our measurements on the Ci-Hv1 E241C proton channel expressed in Xenopus laevis oocytes. Tamra-MTS fluo- 1206-Pos rophore was conjugated to mutated cysteine at positions E241. We analyzed the Optical Sensing of Ion Flux through Biomimetic Carbon Nanotube structure of the channel and recognized two potential quencher amino acids, Channels which we mutated to either alanine or tryptophan, to decrease or increase fluo- Pengyu Zheng1, Aleksandr Noy2, Meni Wanunu1, Yun-Ciao Yao3. rescence quenching properties. Upon mutating one of the two quencher amino 1Department of Physics, Northeastern University, Boston, MA, USA, acids to alanine, we observed a marked decrease in the specific fluorescent 2Lawrence Livermore National Laboratory, Livermore, CA, USA, signal, and mutating both quencher residues to alanine, our fluorescent signal 3Chemistry and Chemical BIology, University of California, Merced, was completely abolished. Tryptophan mutations also supplied VCF signals Merced, CA, USA. but with different shapes and different magnitudes compare to the original Increasing demands for cheaper and more efficient water treatment methods VCF signal based on the natural quenching residues. Our results prove that have never been higher, as shortages of fresh water have become a growing quenching amino acids in the close vicinity of the fluorophore play the most pain in many regions worldwide. In theoretical research and simulations, car- important role in the VCF signal generation. These findings help to deduce bon nanotubes have shown a potential in water treatment, desalination and en- crucial structural and functional information from VCF measurements, and ergy conversion with high ion selectivity and enhanced water transport. In this might suggest that a complex VCF signal does not necessarily represent a com- research, we present an approach to investigate fluid transport across each sin- plex conformational change.

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Posters: Ion Channels, Pharmacology, and excitability. Mutations in KCNQ2 are associated with a wide spectrum of epi- lepsy severity including benign familial neonatal epilepsy and neonatal-onset Disease epileptic encephalopathy. The ever increasing number of ion channel variants presents a formidable challenge for assessing their functional and pharmaco- 1209-Pos logical consequences. We have overcome this challenge by combining high ef- Uncoupling NMDA Receptor Mechanism of Ketamine Block and Proton ficiency electroporation with automated planar patch clamp recording. To Inhibition demonstrate, we report the functional assessment of 10 KCNQ2 variants iden- Jamie A. Abbott. tified in children with epileptic encephalopathy. KCNQ2 and KCNQ3 were co- Anesthesiology, University at Buffalo, Buffalo, NY, USA. expressed in CHO-K1 cells with vectors encoding either EGFP (KCNQ2) or Ketamine, a widely used anesthetic, is currently under renewed inquiry for its mScarlet (KCNQ3) enabling quantification of transfection efficiency by flow rapid and sustained antidepressant effects at sub anesthetic doses. Ketamine ex- cytometry. Approximately 60% of cells were typically co-transfected with erts its effects on the central nervous system (CNS) by blocking open N- both subunits. Whole-cell recordings were performed before and after applica- methyl-D-aspartate (NMDA) receptors with IC50 = 1.0 uM. This overall effect tion of XE-991 (M-current blocker, 20 mM), enabling offline subtraction of is modulated regionally and temporally by dynamic variations in extracellular non-specific currents. The variants displayed a range of functional defects (rela- pH at synapses. An increase in extracellular proton concentrations decrease tive to WT) including severe loss-of-function (T274M, I278T, R291G); mild NMDA receptor open probability (Po) with IC50 = 7.0 in an isoform dependent current reduction (P335L; R529W, R550Q); increased current density manner, and protonated forms of ketamine are more potent blockers of NMDA (P198Q, R207W); hyperpolarizing shifts in voltage dependence of activation receptors. In an effort to separate the effects of proton inhibition and ketamine V½ (R198Q, P335L), and slower activation and deactivation kinetics block on channel activity, we used GluN1-1b containing NMDA receptors with (R207W). Variants Q204H and R573C exhibited WT-like current density reduced proton sensitivity (IC50 = 6.7) expressed in HEK293 cells and quanti- (77-109%) and voltage dependence of activation (DV½<2 mV). These data fied the receptors’ activation mechanism with kinetic modelling from on-cell were collected using just four 384-well planar patch ‘chips’ recorded on 2 single channel recordings. With this approach, we estimated rate constants days. Our results demonstrate that automated electrophysiology can be applied for channel gating and for ketamine association and dissociation at pH 6.8, successfully for the rapid functional assessment of KCNQ2 variants associated 7.2, and 7.6. The activation mechanism of protonated GluN1-1a and GluN1- with epilepsy. Functional information may improve treatment selection and 1b isoforms were indistinguishable. As expected, ketamine reduced the channel prognosis. mean open time with no changes in unitary conductance. Furthermore, keta- mine increased the mean closed time and reduced the number of open compo- 1212-Pos nents. These results quantify the distinct effects of protons and ketamine on Tubular Renal Epithelial Cells are Active Mechanobiological Water NMDA receptor activity, and serve to better understand the complex modula- Pumps tory actions of pharmacologic interventions. Mohammad Ikbal Choudhury1, Yizeng Li1, Panagiotis Mistriotis2, Eryn Dixon3, Debonil Maity2, Rebecca Walker4, Morgan Benson2, 1210-Pos Leigha Martin2, Fatima Koroma2, Feng Qian4, A Mutant Sk Channel Rescued locomotion Defects in C. elegans ALS Konstantinos Konstantopoulos2, Owen Woodward3, Sean Sun1. Model 1Mechanical Engineering, Johns Hopkins University, Baltimore, MD, USA, Young Woo Nam1, Saba Baskoylu2, Hannah Vu3, Rachel Lee3, 2Chemical and Biomolecular, Johns Hopkins University, Baltimore, MD, Pammie Wong3, Anne Hart2, Miao Zhang3. USA, 3Physiology, University of Maryland, Baltimore, MD, USA, 1Sch Pharmacy, Chapman Univ, Irvine, CA, USA, 2Brown University, 4Medicine, University of Maryland, Baltimore, MD, USA. Providence, RI, USA, 3Chapman Univ, Irvine, CA, USA. Autosomal dominant polycystic kidney disease (ADPKD) is a common hered- þ þ Small-conductance Ca2 -activated K (SK) channels mediate medium afterhy- itary disorder caused by mutations in the genes PKD1 and PKD2. A major perpolarization in the neurons which limits the firing frequency of action poten- characteristic of the disease is development and progressive growth of tials and, thus, play a key role in the regulation of neuronal excitability. Given numerous fluid-filled cysts in the renal tubules. Previous studies have shown their importance in neurons, SK channels are potential drug targets for move- that cysts are formed due to rapid cell proliferation and are sustained by hy- ment disorders, including ataxia and Amyotrophic Lateral Sclerosis (ALS). The drostatic pressure generated by the epithelial cells by pumping ions and water þ SK channels are activated exclusively by the Ca2 -bound calmodulin. Here, we into the lumen. However, no work has been done to quantify the fluid pump- report that substitution of a valine to phenylalanine (V407F) in the SK2 channel ing performance of normal and diseased kidney cells. Therefore, we have þ caused a 6 fold increase in the Ca2 sensitivity. Subsequent tests with equiv- measured the fluid flux and hydrostatic pressure difference across a renal alent valine to phenylalanine substitutions in SK1 and SK3 channels also ex- epithelium by using a novel Kidney-on-a-chip microfluidic device with a þ hibited Ca2 hypersensitivity. Additionally, an equivalent phenylalanine volumetric resolution 0.31 mL. The fluid flux across Madin Darby Canine substitution in the Caenorhabditis elegans (C. elegans) SK2 ortholog kcnl-2 Kidney II (MDCK-II) epithelium decreases with an increase in pressure dif- partially rescued locomotion defects in an existing C. elegans ALS model, in ference with a maximum flux of 0.3 mL/min, which is consistent with the re- which human SOD1G85R is expressed at high levels in neurons. This supports sults of our theoretical model for active water pumping. In case of normal the idea that the phenylalanine substitution impacts SK channel function cells derived from human kidneys, the direction of fluid flux was apical-to- in vivo. This work for the first time provides a criticalreagent for future studies: basal. However, the flow direction changed from basal-to-apical in case of þ an SK channel that is hypersensitive toCa2 concentrations with increased ac- cystic cells derived human ADPKD kidneys. The fluid flux in cystic cells in- tivity in vivo. creases with osmotic and drug perturbation from 0.5 mL/min (Control) to 1.5 mL/min (50% osmotic gradient) and 2.5 mL/min (arginine vasopressin (AVP)). 1211-Pos Interestingly, in cystic cells, the stall pressure (DP) doesn’t change with Functional Consequences of Epilepsy-Associated KCNQ2 Variants Deter- perturbation and is measured to be around 200 Pa, indicating a different mech- minend by Automated Electrophysiology anism of active and passive ion transport. As per our knowledge, for the first Carlos G. Vanoye1, Reshma R. Desai1, Shannon L. Gallagher1, time we have shown experimentally, the dynamic nature of fluid flux and stall Dina Sinkim2, Linda C. Laux3, John J. Millichap4, Evangelos Kiskinis5, pressure difference in normal and diseased cells. Alfred L. George1. 1Department of Pharmacology, Northwestern Univ Feinberg Sch Med, 1213-Pos Chicago, IL, USA, 2Departments of Neurology & Pharmacology, A Novel Gain of Function Mutation of Piezo-1 is Investigated in Red Blood Northwestern Univ Feinberg Sch Med, Chicago, IL, USA, 3Department of Cells by High-Throughput Patch Clamp Pediatrics, Northwestern Univ Feinberg Sch Med, Chicago, IL, USA, Andrea Bruggemann1, Giustina M. Rotordam1, Nadine Becker1, 4Departments of Neurology & Pediatrics, Northwestern Univ Feinberg Sch Niels Fertig1, Paola Bianchi2, Markus Rapedius1, Lars Kaestner3. Med, Chicago, IL, USA, 5Departments of Neurology and Physiology, 1Nanion Technologies, Munich, Germany, 2UOS Fisiopatologia delle Northwestern Univ Feinberg Sch Med, Chicago, IL, USA. Anemie, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, The widespread use of genetic and genomic testing has led to an explosive UOC Ematologia, Milan, Italy, 3Theoretical Medicine and Biosciences, growth in the number of ion channel variants associated with human diseases Homburg/Saar and Experimental Physics, Saarland University, Saarbrucken,€ and in reference populations. In epilepsy alone, >2000 variants in ion channel Germany. genes are disease-associated, including >300 KCNQ2 variants. KCNQ2 and During their passage from the bone marrow to peripheral organs, Red Blood KCNQ3 co-assemble to form neuronal M current, which regulates neuronal Cells (RBCs) experience substantial mechanical forces that trigger RBC

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þ volume changes via Ca2þ influx through mechanosensitive channels like the importance of [Na ]i in atrial function, we know little of its regulation in Piezo-1 (1-3). However, direct electrophysiological proof of Piezo-1 activity atrial myocytes. þ in RBCs has been lacking so far as most of the studies come from animal Here we characterize the dynamic regulation of [Na ]i in atrial myocytes models, were recorded under pathophysiological conditions or via Ca2þ imag- from C57BL\6J mice. Whole cell epifluorescence with the Naþ-indicator þ ing (1, 2, 4). In addition, the RBCs’ small and variable size and their large het- SBFI (10 mM) was used to measure [Na ]i, while voltage clamp was used þ þ erogeneity in ion channel expression pose a significant challenge for successful to determine the Na - dependence of the NKA pump current (INKA). [Na ]i patch clamp recordings (5, 7). Here, we used automated patch clamp technol- rises from 9.76 5 0.34 mM at a stimulation rate of 1 Hz to 12.24 5 0.71 ogy to study and characterize different ion channels present in RBCs, aiming mM at 3 Hz. Anemonia sulcata toxin (3nM), which delays the inactivation þ specifically for Piezo-1 activity. We approached the problem of RBC heteroge- of voltage-gated Na channels, thereby increasing the amount of ‘late’ INa, þ neity by upscaling to high throughput automated patch clamp recordings of 384 significantly increased [Na ]i at 1 Hz (15.03 5 1.01 mM) and 3 Hz stimula- þ cells in parallel to separate the cells in Yoda-1 responders and non-responders tion rate (21.29 5 0.69 mM, p<0.01). Na -dependence of INKA revealed an based on automated quality filtering. Thereby, we functionally characterized Imax of 1.16 pA/pF with a kNa of 16.12 mM. Treatment with isoproterenol (1 RBCs from patients carrying a gain of function mutation in Piezo-1 (8), demon- mM) did not alter Imax (1.14 pA/pF) but significantly decreased kNa (5.27 strating that high-throughput patch clamping can provide assays for drug dis- mM). Finally, we used ouabain dose-response curves of INKA, immunocyto- covery and personalized treatment of anaemic disorders such as hereditary chemistry, and Western Blot analysis to characterize NKA subtype (a1,a2) xerocytosis and the sickle cell disease. References: 1.) Danielczok et al., function and distribution. Using these experimental findings to constrain our 2017 2.) Faucherre et al., 2014 3.) Gallagher et al., 2013 4.) Cahalan et al., computational model of atrial EC coupling, we provide the first comprehen- þ 2015 5.) Bouyer et al., 2012 6.) Staines HM et al., 2007 7.) Minetti G et al., sive atrial-specific characterization of [Na ]i homeostasis. Together these 2013 8.) Rotordam MG et al., 2018 works should inform a novel understanding of the altered regulation of þ [Na ]i in atrial disease. 1214-Pos The Molecular Mechanisms of State Dependent hERG Blockade by 1216-Pos Dofetilide A Kinetic Mechanism Underlying hERG Facilitation by a Blocker Kevin R. DeMarco1, John R.D. Dawson1, Borislava Bekker2, Kazuharu Furutani1, Steffen Docken2, Igor V. Vorobyov1, Igor V. Vorobyov3,4, Vladimir Yarov-Yarovoy3, Sergei Yu Noskov5, Colleen E. Clancy1, Timothy J. Lewis2, Jon T. Sack1. Colleen E. Clancy3,4. 1Department of Physiology & Membrane Biology, University of California 1University of California, Davis, Davis, CA, USA, 2Chemistry, American Davis, Davis, CA, USA, 2Department of Mathematics, University of River College, Sacramento, CA, USA, 3Department of Physiology & California Davis, Davis, CA, USA. Membrane Biology, University of California, Davis, Davis, CA, USA, Drugs that block hERG potassium channels attenuate the cardiac IKr current 4Pharmacology, University of California, Davis, Davis, CA, USA, 5Dept responsible for the robust repolarization of the ventricular action potential. Biological Sciences, University of Calgary, Calgary, AB, Canada. Many hERG channel blockers have been shown to increase the risk of The voltage gated potassium channel, KV11.1, is encoded by the human acute ventricular arrhythmia and sudden cardiac death, while others are suc- Ether-a`-go-go-related gene (hERG). It gives rise to a major repolarization cessful in reducing the likelihood of arrhythmia. Recently, we found that current (IKr) in ventricular cardiomyocytes. The hERG channel dynami- several hERG blockers with good clinical safety profiles share a common cally transitions between distinct open, closed and inactivated states, and attribute: They all paradoxically increase hERG current at potentials near possesses unique gating properties, such as fast inactivation at depolarized the threshold for its activation. We refer to this phenomenon as ‘‘facilita- potentials. It is infamous for promiscuously interacting with a diverse set tion.’’ Although the correlation between hERG facilitation and the cardiac of drugs that can adversely interfere with IKr and lead to acquired long safety of drugs has been established, the molecular mechanism underlying QT syndrome (aLQTS) in humans; a surrogate indicator for potentially facilitation remains unknown. Here, we propose a mechanism through deadly arrhythmias. However, not all hERG blockers are proarrhythmic, which facilitation arises from drug effects on hERG channel gating kinetics. and the underlying mechanisms that relate hERG blockade to emergent We focus on the action of nifekalant, because it is a potent hERG channel arrhythmogenicity are not well known. Dofetilide was developed as an blocker and the facilitation induced by nifekalant has been well- antiarrhythmic agent for the maintenance of normal sinus rhythm for characterized at a cellular level. We found that 1) nifekalant accesses the re- people prone to atrial fibrillation, however it is also known to cause aLQTS ceptor site within the pore of the open or inactivated channels at depolarized and ventricular tachycardias. Multi-scale functional modeling in our potentials, 2) upon return to the resting potentials, channels close trapping laboratory has attributed high dofetilide pro-arrhythmia risk to its state- nifekalant inside, 3) trapped nifekalant biases the open-closed equilibrium dependent hERG blockade, i.e. differential binding to the channel open towards the open state, and 4) the kinetics of drug escape from the channel and inactivated conformational states. Here we used molecular dynamics are faster than channel closing rates at potentials where facilitation of (MD) simulations to elucidate the molecular determinants of this phenome- hERG current is observed, thereby drug unbinding reveals channels that non. We developed models of dofetilide in positively charged and neutral have been biased towards the open state. Simulations with a Markov model ionization states and validated them via partitioning simulations between of such nifekalant-hERG interaction successfully reproduce key characteris- bulk solvents and through hydrated lipid membrane, elucidating their ther- tics of hERG facilitation. In conclusion, hERG facilitation arises from the modynamic propensity for hydrophobic and aqueous access to hERG. coupling of the features of the channel gating, slow opening and closing, Multi-microsecond ‘‘flooding’’ MD simulations allowed us to identify en- with a drug action on the gating, similar to a classic ‘‘foot-in-the-door’’ try/egress pathways, and state-specific drug binding sites in hERG. Umbrella mechanism. sampling MD simulations were used to compute potential of mean force (PMF) and diffusion coefficient profiles, from which we estimated drug 1217-Pos binding affinities (KD), that were comparable to experimental IC50 values, Allosteric Modulation via Transmembrane Interfaces in a Pentameric as well as entry/egress rates. These data will be used to fine-tune functional Ligand-Gated Ion Channel models for predicting drug pro-arrhythmia risks and developing safer Rebecca J. Howard1, Yuxuan Zhuang2, Shinjiro Nakamura3, pharmaceuticals. Marie Lycksell1, Helen Kiik4,Urska Rovsnik1, Cathrine Bergh5, Stephanie A. Heusser1, Laura Orellana1, Erik Lindahl1,5. 1215-Pos 1Biochem. & Biophys., Stockholm University, Stockholm, Sweden, Dynamic Regulation of Sodium Homeostasis in Atrial Myocytes 2Chemistry, Uppsala University, Uppsala, Sweden, 3Biotechnology, KTH Libet Garber, Humberto C. Joca, George S.B. Williams, Royal Inst. of Tech., Stockholm, Sweden, 4Biophysics, SciLifeLab, Solna, Christopher W. Ward, W.J. Lederer, Maura Greiser. Sweden, 5Biophysics, KTH Royal Inst. of Tech., Stockholm, Sweden. Univ Maryland, Baltimore, MD, USA. Small-molecule modulators, including drinking alcohol, are known to bind in- þ Intracellular sodium concentration ([Na ]i ) is an important regulator of tersubunit transmembrane sites in pentameric ligand-gated ion channels. How- 2þ 2þ intracellular Ca ([Ca ]i) and provides insight into the activation of the ever, due in part to the controversial functional annotations of published sodium calcium exchanger (NCX), both Naþ channels and the Naþ,Kþ- structures, the molecular basis and mechanistic impact of these interactions 2þ þ ATPase (NKA). In atrial fibrillation, [Ca ]i and [Na ]i homeostasis are remain unclear. Using the bacterial model protein GLIC, we applied a combi- þ altered. We have previously shown that [Na ]i is lower in atrial compared nation of oocyte electrophysiology, structural biology, and molecular dynamics to ventricular myocytes and that rapid atrial pacing leads to an adaptive simulations to characterize alcohol interactions associated with particular func- þ reduction in atrial [Na ]i (Greiser et al, JCI, 2014; 124: 4759-720). Despite tional states. Mutations at the subunit interface stabilized solvent-mediated

BPJ 9367_9369 246a Monday, March 4, 2019 interfacial contacts specific to the apparent open state, a mechanism distinct 1220-Pos from mutations in the channel pore, leading to nonadditive effects on gating. All Optical Interrogation of Voltage Gated Sodium Channels using Next We also found evidence for both potentiation and inhibition via distinct allo- Generation Fast Voltage Sensitive Dyes in a System Suitable for High steric sites, depending on the functional state most favorably bound. Our results Throughput Screening provide structural and dynamic detail for an allosteric model of ion channel Stephen S. Smith, Andrew Blatz, Thomas Lila, James Limberis, gating and modulation, including a critical role for solvation at subunit Jay Trautman. interfaces. Photoswitch Biosciences, Menlo Park, CA, USA. Optical assays are the method of choice for large scale screening due to lower 1218-Pos running cost and ease of execution, however for ion channel targets, like the Repursposing the Kca3.1 Blocker Senicapoc as a Microglia-Targeted voltage-gated sodium channels, optical assays have suffered from not resolving Therapeutic for Alzheimer’s Disease the properties of primary interest like state-dependent block. By combining the Heike Wulff1, Jacopo Di Lucente2, Hai M. Nguyen1, Vikrant Singh1, use of an optogenetic actuator to control the membrane potential, fast voltage Lee-Way Jin2, Izumi Maezawa2. sensitive dyes, and a detector array capable of 10 kHz acquisition, we demon- 1Pharmacology, University of California, Davis, Davis, CA, USA, strate a system well suited to investigate the state-dependent properties of 2Pathology and Laboratory Medicine, University of California, Davis, voltage-gated sodium channels. Using cells engineered to express a channelr- Sacramento, CA, USA. hodopsin optical actuator plus either the Nav1.5 or Nav1.7 sodium channel, Microglia play a pivotal role in the initiation and progression of Alzheimer’s induced action potentials were monitored at high S/N under regimens involving disease (AD). We here tested the therapeutic hypothesis that the Ca2þ-acti- varying stimulation frequencies (0.5 - 8 Hz) or durations of inactivating mem- vated potassium channel KCa3.1 constitutes a potential target for treating Alz- brane voltage potentials. Appropriate isoform selectivity as well as state and heimer’s disease by reducing neuroinflammation. We previously reported that use-dependent pharmacology are shown for local anesthetic and voltage amyloid-b oligomer induced expression of KCa3.1 in cultured primary micro- sensor-binding test compounds. The combination of optical sensors and actua- glia. We here confirmed this result in hippocampal slice cultures. Using the tors offer a promising platform for truly high throughput interrogation of ion small molecule KCa3.1 blocker TRAM-34 we found that pro-inflammatory channel targets that are difficult to screen using preexisting optical and hippocampal long-term potentiation-impairing activities of amyloid-b instrumentation. oligomer required KCa3.1 activity in vitro, in hippocampal slices, or in vivo. The in vivo significance of KCa3.1 is further supported by markedly 1221-Pos enhanced microglial KCa3.1 transcript and protein levels in of both Mechanical Stretch Increases Kv1.5 Potassium Channel Activity through a Alzheimer’s model 5xFAD mice and AD patients. Immunohistochemistry Signaling Cascade Involving N-Terminus of the Channel demonstrated that KCa3.1 was particularly upregulated in microglia associ- Alexandria O. Milton. ated with amyloid plaques. The overexpression was associated with enhanced Queen’s University, Kingston, ON, Canada. activity, as whole-cell patch-clamp studies conducted on microglia acutely The voltage-gated potassium channel Kv1.5 mediates the ultra-rapid delayed isolated from 5xFAD mice showed enhanced KCa3.1 current densities. Since rectifier current (IKur) in the heart, important for human atrial repolarization. TRAM-34 is not suitable for clinical use, we resynthesized senicapoc, a Kv1.5 also plays a role in regulating smooth muscle membrane potential and KCa3.1 blocker that has previously been used in clinical trials for sickle vascular tone in pulmonary arteries. Cardiomyocytes undergo mechanical cell anemia. Senicapoc exhibited excellent brain penetrance and oral avail- load and pulmonary artery pressure varies under certain conditions. There- ability. Treating 5xFAD mice with a three-month oral regimen starting at 6 fore, we investigated the effects of mechanical stretch on Kv1.5 channels. months of age, reduced neuroinflammation, decreased cerebral amyloid Whole-cell voltage clamp and Western blot analysis were used to study load, and enhanced hippocampal neuronal plasticity, compared to vehicle- the current and expression of Kv1.5 channels stably expressed in human em- treated 5xFAD mice. These results provide proof-of-concept data to validate bryonic kidney (Kv1.5-HEK) cells. Low extracellular osmolarity was used to KCa3.1 as a potential therapeutic target for AD. Furthermore, our results induce mechanical stretch by mixing standard cell culture medium with pu- prompt us to propose repurposing of senicapoc for Alzheimer’s disease clin- rified water at a 2:1 ratio, thus decreasing osmolarity from 315 to 211 mOsm/ ical trials. L (defined as low osmolarity, LO). Treatment of Kv1.5-HEK cells with LO 5 Supported by NS100294 and AG043788 from NIH. for 30 min increased cell volume by 40%, and increased IKv1.5 by 45 8%, compared to control cells. Co-immunoprecipitation experiments showed that 1219-Pos Kv1.5 associates with the membrane protein integrin. Previous studies have shown that Focal Adhesion Kinase (FAK) is primarily recruited to sites of Production of Native-Like Refolded NaV1.7 Voltage Sensing Domain as Shown by Toxin Binding Activity integrin clustering, and integrin-activated FAK phosphorylation at Y397 in- Ryan V. Schroder1,2, Ping Wang1, Sebastien F. Poget1,2. duces high-affinity binding of non-receptor protein tyrosine kinase Src to 1Department of Chemistry, College of Staten Island, CUNY, Staten Island, FAK. It has also been shown that Src acts on a specific motif located in NY, USA, 2Program in Biochemistry, Graduate Center, CUNY, New York, the N-terminus of Kv1.5 to suppress channel activity. Our data demonstrated m m NY, USA. that both FAK inhibitor (14 M) and Src inhibitor (PP1, 10 M) abolished Voltage gated sodium channels (VGSCs) are essential to the propagation of the LO-mediated increase in IKv1.5. Furthermore, deletion of the Src- nerve cell impulses and thus communication in the nervous system. The Human targeting region by truncating the N-terminus of Kv1.5 up to residue 209 (DN209) also abolished the LO-mediated Kv1.5 current increase. Our study voltage-gated sodium channel NaV1.7 has been shown to play a vital role in the nerve pathways that induce pain response. Many of the currently available pain reveals a potential pathway linking mechanical stretch and Kv1.5 activity. therapeutic drugs are opiates, derivative molecules of opium. These drugs (Supported by The Natural Sciences and Engineering Research Council of target receptors in the brain that result in many other effects in addition to Canada). pain relief, such as euphoria, which gives such drugs a high risk for misuse. Drugs that target and inhibit NaV1.7 on the other hand should only affect the 1222-Pos pain signal and therefore lead to less addiction problems than opiate-based Modeling Trapping Block of HERG for CiPA: Does the Basal HERG medications. Although the atomic structures of several bacterial VGSCs have Mode Matter? been determined (and that of a bacterial-mammalian chimeric VGSC Brandon Franks1, Mark Nowak1, Brian Panama2, Randall Rasmusson2, 2 construct), the structure of native human NaV1.7 is currently unknown. In Glenna Bett . this study, we expressed, purified and reconstituted the voltage sensing domain 1Cytocybernetics, Buffalo, NY, USA, 2Dept Physiol/Biophys, State Univ (VSD) of repeat II of this channel with the aim of conducting ligand binding New York Buffalo, Buffalo, NY, USA. and structural studies via solution state NMR spectroscopy. We have success- HERG (human ether-a-go-go-related gene) encodes the major ion channel fully expressed the recombinant voltage sensing domain (VSD) of NaV1.7 us- (HERG, Kv11.1) responsible for cardiac repolarization, the delayed rectifier ing the Trp-Delta-Ldr inclusion body system. The fusion protein was cleaved current, IKr. Block or reduction in functional expression of IKr can impair with hydroxylamine using an added NG sequence between the fusion partners. cardiac repolarization, resulting in lengthening of the action potential (AP), The VSD was then refolded using the zwitterionic lipid, DMPC. Micro-scale which can have potentially fatal consequences. Drug block of IKr is a major thermophoresis was used to confirm binding of the recombinant VSD to problem in drug development: up to 70% of promising drug candidates block Protoxin-II, which is a known inhibitor of NaV1.7. We are currently optimizing IKr, and are eliminated from the drug pipeline. Determining drug-channel in- the membrane mimetic system for the recombinant VSD so that it will be suit- teractions for HERG is critically important. HERG binds to a wide variety of able for solution state NMR studies. drug types in a complex manner. Modeling this interaction cannot be done

BPJ 9367_9369 Monday, March 4, 2019 247a with a simple reduction in current flow. This eliminates Hodgkin-Huxley Computational simulations of a neuronal excitability model revealed that the type equations. Markov Models, which assign discrete states to different F302L gain-of-function effect is dominant: a classical Hodgkin-Huxley 2 channel conformations are much better at modelling HERG block by drugs. neuron (1mF/cm ) was complemented with KV1.2 conductance models Li et al (2017) used a 6 state Markov model of HERG and added 3 additional (36mS/cm2) fit to electrophysiological data from either WT or F302L chan- drug bound states. Transition rates into the new drug bound states are depen- nels. HH neurons with KV1.2-F302L were less excitable than those with dent on voltage and drug concentration, and have a fixed drug-trapping rate. KV1.2-WT, having a higher stimulus threshold for tonic firing (WT: 5mA; In developing this model, Li et al fit rate constants to the new states based on F302L: 14mA). Based on our experimental and computational results, it is CiPA HERG block data from the Milnes (2010) protocol to recapitulate the likely that the seizures arise due to inhibitory neuron suppression, increasing degree of trapping type block. We used a 5 state Markov model of HERG at overall circuit excitability. physiological temperature (Adeniran, 2011), added the drug bound states of To rationalize the effects of the conservative mutation F302L on the biophys- Li et al, and determined the response of the model to the Milnes protocol, ical properties of the KV1.2 macromolecule, we are currently investigating which is proposed by CiPA to predict the arrhythmogenic potential of a structural models of KV1.2-WT and KV1.2-F302L in the Resting-VSD / candidate compound. We compared the two models. Despite having very Closed-Pore and Active-VSD / Open-Pore conformations by molecular dy- different time courses during the AP, both base HERG models coupled to namics simulations. the trapping model of Li et al showed functionally similar behavior during the Milnes protocol. 1225-Pos Atomistic Computational Models to Predict Drug-Mediated Cardio- 1223-Pos toxicity Smooth Muscle Kv11.1 Channel Expression is Increased in Pulmonary Subha Kalyaanamoorthy1, Shawn Lamoth2, Xiaoqing (Ervin) Hou3,4, Hypertension Harely Kurata2, Michael Houghton3,4, Khaled Barakat1. Nataliia V. Shults, Vladyslava Rybka, Yuichiro J. Suzuki, 1Faculty of Pharmacy, University of Alberta, Edmonton, AB, Canada, Tinatin I. Brelidze. 2Department of Pharmacology, Faculty of Medicine & Dentistry, University Department of Pharmacology & Physiology, Georgetown University, of Alberta, Edmonton, AB, Canada, 3Department of Medical Microbiology & Washington DC, DC, USA. Immunology, Faculty of Medicine & Dentistry, University of Alberta, Kv11.1 potassium selective channels are essential for the heart repolariza- Edmonton, AB, Canada., 4Li Ka Shing Applied Virology Institute, University tion. Prescription medications that block Kv11.1 channels lengthen the ven- of Alberta, Edmonton, AB, Canada. tricular action potential and cause cardiac arrhythmias. Surprisingly, little is To be biologically active, small molecule drugs must physically fit into their known about the Kv11.1 channel expression in the lung tissues. Thus, we binding site(s) within their targets. However, in reaching their precise bind- characterized the Kv11.1 channel expression in lung tissues. We found ing locations, drugs may interact with a variety of cellular components of that Kv11.1 channels are expressed in smooth muscle cells (SMCs) of large various structures and functions. Recent seminal advances in computer soft- pulmonary arteries in wild-type rats and healthy humans. Interestingly, in ware and hardware render in silico methods increasingly important. They rats with pulmonary arterial hypertension (PAH) and humans with chronic can build detailed atomistic dynamical structural models to predict ligands’ obstructive pulmonary disease (COPD)-associated pulmonary hypertension, off-target interactions. In our lab, we focus on the potential blockade of car- Kv11.1 channel expression was observed not only in large pulmonary ar- diac ion channels by small molecule drugs, a critical event that can lead to teries, but also in small pulmonary arteries. The increase in the expression acquired cardiac long QT syndrome (LQTS) and fatal cardiac arrhythmias. of Kv11.1 channels closely followed the time course of the development We build computational models for the human Ether-a`-go-go-Related of pulmonary vascular remodeling. We further explored the possible role Gene (hERG) channel, the Nav1.5 sodium channel, the Cav1.2 calcium of Kv11.1 channels in pulmonary hypertension using a proliferation assay channel and the KCNQ1/3 and Kir2.1 potassium channels. The ultimate performed on cultured human pulmonary artery SMCs, and found that the goal is to investigate their interactions with drugs in silico and predict the application of Kv11.1 channel blocker dofetilide increased the SMC growth. mode of binding of these drugs and their potential blockage capacity. This Taken together, our findings indicate that Kv11.1 channels are expressed work will provide an overview on the different ion channels models estab- in the lung vasculature and may play a protective role in pulmonary lished in our lab and their success in predicting cardiotoxicity for various hypertension. drugs.

1224-Pos 1226-Pos A De Novo Mutation Associated with Epilepsy Enhances KV1.2 Voltage Voltage- and State-Dependent Blockade of hERG Potassium Channels by Dependence, Suppressing Neuronal Excitability Fentanyl Antonios Pantazis1,2, Maki Kaneko3, Annie M. Westerlund4, Jared Tschirhart, Wentao Li, Jun Guo, Shetuan Zhang. Lucie Delemotte4, Sulagna Saitta3,5, Riccardo Olcese6,7. Dept of Biomedical and Molecular Sciences, Queen’s University, Kingston, 1Department of Clinical and Experimental Medicine (IKE), Linko¨ping ON, Canada. University, Linko¨ping, Sweden, 2Wallenberg Center for Molecular Medicine, The human ether-a-go-go-related gene (hERG) encodes the pore-forming sub- 3 Linko¨ping University, Linko¨ping, Sweden, Center for Personalized unit of the potassium channel IKr, which is important for cardiac repolarization. Medicine, Children’s Hospital Los Angeles, Los Angeles, CA, USA, Drug- or medical condition-mediated disruption of hERG channel function is 4Applied Physics, KTH SciLifeLab, Stockholm, Sweden, 5Department of the primary cause of acquired long QT syndrome, which predisposes affected Pathology, Keck School of Medicine at USC, Los Angeles, CA, USA, individuals to ventricular arrhythmias and sudden death. Fentanyl abuse is 6Department of Anesthesiology & Perioperative Medicine, UCLA, Los associated with sudden death. We used whole-cell patch clamp method to Angeles, CA, USA, 7Department of Physiology, UCLA, Los Angeles, CA, investigate effects of fentanyl on hERG channels expressed in HEK cells. USA. Our results showed that fentanyl blocked hERG current (IhERG)ina An exome-based panel in an infant suffering from epilepsy revealed a concentration-dependent manner. Inactivation gating did not play a role in de novo c.906 T>G change in KCNA2, which encodes the pore-forming sub- fentanyl-mediated hERG blockade. Using a voltage protocol that maximizes unit of KV1.2 channels. The missense mutation results in substitution F302L, the open state of the channel, we found that fentanyl block only developed at the S4 helix of the KV1.2 voltage-sensing domain (VSD). Our previous when channels were open. Fentanyl was trapped by channel closing; upon electrophysiological investigations revealed that KV1.2-F302L channels acti- washout, the blocked current only recovered when channels were open. Fenta- vate up to 2-fold faster, and exhibit a more hyperpolarized voltage depen- nyl block of hERG was voltage-dependent. When hERG tail currents upon 50 dence (13 mV) compared to KV1.2-WT. Voltage clamp fluorometry mV repolarization were analyzed, fentanyl blocked IhERG with an IC50 of 0.92 revealed that augmented channel opening is accompanied by enhanced mM. Using inactivation-deficient hERG channels (by mutation or drug) we VSD activation. examined how fentanyl affected IhERG at different voltages. We found a linear Moreover, KV1.2-F302L channels exhibit accelerated slow inactivation by up relationship between voltage and ratio of block; fentanyl blocked hERG to a to 3-fold and slower inactivation recovery (2-fold at -80mV) than wild- greater extent as voltage increased. When a voltage protocol that mimics a car- type. Their inactivation voltage dependence is shifted to more hyperpolarized diac action potential was used, fentanyl blocked IhERG with an IC50 of 0.27 mM. potentials (DV0.5=-13mV). Fentanyl at a concentration of 0.5 mM prolonged APD90 and blocked IKr re- Investigations at more physiological temperature (33C) recapitulated the re- corded from isolated neonatal rat ventricular myocytes. The concentrations sults of room-temperature experiments: F302L enhances both channel activa- of fentanyl used in this study were higher than seen with clinical use but overlap tion (gain of function) and inactivation (loss of function). with post-mortem overdose concentrations. Fentanyl-mediated hERG block

BPJ 9367_9369 248a Monday, March 4, 2019 may contribute to fentanyl-induced death in high-concentration overdose situ- contraction to the regulation of cancer progression. KCNH channels contain ations, or for individuals with compromised cardiac repolarization. (Supported a Per-Arn-Sim (PAS) domain in their N-terminal and cyclic nucleotide- by Canadian Institutes of Health Research). binding homology (CNBH) domain in their C-terminal regions. These intracellular domains shape the function and contribute to the unique prop- 1227-Pos erties of KCNH channels. The structures of PAS and CNBH domains sug- Photodynamic Modification of Native HCN Channels in Thalamocortical gest that they harbor potential binding sites for small molecules. Here we Neurons describe a surface plasmon resonance (SPR)-based high-throughput Fusheng Wei, Qiang Wang, Ankush Gupta, Qinglian Liu, Lei Zhou. screening method aimed in identifying small molecule binders for PAS Physiology and Biophysics, Virginia Commonwealth University School of and CNBH domains of KCNH channels. The method involves purification Medicine, Richmond, VA, USA. of PAS and CNBH domains of KCNH channels, immobilization of the pu- Previously, we reported photodynamic modification (PDM) of heterologous rified domains on the SPR senor chip and screening small molecules in a expressed HCN channels on membrane patches. In the presence of specific chemical library for binding to the immobilized domains using changes in (FITC-cAMP) and non-specific (Rose Bengal) photosensitizers, light excita- the SPR-angle as a reporter of the binding. The advantages of this method tion transforms the function of HCN channels in an oxygen dependent include low quantity of purified PAS and CNBH domains necessary for manner. Here we extended the study to native HCN channels expressed in tha- the implementation of the screen, direct assessment of the binding between lamocortical (TC) neurons in the Ventrobasal (VB) complex in the thalamus. the PAS and CNBH domains and easiness of assessing KCNH subfamily We first discovered that blue light excitation significant increases the current specificity of the small molecule binders. To demonstrate the effectiveness amplitude and the rate of activation of the hyperpolarization-activated Ih cur- of the method we screened small molecules in the Spectrum Collection Li- rent, which was a reversible process and did not require exogenous photosen- brary and identified a small molecule binder that binds to the PAS domains sitizers. However, when FITC-cAMP, but not FITC alone or FITC and cAMP, in a subfamily specific manner. These results show that the SPR-based was loaded into the cell through whole-cell recording pipette, light excitation method is well suited for identifying small molecule binders of KCNH resulted in long-lasting increases in the voltage-insensitive, instantaneous Iinst channels. component and correspondingly decreases in the Ih component. The Ih and Iinst after PDM can be blocked by Csþ and ZD7288, confirming the specific 1230-Pos involvement of HCN channels. Next, we investigated the impacts of PDM of iPSC-Derived Motor Neurons on the Automated Patch Clamp Platforms native HCN channels on the resting membrane potential (RMP) and input Qube and QPatch resistance (Rin) of VB neurons. As expected, after PDM, there was a signif- Kadla R. Rosholm, Melanie Schupp. icant positive shift in RMP. Importantly, both the long-lasting increase in Iinst Sophion, Ballerup, Denmark. and the positive shift in RMP after light excitation, but not the short-term and Human induced pluripotent stem cells (hiPSCs) can be differentiated into reversible increase in Ih during light excitation, could be blocked by Trolox- multiple cell types, including neurons and cardiomyocytes. This gives rise C, a widely used quencher for singlet oxygen. In summary, we report the to a novel way of establishing human disease models, which in turn can PDM of native HCN channels under more physiological condition which be used for drug development in vitro. Ion channels represent highly attrac- should carry meanings for both basic researches and clinical treatments of dis- tive therapeutic targets in the nervous and the cardiovascular system, eases due to HCN channelopathy. rendering electrophysiological studies of hiPSCs important for their usage in drug discovery. However, such studies have traditionally been limited 1228-Pos by the labor-intensive and low-throughput nature of patch-clamp electro- A Novel High-Throughput Screening Assay for State-Dependent and physiology. Here we use our automated patch clamp systems Qube 384 Subunit-Dependent BK Channel Modulators and QPatch 48 in order to increase throughput and reduce time lines. Our Frank T. Horrigan, Lorie A. Gonzalez, Liang Sun, Michael Bloch, observations include channel expression versus time in culture, the pharma- Shengwei Zou. cological dissection of endogenous ion channels (e.g. Nav and Kv), identifi- Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, cation of ligand-gated receptors, and recordings of action potentials using TX, USA. the current clamp feature. Also, we show the electrophysiology of a spinal BK potassium channels are attractive drug targets for a wide variety of human muscular atrophy (SMA) and an amyotrophic lateral sclerosis (ALS) model. disorders affecting almost every organ system. However, the therapeutic po- The disease model for SMA was derived by mutations in the SMN1 gene tential of many BK channel modulators is limited by lack of tissue specificity, and shows enhanced sodium channel activity but no shift in the normalized reflecting that the pore forming a-subunit (Slo1) is encoded by a single gene. current voltage relationship. ALS was here mimicked by a single point mu- Properties of modulators that can potentially enhance tissue specificity include tation in the superoxide dismutase 1 protein (SOD1), D90A, which had pre- (a) sensitivity to regulatory subunits that are expressed in a tissue-dependent viously been identified in recessive, dominant and seemingly sporadic manner and (b) state-dependent action, conferring sensitivity to tissue-specific pedigrees. Cells carrying this point mutation displayed larger sodium cur- differences in membrane voltage and Ca2þ that activate BK channels. We rents, which eventually led to neurofilament aggregation, neurite degenera- have developed a novel 384-well fluorescent thallium-flux kinetic (FLIPR) tion and other phenotypes. We could confirm that the electrophysiological assay to identify modulators with these properties. The screen includes assays effect could be reversed by point mutation to D90D.Our measurements vali- on a panel of BK channel variants composed of hyperactive human Slo1 mu- date the feasibility of measuring hiPSC ion channel currents using the APC tants (F380Y or R275C) in the presence or absence of different regulatory platforms Qube and QPatch. Altogether, these results can facilitate evalu- subunits (b1, b2, b2aFIW, b4, g1), expressed in U2OS cells with Bacmam ating the use of hiPSC for early drug development and in extension personal virus. Hyperactive Slo1 mutants allow both inhibitors and activators to be medicine. reliably detected under resting cellular conditions. Comparison of R275C and F380Y identifies state-dependent modulators since the voltage-sensors 1231-Pos of R275C are constitutively activated while F380Y are not. A test screen of Insights into Selectivity Filter Gating of K2P Channels from Single- small molecules and arachnid venoms revealed a surprising degree of Channel Recordings variant-selectivity and identified novel modulators with activities that depend Linus J. Conrad1,2, Stephen J. Tucker1,2. on voltage-sensor activation and/or regulatory subunit expression. Thus, the 1Dept of Physics, University of Oxford, Oxford, United Kingdom, 2OXION screen detects modulators that would fail to be detected by conventional Ion Channel Initiative, Oxford, United Kingdom. screens while also providing information about mechanism of action that Polymodal K2P channels are regulated by stimuli such as voltage, membrane should facilitate SAR analysis and development of potent tissue-selective tension, pH and diverse signalling events. TREK subfamily K2P channels, modulators. like some other Kþ channels e.g. BK channels, have been shown to gate pri- marily at the selectivity filter (SF). 1229-Pos The TREK-2 single-channel conductance of 200-300 pS makes it accessible to Identifying Novel KCNH Channel Ligands with Surface Plasmon Reso- study SF gating at high resolution with exceptional signal to noise ratio. The nance Method TREK subfamily in particular is a target for drug discovery because of its Purushottam Tiwari1, Aykut Uren1, Tinatin I. Brelidze2. involvement in pain perception. Activators of these channels are therefore 1Oncology, Georgetown Univ, Washington, DC, USA, 2Dept Pharma/ promising analgesics, through their potential to dampen cellular excitability. Physiol, Georgetown University, Washington, DC, USA. Here, we show that applying the K2P channel activator BL1249, in addition to KCNH family of potassium channels is responsible for diverse physiological affecting open-probability, increases the apparent single-channel conductance functions ranging from the regulation of neuronal excitability and cardiac of TREK-2 and we have investigated the various mechanisms which may

BPJ 9367_9369 Monday, March 4, 2019 249a account for these changes in both conductance and gating. Such studies not tions. (Support: CF Fdn. MCCART17G0, NIH T32 GM008602, NIH F31 only expand our knowledge of the principal mechanisms underlying SF gating HL143863-01). of Kþ channels but may also help aid our understanding of how some Kþ channel agonists exert their effects on the selectivity filter. 1234-Pos Activation of Potassium Channel as a New Strategy to Boost Antitumour 1232-Pos Immune Response Initial Characterization of the Indole-3-Carboxamide Bic-154 as a Fast Seow Theng Ong1, Aik Seng Ng1, Xuan Rui Ng1, Lindsay Kua2, Onset and Reversible ORAI Channel Blocker Fiona Y.X. Lee2, Siqi Tan2, Heesung Shim3, Praseetha Prasannan1, Tetyana Zhelay1, Kalina Szteyn1, Elisa Liardo2, Jae Eun Cheong2, Ramanuj DasGupta2, Iain B.H. Tan4, Heike Wulff3, K George Chandy1, Steffi Koerner2, Anil Ekkati2, Lijun Sun2, J. Ashot Kozak1. Navin K. Verma1. 1Dept Neurosci/Cell Biol/Physiol, Wright State Univ, Dayton, OH, USA, 1Lee Kong Chian School of Medicine, Nanyang Technological University, 2Dept Surgery, Beth Israel Deaconess Medical Center, Harvard Medical Singapore, Singapore, 2Genome Institute of Singapore A*STAR, Singapore, School, Boston, MA, USA. Singapore, 3Department of Pharmacology, University of California Davis, Calcium ion elevations are required for human T-lymphocyte proliferation in Davis, CA, USA, 4National Cancer Centre, Singapore, Singapore. response to antigen recognition by a T-cell receptor. Calcium influx through There have been substantial advances in harnessing the immune system to fight the plasma membrane is necessary for efficient T-cell proliferation and cancers using immunotherapies and engineered T cells. However, tumor- þ effector function. The calcium channels responsible for Ca2 influx in lym- mediated immunosuppression represents a major obstacle to these approaches. þ phocytes have been identified and Orai interacting with ER Ca2 sensor Dying necrotic cells in the tumour microenvironment release substantial þ þ þ STIM were shown to be crucial for persistent calcium mobilization. Loss- amount of intracellular potassium (K ), raising extracellular K ([K ]e)to þ of-function mutations in Orai1 or STIM1 result in severe combined immuno- 25-60 mM. Tumor-infiltrating lymphocytes (TIL) bathed in this [K ]e-rich deficiency (SCID) with muscle hypotonia. Suppression of calcium influx fluid are suppressed by an ‘‘ionic-checkpoint’’ and fail to mount an efficient þ through Orai/STIM channels gives rise to various lymphoproliferative defects. antitumor response. Here, we demonstrate that T cells exposed to [K ]e-rich Thus, deletion of Orai or STIM in mice results in lymphoproliferative disor- media dose-dependently accumulate intracellular Kþ. Presence of high amount þ ders and hair loss. Gain-of-function mutations in Orai or STIM resulting in of [K ]e (50 mM) resulted in significant suppression of T cell functions, constitutively activated channels cause tubular aggregate myopathy (TAM). including proliferation, cytokine secretion, downstream signal transduction Despite the importance of Orai/STIM pathway in multiple signaling pathways (Akt and mTOR pathway) and tumor killing. To test if increased Kþ efflux and diseases, small molecule high affinity specific inhibitors of these channels through Kþ channels would protect TILs from the suppressive effects of are lacking. In this work we examined a class of indole-3-carboxamides for high [Kþ] , we patch-clamped TILs isolated from patients with metastatic colo- þ e their potential to inhibit store-operated Ca2 entry. They were tested in vitro rectal cancer that were expanded ex vivo. We found them to express significant 2þ m þ for their ability to inhibit Orai1 mediated Ca entry and currents. At 10 M numbers of calcium-activated KCa3.1 K channels. Activators of KCa3.1 signif- and higher the less lipophilic BIC-154 was the most potent blocker in the icantly enhanced KCa3.1 channel activity in both the cloned KCa3.1 and primary þ group. We used a constitutively active Orai1 variant implicated in TAM, T cells, rescued T cells from high [K ]e-induced suppression in cytokine and which does not require STIM1 for function. BIC-154 rapidly and reversibly tumor-killing. This study suggests that pharmacological activators of KCa3.1 inhibited Orai1 currents. BTP2 (YM-58483) and 2-APB also inhibited enable T cells to overcome ionic-checkpoint-mediated immune suppression þ þ Orai1 mutant channels in the same cells. TRPM7, a Ca2 /Mg2 permeable and mount effective antitumor activity. channel highly expressed in lymphocytes was insensitive to these compounds tested at 100 mM. Future investigations will test their effectiveness in T-cell 1235-Pos D blastogenesis and proliferation assays. We anticipate that Orai1-specific com- Novel Inhibitors of the Calcium-Activated K Channel KCa3.1 to Treat pounds will be useful for proof of concept studies of the contribution of this Non-Alcoholic Fatty Liver Disease and Liver Fibrosis channel to human disease. Seow Theng Ong1, Gemma Thomas2, Srinivasaraghavan Kannan3, Zhisheng Her4, Xuan Rui Ng1, Xinying Chew2, Hai M. Nguyen5, 1233-Pos Heike Wulff5, Chandra Verma3, Qingfeng Chen4, Mahmood Ahmed2, Human CFTR Channel Function is Regulated by Cholesterol K. George Chandy1. Guiying Cui1,2, Kirsten A. Cottrill1, Kerry A. McGill1,3, Barry Imhoff1,2, 1Lee Kong Chian School of Medicine, Nanyang Technological University, Nael A. McCarty1,2. Singapore, Singapore, 2School of Physical and Mathematical Sciences, 1 2 Department of Pediatrics, Emory University, Atlanta, GA, USA, Children’s Nanyang Technological University, Singapore, Singapore, 3Bioinformatics 3 Hospital of Atlanta, Atlanta, GA, USA, Georgia Inst of Technology, Atlanta, Institute A*STAR, Singapore, Singapore, 4Institute of Molecular and Cell GA, USA. Biology A*STAR, Singapore, Singapore, 5Department of Pharmacology, Cholesterol, a major membrane lipid component, has been known to modulate University of California Davis, Davis, CA, USA. the function of multiple ion channels by specific lipid-protein interactions, by Liver fibrosis resulting from non-alcoholic fatty liver disease (NAFLD) is a physical property changes of the membrane, or by modification of regulatory leading cause of liver-related morbidity and mortality. Thecalcium-activa- proteins associated with the channels in signaling complexes. Previous studies tedKCa3.1 potassium channel is considered a therapeutic target for this disease show that epithelial cells expressing the most common mutant version of the because of its role in myofibroblasts and T cells, which are key drivers of Cystic Fibrosis Transmembrane conductance Regulator (CFTR), F508del- fibrosis. Here, we describe the development of novel KCa3.1 inhibitors. Our CFTR, exhibit increased cholesterol content at the plasma membrane compared starting template nifedipine, a dihydropyridine, blocks KCa3.1 with an IC50 = to wild type control cells; however, neutrophils derived from CF patients show 1.8 mM. Nifedipine analogues were screened for blocking of KCa3.1 using auto- reduced cholesterol levels in the plasma membrane. But whether cholesterol mated planar electrophysiology. Structure-activity-relationship analysis led to directly modulates CFTR channel function remains unknown. To answer this the identification of novel analogues with IC50 in the low nanomolar range. question, we combine heterologous expression in oocytes with more physiolog- Mutagenesis studies showed that Nifedipine bound to the fenestration region ically relevant Ussing chamber recordings utilizing polarized Fischer Rat Thy- of KCa3.1, while the new analogues bound in the channel pore. Molecular roid (FRT) cells to determine the effects of changing plasma membrane modeling and simulations were used to characterize the binding of these com- cholesterol levels on CFTR channel function. Here, we report that cholesterol pounds. In parallel, we tested KCa3.1 inhibitor TRAM-34 in mice reconstituted depletion with methyl-b-cyclodextrin (MbCD) or cholesterol oxidase (CO) has with a functional human immune system and fed an obesity-inducing diet. no macroscopic effect on the magnitude of CFTR-mediated whole-oocyte cur- TRAM-34 prevented the progression of NALFD to liver fibrosis, reduced T rents. However, depletion of cholesterol increased the effect of VX-770, a cell infiltration of the liver and reduced circulating levels of IFN-g and IL- clinically-used potentiator of CFTR, when channels were activated at high 17. These studies indicate that KCa3.1 blockers may have use in the treatment PKA concentrations, but did not change the effect of VX-770 when channels of diet-induced liver fibrosis. were activated at low PKA concentrations. This change in efficacy of VX- 770-mediated potentiation likely reflects the apparent shift in the sensitivity 1236-Pos of WT-CFTR to PKA after depletion of membrane cholesterol. In FRT cells, Structural Modeling of Drug Interactions with hERG Channel in Open P67L-CFTR also exhibited changes in phosphorylation-dependent activation and Closed States after depletion of plasma membrane cholesterol. Sensitivity of WT-CFTR to Aiyana M. Emigh1,2, Kevin R. DeMarco1, Kazuharu Furutani1, Jon T. Sack1, block by GlyH-101 was significantly stronger after plasma membrane choles- Colleen E. Clancy1, Igor V. Vorobyov1, Vladimir Yarov-Yarovoy1. terol depletion. These results demonstrate that changes in the cholesterol level 1Physiology & Membrane Biology, Univ Calif Davis, Davis, CA, USA, of the plasma membrane significantly modulate multiple CFTR channel func- 2Biophysics Graduate Group, Univ Calif Davis, Davis, CA, USA.

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Human Ether-a-go-go-Related Gene (hERG) encodes a potassium-selective decreased. We confirmed that NMN increases cytosolic NADþ levels and voltage-gated ion channel essential for normal electrical activity in the heart. tested the roles of the cytosolic sirtuins (SIRT1 and SIRT2). Inhibiting their Genetic hERG mutations and blockage of the channel pore by drugs can activities with EX5720 and AGK2 abolished the ability of NMN to increase cause long QT syndrome (LQTS). LQTS predisposes individuals to KATP channel density. We performed biotinylation assays and found that arrhythmia and puts them at risk for stroke or sudden cardiac arrest. A major the increased channel density was due (at least in part) to elevated surface problem in development of antiarrhythmic drug therapies is the proclivity for expression. To determine if KATP channel subunits are K-acetylated, we these drugs to promote fatal arrhythmias through hERG blockade. However, immunoprecipitated K-acetylated proteins with anti-K-Ac antibodies and im- not all hERG blocking drugs are pro-arrhythmic, and their differential affin- munoblotted with KATP channel antibodies. Kir6.2 and SUR2A were both ities to discrete channel states and/or their state stability modulations have constitutively K-acetylated, and the K-acetylated levels were decreased by been suggested to contribute to arrhythmogenicity. In this study, we used Ro- NMN in a sirtuin-dependent manner. Kir6.2DC36, expressed in the absence setta density refinement and homology modeling to build complete structural of SUR2A, was also K-acetylated, but not functionally modulated by NMN, models of wild-type and mutant variants of hERG channel voltage sensing suggesting a key role for SUR2A. We are in the process of identifying specific and pore domains in open and closed states based on cryo-EM structures K-residues responsible for function. To determine the pathophysiological rele- of hERG (pdb id: 5VA2) and EAG1 (pdb id: 5K7L), respectively. Here vance, we preformed cellular ischemia assays using isolated rat ventricular we present results from molecular docking studies using RosettaLigand to myocytes. Cardiomyocyte contracture in an oil-overlay ischemia assay was study drug interactions with hERG in open and closed states. Our results mitigated by pharmacological preconditioning with phenylephrine or by will provide structural insights into the molecular and state-dependent drug NMN. The protective effect of NMN was reversed by inhibiting sirtuins or interactions with hERG that play a key role in differentiating safe and harm- by KATP channel block with glibenclamide. In summary, sirtuin-dependent ful hERG blockers. K-deacetylation positively regulates KATP channels and has a cardioprotective role that depends on KATP channels (Supported by TUBITAK- 1237-Pos SBAG117S386). Beneficial Effect of Mitochondrial Calcium Uniporter Overexpression in a Guinea Pig Heart Failure and Sudden Cardiac Death Model 1239-Pos Ting Liu1, Brian O’Rourke2. Probing Kv1.3 Interactome with Proximity-Dependent Biotinylation 1Dept Cardiology, Johns Hopkins Univ, Baltimore, MD, USA, 2Dept Med, Vanessa Checchetto1, Elena Prosdocimi1, Roberta Peruzzo1, Johns Hopkins Univ, Baltimore, MD, USA. Jesusa Capera Aragones2, Luigi Leanza1, Antonio Felipe2, Ildiko` Szabo`1. Introduction: In the heart, excessive mitochondrial Ca2þ (mCa2þ) can cause 1Department of Biology, University of Padua, Padua, Italy, 2Biochemistry permeability transition pore opening and cell death. On the other hand, insuf- and Molecular Biology, Univ de Barcelona, Barcelona, Spain. ficient mCa2þ can also compromise energy supply/demand matching and mito- Protein-protein interactions (PPIs) are central to the proper functioning of chondrial reactive oxygen species scavenging capacity, especially at high the most basic molecular mechanisms underlying cellular life and are often workloads. The effect of increasing mCa2þ uniporter (MCU) expression in perturbed in disease states. Traditional techniques contributed to the the context of heart failure (HF) has not been tested. improvement of knowledge in this field but they demonstrated significant Methods and Results: HF guinea pigs (ACi) were intramuscularly injected limitations. Recently, a new method called BioID (proximity dependent with adenoviral vectors expressing MCU with 3xFlag tag, leading to biotin identification) based on proximity-dependent labeling of proteins an increase of MCU-Flag levels by 30% above endogenous MCU has been developed (Ruox et al., 2012 doi.org/10.1083/jcb.201112098). expression. MCU overexpression restored mCa2þ handling in failing myo- The technique exploits a promiscuous biotinylating enzyme, BirA, that can cytes (1-F/F0 of MityCam at 1hz stimulation in the presence of isoproter- be fused to the protein of interest. In this work, we applied the BioID pro- enol (ISO): 0.2750.02 in Sham, 0.2150.01 in ACi, and 0.2950.02 tocol to study the PPIs of the voltage gated potassium channel Kv1.3, that in ACiþMCU). Sarcomere shortening showed that the contractility of displays subcellular localization to various organelles in addition to being failing myocytes was significantly improved by MCU overexpression during present in the plasmamembrane. Proteins identified by BioID are candidate high work load. MCU overexpression also potentiated cardiac output interactors for the channel and represent the starting point to improve our during b-adrenergic stimulation in perfused ACi hearts (the product of LV knowledge about the role this protein plays in human diseases and to developed pressure and HR was increased by 36522% in ACi vs possibly to identify new pathways in which Kv1.3 is involved at the cellular 152547% in ACi with MCU by ISO compared to baseline). To examined level. the effects of MCU overexpression in vivo, ACi heart was injected with vectors at 2w after aortic constriction, when cardiac decompensation 1240-Pos usually accelerates. Cardiac function worsened in the untreated ACi Inhibition of Connexion Hemichannels by New Aminoglycosides without in the following 2w (FS: 3951% at 2w and 3251% at 4w), whereas Antibiotic Activity MCU overexpression significantly improved cardiac function (3651% Abbey Kjellgren1, Mariana C. Fiori1, Madher N. AlFindee2, at 2w and 4252 at 4w). ECG telemetry recordings also showed a Yagya P. Subedi2, Srinivasan Krishnan1, Cheng-Wei T. Chang2, substantial decrease in the number of premature ventricular beats. Conclu- Guillermo A. Altenberg1. sions: The results indicate that MCU overexpression in ACi hearts 1Dept Cell Physiol/Molec Biophys, Texas Tech Univ Hlth Sci Ctr, Lubbock, enhances contractility and responses to b-adrenergic stimulation in isolated TX, USA, 2Dept of Chemistry and Biochemistry, Utah State University, myocytes and whole hearts while mitigating HF and inhibiting arrhythmias Logan, UT, USA. in vivo. Connexins hemichannels (HCs) from adjacent cells form gap junctional channels that mediate cell-to-cell communication. Abnormal opening of 1238-Pos ‘‘free’’ undocked HCs can produce cell damage and participate in the mech- Sirtuins Positively Regulate KATP Channels, Which Contributes to their anism of disorders such as cardiac infarct, stroke, deafness, skin diseases, Cardioprotective Role and cataracts. Therefore, inhibitors of connexin HCs have great pharmaco- Erkan Tuncay1,2, Hua-Qian Yang2, Ivan Gando2, Belma Turan1, logical potential. Antibiotic aminoglycosides (AGs) have been recently Ravichandran Ramasamy3, William A. Coetzee2. identified as connexin HC inhibitors, but their antibiotic effect is an issue 1Department of Biophysics, Ankara University, Faculty of Medicine, Ankara, for the treatment of disorders where infections do not play a role. Herein, Turkey, 2Dept Pediatrics, New York Univ Sch Med, New York, NY, USA, we synthesized and tested several amphiphilic AGs without antibiotic 3New York University, New York, NY, USA. effect for their inhibition against connexin HCs using a newly developed Sirtuins are NADþ-dependent enzymes that deacetylate proteins specific lysi- cell-based bacterial growth complementation assay. Several leads with su- ne(K) residues and are involved in aging, energy metabolism, inflammation, perior potency than the parent compound, kanamycin A, were identified. and stress resistance. We hypothesized that sirtuins positively regulate KATP Unlike traditional AGs, these amphiphilic AGs are not bactericidal and channels, thereby contributing to stress resistance. We increased cytosolic are not toxic to mammalian cells, making them better than traditional NADþ by preincubation with the NADþ precursor nicotinamide- AGs as HC inhibitors for clinical use and other applications.This work mononucleotide (NMN) (2 mM). After 24 hours, KATP channel currents was supported in part by This work was supported in part by NSF Award were recorded in excised patches from HEK293 cells expressing Kir6.2/ CHE-1429195 for a 500 MHz Bruker NMR, American Heart Association SUR2A, rat cardiomyocytes, or INS-1 cells. In all cell types, KATP current Texas Affiliate Inc. grant 14GRNT18750014, and a TTUHSC Preliminary density was increased by NMN and the inhibitory response of ATP was Data Grant.

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Posters: Cytoskeletal Assemblies & Dynamics Maryland, College Park, MD, USA, 3Institute for Physical Science and Technology, University of Maryland, College Park, MD, USA, 4Department 1241-Pos of Chemistry & Biochemistry, University of Maryland, College Park, MD, Structural Model for Preferential Microtubule Minus End Binding by USA. CAMSAP CKK Domains Semiflexible actin filaments crosslinked by myosins control cell motility and Joseph Atherton1, Yanzhang Luo2, Shengqi Xiang2, Chao Yang3, morphology via dynamic remodeling process - treadmilling. In less chemically Annapurna Vemu4, Marcel Stangier5, Alexander Cook1, Shana Wang1, dynamic reconstituted actomyosin networks, motor proteins induce global geo- Kai Jiang6, Michel Steinmetz5, Antonina Roll-Mecak4, Anna Akhmanova3, metric contraction, creating cluster-like structures. However, filaments tread- Marc Baldus2, Carolyn A. Moores1. mill much faster in living cells and are often found to be abundant at the cell 1Dept Biol Sci, Birkbeck Col London, London, United Kingdom, 2Bijvoet Ctr periphery, frequently forming ring-like structures or thin, contractile sub- Biomolec Res, Utrecht Univ, Utrecht, Netherlands, 3Dept Cell Bio, Utrecht plasma-membrane cortexes. To explore the interplay between treadmilling University, Utrecht, Netherlands, 4NIH, Bethesda, MD, USA, 5Paul Scherrer and myosin induced contractility, we used an advanced computational model Institute, Zurich, Switzerland, 6Wuhan University, Wuhan, China. MEDYAN, which couples stochastic reaction-diffusion scheme with polymer Microtubules are polar polymers, with minus and plus ends exhibiting differen- physics modeling. Our results indicate that the actomyosin network geometry tial dynamics and regulated by different cofactors. The evolutionarily is tightly regulated by filament treadmilling. In our simulation, non- conserved calmodulin-regulated spectrin-associated protein (CAMSAP) family treadmilling and slowly treadmilling actomyosin networks would geometri- are minus end binding proteins that modulate minus end microtubule dynamics. cally collapse and form clusters. On the other hand, enhanced filament tread- All CAMSAPs share a characteristic conserved CKK domain that defines milling generates ring-like structures that are dense and mechanically microtubule minus end specificity. Previously we found that CKK binds micro- contractile with their stability regulated by myosin concentration. We found tubules at an intra-dimer inter-protofilament site and suggested a mechanism of that rapid treadmilling towards boundary helps filaments escape from geomet- specificity based on tubulin polymer conformation. To further investigate this ric collapse, while myosin prevents filaments from growing against the bound- proposed mechanism, we sought to structurally compare a microtubule minus ary by altering their orientation such that those filaments are rescued from end-preferring CKK with a CKK from the protist N.gruberi (NgCKK) that destruction. shows no end preference. Near-atomic resolution structures of 13 and 14 pro- 1244-Pos tofilament human CAMSAP1-CKK (HsCKK) and NgCKK decorated microtu- Simulating Emergent Spatiotemporal Actomyosin Dynamics to Under- bules were determined using a new processing protocol and homogenous stand Spatial Regulation of Non-Muscle Myosin II human embryonic kidney cell tubulin. NgCKK was found to have a similar Callie J. Miller1, Paul LaFosse2, Sreeja Asokan3, Jason Haugh4, fold and microtubule binding site to HsCKK, yet it showed minor loop modi- James E. Bear3, Timothy C. Elston5. fications and subtly altered positioning relative to the microtubule lattice lead- 1Engineering, James Madison Univ, Harrisonburg, VA, USA, 2National ing to a modified interface. Furthermore, unlike HsCKK, NgCKK was found to Institutes of Mental Health, Bethesda, MD, USA, 3Cell Biology and neither compress the microtubule lattice nor impose right-handed supertwist on Physiology, University of North Carolina, Chapel Hill, NC, USA, 4Chemical microtubules. We suggest that these modified structural characteristics prevent and Biomolecular Engineering, North Carolina State University, Raleigh, NgCKK from sensing conformations of tubulin located uniquely at the micro- NC, USA, 5Pharmacology, University of North Carolina, Chapel Hill, NC, tubule minus end and, conversely, identify the features of HsCKK that confer USA. its minus-end binding preference. The punctuated dynamics of cortical actomyosin are critical for cell migration 1242-Pos in applications like cancer, wound healing, or , however the A Dynamic Time Step Method in Cytoskeletal Simulations mechanical role and organization of cortical actomyosin is not well under- Joseph Tibbs1, A. Pasha Tabatabai2, Daniel S. Seara2, Ali Tabei1, stood. We developed a Monte Carlo, particle-based computer simulation Michael P. Murrell3. that resulted in emergent actomyosin asters. In particular, we were interested 1Physics Department, Univ Northern Iowa, Cedar Falls, IA, USA, to understand the role of non-muscle myosin II’s (motor) activation in emer- 2Biomedical Engineering Department, Yale University, New Haven, CT, gent f-actin (filament) aster formation. Our model incorporates activation of USA, 3BBS Program, Yale University, New Have, CT, USA. individual motors through the transition from an inactive folded state to an Computer simulations, such as agent-based modeling, are useful both for active unfolded state, and the bundling of two active motors to form a proc- verifying experimental results and for conducting studies that are not easily essive motor capable of binding to filaments. In addition to considering how accessible experimentally. However, many systems which are biologically changing a single parameter affected the emergent filament asters, we intro- interesting can be extremely computationally expensive to run. One factor duced a spatial gradient of model parameters to mimic spatially controlled affecting the feasible length of a simulation is the time step, which must be activation of motors, or filament polymerization. Recent studies have shown small enough to maintain high resolution and system integrity, but large enough that regulation of motor activity is critical for directed fibroblast migration to shorten wall time. A dynamic time step method resolves this dilemma by in response to a gradient of platelet derived growth factor, and we found adapting the time step during the simulation in response to the state of the sys- that spatially inhibiting motor-filament binding resulted in spatial variations tem. AFiNeS (Active Filament Network Simulation) is an open-source cyto- in filament aster formation. Additionally, motivated from studies with the skeletal simulation package (Freedman, 2017). The purpose of this project small molecule ROCK inhibitor, Y-27632, and Calyculin A, which either was to improve the package by implementing a dynamic time step system, as disrupt or enhance the ability of non-muscle myosin II to exert force, well as a system for detecting unphysical interactions of the system’s compo- and work from the Sellers lab on the biochemical properties of different nents. This has significantly improved the speed with which simulations may be types of co-assembled myosin isoforms, we simulated spatially controlled run while maintaining good resolution, and has shown a large reduction in motor stiffness which directly affects the ability of motors to exert force to certain computational artifacts resulting from using too large a time step. Incor- reorganize filaments. We found that not only was there a change in where as- poration of this method into AFiNeS allows for simulations to be run on larger ters emerged, but that we were able to generate a dynamic pulsatile aster systems, more complicated systems, and systems with faster dynamics, without structure where filament asters would dissipate and new asters would emerge. experiencing unphysical events and while minimizing runtime. Examining Results from our simulation will guide future experimentation for cortical complex cytoskeletal behaviors, such as tearing, aster formation, and collective actomyosin. movement are all applications which would benefit from these improvements. Further, this method can be extended to other agent-based dynamics simula- 1245-Pos tions, within certain computational and logistic constraints. The result is The Physical Bases of Forming a Smooth Boundary between an Expanding simulations which are able to provide biological insights without intense Arp 2/3 Actin Network and a Contractile Actomyosin Network computational overhead. Medha Sharma, Tony Harris. Cell and Systems Biology, University of Toronto, Toronto, ON, Canada. 1243-Pos Smooth boundaries commonly form for cell and tissue morphogenesis. One Rapid Treadmilling and Myosin Motors Synergistically Induce Formation example is the cytokinetic ring which drives cell division, and another is the of Ring-Like Actomyosin Architectures and Cortexes leading edge purse string for wound healing. In both cases, actomyosin net- Qin Ni1, Arpita Upadhyaya2,3, Garegin A. Papoian3,4. works form the smooth boundaries. In addition to these examples, smooth 1Department of Chemical and Biomolecular Engineering, University of boundaries form between actomyosin networks and neighboring Arp2/3 actin Maryland, College Park, MD, USA, 2Department of Physics, University of networks during early Drosophila embryogenesis. Here, nuclei divide

BPJ 9367_9369 252a Monday, March 4, 2019 without cell division beneath the plasma membrane of a single large cell. To We found that irregular kinetochore movements occur for a broader range prevent these dividing nuclei from colliding, a transient nuclear compartment of parameters than regular oscillations. Thus, our work provides an forms by the interaction of an expanding Arp2/3 actin network and a sur- explanation for how regulation of MT dynamic instability contributes to rounding contractile actomyosin network. The interaction forms a smooth kinetochore congression and the accompanying movements around the spin- circular boundary at the cell cortex between the two materials. From this dle center. boundary, the cortex bends and ingresses for compartmentalization. To un- [1] K. Vukusic, R. BuCa, A. Bosilj, A. Milas, N. Pavin, and I.M. Tolic. Micro- derstand the interaction between these two materials and to test the suffi- tubule sliding within the bridging fiber pushes kinetochore fibers apart to segre- ciency and robustness of this interaction, we utilize mathematical gate chromosomes. Dev. Cell. 43: 11-23 (2017). modeling. The contractile actomyosin material is depicted by spring-node [2] A.H. Klemm, A. Bosilj, M. Gluncic, N. Pavin, and I.M. Tolic. Metaphase dynamics such that each myosin node connects randomly with other nodes kinetochore movements are regulated by kinesin-8 motors and microtubule dy- around them and pull each other over these spring-like connections. Our namic instability. Mol. Biol. Cell. 29: 1332-1345 (2018). model mimics reported in vitro behavior of reconstituted actomyosin net- works, including shape changes following the activation of rectangular or 1248-Pos circular networks. The Arp2/3 network is modelled using rules derived Force Regulation of Capping and Arp2/3 Nucleation of Branched Actin from known properties of these branched networks. In silico actin networks Networks also mimic reported in vitro behavior, such as increased density with phys- Tai-De Li1, Peter Bieling2, Dyche Mullins3, Daniel Fletcher4. ical restriction, and their unhindered growth mimics that of Arp2/3 actin net- 1ASRC of CUNY, New York, NY, USA, 2Max Planck Institute of Molecular works without actomyosin borders in the embryo. Starting with networks that Physiology, Dortmund, Germany, 3UC San Francisco, San Francisco, CA, mimic the initially rough-edged networks observed in the embryo, we are USA, 4UC Berkeley, Berkeley, CA, USA. testing how their properties and engagement lead to a smooth boundary be- The assembly of branched actin networks provides the driving force for tween them. numerous cell motilities of all eukaryotic cells. While pushing the cyto- plasm membrane moving forward, the networks are also polymerizing under 1246-Pos a counter force. At the leading edge of the cell motilities, such as Lamelli- Septin Hierarchical Assembly Revealed by High-Speed Atomic Force podium, the pushing force and network formation are dynamically regulated Microscopy(HS-AFM) by nucleation, elongation, and capping of individual growing filaments in Fang Jiao1,2, Kevin Cannon3, Amy Gladfelter3,4, Simon Scheuring1,2. the branched actin networks. Although it has been shown that the motor ac- 1Department of Anesthesiology, Weill Cornell Medicine, New York, NY, tivity and mechanical properties of growing networks adapt to the counter USA, 2Department of Physiology and Biophysics, Weill Cornell Medicine, forces, the force dependence of many other biochemical events and their New York, NY, USA, 3Department of Biology, University of North Carolina molecular mechanisms are still not clear. Here we show how the counter and Chapel Hill, Chapel Hill, NC, USA, 4Marine Biological Laboratory, force regulates capping and nucleation at molecular level. We found that Woods Hole, MA, USA. the capping of filament polymerizing ends (free barbed ends) is force- Septins are GTP-binding proteins that form palyndromic linear hetero- regulated in the same way as for filament elongation. We also discovered oligomeric complexes, which in turn are able to assemble into filaments that counter forces slow the rate of Arp2/3-mediated filament nucleation and higher-order structures, such as filaments pairs and bundles. Septins are via a previously unsuspected mechanism, which involves force-dependent essential for processes that involve cell division, cell polarity maintenance balance of free barbed ends, nucleation promoting factor protein, and and membrane remodeling. Recent studies have provided structural informa- Arp2/3 complex. This work not only uncovers a previously unknown and tion about the different levels of septin organization; however, the assembly functionally significant effects of force in branched actin network assembly, mechanism how septin filaments polymerize and further assemble into higher but also reveals the responsible molecular mechanisms. Based on the newly hierarchical structures, and crucial determinants of septin assembly remain discovered force-dependent capping and nucleation mechanisms, we have unclear. Here we used high-speed atomic force microscopy (HS-AFM) to successfully explained the previously observed force-insensitive filament explore these questions. We image septins from their monomolecular state length and force-induced increase of number of free barbed ends. This to layers of several filaments thickness. We determine that the assembly of work provides the very fundamental molecular mechanisms for cell motil- septin filaments and filament pairs are diffusion-controlled, while multilayer ities and we anticipate our assay to be a start point for more systematic septin assembly is a more complex process, partly diffusion-controlled and studies on the influence of physical perturbations in actin branched network partly dependent on the structural optimization to form pairs and bundles. biochemistry. The KCl concentration in the environment influences septin filament assem- bly, oriented packing, pairing and multilayer formation. Septin filament pair- 1249-Pos ing is pH-dependent around physiological pH. We further propose that Spatiotemporal Organization of Microtubules in Branched Networks filament-pairing is mediated by the face opposite to the C-terminal coiled- Akanksha Thawani1,2, Howard A. Stone1,3, Joshua W. Shaevitz1,4, coil domains. Sabine Petry1,5. 1Princeton Univ, Princeton, NJ, USA, 2Chemical and Biological Engineering, 1247-Pos Princeton University, Princeton, NJ, USA, 3Mechanical and Aerospace Metaphase Kinetochore Movements are Regulated by Kinesin-8 Motors Engineering, Princeton University, Princeton, NJ, USA, 4Physics, Princeton and Microtubule Dynamic Instability University, Princeton, NJ, USA, 5Molecular Biology, Princeton University, Agneza Bosilj1, Anna Klemm2,3, Iva Tolic4, Nenad Pavin1. Princeton, NJ, USA. 1Faculty of Science, University of Zagreb, Zagreb, Croatia, 2Biomedical The mitotic spindle is a self-organized machine that faithfully segregates Center, LMU Munich, Munich, Germany, 3Max Planck Institute of chromosomes. The spindle is composed of microtubules, yet their precise Molecular Cell Biology and Genetics, Dresden, Germany, 4Division of spatiotemporal organization of in the spindle remains unclear. While Molecular Biology, Ruder Boskovic Institute, Zagreb, Croatia. microtubules cannot be resolved within the spindle, we have developed tools During metaphase, sister chromatids are connected to microtubules (MTs) to follow individual microtubules throughout their lifetime using isolated extending from the opposite spindle poles via kinetochores, protein com- nucleation pathways, specifically in branching microtubule nucleation. plexes on the chromosome. Kinetochores congress to the equatorial plane Branching nucleation pathway rapidly amplifies microtubules in the spindle, of the spindle and oscillate around it, with kinesin-8 motors restricting therefore understanding this pathway will inform how the spindle is these movements. Yet, the physical mechanism underlying kinetochore constructed. movements is unclear. We show that kinetochore movements in the fission To study their microtubule architecture, we generated branched microtubule yeast Schizosaccharomyces pombe are regulated by kinesin-8-promoted networks in Xenopus egg extracts and imaged the structures from their MT catastrophe, force-induced rescue and MT dynamic instability [1]. A initiation at high temporal resolution. Surprisingly, we found that new candidate screen showed that among the selected motors only kinesin-8 mo- branches never nucleated near the microtubule plus-ends, while the microtu- tors Klp5/Klp6 are required for kinetochore centering. Our theoretical bule lattice near the minus-ends was most likely to nucleate new branches. model, which uses force-balance equations to calculate movements of Simulations of branched networks, assuming that a single autocatalytic reac- kinetochores [2] and Langevin description of MT dynamic instability shows tion event, showed that this was insufficient to explain the spatial bias that kinesin-8 motors are required for kinetochore centering, whereas in nucleation. Instead, we propose a two-step, nucleator-activator model, sensitivity of rescue to force is necessary for the generation of oscillations. consisting of a binding event of nucleators on the existing microtubules,

BPJ 9367_9369 Monday, March 4, 2019 253a followed by their slow activation to generate new branches. Stochastic sim- 1252-Pos ulations of this model reproduced the spatial profile of nucleation, the prob- Dynamic Hand-In-Hand Interaction between Actin and Spectrin during ability density of microtubule plus-ends and tubulin intensity profiles Mammalian Cell Mechanoadaptation measured in experimental networks. We also demonstrate that branching re- Andrea Ghisleni, Camilla Galli, Qinseng Li, Pascale Monzo, Paolo Maiuri, sults in self-similar structures over time, predicted by nucleator-activator Nils Gauthier. model. Lastly, we vary the concentrations of branching proteins, namely aug- Mechano-oncology, IFOM the FIRC Institute of Molecular Oncology, min and TPX2 that likely mediate the binding and activation step respec- Milano, Italy. tively. We find that the spatial profile of branching nucleation is altered Multicellular organisms have evolved complex mechanisms to sense and when the concentration of augmin and TPX2 are varied, which matches adapt to the surrounding environment by dynamically controlling cell shape our model predictions. In sum, we have determined the spatiotemporal pro- and exert forces. In this context, spectrin is a particularly overlooked file of nucleation in branched microtubule networks via a nucleator-activator mechano-sensing protein able to self-assemble into a non-polarized elastic model. These techniques and model will instruct the architecture of the meshwork engaged in membrane-cytoskeleton connections. Spectrin has mitotic spindle. been described to maintain cell shape in erythrocytes and proposed to be involved in the maintenance of axon rigidity; however, its molecular organi- 1250-Pos zation and dynamics are unknown in most eukaryotic cell types, while the mu- Pivoting of Microtubules Driven by Minus End Directed Motors Leads to rine knockout is embryonically lethal. Specifically we investigate spectrin their Alignment to form an Interpolar Bundle dynamic by different fluorescence microscopy techniques at high spatio- Ivana Ban1, Marcel Prelogovic1, Lora Winters2, Iva Tolic3, Nenad Pavin1. temporal resolution during cell spreading, its role in membrane adaptation 1Faculty of Science, University of Zagreb, Zagreb, Croatia, 2Max Planck 3 during chemical and mechanical perturbations of the cell, and the Institute of Molecular Cell Biology and Genetics, Dresden, Germany, Ruder dominant-negative effect of deletion mutants’ overexpression. Using fibro- Boskovic Institute, Zagreb, Croatia. blasts, we describe how spectrin dynamically interplay with the acto- At the beginning of mitosis, the cell forms a spindle made of microtubules and myosin cytoskeleton and plasma-membrane (PM) with complementary associated proteins to segregate chromosomes. An important part of spindle ar- functions, while immunolocalization screening of different mammalian cell chitecture is a set of antiparallel microtubule bundles connecting the two spin- lines suggests the existence of highly conserved mechanisms between dle poles. A key question is how microtubules extending at arbitrary angles different systems. By applying external mechanical perturbations such as form an antiparallel interpolar bundle. Here we show that microtubules meet cell stretch and compression, we identified a fast elastic response by spectrin, at an oblique angle and subsequently rotate into antiparallel alignment. By while actin reacts with different dynamics. Overall, our results pinpoint a sup- combining experiments with theory, we show that microtubules from each porting mechanism of the lipid bilayer led by spectrin at regions where the pole search for those from the opposite pole by performing random angular actin cytoskeleton is not established, creating a fencing mechanism for actin movement [1]. Upon contact of two microtubules, they slide sideways along remodeling and membrane trafficking; these mechanisms potentially unveil each other towards the minus end, which we interpret as the action of minus why the spectrin family of protein is evolutionary highly conserved and ubiq- end directed Cut7/kinesin-5 motors. In conclusion, random rotational motion uitously expressed in eukaryotic cells. helps microtubules from the opposite poles to find each other and subsequent accumulation of motors allows them to generate forces that drive interpolar 1253-Pos bundle formation. Coordinate Role of Vinculin and Metavinculin in Actin Organization [1] Winters, L., Ban, I., Prelogovic, M., Pavin, N., Tolic, I.M. (2018). Pivoting Sharon Campbell1, Muzaddid Sarker1, Hyunna T. Lee1, Laura Kim2, of microtubules driven by minus end directed motors leads to their alignment to Santiago Espinosa de los Reyes3, Lin Mei3, Andrey Krokhotin1, form an interpolar bundle. bioRxiv https://doi.org/10.1101/347831. Laura Constantini1, Gregory M. Alushin3, Nikolay V. Dokholyan4, Jack D. Griffith5. 1251-Pos 1 Nckipsd Coordinates Arp2/3 and Formin Nucleation of Actin Filaments in Dept Biochem/Biophys, University of North Carolina, Chapel Hill, NC, USA, 2New York Structural Biology Center, New York, NY, USA, the Cell Cortex 3 4 1 2 2 2 Rockefeller, New York, NY, USA, Pharmacol, Penn State College of LuYan Cao , Amina Yonis , Malti Vaghela , Priyamvada Chugh , 5 Pierre Bohec2, Matt Smith2, Genevieve Lavoie3, Ewa K. Paluch2, Medicine, Hershey, PA, USA, Lineberger Comp Cance, Univ North Philippe Roux3, Antoine G. Jegou1, Guillaume Charras2, Carolina, Chapel Hill, NC, USA. Guillaume Romet-Lemonne1. Vinculin (Vcn) is an essential cytoskeletal protein that acts as a scaffold to link 1CNRS, Jacques Monod Institute, Paris, France, 2University College London, transmembrane receptors to actin filaments, thereby playing a crucial role in London, United Kingdom, 3Montreal University, Montreal, QC, Canada. cell adhesion, motility, and force transmission between cells. While Vcn is Modulating the organisation of the cell cortex is essential during the cell cy- ubiquitously expressed, metavinculin (MVcn), a larger isoform of Vcn, is cle and for cell migration. The cortical actin network can be regulated selectively expressed in smooth and cardiac muscle cells. Similar to Vcn, through the control of actin nucleation. There are two main actin nucleators MVcn can directly associate with actin and remodel the actin cytoskeleton. in the cortex: the Arp2/3 complex, which generates branched filaments off However, distinct from Vcn, MVcn contains an additional exon which encodes the sides of existing filaments; and formin mDia1, which can nucleate and a 68-residue insert. Point mutations in the 68-residue insert have been associ- rapidly elongate filaments. However, how these two nucleators are coordi- ated with altered actin organization and heart disease, notably dilated cardio- nated is still unclear. myopathy (DCM) and hypertrophic cardiomyopathy (HCM). To better With a proteomics analysis we reveal that the cell cortex contains several understand the coordinate role of MVcn and Vcn in actin filament assembly actin nucleation promoting factors. One of them is NCKIPSD (also called and the consequences of cardiomyopathy-related mutations, we conducted a se- DIP/Wish/SPIN90), which has been shown to interact with both Arp2/3 and ries of actin co-sedimentation and negative stain EM experiments. Consistent mDia1. Surprisingly, while NCKIPSD was considered as a formin inhibitor, with previous findings, the tail domain of MVcn (MVt) is unable to induce actin we find that the phenotypes of NCKIPSD depletion often mimic those of bundling. In the presence of sub-stoichiometric amounts MVt relative to Vcn mDia1 depletion. tail domain (Vt), MVt inhibits the assembly of actin filaments into parallel bun- Using purified proteins, we characterise the effect of NCKIPSD in vitro on dles. In contrast to wild type MVt, MVt CM mutants induce the formation of branched actin networks containing Arp2/3 activated by VCA (the active disordered higher-order F-actin assemblies. To probe the molecular basis for domain of WAVE), and show that nucleating filaments with NCKIPSD- differences in actin reorganization observed for MVt and MVt CM mutants, Arp2/3 leaves less Arp2/3 available for branching. As a result, NCKIPSD tunes and negative regulation of Vt-mediated actin bunding by MVt, we experimen- the balance of Arp2/3 mediated nucleation and branching. tally examined computational models of actin-induced MVt conformational In addition, we show that mDia1 has a higher affinity for NCKIPSD-Arp2/3 changes. Results from these studies will be presented. generated barbed ends. Moreover, using single filament assays with microflui- dics, we show that the NCKIPSD-Arp2/3 complex is able to recruit mDia1 to 1254-Pos further favour the generation of rapidly elongating, linear filaments. This is an Structure of the Tpm3.1 N-Terminus: A New Target for Anti-Cancer unexpected mechanism where Arp2/3 nucleation is used for the benefit of for- Treatment min elongation. Anita Ghosh1, Miro Janco2, Till Bo¨cking2, Peter W. Gunning3, Overall, our results indicate that NCKIPSD tunes cortex architecture, by using William Lehman1, Michael J. Rynkiewicz1. Arp2/3 to generate unbranched filaments, thereby promoting mDia1 elongation 1Department of Physiology & Biophysics, Boston University School of at the expense of branching. Medicine, Boston, MA, USA, 2ARC Centre of Excellence in Advanced

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Molecular Imaging & EMBL Australia Node in Single Molecule Sci., instability, which in in-vivo are assisted by microtubule-associated proteins University of New South Wales, School of Medical Sciences, Sydney, (MAPs). MT severing enzymes are MAPs which destabilize MTs by Australia, 3University of New South Wales, School of Medical Sciences, removing subunits. A number of severing mechanisms have been proposed Sydney, Australia. such as the unfoldase, the wedge or a combination of both. Here we focused Tropomyosin coiled-coils form head-to-tail polymers along the actin filament on the unfoldase mechanism which involves binding of carboxy-terminal and play a key role in regulation of actin function. Association of different tails (CTTs) of tubulin subunits to the pore loops of the severing enzymes tropomyosin isoforms confer variation in filament function such as mainte- followed by the application of pulling forces by the loops on the subunits re- nance of cell morphology (e.g., non-sarcomeric low molecular-weight sulting in MT lattice breakage. We mimicked the pulling action by applying Tpm3.1 (formerly TM5NM1)) and muscle contraction (e.g., high a force on the C-terminal ends of tubulin dimers in a lattice described at molecular-weight Tpm1.1). Previous work has shown that Tpm3.1 is upregu- coarse-grained level using the Self Organized Polymer model. We deter- lated during tumorogenesis and stabilizes the actin cytoskeleton in tumor mined the role of the two CTTs, the distribution of first breaking force cells. Tumor cell death can be triggered by specifically reducing Tpm3.1 and critical breaking force and the unfolding pathways. Moreover, we stud- levels in vivo, making it an attractive target for cancer treatment. Here we ied the influence of MT finite size effects on MT severing. Our simulations present the 2.3 A˚ crystal structure of the Ala-Ser modified 79 N-terminal res- show multiple pathways leading to loss of single dimer or multiple dimers at idues of Tpm3.1, containing stereotypical two-stranded parallel a-helical different first and critical breaking forces all being clearly influenced by coiled-coils with two anti-parallel coiled-coils in the asymmetric unit. finite size effects. We compare and contrast our results with results from Well-defined electron densities are observed for the core residues. The back- in-vitro assays of MT severing. bone of the parallel Tpm3.1 dimer does not deviate significantly when compared to the crystal structure of a similar fragment of Tpm1.1 (PDB 1257-Pos id: 1IC2). However, unlike Tpm1.1, diffuse density is observed for the N-ter- Katanin Spiral and Ring Structures Shed Light on Power Stroke for minal residues of Tpm3.1 indicating the possibility of an unstructured end. Microtubule Severing This region includes the 6 additional residues absent in Tpm1.1 and present Elena A. Zehr1, Agnieszka Szyk1, Grzegorz Piszczek2, Ewa Szczesna1, in isoforms derived from exon 1b. Additionally, comparison of the solvent Xiaobing Zuo3, Antonina Roll-Mecak1. 1 2 accessible surfaces of Tpm3.1 and Tpm1.1, reveals at least two pockets on Cell Biology and Biophysics, NIH, Bethesda, MD, USA, Biophysics Core, 3 Tpm3.1. The unstructured amino-terminus and the surface pockets could NIH, Bethesda, MD, USA, X-ray Science Division, Argonne National potentially accommodate small molecules affecting the polymerization of Laboratory, Argonne, IL, USA. tropomyosin and therefore, actin stability and function. Considering the Microtubule-severing enzymes katanin, spastin and fidgetin are AAA ATPases role played by this isoform in tumor cell survival and the availability important for the biogenesis and maintenance of complex microtubule arrays in of potential binding sites, we propose to exploit the N-terminal Tpm3.1 axons, spindles and cilia. They use the energy from ATP hydrolysis to break fragment as a target for novel anti-cancer therapies. [Funding: NIH microtubules in the middle. Because of a lack of known 3D structures for (R01HL036153); Australian Research Council/Government sources severing enzymes, their mechanism of action has remained poorly understood. (DP130100936, CRC-P-grant 53981, National Health and Medical Research Here we report the X-ray crystal structure of the monomeric AAA katanin mod- Council (APP1098870)); with support from TroBio Therapeutics Ltd.)] ule from Caenorhabditis elegans and cryo-EM reconstructions of the hexamer in two conformations. The structures reveal an unexpected asymmetric 1255-Pos arrangement of the AAA domains mediated by structural elements unique to Quantifying Dissipation in Actomyosin Networks microtubule-severing enzymes and critical for their function. The reconstruc- Carlos Floyd1, Christopher Jarzynski2, Garegin A. Papoian2. tions show that katanin cycles between open spiral and closed ring conforma- 1Biophysics, University of Maryland, College Park, MD, USA, 2Dept Chem/ tions, depending on the ATP occupancy of a gating protomer that tenses or Biochem, University of Maryland, College Park, MD, USA. relaxes interprotomer interfaces. Cycling of the hexamer between these confor- The self-organization of actomyosin networks is known to be intricately mations would provide the power stroke for microtubule severing. Moreover, controlled by concentrations of filaments and associated proteins, however we show that the elemental step in the severing reaction is the progressive a general physical explanation of the emergence of different dynamical extraction of tubulin subunits out of the microtubule and use structure based states (such as vortices in gliding assays) in these active matter systems functional assays to delineate structural elements essential for tubulin extrac- has not yet been firmly established. It has been hypothesized that the dissi- tion out of the microtubule lattice. pation of free energy by active matter systems is optimized during self- organization, leading to the emergence of more highly dissipative dynamical 1258-Pos states, but this idea has not yet been evidenced in actomyosin systems. Here Length-Dependent Persistence Length for Microtubules Shorter Than 3 we establish a methodology for testing this hypothesis in actomyosin net- Micrometers works using MEDYAN, an agent-based simulation platform for studying Gretchen Niederriter, Douglas S. Martin. active networks. We extend the capabilities of MEDYAN to allow quantifi- Dept Phys, Lawrence University, Appleton, WI, USA. cation of the rates of dissipation resulting from chemical reactions and relax- Microtubules are components of eukaryotic cells which provide structural sup- ation of mechanical stresses during simulation trajectories, and apply these port, transportation pathways, and are involved in separating chromosomes methods here to characterize the trajectory of dissipation rates accompa- during mitosis. Due to their structural role in the cell, the stiffness of microtu- nying the self-organization of small disordered actomyosin networks at vary- bules, or the persistence length, has been extensively studied. Long microtu- ing concentrations of myosin and cross-linkers, as well as the distributions of bules (3 mm-40 mm) have a persistence length that depends strongly on the these dissipation rates. In the data presented here we do not observe network overall microtubule length, but the persistence length of microtubules shorter reorganizations that lead to increases in the total dissipation rate as predicted than 3 micrometers remains poorly characterized. We report the persistence by the dissipation-driven adaptation hypothesis mentioned above, however length for microtubules between 0.15 and 4.5 micrometers. We use a kinesin we discuss possible future experiments utilizing this new methodology gliding assay to measure the persistence length of gliding microtubules; we that could carefully test the applicability of this principle in actomyosin vary the length of the portion of microtubules fluctuating in this assay by vary- networks. ing the density of kinesin. The persistence length for these microtubules be- tween .15 mm and 4.5 mm ranged from 500 mm to 1.8 mm. These results are consistent with theoretical models treating microtubules as an orthotropic Posters: Microtubules, Structure, Dynamics, and thin shell or as an anisotropic bundle of filaments, but not with models treating Associated Proteins microtubules as isotropic thin shells. 1259-Pos 1256-Pos Structural Transformation of Microtubules in the Presence of Cationic Exploring the Unfoldase Mechanism of Microtubule Severing by Coarse- Polymers Grained Simulations Juncheol Lee1, Chaeyon Song2, Jimin Lee1, Herb P. Miller3, Leslie Wilson3, Rohith Anand Varikoti1, Jennifer L. Ross2, Ruxandra I. Dima1. Cyrus R. Safinya4, Myung Chul Choi1. 1Dept of Chemistry, University Cincinnati, Cincinnati, OH, USA, 2Dept 1Department of Bio and Brain Engineering, KAIST, Daejeon, Republic of Physics, Univ Massachusetts Amherst, Amherst, MA, USA. Korea, 2Amore-Pacific Co. R&D Center, Yongin, Republic of Korea, 3Dept Microtubules (MTs) are intracellular assemblies of ab-tubulin dimers, which Molecular, Cellular & Developmental Biol, Univ Calif Santa Barbara, Santa play a vital role in various processes such as cell division and cellular trans- Barbara, CA, USA, 4Dept Matl Res, Univ Calif Santa Barbara, Santa Barbara, port. MTs undergo rapid polymerization and depolymerization i.e., dynamic CA, USA.

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Microtubules (MTs) are cytoskeletal hollow cylinders of 25 nm diameter with Tau is an intrinsically disordered protein (IDP) crucial to neurons of the cen- walls comprised of linear protofilaments (PFs), head-to-tail arrangement of tral nervous system (CNS) despite the lack of a stable tertiary structure. In ab-tubulin heterodimeric proteins. During the cell division, MTs at kineto- neuronal axons one of the most well appreciated and studied functions of chores switch phases between growth and shrinkage, which is governed by tau is the ability to stabilize the cytoskeletal protein, microtubules (MTs), conformational transitions of tubulins. Cationic molecules can trigger the by suppressing dynamic instability. It has been reported in the literature conformational changes of tubulins and assemble tubulins into aberrant as- that alterations to the integrity of tau’s structure can perturb tau-MT associa- semblies, different with MT. We show our recent findings on the tubulin archi- tions causing neurodegenerative tauopathies such as Alzheimer’s and fronto- tectures, built in the presence of synthetic cationic polymers, and the temporal dementia with parksonism-17 (FTDP-17). While the most prevalent underlying assembly mechanism. The assembly structures were studied using hypothesis alludes that misregulation of phosphorylation events can promote synchrotron small angle X-ray scattering (SAXS) and transmission electron tau dysfunction, there is emerging evidence that other post-translational mod- microscopy (TEM). ifications such as acetylation can also perturb tau-MT associations and pro- mote toxic tau-tau interactions. Thus, we hypothesize that post-translational 1260-Pos modifications such as acetylation to specific residues in close proximity to Condensation of Divalent Metail Ions by Map-Tau Remodels Tau- the MT-binding region of tau may influence how tau mediates tau-MT and Microtubule Bundle Architecture tau-tau associations. Furthermore, we demonstrate that purification of site- Bretton Fletcher1, Chaeyeon Song1, Phillip Kohl1, Peter J. Chung1,2, specific mutagenic tau is now readily achievable with expression of mutant Herbert Miller1, Youli Li1, Myung Chul Choi3, Leslie Wilson1, recombinant tau containing a poly-His-tag. Here we probe tau-MT associa- Stuart C. Feinstein1, Cyrus R. Safinya1. tions using transmission electron microscopy (TEM) and small angle x-ray 1 2 University of California, Santa Barbara, CA, USA, Chemistry/James scattering (SAXS). Franck Inst, Univ Chicago, Chicago, IL, USA, 3KAIST, Daejeon, Republic of Korea. 1263-Pos The neuronal microtubule-associated protein Tau binds to and stabilizes individ- Probing Structural Features of Tau Binding to Tubulin and Microtubules ual microtubules (MTs) and mediates MT-MT interactions. In order to under- Ho Yee Joyce Fung, Elizabeth Rhoades. stand the mechanisms governing the formation and stability of Tau-MT Department of Chemistry, University of Pennsylvania, Philadelphia, PA, complexes, we have developed synchrotron small-angle x-ray scattering USA. (SAXS) techniques to characterize nanoscale structural properties of these dissi- Tau is an abundant neuronal protein best known for its association with pative active macromolecular complexes in biological buffer containing GTP. neurodegenerative disorders, including Alzheimer’s disease. However, nor- While fascicles or linear arrays of ordered MTs have been observed in the axons mally tau is microtubule-associated protein with a role in the stabilization of mature neurons, synchrotron SAXS shows that MTs polymerized in the pres- and growth of microtubules. Tau is an intrinsically disordered protein, ence of Tau in vitro primarily self-assemble into hexagonally ordered bundles which has multiple binding sites for tubulin found throughout its (P. J. Chung, C. Song, et al. Nature Communications 2016, 7, 12278. https:// microtubule-binding repeat, and flanking proline-rich regions. Prior work doi.org/10.1038/ncomms12278 and ACS Macro Lett.2018, 7, 228-232. https:// from our lab demonstrated that tau can form ‘‘fuzzy’’ complexes with sol- doi.org/10.1021/acsmacrolett.7b00937). Combining SAXS with transmission þ uble tubulin, likely contributing to its role in facilitating the polymerization electron microscopy, we show that addition of metal ions (e.g. Mg2 ) above of microtubules. By using environmentally sensitive fluorescent probe, threshold concentrations within a biologically relevant regime drastically re- acrylodan, placed at multiple locations throughout one of the microtubule duces the wall-to-wall distance between bundled MTs followed by a remodeling binding repeats we observed a periodic pattern of the tubulin-bound tau transition with increasing time. Our data support a model where Tau’s projection which suggested a non-random structure. Here, we have expanded domain (PD) is in an extended brush-like state at low metal ion concentrations, our studies to include additional repeat regions within the microtubule bind- and, above a threshold concentration, collapses due to cation condensation lead- ing domain. This allows us to contrast structural features of the different re- ing to the structural remodeling of the Tau-MT complex. peats and may support for a hierarchy in tubulin binding sites. Comparison 1261-Pos of these results with the recently published tau-microtubule cryo-EM struc- Higher Order Assembly Structures of Human Tau and Microtubules ture will provide insights into the differences between microtubule-bound Regulated by Ionic Strength and tubulin-bound tau, as well as information on structural features on re- Hasaeam Cho1, Jimin Lee1, Juncheol Lee1, Herbert P. Miller2, gions of tau that were not observed in the cryo-EM structure. Further bio- Keong Sik Jin3, Leslie Wilson4, Stuart C. Feinstein4, Cyrus R. Safinya2, physical characterization of the structural basis of tau binding to tubulin Myung Chul Choi1. and microtubules will advance the understanding of how tau achieves its 1Bio and Brain Engineering, KAIST, Daejeon, Republic of Korea, 2Dept Matl biological function in the cell and how this function may be disrupted in Res, Univ Calif Santa Barbara, Santa Barbara, CA, USA, 3Postech disease. Accelerator Laboratory, Pohang, Republic of Korea, 4Dept Molecular, Cellular & Developmental Biol, Univ Calif Santa Barbara, Santa Barbara, 1264-Pos CA, USA. Impact of AC Electric Fields on Microtubule Dynamics In Vitro Neuronal microtubules (MTs) are anionic protein nanotubes comprised of ab Joseph M. Cleary. heterodimeric tubulins. In neural cells, MTs form ordered bundle structures Pennsylvania State Univ, State College, PA, USA. that function as tracks for transportation through axon. MTs are long-lived Microtubule dynamic instability plays a role in many cellular functions, but with axonal microtubule-associated protein (MAP) Tau. Tau of polyampholitic is of particular importance in the mitotic spindle of dividing cells. A number nature - positively charged tubulin binding region and negatively charged pro- of successful cancer chemotherapies target dynamic microtubules and jection region - and intrinsically disordered protein (IDP) binds to MTs and reg- inhibit proper spindle formation. A novel therapy that was recently devel- ulates tubulin assembly and MT stability. Dysfunction of Tau is correlated with oped to treat glioblastoma involves placing external electrodes on the numerous neurodegenerative diseases including Alzheimer’s, Parkinson’s, skull of the patient that generate moderate amplitude AC electric fields. Pick’s, supranuclear palsy, and fronto-temporal dementia with Parkinsonism In published work in mice, these Tumor-Treating Fields (TTF) have been linked to chromosome 17 (FTDP-17). In this poster, we present our recent find- shown to reduce tumor mass, and to reduce the proliferation rate of cancer ings on higher order assembly structures of human tau and MTs regulated by cell cells in culture. Defective spindles have been observed in treated cells, ionic strength by using synchrotron small angle X-ray scattering (SAXS) and leading to the suggestion that the AC electric fields may act by altering transmission electron microscope (TEM). microtubule dynamic instability. To test this hypothesis, we investigated the effects of 100 - 300 kHz AC fields on the stability of purified microtu- 1262-Pos bules in vitro. Electric fields ranging from 0.1 - 300 V/cm were generated by The Effect of Site-Specific Acetylation Based Tau Mutations on Tau- nanofabricated electrodes deposited on glass, and microtubules were Microtubule Associations observed by fluorescence microscopy. AC electric fields over the ranges Christine Tchounwou1, Bretton Fletcher1, Chaeyeon Song1, and frequencies tested had no apparent impact on the stability of taxol- Phillip A. Kohl1, Peter J. Chung2, Herb P. Miller1, Youli Li1, stabilized microtubules. This result does not support an mechanism by Myung-Chul Choi3, Leslie Wilson1, Stuart C. Feinstein1, Cyrus R. Safinya1. which TTFields directly impact spindle integrity. Current studies are 1Univ Calif Santa Barbara, Santa Barbara, CA, USA, 2Chemistry/James investigating the effects of AC electric fields on the dynamic instability pa- Franck Inst, Univ Chicago, Chicago, IL, USA, 3Korea Advanced Inst of rameters of microtubules grown in the absence of microtubule stabilizing Science and Tech, Daejeon, Republic of Korea. drugs.

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1265-Pos ing on the microtubule part of the complex, causing it to break. The number Microtubule Nucleation and Stabilization by Doublecortin of pushing or pulling Ncd motors was approximated from the microtubule Szymon W. Manka1, Carolyn A. Moores2. length bound by the motors attached to the coverslip and the forces they pro- 1Biol Sciences, ISMB, London, United Kingdom, 2Dept Biol Sci, Birkbeck duced were estimated from forces produced by Ncd motor arrays in laser-trap Col London, London, United Kingdom. assays, reported by other workers. The minimal forces that we obtained for Doublecortin (DCX) is a microtubule (MT)-associated protein indispensable microtubule breakage using these methods are close to those applied by for migrating neurons during brain development. DCX nucleates and stabilises others in microtubule indentation assays by AFM. They are also consistent MTs via homologous DC domains. The C-terminal DC domain (C-DC) is with forces needed to disrupt noncovalent bonds in proteins in force spectros- separated from the N-terminal counterpart (N-DC) by a 42-residue linker. In- copy experiments. These findings provide a first-approximation estimate of tellectual disability (smooth brain) and epilepsy-causing mutations cluster microtubule rupture forces, which is relevant to microtubule mechanical around both DC domains. Thus, both domains are thought to interact with strength during cellular function. This estimate can guide the design of future MTs, but their exact roles remain obscure. For MT nucleation, the naturally experiments to obtain a more precise measure of microtubule breakage bent tubulin dimers associating longitudinally and laterally need to straighten forces. up to form cylindrical lattice. Stabilization of such lattice relies on counteract- ing the natural tendency of tubulin to curl outward. The DCX binding site on 1268-Pos the MT lattice is at the vertex of four tubulin dimers, so DCX binding stabi- Long-Range Mechanical Coupling in the Microtubule Lattice lizes both longitudinal and lateral lattice contacts. But it is neither clear which Maxim Igaev, Helmut Grubmueller. of the two DC domains mediates this MT-stabilizing interaction nor which is Theoretical and Computational Biophysics, MPI for Biophysical Chemistry, involved in MT nucleation. We found that the construct in which C-DC was Goettingen, Germany. replaced with another N-DC showed much lower MT-nucleating activity Microtubules (MTs) are key components of the cytoskeleton and play a central and – in contrast to wild-type DCX – it produced not only the physiological role in cell division and development. Recent structural data suggest that MT 13-protofilament (PF) MT architecture, but also 14-PF MTs. Near-atomic res- instability - in particular, the disassembly process - is accompanied by subtle olution cryo-EM reconstructions enabled visualisation of the structural differ- subnanometer changes in the MT lattice structure. Furthermore, the lattice ences between the DC domains suggesting that N-DC has higher affinity for likely undergoes a fixed sequence of global compaction states (pre-hydrolysis, the mature MT lattice while the newly-formed lattice is predominantly deco- post-hydrolysis, and fully hydrolyzed) prior to catastrophe. However, how rated with C-DC. Molecular modelling suggests that C-DC can bind between exactly the effects of hydrolysis-triggered transitions in one dimer propagate moderately curved PFs, whereas N-DC cannot. The predicted C-DC binding through the lattice, potentially giving rise to long-range mechanical coupling, mode largely overlaps with patient mutations. Taken together, the data sug- is unclear. gest that C-DC facilitates nucleation of physiological 13-PF MTs, while Here, we present accurate atomistic models of complete MT lattices refined N-DC gradually replaces C-DC along the lattice, ensuring robust stabilisation against the most recent high-resolution cryo-EM data, which enabled to of the straight polymer. dissect the mechanics and energetics of lattice compaction as well as the coupling between individual dimers. We show that, in both cases, the nucle- 1266-Pos otide state strongly influences the intrinsic elasticity of the MT lattice. Our Structure and Function of Surface-Bound Tau data suggest that, unexpectedly, GDP-MTs are axially twice as stiff as Zachary J. Donhauser. GTP-MTs and store almost 60% of the per-dimer energy from GTP hydroly- Chemistry, Vassar College, Poughkeepsie, NY, USA. sis as axial strain. Moreover, the compaction/extension of one dimer in GDP- The microtubule associated protein tau has a variety of important cellular MTs modulates the compaction/extension of its nearest neighbors more functions, most notably stabilizing and organizing microtubules in axons; strongly than in GTP-MTs. These findings provide evidence that, in the meanwhile it is also implicated in a host of neurodegenerative disorders GDP lattice, this mechanical coupling might propagate much beyond that are characterized by abnormal aggregation of the protein into paired he- the nearest neighborhood of a tubulin dimer, rendering GDP-MTs more sen- lical filaments and neurofibrillary tangles. Although significant research sitive to lattice defects or external stress and, hence, more susceptible to attention has been paid to the physical characterization of monomers and catastrophe. polymeric assemblies of tau, much of the work in vitro has been focused on the behavior of tau in solution. In order to improve the understanding 1269-Pos of tau’s behavior when it is bound to microtubules, we have used an atomic Multi-Scale Computational Modeling of Tubulin-Tubulin Interactions in force microscopy assay to interrogate tau in physiologically-relevant confor- Microtubule Self-Assembly from Atoms to Cells mations when it is bound to solid supports. AFM force spectroscopy is Mahya Hemmat1,2, Brian T. Castle2, David J. Odde2. used to measure the structure of immobilized tau layers and characterize 1Mechanical Engineering, University of Minnesota, Minneapolis, MN, USA, tau-tau intermolecular interactions in this configuration. Using N- and C-ter- 2Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA. minal truncation mutants we identify regions of the protein that can partici- Microtubules are dynamic filaments that facilitate critical cellular functions pate in complementary dimerization, and confirm that these regions of the such as chromosome segregation, cargo transportation and cell architecture. protein can also form disordered polyelectrolyte brushes. We also compare The question of how to connect across length-time scales, from atoms to the behavior of wild-type tau to that of pseudophosphorylated mutants in cells, to understand the mechanism of microtubule dynamics is yet to be order to study the effect of phosphorylation on the structure of substrate- resolved. In this study, we sought to address this problem by developing a bound tau. novel multi-scale computational approach to study microtubule dynamics. We combine all-atom molecular dynamics simulations, Brownian dynamics 1267-Pos simulations and thermo-kinetic modeling of tubulin assembly, to bridge A First-Approximation Estimate of Forces Required for Microtubule time scales from femtoseconds to minutes and length scales of A˚ ngstrom Breakage to tens of micrometers, so that cellular level behavior can be predicted Sharyn A. Endow1, Piotr E. Marszalek2. from sequence-level information. Using our approach, we conclude that 1Department of Cell Biology, Duke University Medical Center, Durham, NC, the lateral bond is not a significant factor in stabilizing GTP-tubulin in the USA, 2Department of Mechanical Engineering, Duke University, Durham, microtubule lattice through comparing the energetic difference when nucle- NC, USA. otide changes in b-tubulin in all-atom molecular dynamics simulations. In Microtubule breakage has been observed during mechanical bending and addition, we found that the total lateral potential of mean force (PMF) is deformation of microtubules by retrograde actomyosin flow at the leading not significantly different among different nucleotide states of tubulin and edge of migrating cells, but the forces that result in rupture have not yet is calculated to be y11 kBT and the longitudinal bond for the GDP-state been measured directly. In addition to microtubule rupture in vivo, microtu- tubulin is calculated to be y 25 kBT, consistent with previous estimates bule breakage in vitro has been observed in ensemble motility assays of a of longitudinal bond being 3 times stronger than lateral bond. Using molec- bidirectional kinesin-14 Ncd motor. Gliding axoneme-microtubule com- ular dynamics’ interaction energies in our Brownian dynamics simulations of plexes were observed to buckle and break, followed by gliding of the two tubulin dimers yields that the lateral bond is weak on its own, and has a life- parts in opposite directions or with different velocities in the same direction. time of 0.2 ms. Overall, our studies show the ability to predict cellular We modeled the ruptures as due to Ncd motors clamping a region of the level phenotypes from atomic level information sequence and structural in- axoneme-microtubule to the coverslip and other Ncd motors pushing or pull- formation.

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1270-Pos 1272-Pos Tubulin Polymerization-Promoting Protein Family Member 3 (Tppp3) Fa- Mechanical Stability of Microtubule Lattices - Molecular Dynamic Inden- cilitates Microtubule Bundling and Network Formation via its Weak Inter- tation Studies action with Microtubules Lukasz Szatkowski1, Merz R. Dale Jr1, Jennifer L. Ross2, Takayuki Torisawa1,2, Shuji Ishihara3, Kazuhiro Oiwa2. Ruxandra I. Dima1. 1Struct. Biol. Center, Natl Inst Genetics, Mishima, Japan, 2Adv ICT Res Inst, 1Department of Chemistry, University of Cincinnati, Cincinnati, OH, USA, Natl Inst Info Comm Tech, Kobe, Japan, 3Graduate School of Arts and 2Department of Physics, University of Massachusetts Amherst, Amherst, Sciences, The University of Tokyo, Tokyo, Japan. MA, USA. Tubulin polymerization-promoting protein family member 3 (TPPP3) is a Microtubules (MTs) are made of ab-tubulin heterodimers joined longitudinally conserved protein factor found in cytoplasm associated with microtubules into protofilaments (PFs). PFs associate laterally to form the hollow cylinder of in many ciliated cells. The expression level of TPPP3 in mouse tracheal an MT. Most MTs in cells contain 13 PFs, however the most common in vitro epithelial cells (mTEC) was reported to vary in concomitant with the forma- MTs have 13 or/and 14 PFs. The B-lattice (where two adjacent PFs are laterally tion of microtubule networks in the cytoplasm during epithelia development. connected by interactions between the same type of tubulin monomers: a-a/b- These observations suggest that TPPP3 takes part in formation of the micro- b) is dominant in the MT cylinder structure. Additionally, each MT cylinder tubule network. Here, we expressed several TPPP3 constructs and, by using contains a single lateral interaction created by the A-lattice (where two adjacent conventional biochemical measurements and single molecule observations, PFs are connected through a-b and b-a interactions) and called the seam. The examined its effects on cell morphism of HEK293 cells, the binding, the proper functioning of MTs has a key role in the process of cell division, cell bundling, and the elongation of microtubules in vitro. TPPP3 formed micro- motility and cell development due to the fact that MTs are responsible for tubule networks in vitro in a concentration dependent manner and showed cell morphology, cell interior organization and intracellular cargo transport. specific but weak binding (Kd =ca.5mM) to the microtubules. The molec- Dynamic MTs rearrangements by breaking and healing are crucial for accom- ular dissection combined with single molecule observations suggests that the plishing and maintaining their cellular functions. MT breakage can occur ability to dimerize the TPPP3 molecules resides in the N-terminal domain through one of two mechanisms: breaking from high frequency buckling of and the ability to promote microtubule elongation resides in the C-terminal PFs and breaking induced by microtubule-severing enzymes. Due to the high domain of TPPP3. Micro-rheology measurements of TPPP3-microtubule complexity of MTs, experimental techniques are not able to fully answer the network were carried out, in which the Brownian motion of microspheres question which of the A/B MT lattices is the ‘‘sweet spot’’ for the binding of of 0.2 mm-diameter in the microtubule network was measured. The measure- microtubule-severing enzymes. Therefore, a combination of experimental ment showed that the network was crosslinked but remained flexible. This and theoretical techniques is required to solve this problem. crosslinked flexibility enables the network to adopt the optimal mechanical We use computational methods to probe and compare the mechanical stability configuration in response to the external force. These results suggest that of the both MTs lattices using coarse-grained indenting molecular dynamics. during the development of the basal body array in ciliated cells, the loose With the knowledge of how the B-lattice behaves (with/without defects) we but somehow restricted network provides the adaptability of the array to can compare its mechanical stability with that of the A-lattice. Furthermore, the external load. for both lattices computationally predicted breaking forces, bending angles dis- This work was supported by funding from the Dynamic Mechanisms and tributions and MTs length factors are comparable with experimental data ob- Fundamental Technology for Biological Systems and the Creation of Funda- tained from in vitro severing assays and AFM experiments. mental Technologies for Understanding and Control of Biosystem Dynamics, CREST, from Japan Science and Technology. 1273-Pos Modulation of Microtubule Curvature under Different Cellular Condi- tions Revealed by In-Cell Ceryo-Electron Tomography 1271-Pos Julia Mahamid1,2, Saikat Chakraborty2, Wolfgang Baumeister2. Kinetochore-Mediated Multivalency of Ndc80 Complex Controls Microtu- 1 Structural and Computational Biology, European Molecular Biology bule End Dynamics and Force-Generation Laboratory, Heidelberg, Germany, 2Department of Molecular Structural Vladimir A. Volkov1, Pim J. Huis in ’t Veld2, Andrea Musacchio2, 1 Biology, Max-Planck Institute of Biochemistry, Martinsried, Germany. Marileen Dogterom . With persistence lengths in the range of 1–6 mm, microtubules (MTs) are the 1Bionanoscience, Delft University of Technology, Delft, Netherlands, 2 most rigid polymer of all cytoskeletal elements. However, inside cells they Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, often exhibit highly curved shapes. Thus, it is tempting to speculate that the Dortmund, Germany. curvature of MTs in vivo are modulated by cellular factors resulting in During mitosis, a subset of the microtubules in the mitotic spindle connect bending at micrometer length scales. In order to better characterize the curva- to the chromosomes through kinetochores, multi-protein complexes ture of MTs and its potential causes in vivo, we quantified the curvature of assembling on the centromeric DNA. Microtubule ends and kinetochores cellular MTs in their native state from frozen-hydrated HeLa cells in inter- influence each other mechanically: dynamic protofilaments at the end phase and mitosis as model systems using cryo-electron tomography (cryo- of the microtubule bend outwards creating tension while flexible ET). We show that cellular MTs are highly curved beyond the limit posed microtubule-binding elements of the kinetochore resist and are being by the polymer’s intrinsic material’s property. In fact, the apparent persistence stretched by microtubule-generated force. Cell biological experiments iden- lengths measured from the curved MTs are 100-1000 times lower than the tified Ndc80 complex as the crucial force-bearing link between the microtu- persistence length obtained from thermal fluctuations alone, indicating preva- bules and centromeres in the majority of eukaryotes. However the Ndc80 lence of high magnitude of nonthermal forces in vivo. By using specific inhib- complex was shown to not interact with bent tubulin structures, so the mo- itors, we pinpoint the sources of these forces that predominantly include actin- lecular identity of the flexible protofilament-binding connections seen in mediated compressive forces, ATP-dependent motor activities and microtu- fixed cells has been controversial. Here we show that joining 2 or more bule polymerization. MT curvature was also found to vary depending on copies of the Ndc80 complex at their kinetochore-binding sites creates a the cellular state, with mitotic MTs being relatively less curved implying coupler that interacts directly with the shortening microtubule end. We that the magnitude of nonthermal forces have different regimes across cellular used multiple assays to demonstrate that multimerization of Ndc80 is states in vivo. Notably, in-cell cryo-ET provides a glimpse into the native or- essential to capture the power-stroke of the tubulin protofilaments at the ganization of MTs intertwined within the composite network of other cyto- end of a disassembling microtubule. First, a single Ndc80 oligomer skeletal filaments and reveals differences in their organization across travelling with the microtubule end slows down microtubule shortening; different cell cycle stages and cell types. We demonstrate that the presence second, a single Ndc80 oligomer stalls microtubule shortening after the of a heterogeneous elastic actin matrix and its organization is one of the development of the initial force generated by the shortening microtubule main factors that influence the MT curvature in vivo. end; and third, the connection between an Ndc80 oligomer and a microtu- bule end stiffens specifically during the stalled depolymerization of the 1274-Pos dynamic microtubule (as opposed to the wall of the microtubule away Structural Studies of CAP-Gly Domain of Dynactin Assembled with from the end, or a stable, non-depolymerizing blunt end). We further Microtubules by Magic Angle Spinning NMR Spectroscopy investigate molecular requirements for the force-dependent stalling and Changmiao Guo1, John C. Williams2, Tatyana Polenova1. rescue of microtubule depolymerization using additional kinetochore com- 1Department of Chemistry and Biochemistry, University of Delaware, ponents, post-translational modifications, and mutations of flexible regions Newark, DE, USA, 2Department of Molecular Medicine, Beckman Research in Ndc80. Institute of City of Hope, Duarte, CA, USA.

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Microtubules and their associated proteins are central to a broad array of essen- structure that has fascinating dynamics that are a result of its unique struc- tial physiological functions, including mitosis, polarization and differentiation, ture. It has been reported that changes in the tubulin proteins that make up cell migration, and vesicle and organelle transport. They have been extensively the bulk structure of the microtubule can even alter these dynamic properties, studied at multiple levels of resolution. However, there remain significant gaps in particular the polymerization and depolymerization rates. The pathways in knowledge concerning how microtubule-binding proteins bind to microtu- that dictate how these changes in local structure result in system wide bules, how dynamics connect different conformational states and how these in- changes in dynamics, however, has yet to be elucidated by current measure- teractions and dynamics affect the cellular processes. Structures of many ment techniques. This is because previous methods of defining structural microtubule-associated proteins assembled on microtubules are not available properties of the microtubule hinge on static parameters, such as persistence at atomic resolution. length and Young’s modulus. However, using a static measurement makes it We used magic angle spinning (MAS) NMR to solve a structure of the difficult to understand the dynamic properties of the microtubule. Further- cytoskeleton-associated protein glycine-rich (CAP-Gly) domain of dynactin more, the measurements must be spatially resolved to extract the anisotropic assembled on microtubules. We determined the intermolecular interface of behavior of these local changes. Our methods look at the vibrational energy CAP-Gly with polymerized microtubules by direct dipolar contacts between propagation through microtubules as an energy source for the energy inten- CAP-Gly and tubulins using double rotational echo double resonance (dRE- sive dynamics of the microtubule. With our methodology the increased DOR)-filtered experiments. Our results indicate that the structure adopted by spatial resolution accommodates anisotropy along the length of the microtu- CAP-Gly varies, particularly around its loop regions, permitting its interaction bule, and paves the way for developing a dynamic measurement of microtu- with microtubules and other binding partners. This study is the first example of bule mechanics. the atomic-resolution structure of a microtubule-associated protein on polymer- ized microtubules. 1277-Pos We have also characterized the structure and dynamics of the extended CAP- Reconstitution of Pom1 Gradient in Elongated Droplets Gly domain that contains the CAP-Gly domain, basic domain and serine- Renu Vishavkarma. glued proline-rich (SP-rich) domain of p150 subunit by solution NMR and TU Delft, Delft, Netherlands. MAS NMR. Our results indicate that the basic and SP-rich domains contain Fission yeast cells have emerged as the model system to study cell division a second binding site for microtubules in addition to CAP-Gly domain. The because of the similarity with human genes that are responsible for cell division secondary structure prediction is performed based on backbone chemical and cellular organization. It has been proposed and reported that cell size is the shifts from solution NMR and three short segments in the basic domain key regulator of yeast cell’s entry to mitosis. Cell size regulates the gradient of are predicted to contain helical and sheet structures. These segments are DYRK family kinase Pom1 and controls entry to mitotic division. The micro- contained in the rigid regions as revealed by the conformational flexibility tubule based transport is known to be responsible for the polar growth of the analysis and possibly encompass the intermolecular binding sites for yeast cells and gradient formation. According to the proposed mechanism, microtubules. the microtubules based transport leads to the accumulation of the Pom1 kinase and the associated motor proteins at the tip of the cell and through lateral diffu- 1275-Pos sion a gradient is formed. When cell reaches the characteristic size, the gradient Towards an Understanding of Kidney Diseases Associated with Inhibition profile is such that it promotes the localization of SAD kinase Cdr2 in central of Notch Signaling Pathway by Transmission Electron Microscopy part in the cortex and initiates the process of formation of the division plane. Ishara Ratnayake1, Steve Smith1, Indra Chandrasekar2, Our aim is to find minimum set of components required to establish Pom1 Kameswaran Surendran2, Phil Ahrenkiel1. gradient in yeast cells using bottom up approach. Through this approach we 1Nanoscience and Nanoengineering, South Dakota School of Mines and establish a tip tracking system inside the elongated droplets with functionalized Technology, Rapid City, SD, USA, 2Department of Pediatrics, Sanford lipid monolayer. School of Medicine, Sioux Falls, SD, USA. Transmission electron microscopy (TEM) of samples prepared by ultramicrot- 1278-Pos omy provides detailed structural information for interrogation of processes at Computational Study of Abl Pathway Based Axonal Guidance the cellular level. In this work, we are using mouse models and cell culture sys- Aravind Chandrasekaran1,2, Garegin A. Papoian1, Edward Giniger2. tems to understand how Notch signaling suppresses renal tubular cyst forma- 1Chemistry & Biochemistry, University of Maryland, College Park, MD, tion and microadenoma (a tumor) formation. These studies are likely to USA, 2National Institute of Neurological Disorders and Stroke, National reveal how Notch signaling is linked to primary cilia and the cell cycle, and Institutes of Health,, Bethesda, MD, USA. will provide insights into the mechanisms by which Notch functions as a tumor Axonal growth guidance is a tightly regulated process that involves a series suppressor in epithelial cancers. By performing electron microscopy, we are of signaling receptors for axon guidance cues such as Netrin, Slit, and Sem- investigating the structure of cilia to understand their role in Notch signaling aphorin. These signals cascade through non-receptor kinases such as Abelson deficient kidney cells. By analyzing TEM images, we are able to detect normal murine lymphosarcoma (Abl) and Src (for ‘‘sarcoma’’) which results in mod- 9þ0 microtubule doublets within the axoneme of primary cilia in both wild ulation of actin/microtubule (MT) cytoskeletal dynamics. Modulation of type and mutant type cells. After analyzing mutant kidney cells, we detected cytoskeletal network dynamics helps steer axonal growth by varying protru- cilia which have unusual arrangements of microtubules. These abnormal struc- sion rates and direction. Abl kinase signaling pathway has been studied tures may be due to alterations in ‘‘posttranslational modifications’’. We extensively revealing that Abl kinase controls actin dynamics by stimulating believe that the Notch signaling deficiency may alter the posttranslational mod- Arp2/3 activity (through WAVE/SCAR pathway) while inhibiting Enabled ifications of the microtubules in mutant cells. In the future, we plan to deter- (Ena). While Arp2/3 is an actin brancher, Ena is a tetrametric protein that mine the posttranslational modifications in Notch signaling deficient cells by binds strongly to actin filament plus ends in a competitive manner compared performing a quantitative analysis by western blotting. Also, by performing to capping proteins to accelerate filament elongation rate. Taken together, tem tomography, we can confirm that the abnormal structures are real and Abl is thought to activate brancher molecules increasing dendricity of actin not artifacts. network while preventing Ena activity reducing average filament length/turn- over. Given the knowledge of signaling pathway and the essential constitu- 1276-Pos ents, the extent to which Abl affects branching and Ena activities remains Topological Phonons in Microtubules: The Link between Local Structure unknown. We used an advanced active network simulation software, and Dynamics of Microtubules MEDYAN to simulate Abl kinase activity in-silico. Our simulations throw Arooj Aslam1, Ssu-Ying Chen1, Emil Prodan2, Camelia Prodan1. light on how protrusion rates and stability of axonal actin networks depend 1NJIT, Newark, NJ, USA, 2Yeshiva University, New York, NY, USA. on concentrations of Ena and Arp2/3. We also studied how active transport We have developed a model for analyzing thermal energy propagation that controls protrusion of actin networks. Our work lays the groundwork for through a microtubule by tracking it’s movement over time, and extracting addressing the challenge of computational modeling of axonal guidance in a phonon spectrum of energy states and the speed of energy propagation three dimensions at the level of dynamics of individual cytoskeletal through a microtubule. The microtubule is a dynamic self-assembling protein components.

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Posters: Myosins and Smooth Muscle Mechanics, 1281-Pos Tracking of Labeled Motor Domains of Single Full-Length Myosin X on Structure, and Regulation Actin Bundles Xianan Qin1, Harry Chun Man Cheng2, Jing Li1, Hanna Yoo2, Xiaoyan Liu3, 1279-Pos Tianming Lin3, H. Lee Sweeney3, Hyokeun Park1,2. Atomically Detailed Simulation of the Powerstroke in Myosin II by Mile- 1Department of Physics, The Hong Kong University of Science and stoning Technology, Kowloon, Hong Kong, 2Division of Life Sciences, The Hong Katelyn Poole1, Ron Elber1,2. Kong University of Science and Technology, Kowloon, Hong Kong, 1 2 Chemistry, University of Texas at Austin, Austin, TX, USA, Institute for 3Department of Pharmacology and Therapeutics and the Myology Institute, Computational Engineering Sciences, University of Texas at Austin, Austin, University of Florida College of Medicine, Gainesville, FL, USA. TX, USA. Myosin X is an unconventional motor that can move along actin bundles. It is The interaction between actin and myosin II plays an important role in a variety involved in the formation of filopodia in cells, which play important roles in of cellular functions. In particular, myosin II is involved in muscle contraction, migration and cell adhesion. It was found that myosin X monomers dimerize which is attributed to the sliding of thin filament actin past the thick myosin II via an anti-parallel coiled coil. How anti-parallel myosin X moves along actin filaments. Past studies on the structure of myosin have linked severe pathol- bundle has not been clearly understood yet. In particular, the stepping mecha- ogies to defects in myosin, making it important to understand the mechanism nism of single full-length myosin X remains enigmatic. Here, we investigated of the system. In this study, we focus our analysis on the powerstroke of the the stepping mechanism of myosin X along actin bundles by tracking labeled myosin II cross bridge cycle, which is the active process of muscle contraction. motor domain of myosin X with the total internal reflection microscopy. We To do this, we use an algorithm called Milestoning which partitions the dy- observed clear stepping patterns under 2 mM ATP. The step size distributions namics into a sequence of trajectories between ‘‘milestones’’ along the reaction of full-length myosin X were broad and had multiple peaks, which suggests pathway. several possible inter-motor configurations of myosin X. To further understand The structure of myosin II bound to actin in the rigor state was used as a starting the stepping mechanism of myosin X, we labeled two motor domains of myosin point, and a structure for the bound pre-powerstroke state was developed using X using different fluorescent molecules and tracked the motility of myosin X on existing published structures for the unbound pre-powerstroke state as well as actin bundles. We found that the distributions of stepping in two motors were experimental data gathered about the movement of myosin II during the power- broad and had multiple peaks. We also found that the distributions of inter- stroke. With both the beginning and final states of the powerstroke, we can motor distance were broad. The broad distributions of steps with multiple peaks interpolate between these structures to build intermediate states along the reac- and inter-motor distances suggest that myosin X is optimized for moving along tion pathway. actin bundles. A total of 97 all-atom structures along the pathway of the powerstroke were developed to serve as guides and ‘‘anchors’’ along the reaction pathway. Mile- 1282-Pos stoning short trajectories between cells defined by the anchors will allow for the The Role of Non-Muscle Myosin 2A and 2B in the Regulation of Mesen- computation thermodynamics and kinetics of the myosin II powerstroke. This chymal Cell Contact Guidance work will lead to a significant improvement in our understanding of the com- Alexander S. Zhovmer1, Erdem Tabdanov2, Houxun Miao3, Han Wen3, plete powerstroke mechanism, which will in turn facilitate future research on Jinqiu Chen4, Xiaoling Luo4, Xuefei Ma1, Paolo Provenzano2, the effects of structural defects in myosin II on powerstroke function and mus- Robert Adelstein1. 1 2 cle contraction. Cell and Developmental Biology, NHLBI, Bethesda, MD, USA, University of Minnesota, Minnesota, MN, USA, 3NHLBI, Bethesda, MD, USA, 4NCI, Bethesda, MD, USA. 1280-Pos Contact guidance (CG) is important during migration and positioning of Optical Control of Fast and Processive Engineered Myosins In Vitro and in cells inside the growing embryo as well as for tissue repair and cancer pro- Living Cells gression in the adult organism. Mesenchymal cell CG defects are known to 1 1,2 1 Paul V. Ruijgrok , Rajarshi P. Ghosh , Muneaki Nakamura , be induced by the inhibition or ablation of non-muscle myosins 2 (NM2). 1 1 1 1,2 Sasha Zemsky , Robert Chen , Vipul Vachharajani , Jan T. Liphardt , Here we show that loss of the NM2B paralog preserves CG per se but af- 1,3 Zev Bryant . fects adaptive cell morphogenesis. This is manifested by the increased 1Department of Bioengineering, Stanford Univ, Stanford, CA, USA, 2ChEM- 3 CG-mediated linearization of fibroblasts on an anisotropic surface as well H, Stanford, CA, USA, Department of Structural Biology, Stanford, CA, as spontaneous linearization of fibroblasts on an isotropic surface. Lineari- USA. zation is linked to a specific NM2 paralog regulation of actomyosin scaf- Spatiotemporal control of cytoskeletal transport can provide new possibil- folding required for microtubule alignment. Expression of the NM2A but ities for dissecting cellular processes or for constructing complex artificial not the NM2B paralog is associated with accumulation of actomyosin struc- devices. Optogenetic approaches have been used for both controlled recruit- tures, microtubule alignment and cell linearization. Analysis using super- ment of motors to cellular cargos [1] and direct modulation of motor speed resolution live cell imaging provides evidence that the NM2A actomyosin and direction [2]. Here we have worked to create optimized and diversified cytoskeleton serves as an intracellular scaffold for microtubules. The loss engineered myosin motors with velocities that can be optically controlled of microtubules results in a loss of spontaneous linearization and loss of using dynamic changes in lever arm geometry. Previous designs for light- adaptive CG cell morphogenesis. The loss of an actomyosin scaffold results activated gearshifting [2] were non-processive, and suffered from either in a selective loss of spontaneous linearization restricted to the isotropic sur- low velocities (< 10 nm/s) or modest degrees of velocity modulation face, but preserves CG on the anisotropic surface where microtubules are ( 15%) in response to light. These limitations preclude many applications guided by an extracellular scaffold. During CG, the cooperation of microtu- in cell biology, devices, and reconstituted systems. We have now engineered bules and the NM2A paralog is required for cell linearization and directional (i) non-processive myosin motors that combine large optical modulation locomotion whereas the NM2B paralog contributes to cell multi-axiality depths with high velocities and (ii) processive myosin motors with optically that is important for random walk motility or navigation in complex CG controllable directionality. We have characterized a series of optimized con- environments. structs using in vitro motility assays of propelled actin filaments, single- molecule tracking of processive complexes, and live-cell imaging of indi- 1283-Pos vidual motors tagged with fluorescent protein arrays [3]. For non- Electrostatic Interactions Within Loop 1 and the Force Generation Region processive plus-end directed myosins, the most responsive constructs are of Human Cardiac Myosin Affect the Rate of Actomyosin Dissociation and approximately stopped in the dark and reach several microns/second in ADP Release the presence of blue light. We also report on speed modulation in minus- Akhil Gargey1,2, Jinghua Ge3, Alex Grdzelishvili4, Yaroslav Tkachev5, end directed motors. For processive myosins, we demonstrate controllable Yuri E. Nesmelov2,4. bidirectional transport, and observe subcellular localization at filopodial 1Biology, Univ North Carolina at Charlotte, Charlotte, NC, USA, 2Center for tips under optical stimulation. An extended set of optogenetic motors will Biomedical Engineering and Science, University of North Carolina at contribute to an expanded toolkit for programmable control of nanoscale Charlotte, Charlotte, NC, USA, 3Department of Cellular and Molecular transport and force generation. Physiology, Pennsylvania State University College of Medicine, Hershey, [1] P. van Bergeijk, et al. (2015) Nature 518, 7537. PA, USA, 4Department of Physics and Optical Science, Univ North Carolina [2] M. Nakamura et al. (2014) Nat. Nanotechnol 9, 693. at Charlotte, Charlotte, NC, USA, 5Biology, V.A. Engelhardt Institute of [3] R.P. Ghosh et al. (2017) BioRxiv https://doi.org/10.1101/159004. Molecular Biology, Moscow, Russian Federation.

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We have studied the kinetics of ATP induced actomyosin dissociation and ADP time averaged synthetic cryo-EM maps generated from MD overlapped well release in two mutants of beta isoform of the human cardiac myosin S1. The with the cryo-EM data, including the gradual decrease in resolution going mutations were introduced in the Loop 1 (D208Q: K450L) and in the force- from the actin binding interface to the end of the lever arm helix (LAH). generating region (R694N) of the myosin head. Both mutants support myosin Residue-residue cross correlation analysis from MD based on the myo1b C- ATPase activity. We introduced these mutations to remove electrostatic inter- alpha atoms, together with network analysis of correlated motions, were used actions between charged residues in the specified domains of myosin S1. Our to create a dynamic protein structure network depicting the internal dynamic computational analysis showed that in the wild-type myosin, permanent salt coordination within myo1b. This coordination differs among AM.ADPA, bridges are present in the specified regions of the beta isoform. Those salt AM.ADPB and AM states. Among key structural elements associated with bridges are absent in alpha isoform. Since the kinetics of alpha isoform is an the nucleotide binding site [i.e. P-loop, switch-I, switch-II, and helix-loop- order of magnitude faster than the kinetics of beta isoform, we hypothesize helix motif (HH, HF helices, Loop-1)], major communication changes occur that electrostatic interactions in the specified regions play a crucial role in only within the helix-loop-helix motif. At the interface of the N- terminal sub- myosin kinetics regulation. Obtained experimental data were analyzed using domain, converter and LAH, only the interactions of loop-5 with the N-terminal two models, assuming either rapid equilibrium between actomyosin and nucle- extension and the LAH change substantially. Our results provide new details otide or slow dissociation of ADP from actomyosin. We discuss the effect of regarding the allosteric coupling critical for myosin motor domain function mutations on the retention of the nucleotide by actomyosin and the potential that are involved in Mg.ADP release. role of electrostatic interactions in the Loop1 and in the force-generating region of human cardiac myosin on its regulation. We conclude that these salt bridges 1286-Pos are essential for the proper functioning of beta isoform despite their absence in The S217A Mutant Slows the Power Stroke and Phosphate Release in the alpha isoform. Myosin V Laura K. Gunther, Wanjian Tang, Christopher M. Yengo. 1284-Pos Dept Cell/Molec Physiol, Pennsylvania State Univ, Hershey, PA, USA. Targeting Mechanoresponsive Proteins in Pancreatic Cancer: 4-Hydrox- We examined the coordination between lever arm swing and specific yacetophenone Blocks Dissemination and Invasion by Activating MYH14 biochemical steps in the myosin V ATPase cycle. In our previous work Alexandra Surcel1, Eric S. Schiffhauer1, Dustin G. Thomas1, Qingfeng Zhu2, we have examined the recovery and power stroke steps in real time with Kathleen DiNapoli3, Maik Herbig4, Oliver Otto4, Angela Jacobi4, a high resolution FRET system that utilizes FRET between the N-terminus Martin Kr€ater4, Katarzyna Plak4, Jochen Guck4, Elizabeth M. Jaffee5, of myosin V and calmodulin bound to the first IQ domain. We found that the Pablo A. Iglesias6, Robert A. Anders2, Douglas N. Robinson1. power stroke occurs in two steps in myosin V, a rapid rotation of the lever 1Dept Cell Bio, Johns Hopkins Univ School of Medicine, Baltimore, MD, arm that is faster than the observed rate of phosphate release and a slow USA, 2Dept of Pathology, Johns Hopkins Univ School of Medicine, rotation that occurs during the transition between actomyosin.ADP states. Baltimore, MD, USA, 3Dept of Electrical and Computer Engineering, Johns In the current study, we have introduced the mutation S217A into myosin Hopkins Univ, Baltimore, MD, USA, 4TU Dresden, Dresden, Germany, V that is known to slow the phosphate release rate constant in myosin V 5Dept Oncology, Johns Hopkins School of Medicine, Baltimore, MD, USA, and predicted to slow phosphate entry into the phosphate release tunnel. 6Dept of Electrical and Computer Engineering, Johns Hopkins University The mutation also slows the ATP hydrolysis rate constant 10-fold without Whiting School of Engineering, Baltimore, MD, USA. altering the hydrolysis equilibrium constant. We found that the S217A Metastasis is complex, involving multiple genetic, epigenetic, biochem- mutant slows the maximum actin-activated ATPase rate 2-fold and the in vi- ical, and physical changes in the cancer cell and its microenvironment. tro motility sliding velocity 3-fold. We found that the maximum rate of the Cells with metastatic potential are often characterized by altered cellular recovery stroke is 60% faster in S217A than wild-type, which demonstrates contractility and deformability, lending them the flexibility to disseminate priming the active site for ATP hydrolysis is uncoupled from the recovery and navigate through different microenvironments. We demonstrate for stroke in this mutant. We found that S217A slows the fast power stoke the first time that mechanoresponsiveness is a hallmark of pancreatic can- 10-fold, which is similar to the degree of slowing of phosphate release. cer cells. Key mechanoresponsive proteins, those which accumulate in Interestingly, the S217A mutant is more sensitive to phosphate inhibition response to mechanical stress, specifically nonmuscle myosin IIA of in vitro motility. Thus, our work is consistent with a model in which (MYH9) and IIC (MYH14), a-actinin 4, and filamin B, have elevated phosphate entry into the phosphate tunnel is required for the fast power expression in pancreatic cancer. Their less responsive sister paralogs - stroke, while phosphate release from the tunnel is uncoupled from lever myosin IIB (MYH10), a-actinin 1, and filamin A - show a smaller change arm swing. in expression or disappear with cancer progression. We demonstrate that proteins, whose cellular contributions are often overlooked due to their 1287-Pos low abundance, can have profound impact on cell architecture, behavior, Deciphering Load-Induced Anchoring by Myosin VI and mechanics. Here, the low abundant myosin IIC promotes metastatic Rosemarie E. Gough, Natalia Fili, Yukti Hari Gupta, behavior and can be exploited with 4-hydroxyacetophenone (4-HAP), Christopher P. Toseland. which increases myosin IIC assembly, stiffening cells. As a result, 4- Bioscience, The University of Kent, Canterbury, United Kingdom. HAP decreases dissemination and induces cortical actin belts in spheroids, Myosin VI (MVI) is an unconventional myosin with the unique ability to and slows retrograde actin flow. 4-HAP also reduces liver metastases in move towards the minus end of actin filaments. MVI functions in a wide range human pancreatic cancer-bearing nude mice. Thus, increasing myosin of cellular processes, including endocytosis, exocytosis, cell migration and IIC assembly overwhelms the ability of cells to polarize and invade. Spe- transcription. This multi-functionality is achieved through an ability to act cifically, targeting the mechanoresponsive proteins of the actin cytoskel- as both cargo transporter and a load-induced anchor. Although MVI is an eton may provide a new strategy to improve the survival of pancreatic extensively studied molecular motor, many aspects of its regulation remain cancer patients. elusive. Here, we present novel insights into how MVI senses load and the impact it has 1285-Pos upon cellular functions. In particular, we focus upon the regulation of nuclear State Dependent Dynamic Coupling in Myo1B During the Force Sensitive MVI and how load-induced anchoring is critical for stabilising transcription Transition and MgADP Release hubs for gene expression. In this study, we engineered MVI to contain a molec- Ahmet Mentes, Henry Shuman, E. Michael Ostap. ular spring within the tail domain to dissipate load within the protein. We then Pennsylvania Muscle Institute and Department of Physiology, Perelman performed a variety of biochemical and cell-based assays to investigate the School of Medicine, Univ Pennsylvania, Philadelphia, PA, USA. impact on both mechanism and biological function. Myosins adjust their power outputs in response to mechanical loads in an isoform-dependent manner, resulting in their ability to dynamically adapt to 1288-Pos a range of motile challenges. We recently resolved the near-atomic resolution Kinetic Model for Myosin Gating and Backward Stepping Mechanisms structures of one rigor (AM) and two ADP-bound states (AM.ADPA, Mauro L. Mugnai1, Matthew A. Caporizzo2, Yale E. Goldman2, AM.ADPB) of myosin-IB (myo1b) bound to actin, determined by cryo- Dave Thirumalai1. electron microscopy (cryo-EM) to understand the mechanism of mechanical 1Dept Chemistry, The University of Texas at Austin, Austin, TX, USA, force-sensing by myo1b. Atomic models of myo1b flexibly fit with molecular 2Department of Physiology, Pennsylvania Muscle Institute, Perelman School dynamics simulations (MD) were stable over 200 ns for all three states. The of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

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Members of the myosin superfamily form ‘‘processive’’ dimers, meaning expressed in the atria and encodes myosin binding protein H-like (MyBP- they take many consecutive mechanical steps per diffusional encounter HL), a previously unstudied myofilament component with homology to with an actin filament. Processivity requires synchronization between the MyBP-C. Deletion of the Mybphl gene in mice also produces dilated cardio- two heads, or gating, to ensure that the trailing head steps first and binds myopathy and arrhythmias, confirming MyBP-HL’s importance in the heart. forward, which is necessary for unidirectional motility. Synchronization, Using novel tools, we identified MyBP-HL-positive ventricular cardiomyo- which is thought to occur through ‘‘biochemical gating’’ due to inter-head cytes in wild-type mouse hearts, finding that Mybphlþ/ ventricles only tension created by the strain in the two-headed attachment, modifies the have 10% as many MyBP-HL-positive cells. MyBP-HL co-localized with rates for nucleotide binding, hydrolysis and/or release to favor detachment the VCS marker contactin-2 (Cntn2) near the atrioventricular (AV) node of the trailing head. Alternatively, the myosin power stroke may bias reat- and in a subset of Cntn2-positive Purkinje fibers. Surface telemetry revealed tachmentofthefreeheadtobindtoaforwardactinsubunit-aprocesswe third-degree AV block following propranolol administration, and intracardiac refer to as ‘‘mechanical gating’’. Evidence exists to support both the mech- pacing revealed prolonged action potential propagation through the His sys- anisms, but the dominant one is both highly debated and could be myosin tem. In the absence of MyBP-HL, atrial cardiomyocyte morphology is un- isoform specific. Myosin motors also take occasional backward steps that changed despite an increase in total atrial size. Single-cell unloaded may or may not be initiated by ATP binding. To utilize existing experi- shortening measurements showed no difference in contractile function be- mental data and infer the mechanisms governing gating, we present a gen- tween wild-type, Mybphl-/þ, and Mybphl/ atrial cardiomyocytes under eral kinetic model for the processivity of myosin dimers. By including baseline conditions. Mass-spectrometry (MS) of proteins from atrial wild- multiple pathways for forward and backward stepping, and both sponta- type, Mybphl-/þ, and Mybphl/ tissue revealed an inverse relationship be- neous and ATP-induced detachment from actin, the model predicts the con- tween MyBP-HL and cMyBP-C levels. The MS data was combined with sequences of different types of gating on processivity. Further, by fitting super-resolution imaging to model the myofilament occupancy of both these recently published data for myosin VI, we are able to predict which gating proteins. Atrial cardiomyocytes showed MyBP-HL co-localization with mechanisms are compatible with the data. Thus, the kinetic model serves as cMyBP-C in the C-zone of the A-band, but in a position closer to the M- a generalized tool for researchers to use experimentally derived metrics of line. In the absence of MyBP-HL, cMyBP-C is able to occupy additional bind- motility (processivity, backwards stepping, velocity, run length, and their ing sites closer to the M-line than normal. This inverse relationship was nucleotide dependencies) to infer the molecular gating mechanisms of confirmed using a cMyBP-C null mouse model that showed increased each myosin motor. MyBP-HL when cMyBP-C was reduced. These data outline a key role of MyBP-HL in regulating myofilament protein content in atrial and a specific 1289-Pos subset of ventricular cardiomyocytes. Aging Alters Functional Properties of Cell-Matrix Adhesions in Vascular Smooth Muscle Cells 1291-Pos 1 2 1 3 Harini Sreenivasappa , Briana Bywaters , Samuel Padgham , Song Yi Shin , dATP Reduces the Depressive Effect of Acidosis on Cardiac and Slow Jerome P. Trzeciakowski1, Christopher R. Woodman3,4, Andreea Trache1,5. 1 Twitch Skeletal Muscle Dept Medical Physiology, Texas A&M University Hlth Sci Ctr, College 1 2 1 1 2 Saffie Mohran , Mike Woodward , Romi Castillo , Matthew Whithers , Station, TX, USA, Dept Veterinary Pathobiology, Texas A&M University, 1 2 1 3 Valerie Daggett , Edward Debold , Michael Regnier . College Station, TX, USA, Dept Health and Kinesiology, Texas A&M 1 4 Bioengineering, University of Washington, Seattle, WA, USA, University, College Station, TX, USA, Dept Veterinary Physiology and 2 5 Kinesiology, University of Massachusetts, Amherst, MA, USA. Pharmacology, Texas A&M University, College Station, TX, USA, Dept Muscle acidosis occurs under ischemic and/or high workload conditions, Biomedical Engineering, Texas A&M University, College Station, TX, USA. contributing to hypo-contractility. Striated muscle myosin can use 2-deoxy- Vascular smooth muscle (VSM) cell mechanical properties and their ability ATP (dATP) during contraction, resulting in greater force at every level to adapt to external mechanical signals directly contribute to maintain of calcium activation when comparedtoATP.Wepreviouslyreported vascular contractility. Aging is associated with an impaired ability of VSM (Robinson-Hamm 2013, Biophys J, 104:484a) that transgenic mice with cells to develop contractile tension. This study aims to investigate aging ef- elevated muscle dATP have increased exercise tolerance and resistance to fects on integrin-mediated adhesion to the matrix in VSM cells isolated from (in situ) gastrocnemius muscle fatigue. The Debold lab (Longyear 2014, soleus muscle feed arteries from young (4 mo) and old (24 mo) Fischer 344 JAPI 116:1165-74) demonstrated that the acidosis-induced depression in rats. Total internal reflection fluorescence imaging of young and old fixed thin filament velocity in a motility assay was significantly reduced using VSM cells stained with specific antibodies for vinculin, pFAK397, integrins dATP vs. ATP with fast skeletal muscle myosin. Thus, we hypothesized a b 5 and 3 showed that morphology of cell-matrix adhesions is age- that dATP provides a protective effect against reduced contractility in stri- dependent. Young cells presented well-defined streak-like adhesions rich in ated muscle. To test this hypothesis, we are measuring the calcium depen- vinculin distributed all over the basal cell area, while old cells presented dence of force in demembranated rat cardiac and soleus muscle, in smaller adhesions distributed mostly toward the cell edges. Quantitative conjunction with in-vitro motility assays under normal (7.0) and acidic analysis of fluorescence images showed that the protein area for both (6.8) conditions. In preliminary studies, decreasing pH from 7.0 to 6.8 in pFAK and vinculin was two-fold lower in old cells compared with young pCa 5.2 activation solution (50% of Fmax) reduced steady state specific a cells. In addition, integrin 5 localization at cell-matrix adhesions decreased force of cardiac muscle by 57% when ATP was the myosin substrate b in old cells, while no change was found in integrin 3 localization at periph- for contraction. However, when ATP was replaced with dATP as the con- eral adhesions. Moreover, atomic force microscopy functional measurements tractile substrate, the force decreased by only 25%. These data support a b of integrin 5 1 binding to fibronectin show that both adhesion force to the our hypothesis that dATP may alleviate the depressive effects of acidosis matrix and adhesion probability were increased in old cells. Taken together, on myosin crossbridge cycling and muscle contractile capacity. We previ- these data suggest that aging is associated with a reduction in proteins essen- ously reported that dATP alters myosin structure in a manner that may pro- a b tial for VSM cell adhesion, while the increase in integrin 5 1 binding to the mote enhanced binding to actin (Nowakowski 2017, Protein Sci 26:749-62), matrix is most likely a compensatory mechanism to maintain cell-matrix and are currently investigating whether this provides a mechanism for attachment in aged cells. improved resistance to hypo-contraction during muscle acidosis in striated muscle. Supported by R56AG055594 and R01HL128368 to MR and 14GRNT20450002 to ED Posters: Cardiac Muscle Mechanics and * Transgenic mouse data reported at 2013 Biophysical Society meeting by Jac- Structure queline Robinson-Hamm. Study with David Marcinek.

1290-Pos 1292-Pos Myosin Binding Protein H-Like Regulates Myofilament Content in Atrial The Mechanistic Role of Tropomyosin Overlap Dysregulation in Early and a Subset of Ventricular Conduction System Cardiomyocytes Cardiomyopathic Disease Progression David Y. Barefield1, Sheema Rahmanseresht2, Thomas O’Leary2, Melissa L. Lynn1, Teryn A. Holeman1, Lauren Tal-Grinspan2, Jordan J. Sell1, Megan J. Puckelwartz1, Lisa D. Wilsbacher1, Andrea Deranek3, Jil C. Tardiff4. Michael J. Previs2, Elizabeth M. McNally1. 1Sarver Heart Center, Univ Arizona, Tucson, AZ, USA, 2Medicine, Columbia 1Center for Genetic Medicine, Northwestern University, Chicago, IL, USA, University, New York, NY, USA, 3Biomedical Engineering, Univ Arizona, 2University of Vermont, Burlington, VT, USA. Tucson, AZ, USA, 4Medicine, Univ Arizona, Tucson, AZ, USA. A premature stop variant in the MYBPHL gene associates with human dilated An oft-noted component of sarcomeric dilated (DCM) and hypertrophic cardiomyopathy and cardiac conduction system disease. MYBPHL is highly (HCM) cardiomyopathies is that primary mutations can exhibit significant

BPJ 9370_9373 262a Monday, March 4, 2019 phenotypic variability. This variability extends even to families carrying the 1294-Pos same mutation suggesting that independently segregating genetic modifiers Power-Load Characteristics of Human-Derived Engineered Heart Tissue exist that differentially affect susceptible individuals. In addition, recent clin- in Response to Cardiomyopathy Mutations and Myosin-Targeted Drugs ical studies have demonstrated the importance of early stage treatment, Lorenzo R. Sewanan, Stuart G. Campbell. emphasizing the mechanistic role of the primary biophysical disruption Biomedical Engineering, Yale Univ, New Haven, CT, USA. ‘‘domain’’ on pathogenesis. One possible site of primary biophysical disrup- Comprehensive functional characterization of cardiac tissue necessarily in- tion is the crucial tropomyosin (Tm)-overlap which provides the stability and cludes investigation of length and load dependence. Such measurements flexibility required during cross bridge cycling. We hypothesize that the car- have been slow to develop in engineered heart tissues (EHTs), whose mechan- diac thin filament (CTF) has an innate but finite capacity to tolerate changes in ical characterizations have been constrained primarily to isometric force behav- these parameters, beyond which cardiomyopathies result. Highly penetrant iors. A more realistic analog to myocardial function would be isotonic mutations throughout the CTF were studied using differential scanning calo- measurements and resultant force-velocity curves. Here we present the first rimetry to assess changes in thermodynamic stability and flexibility of fully measurements of force-velocity and power-load curves in EHTs. Furthermore, reconstituted CTF’s. N-terminus proximal Tm mutations (E62Q, D84N) we harness the flexibility of human induced pluripotent stem cell-derived car- decreased Tm-cTn stability while increasing flexibility, where C-terminus diomyocytes (iPSC-CMs) in EHTs to study the effects of the myosin activator proximal Tm mutations (D219N, D230N) do the opposite. Strikingly these omecamtiv mecarbil (OM) and the effect of MYBPC3 knockout on power-load proximal-terminal dependent effects occurred independent of late-stage pa- characteristics within a human context. EHTs were generated by seeding hu- thology. Mutations in cardiac troponin T (cTnT), however, only increased man induced pluripotent stem cell-derived cardiomyocytes onto decellularized thermal stability and flexibility when directly adjacent to the overlap. Further- laser cut porcine myocardium as previously described. By simultaneously more, we investigated the role of developmental cTnT isoform switching on measuring force and controlling muscle length, an adaptive iterative control the Tm-overlap to assess its capacity as an independent modifier. Fetal- scheme achieved isotonic contractions at prescribed force values. The scheme cTnT alone had little effect, however, when expressed with D230N-Tm, fila- typically converged to the desired load clamp within four iterations. Force ments exhibited an additive decrease in flexibility of the Tm-overlap. Fetal- clamp was achieved at designated levels of twitch force between 10% and cTnT did not cause additive changes when expressed with D84N-Tm and 80% of maximum, allowing construction of a traditional force-velocity curve. R92L-cTnT. Similarly, mice expressing D230N-Tm and fetal-cTnT exhibited Preliminary study of OM reveals that it impacts power-load characteristics additively reduced % fractional shortening whereas R92L-cTnT did not more potently than isometric force production. Preliminary work on MYBPC3 display additive changes, suggesting fetal-cTnT may be a mutation-specific KO versus a heterozygous MYBP3 control shows that MYBPC3 seems to modifier. Together these data suggest that the Tm overlap represents a sensi- decrease power output at all loads. In summary, we demonstrate a robust tive ‘‘domain’’ of early biophysical dysregulation in cardiomyopathic disease method for the generation of isotonic contraction experiments and force- progression. velocity curves in engineered heart tissues using an adaptive iterative method. We are currently utilizing the technique to further investigate sarcomeric drugs 1293-Pos and sarcomeric protein function, particularly in the context of hypertrophic Myosin and Mybp-C-Mutations in Hypertrophic Cardiomyopathy: Vari- cardiomyopathy. able Effects on Calcium Sensitivity and Contractile Imbalance from Cell to Cell 1295-Pos Mirza Makul1, Ante Radocaj1, Pia Ernstberger1, Judith Montag1, Myofilament Length Dependent Activation in Porcine Cardiac Muscle Kathrin Kowalski1, Britta Keyser2, Andreas Perrot3, Cris G. dos Remedios4, Weikang Ma1, Robert Anderson2, Marcus Henze2, Henry Gong1, Bernhard Brenner1, Theresia Kraft1. Fiona Wong2, Carlos del Rio2, Thomas Irving1. 1Dept Molec/Cell Physiol, Hannover Med Sch, Hannover, Germany, 2Dept 1BioCAT, Illinois Institute of Technology, Chicago, IL, USA, 2MyoKardia Human Genetics, Hannover Med Sch, Hannover, Germany, 3ECRC, MDC, Inc, South San Francisco, CA, USA. Charite Medical Faculty, Berlin, Germany, 4Dept Anat/Hist, Univ Sydney, Myofilament length dependent activation (LDA) is the underlying cellular Sydney, Australia. mechanism for of the Frank-Starling law of the heart. The molecular mecha- Heterozygous mutations of ventricular myosin heavy chain (b-MyHC, nism(s) for LDA are not yet fully understood. Here we used small angle X- MYH7) and myosin binding protein C (c-MyBP-C) frequently cause Hyper- ray diffraction to study the structural differences in skinned porcine cardiac trophic Cardiomyopathy (HCM). We hypothesize that variable levels of muscle fibers at short (2mm) and long (2.2mm) sarcomere length (SL). The mutated protein could result in contractile imbalance of the cardiomyocytes equatorial intensity ratio (I1,1/I1,0), an indicator of the proximity of myosin and eventually trigger cardiomyocyte disarray and fibrosis, hallmarks of heads to the actin filament, increased at longer SL in both relaxed (pCa=8.0) HCM. and contracted (pCa=4.0) muscle. This indicates that in relaxed muscle myosin Here we functionally characterized individual cardiomyocytes isolated from heads were more associated with actin at longer SL, which results in more cardiac myectomy tissue of a HCM-patient with MYH7-mutation Ala200Val, crossbridges attached to actin during contraction. The intensities of the third or- and a HCM-patient with two cMyBP-C mutations, Arg589His and der myosin meridional reflection (IM3), arising primarily from adjacent myosin Glu1096AspfsX93. For the A200V-mutation also the relative expression of crowns on the thick filament, decreased at longer SL indicating a reduction in mutated/wildtype MYH7-mRNA in individual microdissected cardiomyocytes myosin head ordering upon stretch. Interestingly, the stretch-induced increase was determined. in I11/I10 and a decrease in IM3 contradicts previous findings from rodent muscle At full calcium activation the A200V-cardiomyocytes had significantly (Farman et al., AJP Heart. 2011; 300H2155). The third order troponin reflection reduced isometric force (Fmax) and rate constant for force redevelopment (Tn3), however, increased in intensity at longer SL as reported previously (Ait- (kTR), compared to healthy control cardiomyocytes. They also showed Mou et al. PNAS. 2016;113:2306-1). The ratio of the high to the low angle reduced calcium sensitivity which was highly variable from cell to cell peaks in the M3 reflection (RM3), due to the interference effects of myosin with some cells generating substantially reduced force suggesting high heads from each half of the sarcomere, increased upon stretch, in the same di- levels of mutated protein, while others were like controls. The fractions of rection as seen during isometric contraction, indicating a conversion of the mutated/wildtype MYH7-mRNA in individual cardiomyocytes were also myosin heads from the OFF towards the ON state. We propose, therefore, highly variable. Previous analysis of active MYH7-transcription sites in car- that increased contractility upon preload/stretch (i.e., LDA) in healthy cardiac diomyocytes suggested that stochastic, burst-like transcription could pro- muscle results in part from the stretch-dependent recruitment of myosin heads duce variable fractions of mutated and wildtype mRNA and protein from from the folded-back SRX to an extended, less ordered state. Supported, in part, cell-to-cell. by NIH P41 GM103622. The cardiomyocytes with cMyBP-C mutations showed only slightly reduced 1296-Pos Fmax and kTR and no change in calcium sensitivity. Variability of force-pCa curves of these cardiomyocytes was undistinguishable from donor Annulment of Cardiac Muscle Length-Dependent Force Activation in cardiomyocytes. Transgenic Mice Bearing the HcTnT-I79N Mutation 1 2 3 Both HCM-patients exhibited a similar phenotype, with A200V- Maicon Landim Vieira , Bjorn Knollmann , Hyun S. Hwang , 1 4 cardiomyocytes showing large variability in expression levels and measured J. Renato Pinto , P. Bryant Chase . 1Biomedical Sciences, Florida State University, Tallahassee, FL, USA, functional parameters from cell to cell. Assuming that this contractile imbal- 2 ance triggers the HCM-typical cellular/myofibrillar disarray and fibrosis, for Division of Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, USA, 3Nutrition, Food and Exercise Sciences, cardiomyocytes with the cMyBP-C-mutations it is not clear whether another 4 parameter like shortening velocity is variably altered by the mutations from Florida State University, Tallahassee, FL, USA, Department of Biological cell to cell. Sciences, Florida State University, Tallahassee, FL, USA.

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Missense mutation Ile79Asn in human cardiac troponin T (HcTnT-I79N) Here we characterised the OFF-to-ON structural transition in the cardiac thick has been linked to phenotypic outcomes of hypertrophic cardiomyopathy filament induced by cooling from 37Cto9C by X-ray diffraction from intact (HCM) followed by adverse consequences including arrhythmias and sud- and demembranated trabeculae isolated from rat hearts. Thick filament struc- den cardiac death. Little else is known about changes in cardiac muscle me- ture in demembranated trabeculae in relaxing solution in the presence of 3% chanics caused by the mutation. This study investigates cross-bridges dextran T500 at 27-37C was similar to that in intact quiescent trabeculae. kinetics of left ventricular papillary muscle bundles (Ca2þ-sensitivity of iso- Maximal calcium activation at 27C was used as a reference for the ON-state. metric force, sinusoidal stiffness and rate of tension redevelopment (kTR)). Cooling of intact quiescent trabeculae induced a decrease in the intensity Cardiac tissues were harvested from both non-transgenic wild-type (NTg) of the meridional myosin-based reflections and of the first myosin layer mice and transgenic mice bearing the HcTnT-I79N mutation. Sarcomere line, indicating disruption of the OFF-state of the thick filament. This was lengths (1.9, 2.1, and 2.3 mm) were set at pCa 8.0 using HeNe laser. Sinu- accompanied by an increase in the spacing of the M6 reflection associated soidal stiffness (0.2% PTP length oscillation) and kTR were obtained during with the axial periodicity of the thick filament backbone (SM6), a biphasic Ca2þ activation when force was at least 20% of maximum. Increased Ca2þ- change in that of the M3 reflection associated with the myosin motors sensitivity of isometric force was observed in NTg cardiac preparations (SM3), and an increase in the equatorial intensity ratio (I11/I10)associated upon an increase in SL (1.9 vs 2.1 mm, and 2.1 vs 2.3 mm). Interestingly, with the mass distribution of myosin motors within the filament lattice. HcTnT-I79N exhibited an increased Ca2þ-sensitivity at SL 1.9 vs 2.1 mm Cooling of demembranated trabeculae in relaxing solution showed larger only. No further increase was observed at SL 2.1 vs 2.3 mm. This blunt ef- changes in the intensity and spacing of the myosin-based reflections, and fect indicates an abolishment of length-dependent activation at SL 2.1 vs 2.3 SM3, SM6 and I11/I10 in relaxing solution at 9 C were close to the ON- mm. Although HcTnT-I79N mutation exhibited a significant lower maximal state values. kTR, neither NTg nor HcTnT-I79N showed significant changes in maximal The results show that cooling to 9 C induces a structural OFF-to-ON transition kTR regarding length-dependent activation. After compressing the myofila- in the thick filament, and the effect is larger in demembranated than in intact ment lattice spacing using 3% Dextran T500 (SL 2.1 mm), only NTg cardiac quiescent trabeculae. preparations displayed increased myofilament Ca2þ sensitivity. No changes Supported by BHF, UK. in maximal kTR were observed for both NTg and HcTnT-I79N in the pres- ence of dextran. In addition, no significant changes in passive force, 1299-Pos maximal force and cooperativity of thin filament activation were observed Anti-S2 Peptides Modulate Myosin Coiled Coil Structure and Shift between groups. Experiments were carried out at 21C. Analysis of math- Force-PCa Curves in Human Cardiac Muscle ematical modeling of force and sinusoidal stiffness are in progress. NIH- Bertrand C.W. Tanner1, Kenneth S. Campbell2, Motamed Qadan3, HL128683. Negar Aboonasrshiraz4, Dua’a Quedan4, Peter O. Awinda5, Andrea Bernardino-Shaefer3, Douglas D. Root3. 1297-Pos 1Dept Integrative Physiology and Neuroscience, Washington State Univ, Development of an Atomistic Structure of Myosin-Bound Cardiac Thin Pullman, WA, USA, 2Dept Physiology, Univ Kentucky, Lexington, Filament and Free Energy Determination of the Close-to-Open Transition KY, USA, 3Dept Biol Sci, Univ North Texas, Denton, TX, USA, 4Univ Anthony Baldo, Steven D. Schwartz. North Texas, Denton, TX, USA, 5Washington State Univ, Pullman, Chemistry and Biochemistry, The University of Arizona, Tucson, AZ, USA. WA, USA. Myosin represents a superfamily of motor proteins that is diverse both in ho- The binding of myosin heads to the subfragment-2 (S2) coiled coil is a com- mology as well as function and whose association with actin filaments in a mon regulatory mechanism for many types of myosin and growing evidence ATP-dependent manner is often correlated with muscle contraction. With indicates that it also plays a significant role in human cardiac muscle. To test recent advances in Cryo-Electron Microscopy, high resolution images of this hypothesis, anti-S2 peptides were used to either increase (Stabilizer) or various morphologies of myosin interacting with actin has allowed for the decrease (Destabilizer) the stability of the S2 coiled coil and its interaction visualization of the specific interactions between myosin, actin, and tropomy- with myosin heads. The Stabilizer increased the spectroscopically measured osin, but little is still known how these interactions change as myosin pro- alpha-helical content of wild type human cardiac S2 peptides with a gresses through its ATP hydrolysis cycle. Another issue is the lack of a maximum response at one Stabilizer per two S2 peptides. The hypertrophic high resolution structure of human cardiac myosin bound to actin where cardiomyopathy causing E930del mutant interfered with the heptad repeat mutagenic changes in the myosin head have been associated with the devel- of the coiled coil and caused low alpha-helical content which was greatly opment of cardiomyopathies within individuals. In the current study, we increased upon adding Stabilizer up to equal molar concentration with the employ molecular dynamics (MD) simulations as well as other computa- myosin peptides. Dynamics simulations gave a similar result in that the tional methods to study how the beta isoform of cardiac myosin (MYH7) in- wild type human cardiac S2 was stable during a 16 ns simulation, but the teracts with the cardiac thin filament (CTF). Utilizing free energy E930del mutant peptides dissociated during a 16 ns simulation. However, calculations such as the adaptive biasing force (ABF) method, we will probe when two Stabilizers were added, the coiled coil remained intact for the 16 the mean force that a single myosin head must exert on tropomyosin to tran- ns simulation. FRET between two labeled human cardiac myosin S2 peptides sition from the closed to open transition. With these simulations we hope to found that Stabilizer increased coiled coil formation of the E930del, while De- be able to evaluate the overall energetics of the transition, the possibility of a stabilizer decreased coiled coil formation of the wild type. Super-resolution preferential, initial binding site of myosin on the thin filament, and the over- microscopy further demonstrates binding of both the Stabilizer and Destabil- all cooperative effect of myosin binding. Development of this novel atom- izer to the A-bands of myofibrils. When added to human cardiac muscle, the istic myosin-CTF structure provides a unique insight into the specific force-pCa curves were shifted to the right by the Stabilizer or to the left by the structure and dynamics of cardiac myosin and allows for further studies of Destabilizer in a sarcomere length dependent manner consistent with the the development of cardiomyopathies as well as the dynamics of the cross- hypothesis. bridge cycle. 1300-Pos 1298-Pos Impact of Human Beta-Cardiac Myosin Mutation Implicated in Both The Off-To-On Transition of Thick Filaments in Isolated Trabeculae from Hypertrophic and Dilated Cardiomyopathy Rat Heart Induced by Cooling Wanjian Tang, Laura K. Gunther, Jonathan Cooper, Rohini Desetty, Jesus Garcia Ovejero1, Luca Fusi1, So-Jin Park-Holohan1, Christopher M. Yengo. Andrea Ghisleni1, Theyencheri Narayanan2, Malcolm Irving1, Dept Cell & Molec Physiol, Penn State Coll Med, Hershey, PA, USA. Elisabetta Brunello1. Hypertrophic (HCM) and dilated cardiomyopathy (DCM) are autosomal domi- 1Randall Centre of Cell and Molecular Biophysics, King’s College London, nant diseases in which affected individuals are heterozygous for gene mutations London, United Kingdom, 2European Synchrotron Radiation Facility, in sarcomeric proteins, including beta cardiac myosin. HCM is characterized by Grenoble, France. left ventricular (LV) hypertrophy, myocyte disarray, and LV hypercontraction. In isolated thick filaments from cardiac muscle in relaxing conditions the DCM is indicated by LV dilation with impaired contraction of the left or both myosin motors take up a quasi-helical arrangement on the filament surface in ventricles. There is evidence to suggest that mutations which increase myosin a conformation that inhibits their ATPase, known as the super-relaxed or contractility cause HCM, and mutations that reduce myosin contractility cause OFF-state, suggesting that contraction of cardiac muscle requires activation DCM. We examined a missense mutation (R243H) in the switch 1 nucleotide- of the thick filament in addition to calcium-induced activation of the thin sensing region of the human beta-myosin heavy chain (b-MYHC, MYH7) filament. implicated in both HCM and DCM. R243 is highly conserved in myosins

BPJ 9370_9373 264a Monday, March 4, 2019 and participates in a salt-bridge between the switch I and switch II nucleotide 1303-Pos binding regions. We observed very minimal actin-activated ATPase activity for Human Beta-Cardiac Myosin Cardiomyopathy Mutations R712L and the mutant compared to WT (2-fold compared to 100-fold activation, respec- E497D Disrupt a Key Salt-Bridge in the Coupling Domain tively), while the basal ATPase was unchanged. R243H bound to actin in an Bipasha Barua1, Jennifer L. Atherton2, Eva Forgacs2, ATP-dependent manner based on actin-cosedimentation assays. We also Donald A. Winkelmann1. observed no actin-sliding by R243H in the in vitro motility assay. In the mixed 1Dept Pathology and Laboratory Medicine, Rutgers Robert Wood Johnson motility assay, low ratios (%50%) of R243H did not change or only slightly Medical School, Piscataway, NJ, USA, 2Dept Physiological Sciences, Eastern decreased sliding velocities. At higher ratios of R243H (R50%) the velocity Virginia Medical School, Norfolk, VA, USA. decreased dramatically. We speculate that at low ratios of R243H/WT the Mutations in the human beta-cardiac myosin gene (MYH7) are responsible for mutant myosin may cooperatively activate the actin thin filaments to allow a large number of hypertrophic (HCM) and dilated (DCM) cardiomyopathies. more opportunity for WT myosin heads to bind actin without slowing short- Omecamtiv mecarbil (OM) is a small molecule drug that induces allosteric ening velocity, which would correlate with HCM. At higher ratios of R243/ changes in beta-cardiac myosin activity and is in clinical trials for treatment WT, the inhibitory action of R243H may dominate and slow muscle shortening, of systolic heart failure. OM binds in a narrow cleft adjacent to the SH1 helix, which would correlate with DCM. relay helix, and converter domain of beta-cardiac myosin and interacts with res- idues involved in linking changes in the nucleotide pocket to rotation of the 1301-Pos lever arm, known as the ‘coupling region’. Cardiomyopathy mutations Molecular Dynamics Studies of Single Point Mutations in the Cardiac Thin R712L and E497D disrupt a salt bridge between R712 and E497 that is centered Filament in the OM binding pocket and plays a role in coupling the relay helix to a small Allison B. Smith, Anthony Baldo, Natercia Braz, Steven D. Schwartz. beta-sheet of the converter domain. In the present study, we have characterized Chemistry, Univ Arizona, Tucson, AZ, USA. the effect of the mutations and OM on the unloaded shortening velocity of Single amino acid mutations to the cardiac thin filament have been shown to R712L and E497D beta-cardiac HMM mutants. The R712L mutation causes cause genetic cardiomyopathies, a serious and sometimes deadly heart disorder. a 5-fold slowing of motility, and increases the IC50 of OM binding to 30 Experimental groups have extensively studied many of these mutations in an mM from 0.1 mM for the WT cHMM. OM also restores the velocity of attempt to uncover the link between genotype and phenotype, but critical R712L cHMM to 75% of WT cHMM velocity. On the other hand, the atomic level details cannot be determined by these methods. To address this E497D mutation has little effect on the velocity, but increases the IC50 of gap, molecular dynamics (MD) simulations are utilized to model and study OM to 4.5 mM. OM inhibits the velocity of E497D cHMM but only by 2.5- the changes in conformation and dynamics imposed by these single point mu- fold compared to over 25-fold for WT cHMM. In conclusion, the dramatic ef- tations. The goal of this research is to move beyond identifying the effects mu- fect on motility (R712L) and impact on the IC50 of OM (R712L and E497D) tations have on the cardiac thin filament, and to determine a set of guidelines to suggest a central role of this buried salt bridge in coupling the nucleotide state predict the clinical effects unstudied mutations will have on individuals. A set to rotation of the converter domain. of twenty different clinically determined amino acid mutations located in car- diac troponin T and tropomyosin were chosen for analysis, and MD simulations 1304-Pos were performed using the fully atomistic cardiac thin filament previously Isolating the Pathological Contribution of Detyrosinated Microtubules in created by our group. From the simulations, conformation changes imposed Human Myocardial Mechanics by the mutation, both local and distant to the site of mutation, were computed. Matthew A. Caporizzo1, Christina Y. Chen1, Kenneth Bedi2, Steered molecular dynamics along with Jarzynski’s equality were utilized to Kenneth B. Margulies2, Benjamin L. Prosser1. determine the changes in the free energy barrier of calcium binding and disso- 1Physiology, University of Pennsylvania, Philadelphia, PA, USA, 2Hospital ciation due to mutation. Coupling the changes in conformation and calcium dy- of the University of Pennsylvania, Philadelphia, PA, USA. namics data together, subsets of mutations were created to begin the process of Detyrosinated microtubules (dTyr MTs) buckle and bear load during myocyte determining guidelines for mutational predictions. contraction providing a viscoelastic resistance to myocyte shortening. The proliferation of dTyr MTs observed in heart failure increases myocyte visco- 1302-Pos elasticity but the pathological consequences on myocyte and myocardial func- Design of Biocompatible Liquid Cristal Elastomers Reproducing the Me- tion remain to be quantitatively assessed. To determine whether suppressing chanical Properties of Human Cardiac Muscle dTyr MTs will reduce viscoelasticity and improve contractile dynamics pro- Cecilia Ferrantini1, J. Manu Pioner1, Daniele Martella2, Raffaele Coppini3, portional to the severity of disease, we utilize parallel genetic and pharmaceu- Nicoletta Piroddi4, Paolo Paoli5, Martino Calamai6, Francesco S. Pavone7, tical approaches to suppress MT dTyr in freshly isolated human Diederik Wiersma2, Chiara Tesi8, Elisabetta Cerbai9, Corrado Poggesi1, cardiomyocytes and myocardial strips and measure active and passive me- Leonardo Sacconi2, Camilla Parmeggiani2. chanical properties. Compared to myocytes isolated from non-failing human 1Dept Clin/Exp Med, Univ Florence, Florence, Italy, 2European Laboratory hearts, suppression of dTyr MTs reduces viscoelasticity and increases un- for Non-Linear Spectroscopy, Sesto Fiorentino, Italy, 3Dept NeuroFarBa, loaded shortening to a greater degree in failing cardiomyocytes. Manipulation Univ Florence, Florence, Italy, 4Univ Firenze, Firenze, Italy, 5University of of dTyr MT levels does not significantly impact excitation-contraction Florence, Florence, Italy, 6Dept LENS, Univ Florence, Sesto-Fiorentino, coupling and de novo mathematical modelling demonstrates that increased Italy, 7Dept Physics, LENS, Sesto Fiorentino, Italy, 8Dept Sci Physio, Univ contractile dynamics can be attributed to the experimentally measured Degli Studi Firenze, Florence, Italy, 9Dept CIMMBA, Univ Florence, decrease in viscoelasticity. Preliminary results in myocardial strips from Florence, Italy. failing hearts display greater viscoelasticity than non-failing myocardium, Advanced materials, able to work as integrated actuators for the treatment of with depolymerization of MTs in failing myocardium partially restoring pas- muscle injuries, could combine rapid and long-lasting intervention, which is sive mechanical properties. The results indicate that dTyr microtubules the main goal in regenerative medicine. Among them, Liquid Crystalline Elas- constrain myocyte contractile dynamics in heart failure. Moreover, the micro- tomers (LCEs) are biocompatible polymers able to reversibly deform in tubules’ impact in regulating myocardial mechanical properties at rates response to a given stimulus by generating movement. Once stimulated, consistent with diastolic filling is evident in the presence of an intact extracel- LCEs can mimic muscle force production. However, so far their application lular matrix. in biology was limited by the slow response times and the reduced possibility to modulate tension levels during activation. Thanks to a screening of different 1305-Pos monomeric formulations, a palette of biocompatible LCEs is prepared and pre- Predicting and Preventing Myocardial Remodeling in a Murine Model of cisely characterized in isometric conditions in terms of passive and active me- Dilated Cardiomyopathy chanical properties, showing improved muscle-like characteristic. Light Joseph D. Powers1, Galina Flint1, Jil Tardiff2, Michael Regnier1, responsive LCEs stimulated with increasing laser powers, develop progres- Farid Moussavi-Harami3, Jennifer Davis1,4. sively increasing active tensions with fast kinetics of activation and relaxation. 1Bioengineering, University of Washington, Seattle, WA, USA, 2School of Tension levels and contraction time course can be modulate to reproduce twitch Medicine, University of Arizona, Tucson, AZ, USA, 3Cardiology, University contractions of cardiac samples from patients affected by specific diseases. As a of Washington, Seattle, WA, USA, 4Pathology, University of Washington, proof of concept, a LCE-based device able to develop concentric pressure, as in Seattle, WA, USA. cardiac chambers, was demonstrated opening for their use in the preparation of In hearts with dilated cardiomyopathy (DCM), the ventricular walls become contraction assist devices. thin and weak, causing systolic dysfunction, and interventions to prevent the

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DCM phenotype are lacking. However, we recently introduced a metric myopathies, highlighting the importance of primary structural and dynamic capable of predicting the type and severity of myocardial remodeling in pro- ‘‘triggers’’ that lead to disease. To begin to correlate genotype to phenotype, gressive cardiomyopathies (Davis et al. Cell, 2016), which hinges on relating we investigated three cardiomyopathy-linked mutations in the cTnT linker re- force-time integrals of computationally derived twitches of diseased cardio- gion: DCM-associated mutations R173W and R173Q, and HCM-associated myocytes to wild-type. Negative values of this metric (the ‘tension index’) mutation D160E. We coupled computational predictions with in-vitro biophys- correlate with eccentric hypertrophy associated with DCM, and the magnitude ical measurementsto better understand the divergent disease mechanisms. We correlates with the severity of phenotype. Here, we test the hypothesis that an hypothesize that R173Q/W and D160E cause differential structural changes in experimental analogue of the tension index can be generated from twitches of the cTF linker domain leading to atomic and molecular level changes that intact cardiac trabeculae from genetically engineered murine models of DCM, initiate pathogenic remodeling. Differential scanning calorimetry on all 3 mu- and that it will predict the degree of myocardial remodeling. Intact trabeculae tations showed an increased thermal stability required to unfold the Tm:Tn from a DCM model—a calcium-desensitizing tropomyosin mutation complex from actin, suggesting altered protein interactions. To obtain further (D230N)—have significantly decreased twitch force compared to wild-type resolution, molecular dynamics (MD) simulations and time-resolved fluores- trabeculae (1652 versus 3153 kPa, respectively). The corresponding tension cence resonance energy transfer (TR-FRET) studies were employed. Previous index for D230N trabeculae is –6.62x103 (normalized force,ms), predicting studies showed that D160E caused a decreased distance between the linker re- significant eccentric hypertrophy. Indeed, our predictions are verified by echo- gion of cTnT and the tropomyosin C-terminus, resulting in decreased linker cardiographic measurements of ventricular diameters in vivo. In D230N hearts, flexibility. In contrast, MD simulations of the TF containing R173W/Q show the left-ventricular systolic and diastolic diameters are significantly greater an increase distance between the linker region of cTnT and the C-terminus than wild-type (3.1 and 4.3mm, versus 2.4 and 3.8mm, respectively). Further- of tropomyosin and actin 374. Further clarification of these data will be pro- more, intact trabeculae from a double-transgenic (DTG) murine model, vided by ongoing TR-FRET studies to confirm structural distance changes in (D230N plus the calcium-sensitizing troponin mutation, L48Q), produce twitch the liker region in fully reconstituted cTF. This iterative process will allow forces similar to wild-type (3253 kPa). The tension index for DTG trabeculae us to build predictive tools to test cTF mutations associated with these cardio- is –1.48x103 (normalized force,ms), predicting less remodeling than D230N myopathies and eventually to design and implement more precise therapeutic alone. Consistently, systolic and diastolic diameters of DTG hearts are not interventions. different from wild-type (2.5 and 3.9mm, respectively). Our work demonstrates the ability of a trabecula-based tension index to predict organ-level morphology 1308-Pos in DCM hearts and to use these predictions to develop interventions that pre- The Role of GSK3B Mislocalization in Arrythmogenic Cardiomyopathy vent pathological remodeling. Ronald Ng, Stuart Campbell. Yale Univ, New Haven, CT, USA. 1306-Pos Arrythmogenic cardiomyopathy (ACM) is a genetic cardiomyopathy Molecular Dynamics Studies of Myosin Structure with 2-Deoxy-ADP commonly inherited through dominant mutations in desmosomal genes (JUP, Matthew C. Childers1, Weikang Ma2, Valerie Daggett1, Mike Regnier1. DSP, PKP2, DSG2, and DSC2). Characteristic phenotypes of ACM include 1BioEng, Univ Washington, Seattle, WA, USA, 2BioCAT, Illinois Institute of ventricular arrhythmias, fibrofatty replacement of the myocardium, and desmo- Technology, Chicago, IL, USA. somal instability. While mechanistic linkages remain unknown, recent work Myosin uses ATP catalysis to drive muscle contraction by facilitating cross- has identified glucose synthase kinase (GSK3b) mislocalization as another bridge cycling. During the cross-bridge powerstroke, coordinated release of common ACM phenotype. While inhibition of GSK3b has been shown to miti- the byproducts of nucleotide catalysis (Pi, ADP) triggers a series of conforma- gate disease progression, it is unclear what role GSK3b plays in destabilization tional changes in myosin that alter its interactions with actin and ultimately of the desmosome. We hypothesize that a mutation in a desmosomal protein generate force. In addition to ATP, myosins can utilize other nucleotides, causes initial desmosomal instability, and that GSK3b mislocalization to the such as the naturally occurring 2-deoxy-ATP (dATP), to catalyze the power- intercalated disk happens secondary to this insult. To test this hypothesis, we stroke. We have reported that dATP enhances cross-bridge binding and constructed engineered heart tissues (EHTs) from iPSC-derived cardiomyo- cycling dynamics, resulting in stronger, faster contraction especially in cardiac cytes with an ACM-linked point mutation (R451G) in desmoplakin. Our previ- muscle. In a recent study (Nowakowski 2017, Protein Sci 26:749-62) our sim- ous work identified the primary effect of the R451G mutation as causing ulations of myosin in the pre-powerstroke state showed that dADP induces increased susceptibility to calpain mediated degradation. This mutation pro- conformational changes that expose polar residues and enhance its affinity vides, in effect, a means of inducing targeted desmosomal instability by either for actin. In a current study using x-ray structural analysis, we found that allowing or inhibiting calpain activity. After verifying that expression of dATP increases the rate of myosin detachment following tetanic contraction. R451G desmoplakin resulted in unstable desmosomes, GSK3b mislocalization To understand the molecular basis for the enhanced rate of detachment in the was assessed via immunofluorescent microscopy. EHTs were then treated presence of elevated dATP, we performed molecular dynamics simulations of either a calpain inhibitor or a GSK3b inhibitor, and the effects on desmosonal the myosin motor domain modeled in the post-powerstroke conformation in stability, gap junction remodeling, and GSK3b mislocalization were studied. the presence of Mg2þ and either ADP or dADP. We found that dADP was This work will allow for further insights into pathomechanisms underlying more flexible than ADP, more mobile within the nucleotide binding pocket ACM. and had different interactions with myosin relative to ADP. Furthermore, changes in the interactions between dADP and myosin were transmitted to 1309-Pos the actin binding surface. Additionally, we observed partial release of the nu- Isometric and Isotonic Twitch Dynamics in Omecamtiv Mercarbil Treated cleotides from the binding pocket and have delineated the molecular mecha- Intact Rat Cardiac Trabecula nisms by which regulatory loops in myosin control nucleotide release. Our Brianna M. Schick, Alexandra R. Matus, Charles S. Chung. results have revealed molecular mechanisms underlying dATP-enhanced Dept Physiology, Wayne State Univ, Detroit, MI, USA. cross-bridge cycling dynamics. More generally they have yielded insights Contraction and relaxation are coupled in intact cardiac trabeculae. The myosin for the development of novel myosin-targeted therapies for cardiomyopathies activator Omecamtiv Mercarbil (OM) prolongs contraction in unloaded cardi- based the structural advantages produced by dATP in resting and contracting omyocytes and in vivo in canines and humans, but relaxation is not prolonged muscle. as much as contraction is prolonged in vivo. We have previously shown that- in addition to calcium reuptake, thin filament deactivation, and crossbridge 1307-Pos detachment kinetics- fast stretch (strain rate) can accelerate myocardial relax- Computational and Experimental Investigation of Cardiac Troponin T ation. We hypothesized that OM would increase strain rate dependent relaxa- R173Q, R173W and D160E Mutation Specific Correlates to Disease tion. Intact cardiac trabeculae were isolated from wild-type rats, mounted Andrea E. Deranek1, Anthony Baldo2, Steven Schwartz2, Jil C. Tardiff3. between a force transducer and length controller, superfused at 25C, paced, 1Biomedical Engineering, University of Arizona, Tucson, AZ, USA, and isometrically twitched except for load clamped beats. OM was added to 2Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA, the perfusion solution at concentrations up to 2 mM. Both developed isometric 3College of Medicine, University of Arizona, Tucson, AZ, USA. twitch force and rate of force development (dF/dt_max) decreased in rats as Mutations in cardiac troponin T (cTnT), a component of the cardiac thin fila- OM concentration increased. This reduction is consistent with prior reports ment (cTF), are known to cause changes in protein structure and dynamics lead- in rats where alpha-myosin heavy chain is expressed more highly then beta ing to pathological cardiac remodeling observed in patients with hypertrophic myosin heavy chain. Feedback control load clamps were used for loaded short- (HCM) and dilated (DCM) cardiomyopathies. In this complex system, muta- ening and to modify the end systolic strain rate. The rate of loaded shortening tions in cTnT separated by only a few amino acids can cause divergent cardio- were both reduced by OM. OM increased the sensitivity of the relaxation rate to

BPJ 9370_9373 266a Monday, March 4, 2019 strain rate; the increase at 200-400 nM OM matched the effect of a 5% reduc- provide new insight into the molecular basis for cMyBP-C effects on actin tion in preload. This increased sensitivity may be related to an OM-induced structure and function. This work was supported by an NIH R00HL122397 reduction in cooperativity and reduced myosin lever arm motion, but not the to B.A.C. slowed ATPase rate. Dynamic length changes might help prevent diastolic relaxation from becoming adversely prolonged in vivo. 1312-Pos The Hcm-Causing Y235S Cmybpc Mutation Accelerates Contractile Func- 1310-Pos tion by Altering C1 Domain Structure Multi-Timepoint RNA-Sequencing Reveals Differential Gene Expression Chang Yoon Doh, Jiayang Li, Ranganath Mamidi, Julian E. Stelzer. of Transgenic Mouse Models of Hypertrophic and Dilated Cardiomyopa- Dept Physiol/Biophys, Case Western Reserve Univ Med Sch, Cleveland, OH, thies USA. Shivani H. Desai, Jil C. Tardiff, Melissa L. Lynn, Amanda M. Richards. Mutations in cardiac myosin binding protein C (cMyBPC) are a major cause of hypertrophic cardiomyopathy (HCM). In particular, a single amino acid substi- Physiological Sciences, University of Arizona, Tucson, AZ, USA. tution of tyrosine to serine at residue 235 (Y235S) has been linked to HCM with Mutations in cardiac thin filament proteins have been linked to a range of hu- man genetic cardiomyopathies. Two highly-penetrant missense mutations strong disease association. Although cMyBPC truncations, deletions and inser- proximal to the tropomyosin overlap region, D230N in tropomyosin and tions, and frame shift mutations have been studied, relatively little is known R92L in cardiac troponin T, have been linked to dilated cardiomyopathy and about the functional consequences of missense mutations in cMyBPC. In this hypertrophic cardiomyopathy, respectively. We hypothesize that the differen- study, we characterized the functional and structural effects of the HCM- causing Y235S mutation by performing mechanical experiments in tial expression of RNA transcripts in D230N and R92L transgenic mice are detergent-skinned myocardium and molecular dynamics simulations (MDS). modulating cellular pathways, leading to differential cardiac remodeling and cMyBPC null mouse myocardium was virally transfected with wild-type or aggravating the progression of disease. WT Y235S Three sets of mice were studied at three timepoints to assess early, middle, Y235S cMyBPC (KO or KO ). We found that Y235S cMyBPC was and late phases of ventricular remodeling: D230N-transgenic mice, R92L- properly expressed and incorporated into the cardiac sarcomere, suggesting transgenic mice, and non-transgenic sibling control mice at one month, three that the mechanism of disease of the Y235S mutation is not haploinsufficiency months, and six months. RNA was extracted from ventricular tissue and or poison peptides. Mechanical experiments in skinned myocardium isolated from KOY235S hearts revealed hypercontractile behavior compared to KOWT approximately 20 million 50bp single-end reads were obtained for each mouse hearts, evidenced by accelerated rates of cross-bridge kinetics and increased using Illumina HiSeq. The computational workflow included alignment of the 2þ RNA read sequences to the mm10 reference genome using STAR, and the Ca sensitivity of force generation. In addition, MDS revealed that the generation of a transcript expression matrix using HOMER. Data was Y235S mutation causes alterations in important intramolecular interactions, analyzed using R with the edgeR and DESeq2 Bioconductor statistical surface conformations, and electrostatic potential of the C1 domain of packages. cMyBPC. Our combined in vitro and in silico data suggests that the Y235S mu- tation directly disrupts internal and surface properties of the C1 domain of Among the 40,459 expressed transcripts, 34, 22, and 17 were differentially- cMyBPC, which could alter C1 domain ligand-binding interactions. These mo- expressed (DE) between the D230N and WT groups at the one-month, three- month, and six-month timepoints, respectively. In contrast, 20, 17, and 56 tran- lecular changes may underlie the mechanism for hypercontractile cross-bridge scripts were DE between the R92L and WT groups at the one-month, three- behavior which ultimately may result in development of cardiac hypertrophy month, and six-month timepoints. The decrease in DE genes over time with and in vivo cardiac dysfunction. the D230N mice and the increase in DE genes with the R92L mice are funda- 1313-Pos mental to the divergent structural and functional changes associated with each Microtubule Acetylation Regulates Contractile Kinetics of Striated Muscle of these transgenic models. Extensive pathway analysis (Qiagen IPA) is Andrew K. Coleman, Humberto C. Joca, George S.B. Williams, currently underway to identify the differential signaling cascades that charac- W. Jonathan Lederer, Chris W. Ward. terize these divergent patterns of cardiac remodeling and identify novel thera- University of Maryland School of Medicine, Baltimore, MD, USA. peutic targets. Microtubules (MTs) are key cytoskeletal elements with many roles including protein trafficking, defining cell shape, serving as signaling scaffolds, and 1311-Pos providing a means for transmitting mechanical energy for mechanotransduc- Human Cardiac Myosin-Binding Protein C N-Terminal Domains Cooper- tion. Detyrosination and acetylation are tubulin post-translational modifications atively Impact Actin Structural Dynamics (PTMs) shown to regulate MT properties and function. In striated muscle, MTs Rhye-Samuel Kanassatega, Thomas A. Bunch, Victoria C. Lepak, modified by detyrosination and acetylation are present in health and dramati- Brett A. Colson. cally increase with disease. While evidence shows detyrosinated MTs regulate Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA. cytoskeletal stiffness and contractile kinetics, the influence of MT acetylation Cardiac myosin-binding protein C (cMyBP-C) is a thick filament-associated remains undefined. Here we show that MT acetylation regulates cytoskeletal protein that modulates contractility by influencing interactions between stiffness and contractile kinetics independent of detyrosination. Tubulin acety- myosin and actin. Despite being a leading cause of familial hypertrophic lation/deacetylation is regulated by a-tubulin acetyltransferase 1 and histone cardiomyopathy, the mechanism by which cMyBP-C modulates contractility deacetylase 6 (HDAC6) respectively. We show that tubucin, a selective is not well understood. N-terminal domains of cMyBP-C (C0 through C2) HDAC6 inhibitor, increased the level of tubulin acetylation in cardiomyocytes have been shown to interact with the actin-thin filament; however, the and muscle fibers with negligible impact on detyrosination. Using a high-speed cMyBP-C regions involved in the interaction and how cMyBP-C influences sarcomere length and fluorescence acquisition system, we show that increased actin’s dynamic structure remain unclear. Here, we have used cosedimenta- MT acetylation reduces fractional shortening of cardiomyocytes and muscle fi- tion assays for measuring binding kinetics and time-resolved phosphores- bers. This effect was underscored by a reduction in contractile kinetics that cence anisotropy (TPA) for measuring actin rotational dynamics. We occurred independent of any alteration in the calcium transient. Taken together examined the effects of actin interactions with human fragments C0-C1 our evidence shows that acetylation impacts MT properties and myocyte func- and C0-C2 of cMyBP-C. We also evaluated roles of cMyBP-C phosphory- tion independent of detyrosination. We speculate that the level of MT acetyla- lation sites known to influence contractility. We find that C0-C2 binds actin tion may act in concert with detyrosination to regulate myocyte function in with 4-fold increase in apparent affinity compared to C0-C1. TPA of health and disease. labeled-actin showed marked differences in the potency of effects between C0-C2, C0-C1, and phosphorylated cMyBP-C fragments with respect to Posters: Mitochondria in Cell Life and Death decreasing the amplitude of torsional motions, increasing the rate of mono- mer rotation, and increasing relative resilience of the filament. A Hill equa- 1314-Pos tion was used to determine the level of cooperativity of the TPA effects for Mitochondrial Metabolic Function is Affected by Inner Membrane each fragment. Our results demonstrate that C0-C2 exhibits stronger coop- Morphology erativity in propagating its effects on amplitude, rate, and resilience to Nasrin Afzal1, Carmen Mannella2, William J. Lederer2, Mohsin S. Jafri1. more distant actin monomers than C0-C1. Assuming a thin filament cooper- 1School of Systems Biology, George Mason University, Fairfax, VA, USA, ative unit of 7 actin monomers (length of tropomyosin), our TPA analyses 2Center for Biomedical Engineering and Technology, University of Maryland suggest that one molecule of C0-C2 bound to actin propagated effects 2-3 School of Medicine, Baltimore, MD, USA. cooperative units (14-21 monomers), whereas C0-C1 effects were confined The mitochondrial inner membrane (IM) has a dynamic and complex structure to one unit, and phosphorylation reduced cooperativity. These findings that plays a significant role in mitochondrial function and energy metabolism.

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The folds in the IM called cristae have different size and morphology which suggested its involvement in regulation of activity dependent plasticity. How- influence the surface area of the inner membrane, home of critical membrane ever, a direct mechanistic link has yet to be established. Interestingly, Wnt is proteins including ATP synthase and electron transport chain complexes; enriched in cysteine residues which can be targeted by mitochondrial ROS metabolite and ion transporters including the adenine nucleotide translocase, (reactive oxygen species). Based on the rationale that neuronal activity sets the calcium uniporter (MCU), the sodium/calcium exchanger (NCLX); and the pace of aerobic energy conversion and of ROS emission from mitochon- many more. Our multiscale modeling seeks to develop a fundamental under- dria, we generated transgenic fruit flies engineered for targeted manipulation standing of the interplay of the mitochondrial cristae organization and mito- of mitochondrial ROS in vivo. Using this model, we present a preliminary chondrial metabolic function. Our previously published model (Nguyen et study of the effect of specific ROS signaling on motor function and synaptic al., 2007) for mitochondrial energy metabolism was upgraded to take account plasticity. of our new data on both activity-dependent regulation of ATP production, calcium handling, and new information on cristae structure. This spatiotem- 1317-Pos poral model of the mitochondrion was developed using the Virtual Cell plat- Drug Screening and Discovery Strategies at Nanoscale Morphology using form in two and three-dimensional representations. The studies examine the Structured Illumination Microscopy effect of inner membrane morphology and protein localization on metabolic Xintian Shao1,2, Qixin Chen1,2, Peixue Ling1, Jiajie Diao2. function and specifically ATP production and Ca uptake. In this study 1School of Pharmaceutical Sciences, Shandong University, Jinan, China, various aspects of crista structure in two and three dimensions, including 2Department of Cancer Biology, University of Cincinnati College of shape, junctions and fenestrations were targeted. The model predicts spatial Medicine, Cincinnati, OH, USA. gradients of ion and metabolite concentrations for observed (tubular, With the development of technologies such as genomes, proteomes, and lamellar), conformations of cristae and within the mitochondrial matrix. metabolomes, the number of potential therapeutic targets are accessible. The existence of these gradients affects mitochondrial ATP fluxes across However, it is an indispensable puzzle for screening optimal drugs in innu- the inner membrane. The model suggests that mitochondrial crista geometry merable candidate compounds with high specificity and sensitivity for alters the gradients of ion and metabolite concentrations in the intracristal these target. Herein, we propose a strategy using structured illumination space and the matrix. These results provide new insight about metabolic microscopy (SIM) to screen and discover drugs at nanoscale morphology modeling in 2D and 3D structures which can be useful for identifying struc- in living cell. Our results show that this strategy not only can distinguish tural features of mitochondria that influence function in modeling and exper- the interaction behavior between mitochondria and lysosomes such as iment as well. mitochondria-lysosomes contact (MLC) and mitophagy events[1], but also can evaluate the role of different genes (such as ATG13, FIP200 and 1315-Pos PENTA) in these events. Furthermore, it is a visualized evidence to An Automated Method for Segmenting Highly Convoluted Mitochondrial judge the effect of different drugs for MLC and mitophgay events at Inner Membranes from Electron Microscopic Tomograms nanoscale morphology. Our findings thus illustrate a novel perspective Raquel Adams1, Zheng Liu2, Carmen A. Mannella3, W. Jonathan Lederer3, for using SIM to visualize the biological function of candidate drugs at M. Saleet Jafri1. the organelle level in living cells, which can increase the success rate of 1Krasnow Institute for Advanced Study and School of Systems Biology, drug discovery, and even shorten the cycle of novel drug development. George Mason University, Fairfax, VA, USA, 2Tongji University School of [1] Q X Chen, C Z Jin, X T Shao, R L Guan, Z Q Tian, C R Wang, F Medicine, Shanghai, China, 3Center for Biomedical Engineering and Liu, P X Ling, J L Guan, L N Ji, F S Wang, H Chao, and J J Diao, Small, Technology, University of Maryland School of Medicine, Baltimore, MD, 1802166 (2018). USA. The functional importance of the morphology of the mitochondrial inner 1318-Pos membrane (IM) is demonstrated both by the complex machinery required Quantitative Analysis of Interactive Behavior of Mitochondria and Lyso- to maintain normal morphology and by specific changes in shape associated somes using Structured Illumination Microscopy with physiological and pathological states. Accurate computer simulations of Qixin Chen1,2, Xintian Shao1,2, Peixue Ling2, Jiajie Diao1. activities such as ATP production and calcium uptake are best performed in 1Department of Cancer Biology, University of Cincinnati College of 3-dimensional space using realistic membrane surfaces, such as those pro- Medicine, Cincinnati, OH, USA, 2School of Pharmaceutical Sciences, vided by transmission electron microscopic (TEM) tomography. However, Shandong University, Jinan, China. the observed variability in IM shape requires sampling a wide range of crista Super-resolution optical microscopy has extended the scope of cell biology morphologies (from tubular to lamellar in the case of cardiac mitochondria), from the cellular level to the nanoscale, giving researchers the ability to and the complexity and compaction of cristae make segmentation a chal- observe the interactive behavior of single mitochondria and lysosomes[1]. lenge. The traditional method of segmenting the TEM tomographic images However, we are still without an applicable quantitative analysis system to involves manually tracing membrane contours in 2D slices from tomograms, assist us in understanding interactive behavior of mitochondria and lysosomes a laborious technique that is generally more effective for less intricate mem- under super-resolution optical microscopy. Here, we introduce an M-value to brane shapes. Thus, we are developing an automated methodology of seg- quantitatively investigate mitochondria and lysosome contact (MLC) and mi- mentation that employs a novel reproducible sequence of procedures for tophagy under structured illumination microscopy. We found that the M-value membrane segmentation from TEM tomograms of epoxy-embedded and for an MLC is typically less than 0.4, whereas in mitophagy it ranges from 0.5 stained mouse cardiac muscle by Frangi enhancement filtering, a Hessian- to 1.0. This system permits further investigation of the detailed molecular based algorithm that has been used in medical imaging (ocular vasculature mechanism governing the interactive behavior of mitochondria and lyso- enhancement and mapping). We have found the new automated procedure somes. [1] Q X Chen, C Z Jin, X T Shao, R L Guan, Z Q Tian, C R to work well for segmenting intricate membranes against a noisy back- Wang, F Liu, P X Ling, J L Guan, L N Ji, F S Wang, H Chao, and J J ground. The next step is constructing an accurate mesh model of a membrane Diao, Small, 1802166 (2018). of interest using the MATLAB isosurface function. In the current implemen- tation, the procedure does an excellent job of isolating individual membranes 1319-Pos inside crowded mitochondria. However, there is still a need to manually cor- Modeling the Insertion of in the Mitochondrial Outer Mem- rect spurious features, such as random breaks in membrane surfaces due to brane and its Complex Formation with VDAC stain noise. Conditions are being explored to minimize or eliminate these, Nandan Haloi1,2, Po-Chao Wen2,3, Amadou K.S. Camara4, as are automated methods to correct discontinuities. (Supported by NIH grant Wai-Meng Kwok4, Emad Tajkhorshid2,3. U01HL116321.) 1Center for Biophysics and Quantitative Biology, University of Illinois at Urbana Champaign, Urbana, IL, USA, 2Beckman Institute of Advanced 1316-Pos Science and Technology, University of Illinois at Urbana Champaign, Optogenetic Regulation of Mitochondrial Function and Synaptic Plasticity Urbana, IL, USA, 3NIH Center for Macromolecular Modeling and In Vivo Bioinformatics, University of Illinois at Urbana Champaign, Urbana, IL, Kristian M. Zapata, Illya Aronskyy, Stephen Madamba, Pablo M. Peixoto. USA, 4Anesthesiology, Physiology and Pharmacology & Toxicology, Dept Nat Sci, Baruch Coll CUNY, New York, NY, USA. Medical College of Wisconsin, Milwaukee, WI, USA. Previous research has established that synaptic plasticity can be elicited with Hexokinase (HK), a cytosolic protein, interacts with the mitochondrial outer repetitive optogenetic induction of action potentials in fly motor neurons membrane protein VDAC and plays an important role in cell growth and sur- in vivo. Increased levels of the Wnt protein in the presynaptic terminal vival. Overexpression of HK-II in cancer cells increases HK-II-VDAC

BPJ 9370_9373 268a Monday, March 4, 2019 interactions and prevents apoptosis. Previous biochemical studies have shown omyoblasts were infected with control adenovirus or the DEC splice variant of that hydrophobic N-terminal residues of HK are important for VDAC bind- BKCa with a FLAG-tag and R207Q gain-of-function mutation (mBKQ). Cells ing, while several charged residues of VDAC are crucial for the complex for- were treated with or without in vitro hypoxic cardioplegia solution (3 hrs) and mation. However, it was not clear whether HK directly binds VDAC or this reoxygenation (CP/R). Mitochondrial respiration (Seahorse), morphology interaction is facilitated by membrane binding of HK. Herein, we present a (mito-RFP) and ETC supercomplex formation (BN-PAGE) were assessed. computational study modeling membrane insertion of HK-II and the HK- mBKQ was specifically expressed in mitochondria as shown by colocalization II-VDAC complex formation on the membrane surface. To test membrane with Tom20. mBKQ dose dependently increased OCR in control cells. CP/R insertion of HK-II, we used the Highly Mobile Membrane Mimic caused a reorganization of mitochondria promoting fission as demonstrated (HMMM) model of lipids in our MD simulations. Seven out of 10 indepen- with morphometric analysis with mito-RFP, as well as a decrease in mito- dent 200 ns simulations showed that the first 10 amino acids of the N-termi- chondrial supercomplexes. mBKQ expression attenuated mitochondrial nus of HK-II insert into the membrane. Subsequent 200 ns conventional MD morphology changes and preserved SC (p<0.05 Two Way ANOVA, SNK) simulation with a full membrane showed a stable membrane binding of HK- Hypoxic CP/R was also associated with decreased basal and maximal II. The deepest insertion of HK-II is 10 A˚ below the phosphate plane of the respiration which attenuated by mBKQ expression (p<0.05 Two Way membrane. To further study the interaction between the VDAC and HK-II, ANOVA, SNK). Expression of the SC assembly factor COX7RP was simi- we then used atomic resolution Brownian dynamics simulations, where an larly able to attenuate CP/R-induced changes in respiration. Conclusions: implicit membrane was created for HK-II based on the model developed Overexpression of gain-of-function mitochondrial BKCa channels can atten- in our initial phase of the study. From these simulations we found 2 major uate hypoxia-induced deleterious alterations in mitochondrial respiration, distinct binding sites of HK-II in VDAC. The interaction residues of these likely through preservation of mitochondrial structure and higher order ETC two binding sites agree with previous mutational experiments. This study complexes. provides an atomic-scale model of VDAC-bound HK-II at the surface of the membrane. 1322-Pos Single Channel Recordings of mitoBKCa Channel Formed by BK-Dec 1320-Pos Splice Variant Developing Specific Small-Peptide Inhibitors of Mitochondrial VDAC Bogusz Kulawiak1, Shur Karolina Kucman1, Justyna Je˛draszko1, Philip A. Gurnev1, David P. Hoogerheide2, Sergey M. Bezrukov1, Piotr Bednarczyk2, Adam M. Szewczyk1. Tatiana K. Rostovtseva1. 1 2 Nencki Institute of Experimental Biology, Warsaw, Poland, Dept 1Section on Molecular Transport, NICHD NIH, Bethesda, MD, USA, 2 Biophysics, Warsaw Univ Life Sci, Warsaw, Poland. National Institute of Standards and Tech, Gaithersburg, MD, USA. Ischemia of brain and heart tissue is the one of the most common causes of The Voltage-Dependent Anion Channel (VDAC) forms a unique pathway death worldwide. In the inner mitochondrial membrane several potassium across the mitochondrial outer membrane for small ions and water-soluble channels have been identified whose activation lead to cytoprotection dur- metabolites, such as ATP and ADP. Despite VDAC’s involvement in both ing ischemic event. It was found that activation of mitochondrial large normal regulation of respiration and in a wide variety of mitochondria- conductance calcium activated potassium channel (mitoBKCa)preserves associated pathologies, there are, surprisingly, no known pharmacological brain and heart muscle cells. For many years it was speculated that pore- agents targeting VDAC. Emerging data show that VDAC actively controls forming subunit of the channel is encoded by a KCNMA1 gene product. metabolite fluxes via its interactions with cytosolic proteins. Among these, Indeed, recently a BK-DEC splice variant of BKCa-type channels a subunit the small neuronal protein alpha-synuclein (aSyn) is a very potent regulator has been demonstrated to localize in mitochondria. However it was not of VDAC, able to reversibly block this channel at nanomolar concentrations. known whether this isoform is able to form a functional channel in Though generally recognized as a plaque-forming amyloid, aSyn is also mitochondria. known to cause mitochondrial dysfunction. We have shown that the efficiency In our study we used HEK293T cells transfected with cDNA encoding BK- of the aSyn-VDAC interaction is well described by a multistep mechanism of DEC splice variant previously shown to localize in mitochondrial fraction. channel blocking, which includes membrane binding followed by capture in Electrophysiological recordings with use of mitoplast isolated from transfected the VDAC pore. Based on this mechanism, we hypothesized that the architec- cells revealed presence of the large conductance and voltage dependent ion ture of a prospective VDAC inhibitor should fulfill two main requirements: channel. This type of channel was not present in mitoplasts isolated from un- possess a negatively charged disordered polypeptide C- or N-terminus (a transfected cells. We found that recorded channel showed all basic pharmaco- pore blocking domain) and feature a mitochondrial membrane-binding logical properties typical for the mitoBKCa channels described previously. The domain. The hypothesized mechanism of VDAC inhibition is quite general; channel was Ca2þ sensitive and its activity was inhibited by paxilline, well it does not require any specific interaction between the VDAC pore and inhib- known mitoBK channel inhibitor. Additionally, kinetics and conductance itor. Using aSyn as a template, we engineered three peptides and demonstrate Ca of observed channel were very similar to the mitoBKCa channel. Based on that they all characteristically block VDAC reconstituted into planar lipid collected data we conclude that BK-DEC splice variant not only colocalizes membranes in a controllable manner, thereby mimicking the action of aSyn. with mitochondria but forms a functional channel in the inner mitochondrial The VDAC blockage times induced by the synthetic peptides are comparable membrane of HEK293T cells. to aSyn-induced blockages, though the effective solution concentration This work was supported by the Polish National Science Centre grant No.2015/ required for synthetic blockers is 100 times higher than of aSyn (50nM). 18/E/NZ1/00737 and the Nencki Institute of Experimental Biology. Our results demonstrate the general requirements of a VDAC inhibitor that will inform future development of mitochondria-targeting pharmacological tools. 1323-Pos Mitochondrial Potassium Channels: Regulation by Gaseous Transmitter 1321-Pos Adam Szewczyk, Agnieszka Walewska, Daria Rotko, Bogusz Kulawiak, Mitochondrial Gain-Of-Function BKCa Channel Attenuates Mitochon- Piotr Koprowski. drial Dysfunction Associated with Hypoxic Injury Nencki Institute of Experimental Biology, Warsaw, Poland. Thomas Mancini1, Jin O-uchi2, Shanna Hamilton3, Radmila Terentyeva3, Mitochondrial large-conductance calcium-activated potassium channel (mi- Gaurav Choudhary4, Dmitry Terentyev3, Richard T. Clements1. toBK) is one of the main potassium channels localized in the inner mitochon- 1Surgery, Rhode Island Hospital/Brown University, Providence, RI, USA, drial membrane of cardiac and endothelial cells, and neurons. MitoBK is 2Medicine, Lillehei Heart Institute/ University of Minnesota, Minneapolis, formed by a DEC splice variant of KCNMA1 gene and it is a tetrameric protein MN, USA, 3Medicine, Rhode Island Hospital/Brown University, Providence, composed of four a subunits. Various modulators of the activity of mitoBK are RI, USA, 4Medicine, Providence VAMC/Brown University, Providence, RI, known, including activator such as carbon monoxide and inhibitor heme or its USA. oxidized form hemin (Fe(III)-protoporphyrin IX). It is known that the activity Large conductance Caþþ activated Kþ channels in cardiomyocyte mitochon- of mitoBK, similarly to that of plasmalemmal BK channels is inhibited by he- dria have been implicated in cardioprotection. We have previously demon- min. In our study, we performed patch-clamp experiments on mitoplasts strated that BKCa channel openers improve myocardial function in a derived from mitochondria of astrocytoma U-87 MG cells to measure activity cardioplegic arrest model of myocardial stunning. However the mechanism of single mitoBK channels. First, we applied sodium hydrosulfide (NaHS) as a of BKCa-mediated cardioprotection remains unclear and specificity of phar- H2S donor, what had no impact on mitoBK channel activity. Next, we applied macologic agents can be a concern. In this study we determined the effects hemin and other metal ion-substituted protoporphyrins IX followed by NaHS. of adenoviral overexpression of a mitochondrial BKCa channel with a gain Here we found that NaHS activated the hemin-inhibited mitoBK channels but of function mutation (R207Q) in rat cardiomyoblasts. Methods: H9c2 cardi- also for example channels inhibited by Zn(II)-protoporphyrin IX. To address

BPJ 9370_9373 Monday, March 4, 2019 269a modulation of mitoBK by CO, we evaluated the effect of CO-saturated solution 1326-Pos D in the mitoBK single-channel patch-clamp recordings of mitochondria isolated Ca2 -Dependent Mitochondrial Permeability Transition Pore Opening in from human astrocytoma cells. Subsequently, we assessed pharmacological ac- Kidney is Substrate Dependent tion of several structurally different CO-releasing molecules (CORMs), which Namrata Tomar1, Sunil M. Kandel1, Nadezda Zheleznova2, Said H. Audi3, are typically used in the studies of CO regulation of cell membrane BK chan- Allen W. Cowley Jr2, Ranjan K. Dash1. 1 nels. In summary, we observed various and complex effects of H2S and CO do- Department of Biomedical Engineering, Medical College of Wisconsin, nors on the activity of mitoBK. Milwaukee, WI, USA, 2Department of Physiology, Medical College of This work was supported by the Polish National Science Centre (grant no. Wisconsin, Milwaukee, WI, USA, 3Department of Biomedical Engineering, 2015/17/B/NZ1/02496) and grant MSCA-COFUND #665735 (Bio4Med). Marquette University, Milwaukee, WI, USA. Oxidative stress within the kidney is associated with disease states including hy- 1324-Pos pertension, renal diseases. The metabolic functions of the mammalian kidneys Effect of Steroids on Mitochondrial Metabolite Channel Function and result in consumption of nearly 20% of total oxygen utilization. There is limited Lipid Membrane Properties information regarding the effects of oxygen free radicals upon kidney mitochon- William M. Rosencrans1, Maria Queralt-Martin2, Amandine Rovini3, drial functions. Not only mitochondria oxidize numerous metabolic substrates but 2þ Phillip A. Gurnev2, Sergey M. Bezrukov2, Tatiana K. Rostovtseva2. also play a key role in maintaining cellular Ca homeostasis. Failure of cellular 2þ 2þ 2þ 1Physics, Colgate University, Hamilton, NY, USA, 2Section on Molecular Ca homeostasis and excessive mitochondrial Ca (mCa )leadtoopeningof Transport, Eunice Kennedy Shriver National Institute of Child Health and a mitochondrial permeability transition pore (mPTP), which is associated with Human Development, National Institutes of Health, Bethesda, MD, USA, loss of IMM potential and uncoupling of oxidative phosphorylation. We investi- 3Department of Drug Discovery and Biomedical Sciences, Medical gated whether mitochondrial metabolic substrates have a differential impact on 2þ University of South Carolina, Charleston, SC, USA. the Ca -induced mPTP opening in the kidney, which plays a critical role in hy- There is accumulating evidence that endogenous steroids and synthetic pertension. For this purpose, mitochondria isolated from Sprague Dawley (SD) steroid-based drugs are involved in regulating mitochondria physiology. rat kidney outer medulla (OM) were energized using mechanistically distinct The Voltage Dependent Anion Channel (VDAC), the major metabolite chan- substrate combinations, PyruvateþMalate, GlutamateþMalate(GM), Succinate nel in the mitochondrial outer membrane (MOM), mediates the exchange of þRotenone, Succinate, a-KetoGlutarateþMalate, or PalmitoylCarnitineþ 2þ ions and water-soluble metabolites, such as ATP and ADP, across the MOM, Malate. Fluorescence-based analysis using Ca indicator Fura-4F assessed the 2þ thus governing mitochondrial respiration. VDAC reconstituted into planar functional consequences of Ca -induced mPTP opening. Results show that in 2þ lipid bilayers under an applied voltage characteristically switches from a the presence of GM, mitochondria can tolerate significantly greater mCa loads high-conducting ‘‘open’’ state, that allows metabolites to pass, to a variety as compared to that in the presence of other substrates. SuccinateþRotenone addi- of low-conducting ‘‘closed’’ states that prevent metabolite flux. This process, tion significantly delayed mPTP opening over controls (succinate alone). A pore þ 2þ called voltage-gating, involves structural rearrangements of VDAC. Howev- inhibitor CyclosporinA(CsA) and Na /Ca exchanger inhibitor(CGP) were used er, the mechanism behind VDAC gating remains elusive. Recent studies sug- with all substrates for comparisons. The present study reports variations in mito- 2þ gest that steroids may affect VDAC gating through interactions at the chondrial substrates sensitivity to Ca -induced mPTP opening and that the meta- 2þ channel-lipid interface. However, functional data are missing and a molecu- bolic fluxes determine the mitochondrial functional responses to mCa overload. lar mechanism is unknown. Here we evaluate the effect of a neuroprotective The results underscore the effects of various mitochondrial substrates and their hydrophobic cholesterol-derived drug olesoxime, cholesterol, and allopreg- electron flux in regulating mPTP opening, which is a determinant of cell death nanolone on VDAC voltage-gating properties using ion channel electrophys- in many diseases. These data are important in order to understand the different iology. We show that olesoxime enhances VDAC gating while cholesterol pathways/proteins involved in mPTP opening to identify effective interventions and allopregnanolone have no significant effect. Suspecting a bilayer- to prevent/limit it. dependent mechanism of olesoxime action, we examined the effect of ole- soxime on gramicidin A (gA) kinetics. We use gA as a molecular probe of 1327-Pos bilayer properties. We found that olesoxime does not affect gA lifetime while Regulation of the Proton Leak in Mitochondria cholesterol and allopregnanolone increase it. These results indicate that ole- Elena E. Pohl. soxime modulates VDAC gating likely by directly interacting with the chan- Dept Physiol & Biophys, Univ Vet Med, Vienna, Austria. nel, while the other steroids do not. Our study demonstrates a possibility of Mechanisms of proton transport regulation in mitochondria Elena E. Pohl Insti- targeting VDAC for therapeutic outcomes utilizing hydrophobic small- tute of Physiology, Pathophysiology and Biophysics, University of Veterinary molecule drugs. Medicine Vienna Protein-mediated proton transport contributes to the proton- motive force in mitochondria and is therefore important for the variety of 1325-Pos voltage-dependent processes, including the production of ATP and heat. Several membrane proteins such as uncoupling proteins (UCP), ATP/ADP car- Modulation of the Channel Activity of the C-Subunit of the ATP Synthase þ by Polyphosphate and Polyhydroxybutyrate rier (ANT), and phosphate carrier are associated with H transport. It is tightly Giuseppe F. Amodeo1, Magdalena Klim2, Piotr Kurcok2, regulated; however, the underlying molecular mechanisms are highly 1,2 Evgeny V. Pavlov1. debated . I discuss existing models of UCP activation and inhibition, as 1Basic Science, New York University, New York, NY, USA, 2Centre of well as proposed regulators. Polymer and Carbon Materials, Polish Academy of Science, Zabrze, Poland. References C subunit of the ATP synthase has been proposed to be the main constituent 1 Macher, G. et al. Inhibition of mitochondrial UCP1 and UCP3 by purine nu- of the channel part of the mitochondrial Permeability Transition Pore cleotides and phosphate. Biochim Biophys Acta 1860, 664-672, https://doi.org/ (mPTP) complex. Our earlier work demonstrated that when c subunit is 10.1016/j.bbamem.2017.12.001 (2018). co-purified from mitochondria together with polyphosphate (polyP) and pol- 2 Jezek, P., Holendova, B., Garlid, K. D. & Jaburek, M. Mitochondrial uncou- yhydroxybutyrate (PHB), it forms channels resembling native mPTP. Our pling proteins: subtle regulators of cellular redox signaling. Antioxid Redox work is aimed at better understanding the interactions among these three Signal, https://doi.org/10.1089/ars.2017.7225 (2018). macromolecules and their role in the formation of the pore. We extracted c subunit from rat liver mitochondria in physiological conditions and from 1328-Pos mitochondria after the induction of permeability transition by a method Mitochondrial Membrane Potential Oscillations Persist during Reperfu- that excludes formation of complex with polyP. This was followed by the sion After Ischemia in MCU Knockout Cardiomyocytes study of ion channel activity in planar lipid membranes and its modulation Deepthi Ashok, Kyriakos Papanicolaou, Brian O0Rourke. by the addition of synthetic polyP and PHB. We observed channel activity Johns Hopkins University School of Medicine, Baltimore, MD, USA. þ in all preparations. Moreover, the presence of polyP together with Ca2 Ca2þ entry via Mitochondrial Calcium Uniporter (MCU) participates in en- led to the increase in channel conductance from 600 pS up to 1.7 nS. Cir- ergetic adaption to workload under physiological conditions but is thought cular dichroism spectroscopy indicated that, unlike native c subunit being to contribute to cell death during ischemia-reperfusion (I/R) injury. Previous primarily in an a-helical conformation, under our reconstitution conditions work has shown that mitochondrial membrane potential (DJm) instability c subunit possessed a significant amount of b-sheet. We hypothesize that contributes to early-reperfusion arrhythmias and contractile dysfunction; channel activity of the c subunit might be related to its misfolding under however, the role of mitochondrial Ca2þ uptake in triggering DJm oscilla- pathological conditions and that this process can be modulated by polyP tion versus sustained permeability transition pore (PTP) opening is unclear. and PHB. We hypothesized that MCU-mediated Ca2þ uptake is required to trigger

BPJ 9370_9373 270a Monday, March 4, 2019 irreversible DJm loss mediated by PTP but is not required for DJm oscil- quences of calcium overload on mitochondrial ultrastructure. In parallel lation during early reperfusion, involving reactive oxygen species (ROS)- studies, spectrofluorimetry and high-resolution respirometry were used to induced ROS release (RIRR). A spinning-disk confocal microscope was infer the functional changes caused the changes in ultrastructure. Exposing used to image DJm(with TMRM) in monolayers of neonatal mouse ventric- energized mitochondria to permeability transition inducing calcium boluses ular myocytes (NMVM) during I/R (1hr/1hr). Acute MCU knockout (KO) promotes calcium uptake, calcium-phosphate granule (CaPiG) formation was achieved by transducing floxed MCU NMVMs with an adenovirus ex- (50 - 120 nm), and inner/outer membrane (IMM/OMM) rupture. However, pressing CRE-recombinase. DJm response to I/R was analyzed at cellular when mitochondria were treated with CsA, they displayed aberrant morphol- level by segmentation analysis (ImageJ) and a new wavelet transform ogies and, upon calcium administration, greater amount of granules with method was developed to characterize the dominant frequency of DJm sizes ranging from 40 - 200 nm were generated. Although under these oscillation (Matlab). We found that surprisingly, the rate of DJmlossdur- extreme conditions the OMM is lost, CsA-treated mitochondria preserve ing ischemia was greater in MCU-KO compared to control, yet the recovery the IMM and cristae structure. Therefore, they maintain cellular respiration of DJm was more sustained, lacking a late decline after 30 min reperfusion. and can synthesize ATP. These results suggest CsA may interact with DJm oscillations during reperfusion persisted in MCU-KO myocytes and IMM components, namely OPA1 and the MICOS proteins, to enhance cal- wavelet analysis revealed a dominant frequency ranging from 0.022Hz- cium uptake and retention capacity promoting calcium phosphate deposition, 0.0088Hz in both WT and KO. These results suggest that MCU-mediated growth, and overall complex stability. Thus, explaining for the first time, at Ca2þ uptake is not required for RIRR induced DJm oscillation but is the mechanistic level, the efficacy of the drug in delaying permeability tran- involved in late reperfusion-induced sustained DJm collapse; and that sition activation while maintaining the bioenergetics machinery and mito- MCU-mediated Ca2þ uptake during reperfusion contributes to PTP- chondrial network integrity. mediated DJm collapse during reperfusion. However, DJm oscillations 2þ during early reperfusion are independent of MCU-mediated Ca entry, 1331-Pos and likely to underlie mechanical and electrophysiological dysfunction Cardiac Calcium Signaling and Mitochondrial Metabolic Function observed during this period. Nasrin Afzal1, Carmen A. Mannella2, W. Jonathan Lederer2, M. Saleet Jafri1. 1Krasnow Inst, George Mason Univ, Fairfax, VA, USA, 2University of 1329-Pos Maryland School of Medicine, Center for Biomedical Engineering and The MCU Inhibitor Ds16570511 has Off-Target Effects on Mitochondrial Technology, Baltimore, MD, USA. Membrane Potential Mitochondrial calcium uptake is critically important in cellular function, en- Riley Payne1, Carmen Li1, Emily Fernandez-Garcia1, Horia Vais1, ergy production, and initiation of cell death. Even though the large flux of Kevin Foskett1,2. calcium can affect intracellular signals, the amount of calcium uptake by 1Physiology, University of Pennsylvania, Philadelphia, PA, USA, 2Cell and mitochondria is a topic of debate. Here we use computer simulations to study Developmental Biology, University of Pennsylvania, Philadelphia, PA, USA. the effects of calcium sparks observed in cardiac myocytes on mitochondrial Ca2þ uptake into mitochondria occurs through the Mitochondrial Calcium metabolic function, using the Virtual Cell platform (NIH grant # P41 Uniporter (MCU) channel, promoting respiration and ATP production in GM103313). The simulations employ different 2D and 3D representations response to intracellular Ca2þ signals. Nevertheless, excessive matrix of crista geometry, including lamellar and tubular structures, variation in [Ca2þ]([Ca2þ]m) has toxic effects, triggering mitochondrial Permeability crista openings to the intermembrane space (between the inner boundary Transition Pore (mPTP) opening, depolarization of the inner membrane po- and outer membranes), and newly described fenestrations within cristae tential (DJm), and apoptosis. Accordingly, specific inhibitors are of interest (that interconnect matrix compartments). Variable distribution of the mito- as possible therapeutics for cardiac ischemia-reperfusion injury, neurode- chondrial calcium uniporter on the inner membrane is also used. Simulations generative diseases, and cancer. DS16570511 (DS) was recently identified suggest that mitochondrial crista geometry and orientation relative to spark as a specific cell-permeable MCU inhibitor with an IC50 for channel source critically affect the diffusion of [Ca2þ] in the intracristal spaces. Intra- blockade of 7 mM. Using permeabilized cell assays to simultaneously cristal [Ca2þ] exhibits more rapid changes than matrix [Ca2þ] in response to monitor mitochondrial Ca2þ uptake and DJm, we show that whereas DS changes in extramitochondrial [Ca2þ]. The spark dynamics and the distribu- inhibits MCU, it is not specific. DS promotes excessive mitochondrial depo- tion of the mitochondrial calcium uniporter on the inner membrane also can larization in response to elevations in cytoplasmic [Ca2þ]([Ca2þ]c), leading significantly affect mitochondrial matrix [Ca2þ]. By varying key parameters, to overestimates of its inhibitory activity on MCU. At low [Ca2þ]c when the model can be used to predict the average level of matrix [Ca2þ] during MCU is inactive, DS hyperpolarizes DJmbyanunknownmechanism, and after a spark event and its expected effect on respiration (e.g., activation increasing the driving force for Ca2þ uptake into the matrix. Subsequent of dehydrogenases) and adenine nucleotide levels in matrix and intracristal MCU activation in the presence of DS-induced hyperpolarization causes spaces. Extreme conditions leading to calcium overload and triggering of an exaggerated increase of [Ca2þ]m that triggers mPTP opening. With the permeability transition can also be explored and compared to experi- mPTP inhibited by Cyclosporin A, the IC50 of DS for MCU inhibition is mental results. These modeling capabilities, that include cellular calcium reduced to 30 mM. Whole-mitoplast patch clamp electrophysiology with signaling, metabolic characteristic of mitochondria, subcellular DJm experimentally controlled revealed reversible inhibition of MCU cur- of calcium signaling, and nanoscale organization of mitochondria, can rents by 10 mM DS. These findings highlight the importance of screening inform and provoke new future experiments. (Supported by NIH Grant potential MCU inhibitors for off-target effects, particularly those that influ- U01HL116321) ence DJm. 1332-Pos 1330-Pos Mitochondrial Calcium Deregulation in Tau K18-Treated Cortical Neu- Cardiac Mitochondria Ultrastructural and Functional Changes Caused by rons and Astrocytes Massive Calcium Loading Observed using Cryo-EM and High-Resolution Elena Britti1, Noemi Esteras Gallego2, Joaquim Ros1, Andrey Y. Abramov2. Respirometry 1University of Lleida, Lleida, Spain, 2Institute of Neurology UCL, London, Jasiel O. Strubbe1, Jason Schrad2, Kristin N. Parent2, James F. Conway3, United Kingdom. Jason N. Bazil1. Tau protein, encoded by MAPT gene, is a microtubule-associated protein play- 1Pharmacology and Toxicology, Michigan State University, East Lansing, ing physiological role in neurons. Tau aggregation and phosphorylation is MI, USA, 2Biochemistry, Michigan State University, East Lansing, MI, USA, involved in several neurodegenerative diseases such as Frontotemporal Demen- 3Structural Biology, University of Pittsburgh, Pittsburgh, PA, USA. tia, Alzheimer disease and Parkinson disease. Although misfolded tau has Excessive calcium accumulation is the main cause of cardiac tissue and cell strong familial and histopathological (as intracellular tangles) association to death during myocardial ischemia-reperfusion injury (IR injury) and myocar- neurodegenerative disorders the cellular mechanism of tau-induced pathology dial infarction. Calcium dysregulation and overload leads to mitochondrial remains unclear. dysfunction, excessive reactive oxygen species (ROS) production, cata- We used live cell imaging technique to explore effects of 24 and 48 hours in- strophic energy failure, and opening of the mitochondrial permeability tran- cubation of the tau K18 fragment on cytosolic and mitochondrial calcium levels sition pore (mPTP). In the present study we quantified the Ca2þ-phosphate in primary cortical neurons and astrocytes with as genetically encoded calcium granule (CaPiG) size and number distributions in isolated cardiac mitochon- indicators (GECIs) and fluorescent probes for cytosolic and mitochondrial cal- dria exposed to calcium boluses in the permeability transition range in the cium analysis. presence and absence of cyclosporin A (CsA). A time course series of images We found out that incubation of the cortical co–culture with tau K18 fragment 2þ were obtained by electron cryomicroscopy (CryoEM) to visualize the conse- for 24 or 48 hours induced spontaneous oscillations in [Ca ]c of primary

BPJ 9370_9373 Monday, March 4, 2019 271a neurons and astrocytes. These oscillations lead to the increased basal level of courses of oxygen-consumption of various substrates. Different substrates led mitochondrial calcium in both astrocytes and neurons compared to control to different levels of the saturated state3 respiration. On average, it was 8.8 cells. Stimulation of the calcium signal in neurons with glutamate or astrocytes times higher in the heart compared to the kidney. Also, the alterations in the with ATP demonstrated inhibition of mitochondrial calcium efflux. Permeabi- respiratory substrates led to dramatically different respiratory rates/dynamics lisation of control and tau-treated cortical neurons and astrocytes with digitonin in isolated mitochondria from the chosen organs. In the presence of succinate in pseudo-intracellular medium followed by application of micromolar calcium alone, state3 JO2 flux was negligible which increased drastically in presence of confirmed delayed Ca2þ efflux by mitochondria incubated with tau K18 frag- rotenone given to block Reverse-Electron-Transport(RET) in the heart. How- 2þ ment. Thus, misfolded tau K18 induces a [Ca ]c oscillations in neurons and ever in the kidney, the response was similar comparing succinate to succina- astrocytes and mitochondrial calcium overload through inhibition of mitochon- teþrotenone. This may be due to the fact that RET via complex-I has a more drial NCLX which is responsible for Ca2þ efflux that could be a trigger for prominent regulatory role in the heart compared to the kidney. This phenome- neurotoxicity. These results offer a new perspective in understanding the mech- non is applicable not only to OxPhos regulation, but also to other mitochondrial anisms of tau-related neurodegeneration. functions, such as ROS-production and PTP-opening. Our results show that choice of respiratory substrates profoundly affects mitochondrial responses in 1333-Pos a tissue-specific manner. Calcium Stimulation of Mitochondrial Respiration is Substrate Dependent and Tissue Specific 1335-Pos Sunil M. Kandel1, Namrata Tomar1, Nadezhda Zheleznova2, Said H. Audi3, Cyclophilin D Inhibition Rescues Hypoxia-Induced Neonatal Cardio- Allen W. Cowley Jr2, Ranjan K. Dash1. myopathy 1Biomedical Engineering, Medical College of Wisconsin, Milwaukee, WI, Gisela Beutner, George A. Porter, Jr. USA, 2Physiology, Medical College of Wisconsin, Milwaukee, WI, USA, Dept Pediatrics, Univ Rochester, Rochester, NY, USA. 3Biomedical Engineering, Marquette University, Milwaukee, WI, USA. BACKGROUND: Inhibition or deletion of cyclophilin D (CyPD) in neonatal Mitochondrial enzymes, such as pyruvate dehydrogenase, isocitrate dehydro- myocytes closes the mitochondrial permeability transition pore, decreases genase, and alpha-ketoglutarate dehydrogenase, have been shown to be stim- mitochondrial reactive oxygen species production, increases myocyte differen- ulated by calcium ion in isolated enzyme experimental systems. However, tiation and cardiac function in the first week of life. HYPOTHESIS: The the extent of Ca2þ stimulation of these enzymes and the resulting integrated neonatal cardiomyopathy induced by exposing mice to hypoxia is due to stimulatory effect on mitochondrial energy metabolism in in-situ and in-vivo disruption in a CyPD-mitochondrial-ROS-myocyte differentiation axis and states in the heart and kidney remains to be clarified. In this study, we hy- can be rescued by inhibition of CyPD. METHODS: Wild type mice were pothesized that Ca2þ stimulation of mitochondrial respiration and ATP syn- exposed to hypoxia (12% oxygen) or room air (RA) from embryonic day 19 thesis is substrate dependent and tissue-specific. To test this hypothesis, we to postnatal day (P) 7 with or without daily intraperitoneal injections of vehicle isolated mitochondria from heart and kidney outer medulla from Sprague- or 10 mg/kg cyclosporin A (CsA) or NIM811, two inhibitors of CyPD. Mice Dawley rats. Oxygen consumption rates were determined under different res- were examined for cardiac function by echocardiography. Hearts were har- piratory states: leak state (energized mitochondria in absence of ADP) and vested to isolate mitochondria to measure oxygen consumption, electron trans- the ADP-stimulated state (energized mitochondria in presence of saturating port chain enzyme and CyPD enzyme activity, and myocyte differentiation. concentration of ADP) with/without bolus injection of different concentra- RESULTS: Neonatal hypoxia caused significantly decreased cardiac ejection tions of CaCl2 using an Oroboros Oxygraph-2k Instrument with various res- fraction (RA: 83.6 þ/ 4.8 (s.d.), N=31; hypoxia: 74.4 þ/ 7.8, N=31; piratory substrates, including pyruvateþmalate (PM), glutamateþmalate P<0.0001). Treatment with CsA and NIM811 significantly increased ejection (GM), alpha-ketoglutarateþmalate (AM), palmitoyl-L-carnitineþmalate fraction (CsA: 81.4 þ/ 8.9, N=14; NIM811: 80.7 þ/ 5.7, N=16; P<0.05 (PCM), and succinate (SUC). The results demonstrated that the various res- to vehicle: 76.0 þ/ 5.2, N=16) to levels not significantly different from RA piratory substrates yielded differences in ADP consumption rates and hence, mice. Hypoxia also significantly decreased body weight (P<0.0001), but not led to dramatically different respiratory rates in isolated mitochondria from heart weight, leading to increased heart weight/body weight ratio the heart and kidney. Importantly, it was found that Ca2þ significantly (P<0.0001). In contrast, CsA and NIM811 did not increase body weight but increased mitochondrial state III respiration with GM and AM substrates prevented increased heart weight compared to hypoxic controls. Initial exper- compared to the other substrates in both organs. Moreover, Ca2þ effects iment measuring oxygen consumption with malate/glutamate substrates on mitochondrial respiration were found to be biphasic, i.e. a small increases showed that hypoxia may increase V0 and decrease the respiratory index 2þ in [Ca ] had a stimulatory effect while large increases had inhibitory effect. (RCI) ratio while treating hypoxic pups with CsA or NIM811 decreased V0 These results suggest that energized isolated mitochondria in presence of and increased RCI to levels seen in room air treated mice (N=1-2). CONCLU- Ca2þ do not respond the same extent when different substrates are used. SIONS: Hypoxic exposure at birth causes cardiac hypertrophy, decreases car- We conclude that isolated mitochondria require a suitable substrate combina- diac function, and uncouples cardiac mitochondria, while inhibition of CyPD tion, optimal [Ca2þ], and specific tissue source of mitochondria to achieve a rescues these effects. level of energy metabolism and ATP production comparable to in-vivo conditions. 1336-Pos Systemic Metabolomics and Mitochondrial Energetics in High- Compared 1334-Pos to Low-Running Capacity Rats as a Function of Age Differential Regulation of Substrate Dependent Mitochondrial Respiration Miguel A. Aon1, Andrew Lachance1, Sarah J. Mitchell2, Kelsey Bullock2, in the Heart and Kidney Sonia Cortassa1, Steven J. Sollott1. Sunil M. Kandel1, Namrata Tomar1, Nadezda Zheleznova2, Said H. Audi3, 1Lab Cardiovasc Sci, Natl Inst Aging NIH, Baltimore, MD, USA, Allen W. Cowley Jr2, Ranjan K. Dash1. 2Translational Gerontology branch, Natl Inst Aging NIH, Baltimore, MD, 1Biomedical Engineering, Medical College of Wisconsin, Milwaukee, WI, USA. USA, 2Physiology, Medical College of Wisconsin, Milwaukee, WI, USA, Exercise capacity during aging is strongly linked to life quality, morbidity and 3Biomedical Engineering, Marquette University, Milwaukee, WI, USA. mortality. In a selectively-bred rat model of intrinsic aerobic phenotypes, low- It has been recognized that the efficiency of oxidative phosphorylation(Ox- capacity (LCR) compared to high-capacity running (HCR) rats exhibit Phos) depends on the choice of the respiratory substrates, which feed electrons increased rates of metabolic syndrome, adiposity, dyslipidemia, endothelial to the mitochondrial Electron Transport Chain. The substrate combinations dysfunction, and insulin resistance. Previously, we showed that with aging normally represent metabolites that utilize different pathways of mitochondrial HCR rats present greater mitochondrial turnover underlying higher cardiomyo- metabolism. In this study, we analyzed a variety of substrate combinations cyte energetic performance, sustained by preferential selection of lipids oxida- including pyruvateþmalate, glutamateþmalate, alpha-ketoglutarateþmalate, tion with improved control of acetyl-CoA (AcCoA) concentration. All these palmitoyl-L-carnitineþmalate, and succinate5rotenone to determine the sub- positive indexes decline with aging, but they are better preserved in HCR vs. strate combination that maximize respiration and ATP production in mitochon- LCR, parallel with healthier aging and extended longevity. To investigate sys- dria isolated from the heart and kidney. To test mitochondrial function under a temic metabolism in main organs supplying substrates (fatty acids, glucose, ke- specific substrate combination, cardiac and renal mitochondria were isolated tone bodies) to the heart, untargeted metabolomics was performed in liver and from Sprague-Dawley rats and oxygen-consumption rates(JO2) were measured serum of young and middle age female HCR and LCR rats. Multivariate statis- in the presence and absence of ADP that corresponded to leak and ADP- tics indicated a metabolome pattern in liver comprised by higher abundance of stimulated states, respectively. We first measured JO2 at various ADP concen- fatty acids (e.g., palmitic, palmitoleic, oleic) at similar levels of intermediates trations to find the saturated ADP-stimulated respiration. The ADP concentra- from glucose metabolism in HCR vs. LCR. As a function of age, the serum of tion that saturated ADP-stimulated respiration was used to measure the time- HCR rats exhibited an enrichment in fatty acids (e.g., linoleic, palmitic,

BPJ 9370_9373 272a Monday, March 4, 2019 arachidic) consistent with the changes observed in liver’s metabolome. Higher campal cells. ATX treatment demonstrated promise with increased liver AMP levels in HCR vs. LCR suggest activation of AMPK signaling, in cellular growth, which promotes usage of ATP by the cell and ROS pro- agreement with the enhanced autophagy/mitophagy observed in HCR. Impor- duction. This growth is also observed in the presence of Ab and with tantly, the higher relative LCR/HCR fold-changes of acetylated intermediates, other treatments may be useful in the treatment of these two disease in both liver and serum, agrees with the idea of widespread increased acetyla- states. tion, compatible with the higher concentration of AcCoA measured in cardio- myocytes from LCR vs. HCR. Metabolomics reveal systemic, organ-concerted 1339-Pos (liver-serum-heart), metabolic remodeling compatible with higher energetic Mitochondrial Membrane Potential Heterogeneity in Cancer Cells is Inde- performance, better preserved mitochondrial turnover, preferential lipid oxida- pendent of the Cell Cycle and Influences Response to Hyperpolarizing tion, and improved control of acetylation in HCR leading to extended longevity Agents 1 1 1 and better functional capacity. Morgan E. Morris , Diana Fang , Kareem A. Heslop , Charleston F. Christie1, Akos A. Gerencser2, Martin D. Brand2, 1337-Pos Eduardo N. Maldonado1,3. The Complex Crosstalk between Parvalbumin and Mitochondria Regula- 1Drug Discovery & Biomedical Sciences, Medical University of South tion through Changes in Mitochondrial Dynamics Carolina, Charleston, SC, USA, 2Buck Institute for Research on Aging, Lucia Lichvarova1, Thomas Henzi1, Dzhamilja Safiulina2, Allen Kaasik2, Novato, CA, USA, 3Hollings Cancer Center, Medical University of South Beat Schwaller1. Carolina, Charleston, SC, USA. 1Department Medicine, University of Fribourg, Fribourg, Switzerland, Mitochondrial membrane potential (DJ) is sustained by metabolite flux 2University of Tartu, Department Medicine, Institute of Biomedicine and through Voltage dependent anion channels (VDAC). Free a/b-tubulin heter- Translational Medicine, Tartu, Estonia. odimers decrease VDAC conductance and high free tubulin in cancer cells Parvalbumin (PV) is a cytosolic Ca2þ-binding protein, or better perceived as decreases DJ. The small molecule erastin antagonizes the inhibitory effect Ca2þ signal modulator, that together with mitochondria and ER compartments of tubulin on VDAC and increases DJ. We found higher erastin-induced play an important role in Ca2þ signaling, buffering and sequestration. More- hyperpolarization in low DJ compared to high DJ cells. We hypothesized over, antagonistic regulation of PV expression levels and mitochondrial vol- that heterogeneity of DJ is independent of cell cycle and determines the ume occurs in several in vivo and in vitro model systems. The aim of this magnitude of drug-induced hyperpolarization. Our AIM was to quantitate study was to examine mitochondrial dynamics (fusion, fission, mitophagy) DJ in cancer cells in relation with the cell cycle and after mitochondrial in genetically modified MDCK cell lines characterized by PV overexpression hyperpolarizing agents. Methods: Confocal fluorescence microscopy as- or PV downregulation. Cell morphology was assessed by live-cell confocal sessed DJ (TMRM, rhodamine 123) and plasma membrane potential (Di- imaging and 3D-reconstruction using Imaris software. Mitochondrial fusion BAC4(3), PMPI). HepG2 and Huh7 human hepatocarcinoma cells and was analyzed as fluorescence change of the photo-convertible protein HCC4006 human lung adenocarcinoma cells were used to measure DJ in mEOS2. Selective removal of damaged mitochondria by the process of auto- relative and absolute values. HepG2 were synchronized in G1 by serum phagy (mitophagy) was quantified from TEM images, as well as from starvation, early S by double thymidine block, and G2 by double thymidine confocal images showing fluorescence of YFP-Parkin, GFP-LC3-C and block followed by nocodazole. Results: HepG2, Huh7, and HCC4006 mKeima proteins. Transcript and protein expression levels of selected genes cells displayed wide spectrum of DJ with median values of 20%, 25%, were evaluated by RT-qPCR and Western blot, respectively. Volumetric ana- and 29% of maximal constitutive DJ respectively. HepG2 DJ range lyses revealed the antagonistic regulation of PV and mitochondrial volume in was 120 mV to 230 mV. DJ heterogeneity did not correlate with differ- MDCK cells. Control (PV-negative) cells showed typical epithelioid ences in plasma membrane potential. After synchronization in S, G1, and morphology with elongated mitochondria and frequent fusion-fission events. G2, DJ remained heterogeneous. The hyperpolarizing effect of erastin, PV-overexpression resulted in smaller, roundish PV-MDCK cells and shorter paclitaxel (decreases free tubulin), and oligomycin (inhibits complex V) mitochondria, the latter related to reduced fusion rates and decreased expres- was higher in low DJ cells compared to high DJ cells (288% vs 148%, sion of genes involved in mitochondrial fusion. They also exhibited increased 334% vs 141%, and 137% vs 21% increase, respectively). Conclusion:Het- mitophagy, a likely cause for the decreased mitochondrial volumes and the erogeneity of DJ is independent of cell cycle in cancer cells. Constitutive smaller overall cell size. PV down-regulation by an shRNA approach in DJ determines the magnitude of response to hyperpolarizing agents PV-MDCK cells reverted the mitochondrial morphology to the condition pre- with different mechanisms of action, suggesting a cell specific modulation vailing in parental MDCK cells, resulting from faster mitochondrial move- of DJ. ment and augmented fusion rates. Hence, PV-modulated, bi-directional and reversible mitochondrial dynamics are key to regulation of mitochondrial 1340-Pos volume. St. John’s Worth Extract Induces Apoptosis and Inhibits Cancer-Related Inflamation in Basal Cell Carcinoma Cell Lines 1338-Pos Ebru Celik1, M. Salih Celik2, H. Mahir Kaplan3, Ergin Singirik3. The Effects of Astaxanthin on Amyloid Beta Challenged Hippocampal Cell 1Department of Dermatology, Hatay Mustafa Kemal University, Faculty of Growth and Mitochondrial Function During Hypoglycemia Medicine, Antakya, Turkey, 2Medical Faculty Student, Hatay Mustafa Kemal Marie L. Kelly-Worden, Emma Cieslik, Julie A. Griffith. University, Faculty of Medicine, Antakya, Turkey, 3Department of Dept Biology, Ball State Univ, Muncie, IN, USA. Pharmacology, Cukurova University, Faculty of Medicine, Adana, Turkey. In the next 30 years, the amount of individuals diagnosed with Alz- Basal Cell Carcinoma (BCC) is the most commonly diagnosed skin cancer in heimer’s disease is expected to reach 30 million while the rate of diabetes the world. It can be treated through standard surgical excision in most of the mellitus is also projected to rise as well. In diabetes, hypoglycemia is a patients, but some patients can not be tolerate surgical procedures; therefore, common consequence due to therapy. Previous research has shown a po- it should be used non-surgical treatment for these kinds of patients. St. John’s tential link between Alzheimer’s disease and diabetes. This study sought worth/Hypericum perforatum (HP) plant has biological active contents and is to determine if Astaxanthin, ATX, could prevent mitochondrial dysfunc- also used by local people in treatment of some diseases. The content of HP tion from the compounded effects of amyloid b (Ab) plaque and hypogly- affects apoptosis. Apoptotic pathways are significant in cancer development cemia. Growth patterns, ATP production, and ROS generation were and chemotherapeutic resistance. Furthermore, it is reported that anti- examined in 2 mM, 5 mM, 25 mM (hypoglycemic groups) and 2 mM or inflammatory agents are a potential target for cancer. 5 mM glucose, and then treated with or without ATX or Ab. When hypo- Thus, we studied if HP extract affects apoptotic pathways mediators; caspase-3, glycemic groups were treated with ATX, their growth patterns were either bax, bcl-2, gadd153, grp78, AIF and cell cycle G2/M checkpoint kinase; wee 1, comparable to or increased. ATX and Ab monomers treated cells demon- and also inflammation mediators; iNOS, COX-2, cPLA2, NFkB expression in strated increased growth patterns over hypoglycemic cells treated with Ab treated BCC cells. monomers. Ab alone treated groups overall had significantly less growth Human basal cancer cell lines were obtained from ATCC, then were grown than controls (p < 0.05). Hypoglycemic groups produced overall low on Dulbecco’s Modified Eagle’s Medium (DMEM). Cell viability assay and levels of ATP when ATP production was analyzed. Cells cultured with cell homogenization were performed. One of the two BCC cell lines was Ab demonstrated low levels of average fluorescence generated by ROS given HP extract, whereas the other was not given. Then their activity of production using the MitoSox assay while ATX groups actually produced apoptotic pathways, cell cycle, inflammation mediators were analyzed by higher to normal levels of ROS. Cells grown in the presence of Ab and ELISA in these cultured cells. The data were analyzed statistically. ATX generally produced more ROS than just Ab groups. Thus, hypogly- It was detected that HP extract treatment increased expression of caspase-3, cemia does appear to compound the effects of Ab monomers on hippo- bax, gadd153, grp78, AIF apoptotic proteins and cell cycle G2/M checkpoint

BPJ 9370_9373 Monday, March 4, 2019 273a kinase- wee 1 although it decreased bcl-2 antiapoptotic protein and iNOS, 1343-Pos COX-2, cPLA2, NFkB inflammatory mediators in BCC cells. Combinational Genetically Encoded Toolbox for Cell-Surface Mucin In this study, it is asserted that HP extract will be able to affect in the treatment Biopolymer Engineering of BCC due to characteristic feature of anti-inflammatory and wiping out can- Hao Pan1, Matthew Paszek2. cer cells by affecting several intracellular pathways. 1Biophysics, Cornell Univ, Ithaca, NY, USA, 2Cornell University, Ithaca, NY, USA. 1341-Pos Few approaches exist for the stable and controllable synthesis of bottlebrush Loss of Mgr2P Destabilizes the TIM23 Channel and Reduces Mitochon- biopolymer coatings on eukaryotic cells. Inspired by cellular mucins, we con- drial Emission of Reactive Oxygen Species structed a mix-and-match catalogue of cDNA bricks for biosynthesis of bottle- Oygul Mirzalieva1, Shinhye Jeon2, Kevin Damri1, Ruth Hartke1, brush biopolymers of tunable size, glycan side-chain spacing, and charge Layla Drwesh3, Keren Demishtein-Zohary3, Abdussalam Azem3, density. Fusing the polymer cDNAs to additional bricks for leader tags, optical Cory D. Dunn4,5, Pablo M. Peixoto1,6. reporters, membrane anchors, and cytoplasmic motifs, we can generate a li- 1Dept Nat Sci, Baruch Col CUNY, New York, NY, USA, 2Dept Nat Sci, City brary of over 400 cDNAs, each encoding a polymer coating with unique chem- University of New York (CUNY-Baruch College), New York, NY, USA, ical, physical, and optical properties. Notably, we found that we can tune the 3Department of Biochemistry and Molecular Biology, The George S. Wise number of negatively charged sialic acids on our biopolymers through rational Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel, 4Institute of design of cytoplasmic motifs that impair or promote intracellular recycling. Biotechnology, Helsinki Institute of Life Science, University of Helsinki, Applying this biology-by-parts approach, we have generated new anti- Helsinki, Finland, 5Department of Molecular Biology and Genetics, Koc¸ adhesive coatings on cells and tuned the diffusion and transport of transmem- University, Istanbul, Turkey, 6Graduate Center, CUNY, New York, NY, brane proteins on the cell surface. We have also found that clustering of serine USA. or threonine contributes to the extension of O-glycans. Thus, we expect that our The TIM23 complex is a hub for translocation of preproteins into or across the parts inventory can be broadly used to study in-vivo glycosylation, engineer the mitochondrial inner membrane. This dual sorting mechanism is currently be- adhesive properties of cells in biotechnology, as well as the transport and func- ing investigated, and in yeast it seems to be regulated by a recently discovered tioning of native receptors on the cell surface. subunit, the Mgr2 protein. Deletion of Mgr2p has been found to delay protein translocation into the matrix and accumulation in the inner membrane. This 1344-Pos result and other findings suggested that Mgr2p controls the lateral release of Creation of Scaffold-Free Human-Induced Pluripotent Stem Cell-Derived inner membrane proteins harboring a stop-transfer signal that follows an Cardiomyocytes (hiPSC-CMs) Cell Sheets for Drug Screening and Regen- N-terminal acid presequence. However, the mechanism of lateral release is un- erative Medicine known. Also unknown is whether the presence or absence of yeast Mgr2p Xie He, Ana Da, Silva Costa, Francis L. Burton, Godfrey L. Smith. might modulate reactive oxygen species (ROS), like its mammalian counter- Institute of Cardiovascular and Medical Sciences, University of Glasgow, part, Romo1. Here, we used patch clamp electrophysiology and spectro- Glasgow, United Kingdom. fluorimetry to investigate the effect of Mgr2p deletion upon the channel We describe a method to create a liquid surface to support self-assembly of activity of TIM23 and upon emission of ROS from mitochondria. Our results cells, the curvature of which can be altered to produce cell sheets with different suggest that Mgr2p deletion both destabilizes the TIM23 channel and de- shapes. The assembly comprises a removable polydimethylsiloxane (PDMS) creases ROS emission. However, these two effects seem independent from cell mold and a standard 96-well polystyrene (PS) microplate. The cell mold each other, because ROS emission was not affected in the presence of prese- is inserted into each well of the plate to create an air-filled chamber between quence peptides. PDMS and PS. The sealed volume of air in the chamber provides mechanical support for the liquid inside the PDMS cell mold, allowing the bottom surface of the suspended liquid to be flat which we show to provide a supporting surface Posters: Emerging Techniques and Synthetic for self-assembly for cell cultures. Thereby, small scale scaffold-free cell sheets Biology can be produced easily and repeatably. The size of the cell sheets can be varied from 1 to 3 mm depending on the size and shape of PDMS cell mold used. The 1342-Pos thickness of the cell sheet ranges 0.06-0.1 mm day 1 after plating, representing Characterizing DNA Nanotube Networks Assembled via Y-Junction DNA 3-5 cell layers. The electrophysiology and contractility of the 3D culture layer Origami Seeds containing commercially available human-induced pluripotent stem cell- Michael S. Pacella1, Ruby Liu2, Jasen Zhang1, Tiffany Hou1, derived cardiomyocytes (hiPS-CMs) was investigated using the CellOPTIQÒ Jonathan Gunn1, Paul Vallejo1, Pragya Singh1, Marc Bordui1, platform (Clyde Biosciences Ltd). Visible scaffold-free hiPS-CMs cell sheets Altarash Barthakur1, Rebecca Schulman1. formed after 24 hours of the cell seeding showed spontaneous rhythmic electri- 1Chemical and Biomolecular Engineering, Johns Hopkins University, cal and contractile activity; by comparison, traditional 2D hiPS-CMs cell cul- Baltimore, MD, USA, 2Biomedical Engineering, Johns Hopkins University, ture required several days before comparable activity was evident. The 3D Baltimore, MD, USA. culture layer formed with the present method is thicker and more uniform DNA nanotechnology offers a means to synthesize custom nano-structured than the traditional 2D monolayer cell culture. Scaffold-free 3D cell sheets materials from the ground up in a hierarchical fashion. While the assembly made with the method therefore constitute pure tissue-like cell structures which of DNA nanostructures from small (nanometer-scale) monomeric compo- are suitable for high throughput drug screening and have potential also for nents has been studied extensively, a general model for the hierarchical as- regenerative medicine. sembly of rigid or semiflexible units into multimicron-scale structures remains elusive. To study such hierarchical assembly, we have developed a 1345-Pos system for assembling extend networks of semiflexible DNA nanotubes. Integration of Reaction-Diffusion Master Equation and Brownian Dy- These nanotubes assemble from nanometer scale tiles into materials via the namics Methodologies to Simulate a Minimal Cell nucleated growth from sites on rigid, Y-shaped nanotube seeds. In this pro- Tyler M. Earnest1, Michael J. Hallock2, Zaida Luthey-Schulten1. 1 cess, nanotubes first grow from these Y-shaped seeds to form 3-armed nano- Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, USA, 2 tube architectures. These architectures then in turn assemble into networks School of Chemical Sciences, University of Illinois at Urbana-Champaign, that include as many as 80 seeds and can extend over areas as large as 900 Urbana, IL, USA. mm2. We measure the kinetics of network growth and find that the assembly Simulating whole-cells at the genome scale involves the integration of models of these networks can be explained by a stochastic model of hierarchical as- which operate at incompatible length, time, and concentration scales. Hybrid sembly that assumes a single joining rate between DNA nanotube ends. simulation schemes are necessary to bridge these scales. Progress is presented Because the number of nucleation sites on the seeds and their spatial arrange- on a method combining the reaction-diffusion master equation (RDME) with ment can be systematically varied by design, this system allows the assembly Brownian dynamics (BD) into Lattice Microbes, a GPU-based RDME simula- of a wide variety of networks and characterization of the assembly mecha- tion code. RDME is used for protein diffusion, while BD is used for diffusion of nisms that lead to different types of material architectures at length scales particles larger than the subvolume size (). The BD integration is per- of tens to hundreds of microns. Further, by activating/deactivating the incor- formed on the GPU, allowing both simulations to progress in lockstep. Owing porated Y-shaped DNA origami junctions via strand displacement we are also to the dense molecular crowding observed in living cells, accounting for able to direct networks to change form, suggesting a model system for under- excluded volume interactions is necessary to predict the spatial distribution standing not only the formation of filament networks at this length-scale but and diffusion time scale of chemical species. Particles interact through a shared also their reconfiguration. excluded volume field which influences the spatial transition rate of RDME

BPJ 9370_9373 274a Monday, March 4, 2019 particles as well as the friction coefficient of BD particles. Integration of the nature of these synthetic linkages should be a powerful tool for biophysical methods shows little impact on the time-to-solution compared to a pure analysis and extend the possible architectures in synthetic biology. RDME simulation. A proof of principle is presented using the particle densities and geometry of a 500-nm diameter minimal cell. This minimal cell, named 1349-Pos JCVI-syn3A, with a 543-kbp genome and 493 genes, provides a versatile plat- A Semi-Synthetic Approach to Engineer Ligand- and Voltage-Gated Ion form to study the basics of life. This computational methodology will become a Channels in Live Cells major component of the goal to simulate JCVI-syn3A at a spatially resolved, Keith K. Khoo, Iacopo Galleano, Stephan A. Pless. stochastic level. Dept Drug Design/Pharmacol, Univ Copenhagen, Copenhagen, Denmark. Site-directed insertion of non-canonical amino acids (ncAAs) or post- 1346-Pos translational modifications (PTMs) is often challenging, particularly in large Optogenetic Delineation of Receptor Tyrosine Kinase Subcircuits in PC12 membrane proteins, such as ion channels. Amber-codon suppression mutagen- Cell Differentiation esis is routinely used for this purpose. However, this approach has limitations John Khamo1, Vishnu Krishnamurthy1, Qixin Chen2, Jiajie Diao2, regarding the side chain identity that can be introduced, as well as the efficiency Kai Zhang1. of incorporation. For this reason, it would be valuable to develop a method that 1Dept Biochemistry, Univ Illinois at Urbana-Champaign, Urbana, IL, USA, overcomes these issues. Here, we present the development of a intein-based 2Dept Cancer Biol, Univ Cincinnati, Cincinnati, OH, USA. approach for the incorporation of synthetic peptides into ion channels expressed Nerve growth factor elicits signaling outcomes by interacting with both its in live cells. The approach utilizes split inteins, which can seamlessly join high-affinity receptor, TrkA, and its low-affinity receptor, p75NTR. Although selected protein segments, to replace selected peptide segments within ion these two receptors can regulate distinct cellular outcomes, they both activate channels with synthetic peptides carrying the desired ncAAs and/or PTMs. the extracellular-signal-regulated kinase pathway upon stimulation with ligand. We demonstrate the successful implementation of this approach by inserting To delineate TrkA subcircuits in PC12 cell differentiation, we developed an op- non-canonical lysine analogs into the extracellular binding pocket of P2X2 togenetic system where light was used to specifically activate TrkA signaling in receptors and by inserting PTMs into intracellular linkers of the cardiac the absence of nerve growth factor. By using tyrosine mutants of the optoge- voltage-gated sodium channel, Nav1.5. Correct reconstitution of full-length netic TrkA in combination with pathway-specific pharmacological inhibition, ion channels and the impact of the mutations on channel function were verified we find that Y490 and Y785 each contribute to PC12 cell differentiation by Western blots and electrophysiology. The technology has the potential to through the extracellular-signal-regulated kinase pathway in an additive complement existing ribosome-dependent methods to incorporate ncAAs and manner. Optogenetic activation of TrkA eliminates the confounding effect of PTMs, especially for those that cannot currently be incorporated using existing p75NTR and other potential off-target effects of the ligand. This approach methods. Additionally, the approach offers a unique way to introduce combina- can be generalized for the mechanistic study of other receptor-mediated tions of multiple ncAAs and/or PTMs, thus enhancing the precision with which signaling pathways. we can study ion channel function and pharmacology. 1347-Pos 1350-Pos The Proposed Mechanism Behind Lyse-It(R): A Rapid Sample Preparation Removal of Colour from Textile Industrial Effluent using Modified (Epox- Technique idized) and Unmodified Rubber (Hevea Brasiliensis) Latex Tonya M. Santaus, Christopher D. Geddes. Sarah O. Oni1,2, A.K. Akinlabi2, A.A. Adeagbo1. Dept Chem/Biochem, Univ Maryland Baltimore County, Baltimore, MD, 1Department of Chemical Sciences, Lead City University, Ibadan, Nigeria, USA. 2Department of Chemistry, Federal University of Agriculture, Abeokuta, Lyse-ItÒ is a microwave-based platform that focuses microwaves for cellular Nigeria. lysis, DNA/ RNA/ protein extraction and fragmentation, and nuclease degrada- Discharge of colored effluent from textile industries interfere with the photo- tion and inactivation. DNA/RNA and protein fragmentation cannot be simply synthetic activities of aquatic life. A large number of dyes and their metabo- explained by rapid focused heating alone; therefore, we have investigated reac- lites have been reported to be toxic and carcinogenic to humans and other tive oxygen species, ROS, as contributing factors. living things. The removal of this color dye from waste effluents becomes The mechanism starts with the rapid heating and lysis of the sample which re- environmentally important. The purpose of this research is to investigate leases intact intracellular components such as genomic DNA/RNA and pro- how to remove color from textile industrial effluent using modified and un- teins. Subsequently, at higher and longer both microwave powers and times, modified natural rubber latex. Epoxidized rubber latex (ERL) and unmodified DNA/RNA and proteins get fragmented into tunable fragment sizes which rubber latex (URL) were the absorbents used on textile industry effluent. get progressively further fragmented over time. We postulate that this fragmen- Adsorption experiments were carried out on the adsorbent and adsobate, while tation is due to the generation of ROS, which in the literature, has been shown varying its contact time between 30 and 180 minutes in 30-minute increments. to cause oxidative damage, such as DNA fragmentation and protein degrada- The acidity of the solution varied from pH of 2 to 12. The effect of the pH tion. In this paper, we present data demonstrating the release and fragmentation showed that adsorption of dyes was pH dependent. Better adsorption was ob- of DNA and proteins and the generation of ROS using both fluorescent probes tained in an acidic medium as compared to an alkaline medium. It was also and metal complexes. Additionally, we demonstrate that the released and frag- noticed that adsorption decreased as contact time increased. The kinetics mented DNA is viable for Polymerase Chain Reaction (PCR). study revealed pseudo 1st order and 2nd order and temkin showed that adsorp- tion process took place. The results showed that adsorbents prepared from 1348-Pos rubber latex could be good adsorbents for the removal of dye from textile in- Spy and Snoop Superglues Enhance Anchoring and Team-Building in dustrial effluent. Biophysics and Synbio Mark Howarth. 1351-Pos Department of Biochemistry, Oxford University, Oxford, United Kingdom. Controlled Stirring of Biological and Bio-Mimetic Microdoplets Biophysicists often need robust linkage or anchoring of component parts. Even Pierre-Yves Gires, Mithun Thampi, Matthias Weiss. the best non-covalent interactions may break in milliseconds with force, so irre- Dept. Exp. Physics I, Univ. Bayreuth, Bayreuth, Germany. versible interaction systems bring important advantages. We previously engi- Producing and manipulating droplets of biological and bio-mimetic fluids in neered a genetically-encoded unbreakable interaction based on the microfluidic platforms is a versatile tool for Synthetic Biology. Here we report pathogenic bacterium Streptococcus pyogenes: a peptide tag (SpyTag) upon on an approach to achieve a controlled stirring of aqueous microdroplets by mixing forms an irreversible amide bond to its protein partner (SpyCatcher). encapsulating magnetic nano stir bars. The nanometric diameters were pro- This protein padlock has been applied for single-molecule AFM in mechanobi- duced from 30 nm iron oxide beads aligned in a magnetic field and strengthened ology, creating living biomaterials with amyloids, and making ultra-stable en- by a silica coating, the final cylindrical structures reaching lengths in the range zymes for nutrition and bioremediation. We now have other peptide/protein 1-20 mm. They were then included in microdroplets with sizes in the range 10- tags forming unbreakable linkages, including SnoopCatcher and SnoopTag/ 100 mm and varying composition, produced at a PDMS microfluidic junction DogTag/SnoopLigase. We also have new generations of SpyTag/SpyCatcher within a hydrophobic continuous phase. Proper stirring upon applying an from Rosetta-based design, along with evolution on phage or intimin-display external rotating magnetic field was monitored via quantitative microscopy. on E. coli. We will describe their use for programmable synthesis of polypro- In addition, the impact of stirring both on the diffusional transport at small Pec- tein teams for modulation of signal transduction on cancer cells and how nano- let numbers and on the formation of a mitotic spindle in Xenopus egg extract assembly stimulates the immune system against malaria. The simple modular was studied.

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Posters: Neuroscience: Experimental 1354-Pos Study of Biophysical Parameters in Rubi-Gaba Uncaging using Non- Approaches and Tools Linear Photoactivation and Electrophysiology in Cerebellar Granule Cells Marco Cozzolino1,2, Virginia Bazzurro2, Elena Gatta2, Paolo Bianchini1, 1352-Pos Alberto Diaspro1,2, Mauro Robello2. Morphology of Human Cystatin C Oligomers - Towards the Construction 1Nanoscopy, Istituto Italiano Tecnologia, Genova, Italy, 2Physics, Universita` of Hcc Nanodetector degli Studi Di Genova, Genova, Italy. Maciej Kozak1,2, Zuzanna Pietralik1, Augustyn Molinski1,3, RuBi GABA is a photoactivatable molecule whose activity can be controlled by Aneta Szymanska4, Veronica Lindstro¨m5, Anders Grubb5, Micha1 Taube1, a pulse of light. These probe compounds are prepared via covalent appendage of Kosma Szutkowski3. a light-sensitive protecting group ‘‘the cage’’ (RuBi) to a signaling molecule 1 Department of Macromolecular Physics, Adam Mickiewicz University, (GABA). With the cage bound, the signaling molecule is unable to activate its 2 Poznan, Poland, Joint Laboratory for SAXS studies, Adam Mickiewicz receptor [Diana D., J Am Chem Soc., 2014]. One photon (UV-VIS light) and 3 University, Poznan, Poland, NanoBioMedical Centre, Adam Mickiewicz two-photon (700-900nm) absorption can be used to break the ‘‘cage’’ binding. 4 5 University, Poznan, Poland, Gdansk University, Gdansk, Poland, Division The uncaged molecule becomes active and can bind on GABAA receptor site of and Pharmacology, Lund University, Lund, Sweden. [Rial Verde E.M., Front Neural Circuits., 2008]. We described a novel approach Human cystatin C (HCC) is a commonly known inhibitor of cysteine proteases to control the neurotransmitter release in time, space and amplitude of intensity. and also macromolecule used as a marker in the clinical diagnosis of the kidney In particular, uncaging method and fluorescence microscopy coupled to patch function. This protein exhibits a strong tendency to misfolding via the domain clamp technique provide a useful approach to detect a selected biological target swapping mechanism and then it is able to form oligomers, fibrils and amyloid in a temporally and spatially confined way. We analyzed how the change of deposits that can be built in brain blood vessels. The aim of our study was pre- physical parameter such as uncaging distance, exposure time, laser power, linear cise characterisation of morphology of these oligomers obtained in various con- and non-linear photoactivation influence the measurements and we have deter- ditions. Different types of HCC oligomers and aggregates were generated using mined how these parameters change the modality and efficacy of the GABA native cystatin C and its covalently stabilised variant. These HCC oligomer release and consequently the GABAA response. Specifically, localization preci- species were generated in different compositions of solution, pH values, ionic sion can be improved using advanced fluorescent optical methods, in particular, strength, and also using various temperature and agitation conditions. The we used the super-resolved and non-linear fluorescence microscopy [Diaspro A. micromorphology of obtained HCC aggregates and oligomers was described et al. QRB, 2006 ]. This allows exploring the release of caged GABA topically on the basis of electron microscopy images and topographic data from the applied in situ at defined concentration and in a specific region of neuronal cells atomic force microscopy. In parallel the process of oligomer formation was for mapping the localization and the functional distribution of GABAA receptors characterised using 1H NMR diffusometry. In the next step, these HCC oligo- in cerebellar granule cells in vitro. Finally, we are able to explore the responses mers were selected as targets for nanosensing studies. At this stage, preliminary generated by specifics drugs in different regions of neuron. construction studies of a nanosensor selective for detection of HCC variants were initiated. For this research stage the initial immobilisation protocol of an- 1355-Pos tibodies, selective to HCC oligomers, on the gold nanoparticles was tested. The Engineering Novel Genetically Encoded Voltage Indicators Based on work was supported by the grant POLTUR2/3/2017 from National Centre for Intra-Protein Electron Transfer Research and Development (Poland) and grant 117Z009 from TU¨ BITAK_ Martin J. Iwanicki1, Sohini Mukherjee1, Christopher C. Moser1, (Turkey). Brian Y. Chow2, Bohdana M. Discher1. 1Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA, 2Department of Bioengineering, University of 1353-Pos Pennsylvania, Philadelphia, PA, USA. Controlled Photosensitizing Activity of Oligomeric P-Phenylene Ethyny- b Developing technologies to optically record neuronal voltage signaling events lenes on Amyloid- Fibrils on the sub-microsecond scale is crucial in understanding interneuronal commu- 1 1 2 Adeline M. Fanni , Florencia A. Monge , Julia J. Hammond , nication. To image voltage signaling, early probes were organic voltage- Jennifer S. Martinez3, David G. Whitten4, Eva Y. Chi4. 1 sensitive dyes. These dyes tend to be exceptionally sensitive to changes in mem- Center for Biomedical Engineering, Biomedical Engineering Graduate brane potential on appropriate timescales, but cannot be targeted to a specific Program, University of New Mexico, Albuquerque, NM, USA, 2Rose- 3 membrane or cell. Genetically encoded voltage indicators (GEVIs) were engi- Hulman Institute of Technology, Terre Haute, IN, USA, Center for neered to introduce targetability. GEVIs are typically derived from a voltage- Integrated , Los Alamos National Laboratory, Los Alamos, 4 sensing domain of a natural protein, such as voltage-gated ion channels, NM, USA, Department of Chemical and Biological Engineering, University rhodopsin proteins, and voltage-sensitive phosphatases. While they can be mem- of New Mexico, Albuquerque, NM, USA. brane- and cell-specific, they tend to be slower and not as sensitive as organic Alzheimer’s disease is an incurable disorder believed to be caused by amyloid voltage-sensitive dyes. Here, we present our progress on the development and protein misfolding and aggregation. Among the therapeutic targets that have characterization of de novo designed GEVIs (dnGEVIs) to improve upon the been evaluated, disassembly and clearance of amyloid aggregates remain an speed and sensitivity of current GEVIs. Our dnGEVIs are based on artificial attractive approach. Photodynamic therapy (PDT), which has been mainly 4-a-helical bundle proteins, called maquettes. These maquettes can be designed investigated in oncology, may be used to trigger the clearance of amyloid aggre- to attach various natural cofactors and can either be water-soluble or transmem- gates through their oxidation. In PDT, a photosensitizer is used to generate reac- brane constructs. Our current designs for dnGEVIs are fusion constructs of trans- tive oxygen species upon light exposure. However, off-target oxidation is membrane maquettes and fluorescent proteins, whose fluorescence is modulated problematic as the photosensitizer activity is always turned-on under photora- by the redox state of heme embedded in the maquette. The heme cofactors in the diation. We have recently shown that oligomeric p-phenylene ethynylenes maquette are positioned to enable electron transfer as a function of change in (OPEs) selectively bind to the aggregate conformation of amyloid proteins b b membrane potential. Biophysical in vitro characterization of prototype dnGE- such as amyloid- (A 40) and display fluorescence turn-on upon binding. Addi- VIs expressed in E. coli reveals expected energy transfer quenching between tionally, OPE photosensitization was found to be activated with fluorescence heme and the fluorescent protein upon heme reduction. In vivo expression of turn-on. In this study, we are evaluating the capability of OPEs to selectively b dnGEVIs in mammalian cells proves appropriate trafficking to the cell mem- photo-oxidize A 40 fibrils upon binding and compare it to a well-known photo- brane. Currently, optimization of maquette sequences are underway to improve sensitizer Methylene Blue. The selective oxidation of fibrils over monomers are the detection of membrane potential changes. investigated by a combination of characterization methods including DNPH dot blot, mass spectrometry, and amino acid analysis. The effects of oxidation on 1356-Pos amyloid fibril morphology and secondary structures are monitored by TEM im- Probing the Biophysical Mechanisms of Infrared Neural Stimulation with aging and circular dichroism spectroscopy. The effect of oxidation on amyloid Nonlinear Raman Imaging fibril toxicity is additionally evaluated. Our results show that an anionic OPE Wilson R. Adams1, Manqing Wang2, Rekha Gautam1, E. Duco Jansen1,3, selectively sensitized the oxidation of Ab40 fibrils, but not monomers. The Anita Mahadevan-Jansen1,3. likely oxidization sites are Histidine 13, Histidine 14 and Methionine 35. The 1Biomedical Engineering, Vanderbilt University, Nashville, TN, USA, oxidation of Ab40 fibrils also led to their disassembly into shorter aggregates. 2Bioengineering College, Chongqing University, Chongqing, China, Our findings demonstrate that OPEs can selectively oxidize amyloid fibrils 3Department of Neurosurgery, Vanderbilt University Medical Center, with minimal off-target oxidation. This investigation contributes to a better un- Nashville, TN, USA. derstanding of the controllable photosensitizer activity of OPEs and could lead Infrared neural stimulation is a label-free optical neuromodulation technique to the development of a new therapeutic approach for amyloid proteinopathies. that utilized fast thermal gradients to achieve spatially selective neural

BPJ 9374_9377 276a Monday, March 4, 2019 activation. Despite extensive work over the last 15 years, the biophysical mech- mM) elicited inward currents of 42585430 (n=19) and 4558 pA (n=18), anisms of INS are still debated. Multiple downstream molecular effectors have respectively, consistent with a GABAergic neuron phenotype. In current clamp been implicated, however the ability of IR to evoke cellular responses in range mode, expression of background currents hyperpolarized cells to physiological of cell phenotypes suggests that the mechanism has a universal basis. While cell levels and stabilized RMP. APs were triggered by stimulating with 0.5-1.0 nA membrane dynamics and nanoporation have been explored, limitations in im- pulses for 0.3-1.5 ms at 0.5 Hz. Untreated iPSCD neurons had a RMP of aging technology make it difficult to study such fast photothermal processes 3451 mV (n=52) and stable APs could not be elicited by pacing. Back- such as INS. Electrophysiology and electrodynamic modeling suggests that ground current was electronically added in the form of either a non-linear þ pulsed IR light nonspecifically depolarizes irradiated cells by thermally- inwardly rectifying K current, (modeled after IK1) or a non-linear outwardly mediated transient alterations in cell membrane structure (e.g. trans-cis isomer- rectifying Kþ current (modeled after the Goldman-Hodgkin-Katz equation, ization of fatty acid chains). While the electrodynamic model fits experimental IGHK)). With the electronically expressed current, RMP became more hyperpo- data well, the hypothesis lacks direct experimental evidence of lipid structural larized: 8451 (n=14) vs. 6351 mV (n=38) in control. The lower resting changes. Nonlinear Raman imaging (NRI), (e.g. coherent anti-Stokes Raman potential was accompanied by the ability to evoke stable APs with character- scattering (CARS) and stimulated Raman scattering (SRS)) is a rapidly istic after-hyperpolarization. These findings illustrate that the non-linear evolving technique that is particularly good for observing lipid dynamics based properties of leak current rectification are critical for setting RMP and on vibrational spectra changes. We have developed a platform to observe vibra- electronic expression of these background currents via dynamic clamp im- tional spectral changes during INS to study lipid dynamics in cellular model proves functional responses (phasic firing, post synaptic summation, after- systems. We will present our methods on validating NRI to identify vibrational hyperpolarization and long-term potentiation) of hiPSCD neurons. spectral changes to pulsed IR light in microbeads, application towards several live neural cell models, and the biophysical implications of the results. A 1359-Pos deeper understanding of biophysical mechanisms underlying INS will provide Cholesterol Functionalization of Gold Nanoparticles Enables Neural the basis for optimizing delivery of infrared light for neuromodulation and shed Photo-Activation 1 2 3 3 light on novel neuromodulation technologies. Joao A. Carvalho-de-Souza , Okhil K. Nag , Eunkeu oh , Alan J. Huston , Igor Vurgaftman3, David Pepperberg4, Francisco Bezanilla1, 1357-Pos James B. Delehanty2. Effect of Amyloid Fibril Oxidation on its Seeding Potency 1Dept Biochem/Molec Biol, Univ Chicago, Chicago, IL, USA, 2Center for Daniel C. Okoye1, Adeline Fanni2, David G. Whitten3, Eva Y. Chi3. Biomolecular Science and Engineering, Naval Reserach Laboratory, 1Center for Biomedical Engineering, Post Baccalaureate Scholar Program Washingto, DC, USA, 3Optical Sciences Division, Naval Research (PREP), University of New Mexico, Albuquerque, NM, USA, 2Center for Laboratories, Washington, DC, USA, 4Dept. Ophtalmology, Univ. Illinois at Biomedical Engineering, Biomedical Engineering Graduate Program, Chicago, Chicago, IL, USA. University of New Mexico, Albuquerque, NM, USA, 3Department of Gold nanoparticles (AuNPs) attached externally to the plasma membrane of Chemical and Biological Engineering, Center for Biomedical Engineering, neurons enable the generation of action potentials (APs) in response to brief University of New Mexico, Albuquerque, NM, USA. pulses of light. Recently described functionalization techniques facilitate the Extracellular plaques composed of b-sheet rich amyloid-beta (Ab) fibrils are stable binding of AuNP bioconjugates directly to ion channels in neurons believed to cause neurodegeneration and cognitive decline in Alzheimer’s dis- that can enable robust AP generation mediated light. However, functionaliza- ease. Fibrils can form through a primary nucleation-dependent aggregation tion that affords AuNP binding to the plasma membrane in a non-protein- pathway or secondary nucleation mechanism in which preformed fibrils cause specific manner could represent a simple, single-step means of establishing Ab monomers to misfold and aggregate into amyloid fibrils. As Ab aggregation light-responsiveness in multiple types of cells contained in the same tissue. is believed to occur during the prolonged asymptomatic stage before neurode- Based on the ability of cholesterol to bind to and insert into plasma mem- generation, it is important to gain insights into the aggregation process. Photo- branes, we have tested whether AuNP functionalization with linear dihydroli- dynamic therapy utilizes photosensitizing molecules to produce reactive poic acid-poly(ethylene) glycol (DHLA-PEG) chains that are distally oxygen species, which in turn can oxidize an array of biomolecules including terminated with cholesterol (AuNP-PEG-Chol) can enable light-induced AP proteins. We have recently shown that a novel Ab fibril sensor, the anionic phe- generation in neurons. Dorsal root ganglion (DRG) neurons of rat were nylene ethynylene oligomer OPE1-, exhibits controllable photosensitizing ac- labelled with AuNP-PEG-Chol conjugates consisting of 20 nm diameter spher- tivity. When bound to Ab fibrils, OPE1- becomes highly fluorescent, ical AuNPs. Voltage recordings showed that DRG neurons labeled in this sensitizes the production of singlet oxygen, and induces fibril oxidation upon manner exhibited a capacity for AP generation in response to microsecond irradiation. As the singlet oxygen species is short-lived, the controlled photo- and millisecond pulses of 532 nm light. This likely reflected the AuNP- sensitization thus lowers off-target oxidation. In this study we evaluated the ef- PEG-Chol’s ability, upon plasmonic light absorption and resultant slight and fects of OPE1- induced Ab40 fibril oxidation on its seeding potency. Ab40 rapid heating of the plasma membrane, to induce a concomitant depolarizing aggregation seeded by non-oxidized and oxidized fibrils were characterized capacitive current. Notably, AuNP-PEG-Chol delivered to the DRG neuron by Thioflavin-T fluorescence assay and compared. Morphologies of the fibrils by inclusion in the buffer contained in the recording pipette/electrode enabled produced by both seeding conditions were imaged by transmission electron mi- similar light-responsiveness, consistent with the activity of AuNP-PEG-Chol croscopy and fibril secondary structures were analyzed by circular dichroism bound to the inner leaflet of the plasma membrane. Our results demonstrate spectroscopy. Our results thus far show that oxidation of the fibril seeds does the ability of AuNP-PEG-Chol conjugates to confer timely stable and direct not alter Ab40 aggregation rates or its seeding potency after 3 days of incuba- responsiveness to light in neurons. Further, this strategy represents a general tion. Furthermore, fibrils produced in the presence of oxidized and non- approach for establishing excitable cell photosensitivity that could be of sub- oxidized fibril seeds showed similar morphologies on TEM images. We plan stantial advantage for exploring a given tissue’s suitability for AuNP- to further investigate the effect of oxidation on fibril cell toxicity. Results mediated photo-control of neural activity. from this study contribute to a better understanding of amyloid protein aggre- gation via secondary nucleation pathway, which is important in understanding Alzheimer’s disease process. Posters: Single-Molecule Spectroscopy II 1358-Pos 1360-Pos A Neuronal Dynamic Clamp System for Action Potential Recording in Hu- Optical Tweezers with IRM, TIRF and Widefield: Studying Cytoskeletal- man Induced Pluripotent Stem Cell Derived Neurons processes with or without the Need for Fluorescence Labeling Mark Nowak1, Brian Panama2, Brandon Franks1, Randall Rasmusson2, Trey Simpson1, Sara Tafoya1, Ernie Au1, Ali Raja1, Willem Peutz1, Glenna Bett2. Andrea Candelli2, Gerrit Sitters2. 1Cytocybernetics, Buffalo, NY, USA, 2Dept Physiol/Biophys, State Univ 1LUMICKS USA INC., Boston, MA, USA, 2LUMICKS, Amsterdam, New York Buffalo, Buffalo, NY, USA. Netherlands. Human induced pluripotent stem-cell derived (hiPSCD) neurons are an impor- Microtubules and actin filaments are dynamic cytoskeletal structures that tant model system for studying neuronal electrophysiological properties. How- interact with motor proteins and play a fundamental role in many essential bio- ever, hiPSCD neurons have a depolarized resting membrane potential (RMP), logical processes including cell division, cell migration, and mechanosensing. resulting in the inability to evoke stable action potentials (APs), due to mini- Single-molecule analysis played a central role in revealing many aspects of mal/absent expression of background currents. We used the Cybercyte (Cyto- these complex and dynamic interactions. During a typical dynamic single- cybernetics) dynamic clamp system to electronically express background molecule experiment, cytoskeletal filaments are imaged for an extensive period currents, in real time, in hiPSCD GABA Neurons (Cellular Dynamics) under of time using fluorescence methods. It can be highly desirable to study these whole-cell ruptured voltage clamp. GABA (100 mM) and glutamate (100 individual protein filaments with high contrast and at high temporal resolution,

BPJ 9374_9377 Monday, March 4, 2019 277a but without the need for fluorescence labeling, to make the assay set-up less ing data, the model was used to correctly predict the rates of binding and release laborious, costly and - in some experiments - prevent inducing phototoxicity. of fluorescently labeled ATP without any adjustable parameters. This initial Here we present an experimental arrangement that includes optical tweezers in success of the model prompted its further development to other systems and ex- combination with Interference Reflection Microscopy (IRM) and Total Internal periments. In particular, the idea of group transfer elastically coupled to confor- Reflection Fluorescence (TIRF) Microscopy. Interference Reflection Micro- mational change monitored by the probe is applied and extended to (1) scopy is a recently introduced imaging method that allows visualizing biolog- calculate power stroke rate constants in two different force-spectroscopy exper- ical structures in 3D without the need of fluorescence labeling and with iments on single and double-headed myosin V; to (2) extract rate constants for sensitivity exceeding DIC microscopy. Total Internal Reflection Fluorescence hydrolysis and synthesis from controlled rotation data on F1-ATPase; and to (3) microscopy provides high resolution fluorescent imaging of specimen near the account for ‘‘dynamical effects’’ due to the internal and probe-related friction working surface with high signal to noise ratio resulting in improved single- in the single-molecule imaging experiments. molecule surface assays. An important aspect of the analysis is the statistical modeling of biasing effects In addition, we demonstrate the faster 2D imaging capabilities obtained - of finite time resolution in the photon-counting trajectories and the finite compared to confocal and STED techniques - by introducing widefield micro- response time of the imaging probes. scopy methods to our system. In this work, we will discuss the experimental design and show an overview of 1364-Pos the latest results obtained using this single-molecule approach. Interactions between a Bioflavonoid and G-Quadruplex DNA at the Ensemble and Single-Molecule Level 1361-Pos Sneha Paul, Anunay Samanta. A Novel Cuvette-FCS Setup for Single Molecule Measurements of Protein Chemistry, University of Hyderabad, Hyderabad, India. Unfolding and Early Stages of Protein Aggregation G-Quadruplex (GQ) DNA has garnered the attention of the scientific commu- Kanchan Garai, Bankanidhi Sahoo, Timir Sil. nity due to its role in important biological processes like telomerase activity in- Tata Institute of Fundamental Research, Hyderabad, India. hibition, regulation of gene expression and genomic stability. This has Fluorescence correlation spectroscopy (FCS) is a single molecule technique triggered studies on understanding the interactions of GQ with small molecules with wide spread applications in measurements of biophysical properties, of therapeutic significance. We have studied the interactions of bioflavonoid e.g., hydrodynamic size and conformational dynamics of biomolecules. How- kaempferol (3,4’,5,7-tetrahydroxyflavone, KF), known for its anti-cancer prop- ever, conventional FCS setups that use high numerical aperture objectives have erties, with a c-myc promoter-based GQ at the ensemble and single-molecule limitations in performing certain experiments such as those requiring agitations level. The binding constant (Kb) is estimated using steady state and time- such as stirring or heating, and those involving measurements in solvents with resolved fluorescence measurements. Upon binding no structural perturbation mismatch of refractive indices. Recently using an extra-long working distance of GQ is observable from the circular dichroism (CD) data. However, an in- objective (ELWD) we have developed an FCS setup that is suitable for per- crease in melting temperature of GQ upon binding suggests stabilization of forming measurements inside regular cuvettes. The radial resolution and the the DNA structure by the ligand, an observation substantiated by the ensemble confocal volume achieved in this setup are approximately 340 nm and 1.8 fL FRET experiments. Fluorescence correlation spectroscopy (FCS) measure- respectively. The signal to noise ratio in the autocorrelation data and counts ments, which probe the interactions at the single-molecule level, reveal a pro- per molecule obtained from an aqueous solution of rhodamine B are greater gressive increase in the diffusion time of KF with increase in the amount of GQ than 150 and 60 kHz respectively. We demonstrate that cuvette-FCS can be confirming the binding event. The rate constants of the association (kþ) and coupled to accessories such as autotitrators and magnetic stirrers for fully auto- dissociation (k-) processes have been estimated from the FCS time profiles. mated measurements of unfolding of proteins mediated by chemical denatur- The nature of the binding interactions and possible binding site of KF in GQ ants such as urea. Furthermore, scanning-FCS measurements in this setup have been determined from the molecular docking studies. The above findings allows measurements of time evolution of the size distribution of the early stage are expected to enhance our understanding of flavonoid-GQ DNA interactions aggregates of amyloid proteins. Hence, cuvette-FCS is a suitable technique for for development of therapies involving quadruplex DNA. performing single molecule measurements in the experiments that are regularly performed in spectrofluorometers but are generally avoided in microscope- 1365-Pos based FCS. A Single-Molecule Investigation on Interfacial Base-Stacking Interaction using a Centrifuge Force Microscope 1362-Pos Jibin Abraham Punnoose. Immunoassay Detection using Direct Single-Molecule Counting The RNA Institute, SUNY at Albany, Albany, NY, USA. Patrick J. Macdonald, Qiaoqiao Ruan, Sergey Y. Tetin. Single-molecule force experiments can be critical in providing a mechanistic Abbott Laboratories, Abbott Park, IL, USA. understanding of biomolecular interactions. The recently developed Centrifuge Single-molecule methods have a great deal of specificity for studying complex Force Microscope (CFM) enables massively parallel single-molecule force systems and dynamics, but they also offer high sensitivity for basic enumera- manipulation with a low cost and easy to use instrument. Experiments are per- tion. We apply single-molecule TIRF to immunoassays by simply and literally formed by subjecting surface tethered microspheres to centrifugal force while counting the number of target molecules captured on a streptavidin surface. We observing their motion with a microscope objective coupled to a CMOS cam- demonstrate that utility of using single-molecule counting on eluted detection era. In this project, we have developed a plug-and-play CFM module that is conjugate, the capture and sandwich formation portions of the assay having self-contained in a commercial centrifuge bucket, and is able to live-stream im- completed on microparticles or other systems. This approach is simple and ages to an external computer during . The whole system effective, and creates the opportunity for a universal detection platform that including data acquisition is controlled wirelessly through a LabVIEW inter- can be used to perform a variety of assays. We take advantage of the low vol- face. Using this system, we have been investigating the influence of the inter- ume requirements of single-molecule detection and apply a sample reloading facial DNA base stacks on the stability of DNA structures held together by approach to concentrate sample onto the detection surface. Due to the high af- sticky-end hybridization. Through systematic elimination of interfacial base- finity of the streptavidin-biotin reaction, concentration through reloading is stacks on one or either strand we were able to analyze the contribution of both quick and robust. These findings are demonstrated on both model and DNA base-stacking. DNA duplexes held together only by base-pairing showed HIV p24 antigen assay systems. Single-molecule detection techniques do not significantly faster force-dependent dissociation rates compared to those held need to be complex to exhibit both power and flexibility, and so can become together by the same number of base-pairs with interfacial base-stacks. The valuable in the field of immunoassay diagnostics. high-throughput CFM can collect hundreds of data points in a single minutes-long experiment, enabling us to probe many different duplex variants 1363-Pos and conditions. Additionally, the simple operation of our instrument facilitates Model for Concerted Power Stroke Generation in Single Myosin V and F1- use by undergraduate researchers, introducing a new generation to biophysics. ATPase Imaging Trajectories Sandor Volkan-Kacso, Rudolph Marcus. 1366-Pos Chemistry & Chemical Engineering, Caltech, Pasadena, CA, USA. Quantitative Measurements of Single-Molecule FRET between Quantum The idea of a concerted mechanism of molecular group transfer and large Dots and Organic Dyes conformational change in power stroke generation serves as the basis for an Nooshin Shatery Nejad, Candice M. Etson. elasto-chemical theory of the rate constants that dictate the stepping kinetics Physics, Wesleyan Univ, Middletown, CT, USA. in single F1-ATPase motors. The theory was employed to calculate the rate Single-molecule Fo¨rster resonance energy transfer (FRET) is a powerful tech- and equilibrium constant dependence on the rotor angle in experiments with nique for measuring distance changes on the molecular scale. FRET efficiency magnetic tweezers. Using independent biochemical and single-molecule imag- is highly sensitive to distance, with half-maximal energy transfer occurring at

BPJ 9374_9377 278a Monday, March 4, 2019 an inter-dye distance that depends on properties of the donor and acceptor mol- the single-molecule interface (‘tuba’). Therefore, the beautiful music of a single ecules including their spectral overlap and orientation, as well as the quantum molecule will be played with a typical rhythm and melody. yield of the donor. Thus, the choice of dye pair is critical to the success of any experiment. Most organic dyes that are used for single-molecule imaging have 1369-Pos bleaching lifetimes in the tens of seconds at most, which limits their use to rela- A Novel Approach for Single Molecule Observation of DNA Loops Formed tively fast processes and short experiments. Quantum dot nanocrystals can emit by ToxR-RNA Polymerase Complex, LacI, and DmpR for thousands of seconds without entering a long-lived dark state, which should Xuelin Jin, Kyubong Jo. make them good candidates for use as a FRET donor for longer single-molecule Department of Chemistry, Interdisciplinary Program of Integrated experiments. However, there has been little quantitative study of the energy Biotechnology, Sogang University, Seoul, Republic of Korea. transfer efficiency between such pairs at the single-molecule level. In this DNA looping gets involved in DNA transcription, replication and recombina- work, we immobilize Qdot-streptavidin conjugate donors on a functionalized tion. These DNA loops, that are formed by interaction between proteins, pro- coverslip and use dsDNA to couple them to organic dye acceptors. We image tein complexes, and DNA molecules, prominently regulate gene expression. our FRET pairs simultaneously by use of a custom-built total internal reflection DNA looping has been extensively studied; however, there are a limited num- (TIRF) microscope. We vary the length of the duplex DNA between our donor ber of tools to observe DNA looping phenomena. Current approaches for obser- and acceptor and quantify the energy transfer efficiency over a range of vation of DNA looping are mutation of protein binding sites succeeded by nanometer-scale distances. We also explore the effects of different coupling transcriptional regulation analysis, DNase footprinting, EMSA (electrophoretic chemistries, which can impact the orientation and absolute distance between mobility shift analysis), electron microscopy, TPM (tethered particle motion), the donor and acceptor. We hope to develop the use of quantum dots as donors and AFM (atomic force microscopy). We developed a novel approach to for quantitative single-molecule FRET experiments. directly visualize the DNA looping phenomena. We utilized a fluorescent mi- croscope for visualization of DNA loops, formed by ToxR-RNA polymerase 1367-Pos complex, LacI, and DmpR; furthermore, we quantitatively analyzed the Versatile Tools Towards Real-Time Single-Molecule Biology DNA looping efficiency by counting DNA molecules at single molecule level. Trey Simpson1, Sara Tafoya1, Ernie Au1, Ali Raja1, Willem Peutz1, We used a flowcell system to synthesize substrate DNA molecules containing Andrea Candelli2. ToxR-RNA polymerase complex binding sites, LacO sites, and DmpR binding 1LUMICKS USA INC., Boston, MA, USA, 2LUMICKS, Amsterdam, sites. As a result, we observed bright dots at the DNA looping position on the Netherlands. DNA backbones. The dots result from comparatively higher density of DNA at Biological processes performed by proteins interacting with and processing DNA looping sites compared with DNA backbones. This approach has advan- DNA and RNA are key to cell metabolism and life. Detailed insights into these tages over other approaches in simplicity and sensitivity; therefore, the processes provide essential information for understanding the molecular basis approach will be used for detection of DNA looping. Moreover, this approach of life and the pathological conditions that develop when such processes go will also be applied in studies of new therapeutics for genetic disease caused by awry. The next scientific breakthrough consists in the actual, direct, real-time gene expression errors and incorrect DNA looping. Key Word: DNA looping, observations and measurements of the individual mechanisms involved, in or- fluorescent microscopy, flowcell, ToxR, RNA polymerase, LacI, DmpR der to validate and complete the current biological models. 1370-Pos Single-molecule technologies offer an exciting opportunity to meet these chal- Detailed Kinetics of RNA Folding Pathways and Thermodynamic Origins lenges and to study protein function and activity in real-time and at the single- of Crowding by Single-Molecule FRET molecule level. Hsuan-Lei Sung1,2, Abhigyan Sengupta2, David J. Nesbitt1,2. Here, we present our efforts for further enabling discoveries in the field of 1University of Colorado, Boulder, CO, USA, 2JILA, Boulder, CO, USA. biology and biophysics using both the combination of optical tweezers with Specific three dimensional folding is required for RNA function while RNA single-molecule fluorescence microscopy (C Trap).We show the latest applica- can be a highly dynamic system that actively samples different configurations tions of these technologies that can enhance our understanding not only in the under thermal fluctuations. For instance, in gene regulation of RNA riboswitch, field of DNA/RNA-protein interactions but also in the fields of molecular mo- the prompt structural transition in response to ligand is crucial for triggering tors, protein folding/unfolding, cell membranes and genome structure and transcriptional termination, making the time scale of folding and ligand organization. response exceedingly relevant to RNA functions. In single-molecule FRET These experiments show that the technological advances in hybrid single- (smFRET) experiments, the conformational changes of RNA are visualized molecule methods can be turned into an easy-to-use and stable instrument by the time-dependent energy transfer to determine folding and unfolding rates. that has the ability to open up new venues in many research areas. Such kinetic information allows us to construct a model for manganese ribos- witch folding and explore the detailed folding mechanisms, where Mg2þ bind- 1368-Pos ing is found to pre-fold the RNA, followed by ligand (Mn2þ) association to A Single Biomolecule Interface for Advancing the Sensitivity, Selectivity further stabilize the folded structure. Moreover, the thermodynamic origins and Accuracy of Single-Molecule Detection can be obtained from temperature dependent folding. The manganese ribos- Yi-Tao Long, Yaqian Wang, Meng-Yin Li, Jie Yang. witch folding is found unexpectedly promoted by heat and the process is endo- East China Univ Sci Tech, Shanghai, China. thermic with net entropy gain, much contradictory to the conventional idea of The performance of a single-molecule sensor is mainly determined by its inter- folding into a more order state. In molecular crowding study where we try to face which is comprise of reactive molecules for the recognition, active mole- understand the influence of fundamental excluded volume on RNA structures cules for the signal responses and/or the functional materials for the signal in highly concentrated intracellular environments, the chemically inert crow- enhancement. Here, we proposed the single-biomolecule interface which is ders are shown to entropically favor the more compact folded structure of assembled from several membrane protein monomers. Therefore, the side chain RNA; a simple physical picture of osmotic pressure is employed to explain of every single amino acid groups could be designed respectively by mutagen- the crowder size dependence. Lastly, a hydraulic pressure generator is used esis to achieve site-directed modification for enhancing sensing of single moel- to apply pressure (up to 5 kbar) to RNA sample through capillary. The free vol- cule. The single molecule interface has been achieved in biological nanopore ume change (pDV) during RNA folding may be related to structural response sensors. Recent attempts of improving the selectivity and sensitivity of aeroly- under crowding conditions. sin nanopore interface by designing the mutants have been achieved notably. To study the selectivity, we replaced the positively charged R220 at the 1371-Pos entrance of the pore with negatively charged glutamic acid, resulting in barely Ftsn Bridges the FtsZ-Treadmilling and Septal Peptidoglycan Synthesis no current blockages for sensing negatively charged oligonucleotides. For the Zhixin Lyu1, David Weiss2, Jie Xiao1. 1 sensitivity, we substituted the positively charged lumen exposed amino acid Biophysics and Biophysical Chemistry, Johns Hopkins University, 2 K238 located at trans-ward third of the b-barrel stem with glutamic acid, Baltimore, MD, USA, Microbiology, University of Iowa, Iowa, IA, USA. neutral Phenylalanine and Glycine. Particularly, the excellent ability of FtsN is the last essential protein that is recruited to the septum before the onset K238G mutant aerolysin ensures the detection of methylated cytosine in a of cell constriction. It is believed that FtsN triggers the septal peptidoglycan random sequence, which is hard for WT aerolysin to achieve. The single- synthesis so as to initiate the cell constriction. However, the mechanisms biomoelcule interface could offer the possibility to study the complicated behind it are unclear. Here we studied the dynamics of FtsN by using the com- and important interactions between biomolecules. We illustrate that a single- bination of single molecule tracking (SMT), total internal reflection fluores- molecule interface likes a tub. As the single target molecule lows into the cence structured illumination microscopy (TRIF-SIM), and fluorescence ‘tuba’, its dynamic structural information could be performed by modulating recovery after photobleaching (FRAP). Mutagenesis was also utilized to study the interactions (‘buton’) between ‘molecule low’ and the resonance space at the interaction partner which contributed to FtsN’s dynamics.

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1372-Pos from tens of minutes to hours. Over such long data acquisition times, sample Exploring Conformational Dynamics in EGFR using Single-Molecule drift becomes a critical problem reducing resolution of the reconstructed image Spectroscopy and cannot be neglected. We present here a simple method to prevent long-term Shwetha Srinivasan1, Raju Regmi1, Steven Quinn1, Wei He2, drift using periodic registration to bright-field reference image of the cell at the Vandna Kukshal3, John Monsey3, Kermit L. Carraway III,4, Ron Bose3, desired focal plane. We combined this with a post-collection correction based Matthew A. Coleman2, Gabriela S. Schlau-Cohen1. on nearest-neighbor distributions that removes residual drift that happens while 1Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA, collecting data between registrations and corrects small remaining registration 2Lawrence Livermore National Laboratory, Livermore, CA, USA, errors beyond the limit of the bright-field registration. This hardware/software 3Washington University School of Medicine, St. Louis, MO, USA, combination does not require any specialized hardware or fiducials. We demon- 4Chemistry, University of California Davis School of Medicine, Sacramento, strate robustness of this approach in 2D and 3D using DNA-PAINT imaging of CA, USA. several cellular structures in fixed cells. Epidermal growth factor receptor (EGFR), a canonical example of receptor tyrosine kinase, plays a significant role in signaling leading to cell survival, pro- 1375-Pos liferation and differentiation Overexpression or mutations in EGFR cause Multimodal Label Free Stokes/Mueller Matrix and Non Linear Scanning several diseases including cancer. Membrane receptors are highly dynamic Microscopy 1 1,2 1 and entail conformational changes on ligand binding to transmit signal. So Aymeric Le Gratiet , Riccardo Marongiu , Paolo Bianchini , 1,2 far, spatio-temporal dynamics of such a biologically and medically relevant alberto diaspro . 1Nanoscopy and NIC@IIT, Istituto Italiano di Tecnologia, Genoa, Italy, protein has been largely unexplored at the single-molecule level which can 2 be employed to advance our understanding of heterogeneity, indispensable to Department of Physics, University of Genoa, Genoa, Italy. biological systems in addition to obtaining information on dynamics. The spe- Scanning microscopy techniques based on the measurements of polarimetric cific aim of this research is to explore the conformational changes of the intra- contrasts as proved to be an important tool to investigate the full optical cellular domain of EGFR upon ATP binding and phosphorylation using single- properties of a sample without using fluorescence label. It have demonstrated molecule Fo¨rster Resonance Energy Transfer (smFRET). Using smFRET, their efficiency for quantitative methods for ophthalmology and biomedical conformational dynamics under physiological conditions inaccessible using diagnosis. Among these techniques, Mueller polarimetry is a label-free contemporary techniques that provide an ensemble picture or require cryogenic method allowing the acquisition of all the physical effects induced by the samples can be explored. The intracellular kinase domain of EGFR contains the interaction of the polarized light with a medium. This interaction can be ATP binding site and phosphorylates the tyrosines present in the C-terminal tail summarizes in a single 4x4 elements Mueller-matrix by comparing the inde- of EGFR. Several signaling proteins bind to the phosphorylated tyrosines pendent coding and decoding polarization states after the medium. In this thereby propagating the signal from the intracellular domain of EGFR to the way, it gives access to polarimetric parameters such as dichroism, birefrin- further interior of the cell. We have prepared fluorescently labeled full- gence and scattering. However, early researches have used this technique length EGFR in nanodiscs to provide a native environment. Our results indicate for its sensitivity capability to the spatial organization at the single molecular that the C-terminal tail of EGFR is highly dynamic and accesses different con- level of biological structures such as chromatin-DNA. In a first step, formations upon phosphorylation. Further single-molecule studies on solely the we have developed a scanning Stokes/Mueller-matrix microscope using C-terminal tail of EGFR has helped us tease out the differences in dynamics in four channels detection methods allowing fast polarization detection. the absence and presence of phosphorylation which will be key to understand- Using Stokes-Mueller formalism and specific mathematical decomposition, ing the binding of the signaling proteins. Mueller-matrix images of a non-label sample are proposed giving access to the structural organization and orientation of the molecules under illumina- 1373-Pos tion. Then, this polarimetric technique has been coupled with non-linear con- Single-Molecule Analysis of Spliceosome Activation Kinetics Reveals trasts such as SHG and fluorescence, available into the same confocal Multiple Intermediate States microscope without moving part allowing a multimodal imaging quantitative Xingyang Fu, Aaron A. Hoskins. analysis of the same biological sample. As a proof of principle, we have University of Wisconsin-Madison, Madison, WI, USA. imaged biological structures (collagen fibers and muscles) without any labels, During pre-mRNA splicing, the spliceosome needs to be activated in an ATP- in transmission and in backscattering configuration for thin and thick samples dependent manner in order to carry out the catalytic steps of the splicing re- respectively. action. During activation, the protein composition of the spliceosome changes 1376-Pos dramatically and includes loss of the B complex proteins (Spp381, Snu23 and Challenges and Opportunities for Characterizing the Assembly of Nuclear Prp38) and Snu66. Using colocalization single molecule spectroscopy Envelope Proteins by Fluorescence Fluctuation Spectroscopy (CoSMoS), we show that Spp381, Snu23, and Snu66 release is dependent Jared Hennen1, Kwang Ho Hur1, John Kohler1, G.W. Gant Luxton2, on ATP by carrying out reactions at both high and low ATP concentrations. Joachim D. Mueller1. By comparing the characteristic dwell times for these proteins at high ATP, 1Dept Physics, Univ Minnesota, Minneapolis, MN, USA, 2Dept Genetics, we found that Spp381 and Snu23 behave similar to each other and with ki- Cell Biol, and Development, Univ Minnesota, Minneapolis, MN, USA. netics distinct from Snu66. Specifically, at both ATP concentrations, Snu66 The nuclear envelope (NE), consisting of two membranes separated by a thin shows much longer-lived populations than Spp381 and Snu23. This suggests perinuclear space, has proven to be a challenging environment for measuring that Snu66 is bound more stably to the spliceosome than the other two. protein dynamics in living cells. First, exogenously expressed, fluorescently These results are consistent with previous biochemical and structural studies tagged proteins must be properly localized to the NE. Furthermore, undulations suggesting that Spp381, Snu23, and Prp38 form a protein subcomplex of of the nuclear membranes complicate the application of fluorescence fluctua- the spliceosome. Together, our results suggest that this subcomplex and tion spectroscopy (FFS) to proteins located within the perinuclear space by Snu66 likely have two different roles in the activation step and give rise to adding a slowly fluctuating signal. While this signal is absent for proteins asso- multiple activation intermediates as they are sequentially released from the ciated with the nuclear membranes, distinguishing soluble from membrane- spliceosome. associated protein complexes by FFS presents a significant challenge since Posters: Optical Microscopy and Superresolution the diffusion time depends strongly on molecular size within the perinuclear space. Some proteins, such as the AAAþ ATPase TorsinA, may exist in Imaging II both a membrane-associated and soluble form depending on the conditions in the cell. Determining the extent to which a protein associates with the mem- 1374-Pos brane can provide valuable insight into its function within the cell. While diffu- Simple and Fiducial-Free Drift Correction for Super-Resolution Imaging sion time alone is insufficient to differentiate these populations, we explore of Cellular Structures whether the dependence of diffusion time on temperature can provide insight Michael J. Wester, Sandeep Pallikkuth, Hanieh Mazloom-Farsibaf, into a protein’s localization within the NE. In addition, we explore how spatial Mohamadreza Fazel, Keith A. Lidke. correlations can be used to study the dynamics of proteins within the NE and Physics And Astronomy, Univ New Mexico, Albuquerque, NM, USA. the membrane undulations we previously reported. Finally, z-scan FFS pro- Super-resolution fluorescence imaging using single molecule localization mi- vides a means to quantify the localization of proteins to the NE. These exam- croscopy (SMLM) can provide spatial resolution higher than the one imposed ples highlight the ways in which FFS techniques can be used to overcome the by diffraction limit. This virtue has made SMLM a powerful imaging tool for challenges of quantifying protein behavior in the NE of living cells. This work biomedical research. But this feature comes at the cost of having to acquire has been supported by a grant from the National Institutes of Health (R01 several thousands of images of the same cell, over measurements times ranging GM64589).

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1377-Pos DNA damage and the onset of genomic instability [Negrini et al, Nature Rev Quantitative Super-Resolution Microscopy of Endogenously Tagged Auto- Mol Cell Biol (2010)]. Genome-wide sequencing methods are shedding light phagy Proteins in Mammalian Cells on the series of molecular events (e.g. replication stress) that follow oncogene Chiranjib Banerjee. activation and produce genomic damage [Macheret and Halazonetis, Nature School of Physics and Astronomy, University of Minnesota-Twin Cities, (2018)]. However, how these events are unfolding in the context of the crowded Minneapolis, MN, USA. three-dimensional nucleus and which is the role of nanoscale chromatin archi- Quantitative Photoactivated Localization Microscopy (qPALM) is evolving tecture remain to be determined yet. into a powerful technique for biological and biomedical science to study Here we discuss the application of advanced fluorescence microscopy to inves- the oligomeric states of signaling proteins as well as the molecular composi- tigate chromatin alterations in a model of oncogene activation. The in vitro tion of nanoscopic protein assemblies and intracellular organelles that are too model is an established cell line bearing an inducible PML-RAR-alpha onco- small to be resolved with conventional fluorescence microscopy. The single gene. Our goal is to study the spatial distribution of replication and transcription molecule detection using qPALM in principle gives access to the location and the chromatin nanoscale architecture concerning the expression of the on- of every tagged protein and thus allows for counting of individual protein coprotein and the generation of DNA damage. species. However, in practice sophisticated data analysis, controls and cali- To this aim, we take advantage of several advanced fluorescence microscopy bration standards are required to account for artifacts stemming from the pho- techniques. In order to improve the spatial resolution, we use our recently tophysical properties of the used probes. A second outstanding problem is developed method that improves the STED imaging of nuclear foci [Sar- associated with non-physiological copy numbers and behaviors of proteins mento et al, Nat Commun (2018)]. In order to measure nanoscale chromatin transiently expressed or overexpressed in mammalian cells. To overcome compaction, we use a novel FRET assay [Pelicci et al, Biophys J supp this problem in this work, we have applied TALEN and CRISPR-cas9 (2018)]. Finally, in order to analyze the relative spatial distribution of genome editing tools to develop human cell lines where autophagy proteins different nuclear structures, we use a combination of particle localization al- are expressed at endogenous levels with photoswitchable tags, such as gorithms and image cross-correlation spectroscopy. Such a combined mEOS2 and PamCherry. Using the cells, we have conducted qPALM and approach offers an essential investigative window on the relationship be- found that ULK1, the central protein kinase for autophagy induction, un- tween chromatin alterations and oncogene activation. [Work partly funded dergoes a transition of its oligomeric state when cells are situated in by AIRC-Cariplo TRIDEO-17215]. autophagy-inducing conditions. We were able to resolve autophagosomal initiation sites that have not been seen before as well as to quantify the 1380-Pos numbers and oligomeric states of ULK1. This work demonstrates a high po- Dynamics of Activating and Inhibitory Receptors in Murine Natural Killer tential of qPALM in elucidating the dynamic changes of autophagy proteins Cells Revealed by 2D Pair Correlation Function Analysis at unprecedented levels of resolution. Per Niklas Hedde1, Elina Staaf2, Sunitha Bagawath Singh2, Sofia Johansson2, Enrico Gratton1. 1378-Pos 1Dept Biomed Eng, UCI, Irvine, CA, USA, 2Karolinska Institute, Stockholm, Quantifying Intracellular Mass Generation using Quantitative Phase Sweden. Microscopy Natural Killer (NK) cells are innate immune cells that mediate anti-viral im- Soorya Pradeep, Thomas A. Zangle. munity and protect against cancer. Their killing threshold is determined by a Chemical Engineering, University of Utah, Salt Lake City, UT, USA. balance between inhibitory and activating membrane receptors. By expressing Conventionalapproachestostudycellgrowthrelyonpopulationorwhole self MHC class I specific inhibitory receptors they become educated, i.e., they cell average measurements. This makes it difficult to assess the impact of learn to recognize self from non-self. Educated NK cells are more efficient in new and existing cancer therapies or other cell perturbations on specific killing aberrant cells. However, the mechanism for this functional difference mechanisms of biomass generation within cells. In this work we between educated and uneducated NK cells is still unclear. In a previous study, will show how a simple mass balance in discretized portions inside a we found that murine educated NK cells exhibited faster movement of the acti- growing cell can be used to localize the regions of biomass generation. vating receptor NKp46 and confinement of the inhibitory receptor Ly49A by The mass distribution inside cells is obtained using a quantitative phase im- means of image mean square displacement analysis. Yet, these values repre- aging (QPI) microscope. QPI is a label-free, non-invasive technique which sent a spatial average over a region of interest at the cell membrane. Here, measures the phase shift of light passing through a sample. The phase shift we utilized the 2D pair correlation function approach in order to study the of light depends on the refractive index and thickness of the sample, which spatial distribution of obstacles to NK cell receptors at the immune synapse. can be used to calculate mass at each point within the cell. Previous appli- With this approach, it can be seen that the motion of activating receptors is cations of QPI have typically integrated over the cell area to obtain total cell much more restricted in uneducated vs. educated cells. This further supports mass. This approach can be used to precisely measure the impact of our hypothesis that the faster and more dynamic movement of NKp46 in drugs on cell growth, but ignores data about the changing distribution of educated NK cells may facilitate a swifter response to interactions with target mass within the cell. The approach we present here is to discretize the cells. cell into small volumes and track the mass within each over time. The ve- Work supported by NIH grants P50 GM076516 and P41 GM103540, Veten- locity of each discrete element tracked is obtained using a particle image ve- skapsra˚det, Vinnova the Swedish agency for innovation systems, Selma Ander- locimetry (PIV) algorithm. By removing the component of changing cell sons stipend fund, Hierta-Reitzius stipend fund, The Swedish Foundation for mass due to motion, any change in mass in each discretized volume repre- International Cooperation in Research and Higher Education. sents generation or loss of biomass. Our method therefore reconstructs the pattern of mass generation or depletion inside a cell. Future applications 1381-Pos of this work may include studying growth dysregulation of cancer cells Type 3 Secretion System Chaperone-Effector Dynamics in Live Yersinia or the impact of drug treatments on the sub-cellular components within a enterocolitica cell. Alecia M. Achimovich1, Alma Rivera2, Julian Rocha2, Andreas Gahlmann1,2. 1379-Pos 1Dept Molecular Physiology & Biological Physics, Univ Virginia, Chromatin Alterations in a Model of Oncogene Activation Studied by Charlottesville, VA, USA, 2Dept Chemistry, Univ Virginia, Charlottesville, Advanced Fluorescence Microscopy VA, USA. Luca Lanzano01, Michele Oneto1, Isotta Cainero1,2, Simone Pelicci1,2, The Type 3 Secretion System (T3SS) is a dual membrane-spanning machine Maria Sarmento1, Lorenzo Scipioni1, Mario Faretta3, Laura Furia3, used by many Gram-negative bacterial pathogens as a mechanism of promot- Gaetano Ivan Dellino3, Pier Giuseppe Pelicci3, Paolo Bianchini1, ing host infection. After initial infection, needle-like complexes, called injec- Alberto Diaspro1,2. tisomes, pump effector protein into host cells to alter host cell function. A 1Nanoscopy and NIC@IIT, Istituto Italiano di Tecnologia, Genoa, Italy, key determinant of virulence via the T3SS is the temporal regulation of 2Department of Physics, University of Genoa, Genoa, Italy, 3Department of the secretion hierarchy: early (needle proteins), middle (pore proteins), and Experimental Oncology, European Institute of Oncology, Milan, Italy. late (effector proteins) secretion substrates are secreted in three separate Defects in the global organization of chromatin are relevant in many patholog- phases. Proteomic experiments suggest that the cytosolic components of ical processes, including cancer. The activation of oncogenes has been recog- the injectisome distinguish between secretion substrates by functioning as nized as a triggering event of alterations in the dynamics of replication, a sorting platform. However, a mechanistic understanding of how these reg- transcription and DNA damage response (DDR), that may lead to increased ulatory proteins switch secretion substrates selectivity is still lacking. Here,

BPJ 9374_9377 Monday, March 4, 2019 281a we utilize single-molecule microscopy to localize and track fluorescently 1384-Pos labeled chaperone and effector proteins in live Yersinia enterocolitica cells, Z-Scanning in Volumetric 2-Photon or Light-Sheet Microscopy with a Fast a human pathogen in which the three phases of secretion can be physically Voice-Coil Driven Focusing System and chemically controlled. By combining single-molecule tracking with bac- Gert Rapp1, Christian Schulze2, Thomas Oertner2, Florian Huhn1. terial genetics, we determine the prevalent diffusive states of chaperone pro- 1Rapp Opto Electronic, Hamburg, Germany, 2Institute for Synaptic teins and chaperone:effector protein complexes in the presence and absence Physiology, Center for Molecular Neurobiology Hamburg (ZMNH), of their (putative) cytosolic binding partners. The results allow us to test Hamburg, Germany. whether the temporal hierarchy of secretion is regulated by cytosolic T3SS Fast volumetric sampling of biological specimens with laser scanning micro- components that interact with chaperone:effector substrates either at the in- scopes is required in many fields of neurobiology and medicine. When image jectisome or while diffusing freely in the cytosol. Understanding the mech- acquisition in the lateral (x,y) plane reaches frame rates up to 100Hz by means anism of temporally-ordered secretion may reveal new strategies for of resonant scanner systems, a jump between two z-layers in the image stack targeted anti-virulence therapies and enable the controlled use of T3SSs should also be performed in a few milliseconds, a time scale that cannot be for therapeutic purposes. reached by moving the objective or the specimen. Several proposed remote focusing systems only translate a small mirror with a mass below 1g and there- 1382-Pos fore rely on fast linear positioners. We present measurements with a fast remote Label Free Microscopy with Ptychography focusing system on a two-photon-microscope using a voice coil driven linear Nicholas S. Anthony1, Paolo Bianchini1, Alberto Diaspro1,2. stage that we developed. The voice coil stage has a travel range of 1000mm, 1Nanoscopy and NIC@IIT, Istituto Italiano di Tecnologia, Genoa, Italy, m m 2 a10 m step response time of 1ms and a position accuracy better than 1 m. Department of Physics, University of Genoa, Genoa, Italy. We characterize the dynamic properties of the voice coil stage and describe The desire for new methods that can explore the function of biological systems the remote focus system. As proof of concept, we show its applicability for without the need for sample modification, or the addition of fluorescent tags, is 2-photon calcium imaging of neuronal activity in the CA1 region of rat hippo- steadily emerging. The development of so called ‘label free’ is campus. The remote focusing system is compatible with the open-source soft- important for the exploration of the natural state of biological systems, without ware ScanImage (HHMI Janelia Research Campus/Vidrio Technologies, alteration and without potentially affecting the processes being explored [1]. LCC). Label free techniques utilize contrast from novel processes to provide a means of imaging without external modification. However, the imaging of weakly in- 1385-Pos teracting samples is not trivial and has required significant development or Post-Expansion Storm Enables Imaging Beyond Super-Resolution Limits modification of current techniques to produce suitable resolution and contrast. Xiaoyu Shi1, Arthur Tran2, Xiaomeng Wang2,3, Ian Seiple2, Bo Huang1. Ptychography is a diffractive imaging technique that uses multiple diffracto- 1Dept Pharm Chem, Univ Calif San Francisco, San Francisco, CA, USA, grams collected from spatially overlapping regions of a sample to retrieve a 2Univ Calif San Francisco, San Francisco, CA, USA, 3Bates College, samples’ complex transmission function, providing images of its amplitude Lewiston, ME, USA. and phase. This overlap aides not only in the unambiguous retrieval of the Applications of Expansion Microscopy (ExM) have rapidly narrowed down the transmission function but can also be used to recover the quantitative phase, resolution gap between superresolution microscopy and electron microscopy with great potential as a contrast mechanism for cellular imaging [2, 3]. (EM), since the invention of ExM in 2015. With typical 4x expansion of cells Here we demonstrate how the quantitative phase information from ptychogra- and tissues in hydrogel, super-resolution microscopy should permit the ultimate phy, and super-resolution techniques, can be used to obtain high contrast im- resolution of a few nanometers. What seriously limits the application of expan- ages of biological molecules at high resolution, without the need for sion super-resolution microscopy is the low labeling efficiency caused by a fluorescent tags or sample modification. We also outline the benefits of inte- huge loss of fluorescence during polymerization. To overcome this bottleneck, grating ptychographic imaging with other optical microscopies, improving we designed novel trifunctional chemical linkers that survive the polymeriza- contrast and providing an additional source of information. tion and enable post-expansion fluorescence labeling. With the help of these [1] Magidson & Khodjakov, Methods in Cell Biology, 2013 linkers, we developed post-Expansion labeling Stochastic Optical Reconstruc- [2] Marrison et al., Scientific Reports, 2013 tion Microscopy (postExSTORM) with zero fluorescence loss, enabling the [3] Suman et al., Scientific Reports, 2016 visualization of preserved ultrastructures of macromolecular assemblies with 5 nm resolution. We demonstrated the power of postExSTORM by localizing 1383-Pos alpha tubulins in the hollow structure of microtubules, and resolving the cla- Understanding Complex Single Molecule Emission Patterns with Deep thrin lattice of clathrin-coated pits, in situ. This method provides single- Learning protein resolution for structural biological studies. Peiyi Zhang1, Sheng Liu1, Abhishek Chaurasia2, Donghan Ma1, Michael J. Mlodzianoski1, Eugenio Culurciello2, Fang Huang1. 1386-Pos 1Weldon School of Biomedical Engineering, Purdue University, West Two-Photon Imaging of NADH in SKH1 Mice Reveals Changes in Kerati- Lafayette, IN, USA, 2School of Electrical and Computer Engineering, Purdue nocyte Metabolism with Chronic UVA Exposure University, West Lafayette, IN, USA. Dominick Myers1, Katie D. Sotelo1, Marifel Frances Gabriel1, Analyzing single molecule emission patterns plays a critical role in retrieving Kelsey A. Jackson1, Brooke L. Yang1, Ben G. Huerter1, Dan L. Che1, the structural and physiological information of their tagged targets and, further, Molly Myers1, Duyen Nguyen1, Laura A. Hansen2, Michael G. Nichols3. understanding their interactions and cellular context. These emission patterns 1Creighton University, Omaha, NE, USA, 2Biomedical Sciences, Creighton of tiny light sources (i.e. point spread functions, PSFs) simultaneously encode University, Omaha, NE, USA, 3Physics, Creighton University, Omaha, NE, information such as molecule’s location, orientation, environment within the USA. specimen and the paths the emitted photons took before captured by the cam- Cancer development is characterized by altered cell metabolism. Alterations of era. However, to date, retrieving multiple classes of information beyond 3D po- growth-factor signaling pathways adjust the metabolic phenotype to better sup- sition from complex or high-dimensional single molecule data remains port tumorigenesis and disease progression, which affects the concentration and challenging, due to the difficulties in perceiving and summarizing a compre- binding characteristics of endogenous coenzymes such as nicotinamide adenine hensive yet succinct model. We developed smNet, a deep neural network dinucleotide (NADH) and flavoproteins. To characterize metabolic changes that can extract multiplexed information near the theoretical limit from both induced by chronic UVA exposure in vivo, we designed and constructed a complex and high-dimensional point spread functions. Through simulated multiphoton laser scanning imaging platform specifically for in situ metabolic and experimental data, we demonstrated that smNet can be trained to efficiently imaging of NADH within the skin of anesthetized mice. Age matched SKH1 extract both molecular and specimen information, such as molecule location, hairless mice were divided equally into sham and UV treatment groups, with dipole orientation and wavefront distortions from complex and subtle features the treatment group receiving daily illumination of 15 kJ/m2 UVA. NADH pha- of the PSFs, which otherwise are considered too complex for established algo- sor FLIM was used to characterize metabolism of keratinocytes as a function of rithms. The capability of smNet in extracting sample induced aberration cell layer within the epidermis in both groups over a period of months. Early through the raw single-molecule blinking data itself allows wavefront measure- changes in the enzyme-bound-to-free ratio of NADH were clearly evident prior ment deep into the specimen without guide star and will further allow contin- to the onset of disease, with UVA- treated mice showing significantly greater uous feedback to a wavefront-control element during single molecule imaging proportion of enzyme-bound NADH. Basal keratinocytes generally had a lower of a living specimen. We expect that smNet will pave the way for multiplexed bound-to-free ratio than keratinocytes from stratum spinosum or granulosa physical and physiological measurements through the emission pattern of a sin- layers. Keratinocytes in vivo had a relatively greater fraction of bound gle molecule. NADH compared to squamous cell carcinoma cells in vitro. Changes in skin

BPJ 9374_9377 282a Monday, March 4, 2019 metabolism with normal aging was also clearly evident. This study demon- also be used in a super-resolution configuration as per the ISM [1] principle, strates the feasibility and challenges of phasor FLIM metabolic imaging for while the arrangement of the detectors allows for the implementation of long term longitudinal studies. advanced state-of-the-art spatiotemporal correlation techniques. We will pre- This study was made possible by grants from the National Institute for sent a novel application of the spot-variation FCS technique [2] in super- General Medical Science (NIGMS) (5P20GM103427), a component of the resolution, as well as the implementation of the 2D-pCF [3], iMSD [4] and National Institutes of Health (NIH), and its contents are the sole responsibility number and brightness [5] analysis using this fast detector array. This simulta- of the authors and do not necessarily represent the official views of NIGMS neous analysis of the same dataset provides biophysical information regarding or NIH. the fluorescent probe and the surrounding environment, such as diffusion coef- ficient, concentration, diffusion modality, environment organization, direction 1387-Pos and anisotropy of molecular flows, diffusion connectivity and oligomerization Computational Correction of Spatially-Variant Optical Aberrations in 3D state in a single analysis in a few seconds. Single-Molecule Localization Microscopy Using CCA we obtained structural and dynamic fingerprints of several subcel- Ting Yan, Charles Richardson, Mingxing Zhang, Andreas Gahlmann. lular compartments of living NIH-3T3 cells, namely structured and unstruc- Dept Chemistry, Univ Virginia, Charlottesville, VA, USA. tured cytoplasm, the nucleoli and heterochromatin- and euchromatin-rich Single-molecule localization microscopy has the ability to measure spatial regions in the nucleus. CCA provides a new methodology for the investigation proximity between individual molecules with tens of nanometers precision. Ex- of sub-resolution organelle structure and dynamics as well as insights on chro- tracting meaningful biological results, however, requires fully characterizing matin compaction states in living cells. The parallel acquisition of numerous the distribution of molecular behaviors, which in turn, necessitates analyzing biophysical parameters opens the doors to new multidimensional characteriza- large numbers of individual measurements. Making large numbers of replicate tion and classification methods that can be applied to virtually any fluorescent measurements in a single imaging session has been made possible in recent or fluorescently-labeled probe diffusing in complex environments. [Work sup- years by large area detectors that afford an ultrawide field-of-view as well as ported by NIH P41 GM103540 grant] fast frame rates. A remaining barrier to ultrawide-field imaging is that optical [1] Sheppard et al Opt.Lett. (2013) aberrations become pronounced when imaging far away from the central opti- [2] Wawrezinieck et al Biophys.J. (2005) cal axis, which can compromise the precision and accuracy of point-spread- [3] Hinde et al PNAS. (2010) function (PSF) fitting across the field-of-view. Here, we present a computa- [4] Di Rienzo et al Nat.Comm. (2014) tional phase retrieval routine based on vectorial PSF models to account for [5] Digman et al Biophys.J. (2008) the spatially-variant aberrations in two color channels of a 3D single- molecule localization microscope. By computationally correcting the aberra- 1390-Pos tions during data post-processing, we are able to localize emitters in an ultra- Localization Error and Fitting Model Evaluation in Single Particle wide filed-of-view with improved precision and accuracy compared to Tracking approaches based on analytical PSF models. The use of a spatially-variant Francesco Reina1, James Ross2, Mark Howarth3, Christian Eggeling1,4, PSF model enables accurate emitter localization in x, y and z over the entire B. Christoffer Lagerholm5. field-of-view, so that the reconstructed super-resolution images and single- 1Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, molecule trajectories accurately reproduce the relative spatial arrangement United Kingdom, 2University of Oxford, Oxford, United Kingdom, 3Dept among all localized emitters. Biochemistry, University of Oxford, Oxford, United Kingdom, 4Institute of 5 1388-Pos Applied Optics, Friedrich-Schiller University, Jena, Germany, Wolfson An Alternative Framework for Fluorescence Correlation Spectroscopy Imaging Centre, University of Oxford, Oxford, United Kingdom. Sina Jazani1, Ioannis Sgouralis1, Omer M. Shafraz2, Sanjeevi Sivasankar2, Single Particle Tracking (SPT) is a powerful technique for uncovering mo- Steve Presse3. lecular dynamics in lipid membrane biophysics, as well as in many other 1Center for Biological Physics, Department of Physics, Arizona State fields. Thus far, it has been successfully used to discover non-Brownian mo- University, Tempe, AZ, USA, 2Department of Biomedical Engineering, tion phenomena such as transient confined (also known as hop) diffusion of 3 lipids and membrane proteins in the plasma membrane by accessing high University of California, Davis, CA, USA, Center for Biological Physics, 3 4 Department of Physics and School of Molecular Sciences, Arizona State framerates (10 -10 fps) and claimed localization accuracies in the nano- University, Tempe, AZ, USA. metre range. However, most of literature reporting high-speed SPT data re- Fluorescence correlation spectroscopy (FCS) is a flexible and widely used tool lies on analysis protocols that are very susceptible to underestimations of the routinely exploited in vivo. While FCS provides estimates of dynamical quan- effects of localization errors; whose consequences are particularly pro- tities, such as diffusion coefficients, it demands high molecular concentrations, nounced at high framerates. Therefore, we propose a different approach to high signal to noise ratio and long time traces, typically in the minute range. estimate such errors, and a statistically-driven procedure for determining This is in contrast to data acquisition which, in principle, provides information the appropriate mode of motion fitting model. Using Interferometric Scat- on microsecond timescales that has, thus far, evaded analysis. To overcome tering (iSCAT) microscopy, we were able to collect high sampling frequency these limitations, we adapt novel tools inspired by Bayesian non-parametrics. data of immobile and moving particles in different situations, to use for our Within this principled and generalizable framework, which starts from the analysis. First, we compare the effect of Signal-to-Noise Ratio on the local- direct analysis of photon counts, we achieve the same accuracy as FCS, but ization precision of different immobile particles (gold nanoparticles, single with much shorter traces, even at nearly single molecule concentrations. Our proteins). Next, we show the effect of camera blur and localization error new analysis extends the capability of confocal microscopy based approaches, on the same particles diffusing on quasi-homogeneous Supported Lipid Bila- by (i) probing processes several orders of magnitude faster in time; (ii) reducing yers (SLB), to develop an error correction method and a fitting goodness phototoxic effects on living samples induced by long periods of light exposure; discrimination method of fitting SPT data. These last results are compared (iii) tracking instantaneous molecules concentration; (iv) estimating the molec- to Fluorescence Correlation Spectroscopy (FCS) measurements as a control ular and background photon emission rates; and (v) incorporating demanding for the values of diffusion rate. Finally, we acquired data of gold illumination profiles, such as those arising in two-photon excitation and -tagged lipids diffusing on epithelial cell membranes, and apply TIRF microscopy or even Airy patterns by changing the specified point spread the newly developed method to these data, to determine which model is the function. best statistical fit for cell membrane dynamics. In conclusion, the analysis workflow we propose, based on statistical methods rather than previous 1389-Pos knowledge, could increase reliability and accuracy of future SPT measure- Airyscan Cca Provides Structural and Dynamics Fingerprinting of Subcel- ments at high spatiotemporal resolution. lular Compartments in Living Cells Lorenzo Scipioni1, Luca Lanzano02, Alberto Diaspro3, Enrico Gratton1. 1391-Pos 1Biomedical Engineering, University of California, Irvine, Irvine, CA, USA, Multifocus Structure Illumination Microscopy 2Nanophysics, Istituto Italiano Tecnologia, Genoa, Italy, 3Dept Nanoscop, Juliana Hernandez, Sara Abrahamsson. Inst Italiano d Tecnologia, Genoa, Italy. Electrical and Computer Engineering, University of California Santa Cruz, In this work we will present our current advances in the development of the Santa Cruz, CA, USA. CCA (Comprehensive Correlation Analysis) technique using the Zeiss Airy- Multifocus structure illumination microscopy allows for live super resolution scan detector. This detector consists of 32 GaAsP PMT arranged in a hexagonal 3D cell imaging for biological research applications. Using fastSIM (Hui- pattern, featuring a fast temporal sampling (down to 1.28 ms per frame). It can Wen Lu-Walther et al 2015 Methods Appl. Fluoresc. 3 014001) we acquire

BPJ 9374_9377 Monday, March 4, 2019 283a images with high temporal and spacial resolution. We combine this method 1395-Pos with Multifocus microscopy (Abrahamsson et al 2015 Opt. Express 23, Autofluorescence Suppression by Optically Controlling Dark States of 7734-7754), which allows us to image 25 focal planes simultaneously for Photoswitchable Fluorescent Proteins on Commercial Microscopes fast volumetric imaging. Yen-Cheng Chen1, Chetan Sood1, Robert M. Dickson2, Gregory B. Melikyan1. 1392-Pos 1Department of Pediatrics, Emory, Atlanta, GA, USA, 2Department of Lens Characterization for Multifocus Microscopy Applications Chemistry & Biochemistry, Georgia Tech, Atlanta, GA, USA. Brandon J. Lynch. Inhomogeneous autofluorescence of live cells generates false positive signals Electrical Engineering, University of California Santa Cruz, Santa Cruz, CA, that confound fluorescence imaging of dim objects. The ability to optically con- USA. trol the photophysical bright and dark states of photoswitchable fluorescent pro- Multifocus Microscopy(MFM) has provided the opportunity to 3D image bio- teins (PS-FPs) provides an alternative means to suppress the same emission- logical events at speeds limited by the camera readout of a single image. High wavelength autofluorescence. Previous demonstration of synchronously ampli- speed MFM has been made possible by utilizing diffractive fourier optics to fied fluorescence image recovery (SAFIRe) shows >10-fold suppression of au- multiplex light from different focal planes on a single camera sensor to pro- tofluorescence on custom-built microscopes. We further adapts SAFIRe to duce an instantaneous 3D image from the resulting 2D focal stack. When commercial microscopes, including laser scanning confocal microscope (Zeiss designing imaging systems for MFM applications, there is a strong desire 700), wide-field fluorescence microscope (GE DeltaVision), and spinning disk to prevent distortion and reduce chromatic and spherical aberration in the confocal microscope (PerkinElmer UltraVIEW VoX), to suppress confounding 2D focal stack of images. In addition to improving image quality, imaging autofluorescence signals by optically controlling the photophysical dark states small singular biomolecules requires maximization of photon collection effi- of the photoswitchable protein rsFastLime. The recovery of rsFastLime fluores- ciency. It is the goal of this project to determine if large acromatic doublet cence allows us to discriminate the relatively dim HIV-1 virus-like particles lenses could be replaced with camera objectives to improve MFM system (VLPs) with >10-fold signal-to-background ratio improvement and <1 second performance. time resolution sufficient for real-time live cell imaging. This cell-type inde- pendent method of background suppression enables more reliable single virus 1393-Pos tracking in living cells. Structured Illumination Reveals Reduced Chromatin Cohesion in Cells 1396-Pos with DNA Damage Ultra-Sensitive Detection of Peripheral Membrane Protein Binding to the Keith Bonin1, Amanda Smelser2, Naike Salvador Moreno2, Plasma Membrane of Living Cells George Holzwarth1, Dave Segall1, Pierre-Alexandre Vidi2. Isaac Angert1, John Kohler1, Siddarth Reddy Karuka1, Morgan E. Meissner2, 1Dept of Physics, Wake Forest University, Winston-Salem, NC, USA, 2Dept Louis M. Mansky3, Joachim D. Mueller1. of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, NC, 1School of Physics and Astronomy, University of Minnesota, Minneapolis, USA. MN, USA, 2Molecular, Cellular, Developmental Biology and Genetics We have developed a novel structured illumination microscopy method to Graduate Program, University of Minnesota, Minneapolis, MN, USA, map chromatin motions in mammalian cell nuclei. The method relies on a 3Department of Diagnostic and Biological Sciences, School of Dentistry, diffractive optical element that generates a lattice of photoactivated spots University of Minnesota, Minneapolis, MN, USA. in cells expressing histones tagged with photoactivatable GFP. Photoacti- Delicate and transitory protein engagement at the plasma membrane (PM) is vated spots correspond to chromatin microdomains that can be tracked crucial to many cellular functions and disease states, including cell motility, with time-lapse imaging. Chromatin motions are thought to influence most signal transduction, and the budding and entry steps in the life cycle of many if not all genomic functions including gene expression, DNA replication, viruses. We describe quantification of PM binding by the use of two-photon and DNA repair. Yet the mechanisms governing chromatin diffusion are dual color z-scan (DC z-scan) deconvolution in cells co-expressing a fluores- not well understood, particularly in the context of the DNA damage response. cently labeled protein of interest along with a soluble fluorescent protein Most studies in yeast have shown increased chromatin motions after DNA with a distinct color that serves as a reference to delineate the cytoplasmic cleavage, proposed to facilitate homology search during homologous recom- extent. We show that this technique can detect very small amounts of PM bind- bination. However, it is unclear whether chromatin dynamics in yeast applies ing that are otherwise extremely difficult to discern. Due to the nature of the to mammalian cells, given the fundamental differences between the two or- two-photon excitation, the DC z-scan PM binding assay technique attains ganisms in terms of nuclear organization and DNA repair pathway usage. very low levels of systematic artifacts, for which we provide observational up- Our approach has led to the discoveries of (1) correlated motions for neigh- per bounds. We further extend the technique to interrogate the passage of retro- boring chromatin domains, and (2) reduced chromatin cohesion in cells with viral assembly proteins through the actin cortex, and suggest that the dense DNA damage. A unique feature of the method is to provide spatial informa- cortical meshwork may present a barrier to the assembly and budding of retro- tion on chromatin motions, used to compare DNA damage sites to undam- viruses such as human immunodeficiency virus. This work is supported by aged nuclear regions and to understand chromatin dynamics within and grants from the National Institutes of Health (RO1 GM64589, RO1 across chromosome territories. We anticipate that this approach will yield GM098550, RO1 GM124279). a better and dynamic understanding of the higher organization of the genome. 1397-Pos Active PSF Shaping and Adaptive Optics Enable Volumetric Single Mole- 1394-Pos cule Super-Resolution Microscopy through Brain Sections Photoswitching Anisotropy FRET for Monitoring Homo-Oligomerization Michael Mlodzianoski1, Paul Cheng-Hathaway2, Sheng Liu1, of Proteins Shane Bemiller3, Tyler McCray3, David Miller1, Bruce Lamb3, Namrata Ojha, Kristin Rainey, George H. Patterson. Gary Landreth2, Fang Huang1. NIBIB NIH, Bethesda, MD, USA. 1Dept Biomed Eng, Purdue Univ, West Lafayette, IN, USA, 2Department of The monitoring of protein oligomerization has benefited greatly from Fo¨rster Anatomy and Cell Biology, Indiana University School of Medicine, Resonance Energy Transfer (FRET) measurements between donor-labeled Indianapolis, IN, USA, 3Stark Neurosciences Research Institute, Indiana molecules and acceptor-labeled molecules. Although the donors and acceptors University School of Medicine, Indianapolis, IN, USA. are typically fluorescent molecules with different spectra where the donor emis- Three dimensional Single Molecule Switching Nanoscopy (SMSN) imaging sion overlaps with the acceptor absorption, homo-FRET can occur between beyond the coverslip surface poses significant challenges. One of the major ob- fluorescent molecules of the same type if the emission spectrum overlaps stacles is that the sample-induced aberrations distort and blur single molecule with the absorption spectrum. Since molecules undergoing homo-FRET do emission patterns, known as Point Spread Functions (PSFs). These distortions not display marked changes in fluorescence spectrum or lifetime, anisotropy and blurring make three-dimensional (3D) inference of single molecule posi- measurements are normally employed. Here, we describe the use of monitoring tions unreliable. Adaptive Optics (AO) assisted SMSN developments helped anisotropy changes in photoswitchable fluorescent proteins as a way to observe push the volumetric 3D-SMSN imaging depth to a whole cell sample, however, homo-FRET. Similar to photobleaching measurements, these offer the capa- to date robust 3D SMSN reconstruction of more than a couple layers of cells or bility to estimate anisotropy in the same specimen during homo-FRET and tissues remains a practical challenge due to the permanent information loss non-FRET conditions. We demonstrate photoswitching anisotropy FRET (psA- caused by aberrations induced by fixed or living specimens. Here, we introduce FRET) with test chimeras as well as several oligomeric complexes inside living a method combining adaptive PSF engineering and an efficient adaptive optics cells. routine to allow simultaneous correction of sample-induced aberrations and

BPJ 9374_9377 284a Monday, March 4, 2019 enforcement a consistent PSF response through large depths, therefore permit- Self-association of proteins forming dimers or oligomers is a common occur- ting robust 3D-super-resolution volumetric imaging of fluorescence stained rence in biological systems. Probing the dimerization status can be performed whole-cell samples or tissues. We show that our method allows imaging by various size- and mass-selective methods (e.g. analytical ultracentrifugation, through 30 mm brain sections from the mouse frontal cortex by reconstructing gel filtration, ). Every method has its strengths but generally the detection fibrillar amyloid-b plaques found in Alzheimer’s disease. directly in the solution by optical means is preferred. Metal nanoparticles (NP) can be observed in a light microscope and their surface can be function- 1398-Pos alized with different chemical compounds, like dyes or affinity tags. For 3D Microscopy-Independent Approach for Obtaining 3D Super- example, nickel-chelated N-nitrilo-triacetic acid (Ni-NTA) functionalized Resolution Information in Rotationally Symmetric Bio-Structures metal nanoparticles (Ni-NTA NP) can be site-specifically attached to His- Andrew Ruba, Wangxi Luo, Joseph Kelich, Weidong Yang. tagged proteins, forming a thin single protein layer on a NP. Additional func- Temple University, Philadelphia, PA, USA. tionalization of the Ni-NTA NP with Raman or fluorescent dyes provide Currently, it is highly desirable but still challenging to obtain high-resolution furthermore the ability to observe red-shifted emissions, like surface- < ( 50 nm) three-dimensional (3D) super-resolution information of structures enhanced Raman scattering (SERS) or surface-enhanced fluorescence (SEF), in fixed specimens as well as for dynamic processes in live cells. Here we intro- especially if plasmonic metals are used as NP material. Those effects are large duce a simple approach, without using 3D super-resolution microscopy or real- if single NPs are near to each other upon laser irradiation. In case the NP sur- time 3D particle tracking, to estimate 3D sub-diffraction-limited structural or face attached proteins form stable dimers or oligomers, the functionalized NPs dynamic information in rotationally symmetric bio-structures. This is a post- will be concomitantly linked and in close proximity to each other, forming NP localization analysis that transforms 2D super-resolution images or 2D dimers or oligomers itself. The new formed NP-complexes can be detected by single-molecule localization distributions into their corresponding 3D spatial dynamic light scattering (DLS), asymmetric flow field fractionation (AFFF) or probability distributions based on prior known structural knowledge. This anal- observed optically on the single-particle level by red-shifted emissions upon ysis is ideal in cases where the ultrastructure of a cellular structure is known but laser irradiation. Observing the red-shifted emissions, inelastic scattering the sub-structural localization of a particular (usually mobile) protein is not. (Raman/Mie) or fluorescence, concomitantly with elastic (Rayleigh) emissions The method has been successfully applied to achieve 3D structural and func- via two separate imaging channels in a light-sheet illumination approach allows tional sub-diffraction-limited information for 25-300 nm subcellular organelles the check of the NP-dimerization/oligomerization status. This indicates protein that meet the rotational symmetry requirement, such as nuclear pore complex, interaction in the native suspension environment and enables determination of primary cilium and microtubule. Herein, we will provide comprehensive ana- the ratio of non-oligomerized to oligomerized protein in a microscope image or lyses of this method by using experimental data and computational simulations. video. This approach paves the way for fast optical determination of protein- 1399-Pos protein or protein-ligand interactions. Restricting Diffusive Exchange In Vitro Demonstrates Inos Modulates Posters: EPR and NMR: Spectroscopy and Hypoxic Gradients in the Tumor Microenvironment Caroline Gilmore1, Veena Somasundaram2, David Scheiblin3, Imaging William Heinz3, Stephen Lockett3, David Wink1. 1Optical Microscopy and Analysis Laboratory, Frederick National 1401-Pos Laboratory for Cancer Research, Frederick, MD, USA, 2Cancer and Confidence Analysis of DEER Data and its Structural Interpretation with Inflammation Program, Center for Cancer Research, National Cancer Ensemble-Biased Metadynamics Institute, National Institute of Health, Frederick, MD, USA, 3Optical Eric J. Hustedt1, Fabrizio Marinelli2, Richard A. Stein3, Jose D. Faraldo- Microscopy and Analysis Laboratory, Frederick National Laboratory for Go´mez4, Hassane Mchaourab5. Cancer Research, Leidos Biomedical Research Inc. for the National Cancer 1Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Institute, Frederick, MD, USA. TN, USA, 2TMB, NHLBI NIH, Bethesda, MD, USA, 3Vanderbilt Univ, The diffusion of metabolites in tissues produces gradients and heterogeneous Nashville, TN, USA, 4National Institutes of Health, Bethesda, MD, USA, microenvironments. As tumors grow, they eventually are no longer fully 5Dept Physiol/Biophys, Vanderbilt Univ, Nashville, TN, USA. oxygenated by diffusion from normal vasculature. This leads to hypoxic re- Given its ability to measure multicomponent distance distributions between gions within the tumor microenvironment that affect cellular processes and electron-spin probes, Double Electron-Electron Resonance spectroscopy signaling pathways. Alterations in these pathways can lead to events such as (DEER) has become a leading technique to assess the structural dynamics of angiogenesis and metastasis. We utilized a chamber system for cell culture biomolecules. Recently, we have published two new methods for advancing that forms cell-generated hypoxic and metabolic gradients in two-dimensions the rigorous interpretation of DEER data (https://doi.org/10.1016/j.bpj.2018. by restricting the diffusive exchange of oxygen and metabolites to a monolayer 08.008). First, building upon a model-based approach in which the distance of cells in a small volume – analogous to diffusion between a capillary and probability distribution is represented as a sum of Gaussians or other functions, nearby tissue. Triple-negative mouse breast cancer cells (4T1s) were cultured we use propagation of errors to calculate an associated confidence band for the in the chambers and live-cell imaging was used to quantify the evolution and distance distribution. This approach considers all sources of uncertainty, extent of the oxygen concentration gradient. Cells migrated along the hypoxic including the experimental noise, the uncertainty in the fitted background gradient and formed a millimeter-scale disk of viable cells that was stable for at signal, and the limited time-span of the data collection. The resulting confi- least one month. We quantified the spatial distribution of biomarkers such as dence band reveals the most and least reliable features of the probability distri- HIF-1a, EMT markers, and proteins involved in inflammation by multiplexed, bution, thereby informing the structural interpretation of DEER experiments. large-area immunofluorescence microscopy. The distribution of these markers Second, to facilitate the interpretation of distance distributions obtained from correlated with the oxygen gradient. With this model, we studied changes in DEER experiments, we generalized the molecular-simulation method known cellular signaling pathways as a function of cell position within the hypoxic as Ensemble-Biased Metadynamics. This method, originally designed to gradient. Macrophages stimulated with lipopolysaccharide (LPS), an inflam- generate maximum-entropy structural ensembles consistent with one or more matory cytokine, upregulated inducible nitric oxide synthase (iNOS), and nitric probability distributions, has been modified to account for the uncertainty in oxide (NO) production has been linked to decreased oxygen consumption in those target distributions as dictated by their confidence bands. After careful hypoxic environments in vitro. We investigated the interaction between benchmarks, the proposed techniques have been demonstrated using DEER re- iNOS in activated ANA-1 macrophages and hypoxia and demonstrated that sults from spin-labeled T4 lysozyme. treatment with LPS increased iNOS expression and altered the magnitude This work is now being extended to allow the determination of confidence and spatial extent of hypoxic gradients. Funded by NCI Contract No. bands for distance distributions obtained from the global analysis of multiple HHSN261200800001E. data sets. https://lab.vanderbilt.edu/hustedt-lab/software/ 1400-Pos https://github.com/Colvars/colvars Protein Dimerization Probed with Site-Specific Attached Single Nanopar- ticles 1402-Pos Joerg Wissler1, Sandra B€acker2, Alessandro Feis3, Shirley Knauer2, Electron Paramagnetic Resonance Elucidates the Structural Mechanism Sebastian Schlucker€ 1. by Which SERCA is Activated by DWORF 1Faculty of Chemistry, Physical Chemistry I, University of Duisburg-Essen, Mark D. Rustad, Peter D. Martin, Daniel R. Stroik, Christine B. Karim. Essen, Germany, 2Center of Medical Biotechnology, Molecular Biology II, BMBB, Univ Minnesota, Minneapolis, MN, USA. University of Duisburg-Essen, Essen, Germany, 3Dipartimento di Chimica, We have used electron paramagnetic resonance (EPR) spectroscopy to examine Universita’ di Firenze, Firenze, Italy. the protein-protein interaction of a recently discovered 34-codon micropeptide,

BPJ 9374_9377 Monday, March 4, 2019 285a dwarf open reading frame (DWORF), with the sarco/endoplasmic reticulum flow-NMR potential to explore water-proton interactions. Meanwhile, due to calcium ATPase (SERCA) in the presence and absence of phospholamban generally high water concentration in aqueous solutions of biomacromolecules (PLB) in lipid vesicles. Heart failure is one of the leading causes of death in and long relaxation times of water, many existing limitations of flow-NMR are developed countries. A characteristic pathology of cardiac disease is inade- lifted when it comes to study water-protein interactions mediated by relaxation. quate activity of SERCA, the integral transmembrane Ca2þ pump, so SERCA We report here the results obtained by a newly designed low-field benchtop 1 activation is an important goal in developing treatments for heart failure. PLB flow-NMR instrument, which can monitor R2( H2O) within the flow rates is known to decrease SERCA’s Ca2þ affinity through allosteric interactions. ranging from 0 to 50 mL/min. In our experiments, we demonstrated that 1 However, it has been shown that co-expression of DWORF with PLB and R2( H2O) in a flow depends on a flow rate in accordance with theoretically pre- SERCA, in HEK293 cells, restores SERCA activity (Nelson et al., 2016). It dicted residence time component. We also showed the sensitivity of water flow- has been proposed that this relief of SERCA inhibition is due to direct compe- NMR to the pH and ionic strength/conductivity of buffered aqueous solutions. 1 tition by DWORF for PLB binding to SERCA. To test this hypothesis in a R2( H2O) was also found sensitive to the concentration of proteins in solution membrane of defined protein and lipid composition, we co-reconstituted puri- in the range from 0 to 50 mg/mL and flow rates from 0 to 50 mL/min. Our re- fied SERCA and spin-labeled PLB, with and without DWORF, in lipid vesicles. sults pave the way for the application of water-proton flow-NMR as a noninva- We then performed NADH-coupled Ca-ATPase assays to determine SERCA sive process analytical technology in continuous biomanufacturing. In a activity, and EPR experiments to determine the binding of PLB to SERCA. product flow, this unique technique could detect in-line concentration changes The sensitivity of EPR to structural dynamics, using stereospecifically attached and protein aggregates. spin labels, made the experiments extremely sensitive to PLB immobilization by SERCA. The results indicate that DWORF’s restoration of SERCA activity 1405-Pos correlates with some direct displacement of PLB from SERCA, but the frac- Low-Field NMR in Nondestructive Quantitative Inspection of Drug tional relief of inhibition exceeds the fractional PLB displacement, suggesting Products a more complex mechanism. These results provide direct insight of the equilib- Katharine T. Briggs, Marc B. Taraban, Yihua B. Yu. rium dynamics of SERCA and its regulators PLB and DWORF, providing valu- Dept. Pharm. Sci., Univ. Maryland Sch. Pharmacy, Baltimore, MD, USA. able information for the development of novel therapeutic remedies for cardiac Batch-to-batch inference-based quality control of drug products has historically pathologies. been very effective. However, infrequent incidences of drug defects may be missed by traditional batch-to-batch testing, especially for complex drugs, 1403-Pos e.g. biologics. Compared to small molecule drugs, complex drugs have many Site-Directed Spin Labeling EPR Studies on the Catalytic Aspartate Loop more factors involved their production and manufacturing, and are more prone of Exou Upon Interaction with Ubiquitin and Membranes to degrade with temperature stresses or agitation during transportation and stor- Samantha Kohn1, Jimmy B. Feix2. age. Relying on batch testing, the sole inspection method of released drug prod- 1Medical Coll of Wisconsin, Milwaukee, WI, USA, 2Dept Biophysics, Med ucts is human visual inspection. Here we investigate the use of low-field NMR Coll Wisconsin, Milwaukee, WI, USA. (23 MHz) to inspect four types of drug products, each addressing a different ExoU is a potent virulence factor produced by the opportunistic pathogen Pseu- potential quality control problem: 1. Novolog FlexPenÒ (wrong dosage), 2. domonas aeruginosa. ExoU functions as a patatin-like phospholipase with a FerrlecitÒ vs. generic (false equivalence), 3. AlhydrogelÒ (uneven filling of catalytic serine-aspartate dyad. Phospholipase activity requires a novel non- alum adjuvant), 4. DaptacelÒ, Engerix-BÒ, and VaqtaÒ vaccines (freeze/ covalent interaction with a ubiquitin cofactor. Two crystal structures of thaw stress). In every case, during our experiments the product (vial or pen) re- ExoU in the presence of its chaperone SpcU have been reported. However, mained sealed without any tampering; low-field NMR as a method for drug both structures are of an inactive conformation with approximately 25% of res- product inspection is non-destructive. The water proton transverse relaxation 1 idues unresolved, including a twenty-residue loop containing the catalytic rate, R2( H2O), was found to be sensitive to concentration changes in aluminum aspartate. The structure of the active conformation remains unknown. Conse- hydroxide adjuvants, and insulin protein. Furthermore, differences in brand 1 quently the activation mechanism and details of the ExoU-membrane interac- name and generic drug products were detected by the R2( H2O), where proper- tion remain poorly understood. This study focuses on elucidating the local ties measured by other analytical methods failed to detect differences. These 1 structural environment of sites near the catalytic aspartate for ExoU alone findings suggest that low-field H2O NMR could serve as a non-destructive in- and upon interaction with ubiquitin cofactor and membrane substrate. Site- spection method for drug products, such as insulin pens and vaccines. directed spin labeling (SDSL) in conjunction with electron paramagnetic reso- nance (EPR) spectroscopy was utilized to assess the motional dynamics of spin- 1406-Pos labeled sites near the catalytic aspartate upon interaction with diubiquitin Quantitative Binding of Divalent Metal Ions to DNA Hairpin Loops (diUb) and phospholipid membranes. Spin labels introduced at sites near the Harrison Russell1, William Gunderson2, Julie Gunderson1. catalytic aspartate were immobilized even for ExoU alone in solution (Apo 1Physics, Hendrix College, Conway, AR, USA, 2Chemistry, Hendrix state), suggesting these sites form tertiary contacts that are not apparent in College, Conway, AR, USA. the reported crystal structures. Changes in spin label motion were observed Slipped-strand DNA structures can form when complementary repeating se- in the presence of diUb and membranes combined (Holo state), but not with quences on a single-strand pair up to form thermodynamically stable hairpins. either diUb or liposomes alone, indicating that interactions with both diUb The formation of these hairpin structures is believed to contribute to the expan- and phospholipid substrate are required to form the active conformation. Power sion of nucleotide repeat tracts, which are mutations associated with the devel- saturation EPR spectroscopy was used to investigate ExoU-membrane interac- opment of many hereditary and anticipative neurodegenerative diseases in tions, and in contrast to previous studies of sites surrounding the catalytic humans. Mg(II) is a divalent cation that is known to play important structural serine, penetration of the lipid bilayer was not observed for sites near the cat- roles within DNA molecules, but the number of Mg(II) binding sites and the alytic aspartate. These studies provide new insights into the structural organi- affinity of Mg(II) to slipped strand DNA structures are not known. The objec- zation of ExoU in its catalytically-active state. tives of this study are to determine the stoichiometry of interaction and the dissociation constant (KD) for Mg(II)/hairpin DNA binding. Mg(II) is spectro- 1404-Pos scopically silent and cannot be observed directly. To determine the binding Water Proton Flow-NMR—A Novel Tool for Real-Time In-Line Process characteristics of Mg(II) to DNA hairpins, competitive titrations with Mn(II) Monitoring in Biomanufacturing were performed. Concentrations of Mn(II) were determined using electron Marc B. Taraban, Katharine T. Briggs, Yihua Bruce Yu. paramagnetic resonance (EPR) spectroscopy, and the number of Mg(II) binding Dept Pharm Sci, Univ Maryland, Baltimore, MD, USA. sites and the KD for Mg(II) were calculated using a binding isotherm. The re- Due to serious limitations, such as experiment duration, relaxation parameters, sults suggest that Mn(II) binds to DNA hairpins with a significantly higher af- flow-NMR has limited applications to study structure and properties of a solute, finity than Mg(II). especially, in the case of biomacromolecules, or in the manufacturing of bio- logics. Unlike conventional structural applications of NMR spectroscopy tar- 1407-Pos geted towards complex solute biomacromolecules, its approaches directed to Highly Sensitive Resonators for EPR Spectroscopy of Submicroliter/Sub- study the interactions between solute and solvent are less explored. However, micromolar Biomacromolecular Samples in aqueous solutions, water molecules bear useful information about the state Nandita Abhyankar1, Amit Agrawal1, Robert McMichael2, of solute molecule, in particular, biomacromolecules, e.g., proteins, actively in- Szalai Veronika2. teracting with water molecules. We have already demonstrated that water pro- 1Institute for Research in Electronics and Applied Physics, University of 1 2 ton transverse relaxation rate, R2( H2O), is a powerful indicator of protein Maryland College Park, College Park, MD, USA, National Institute of aggregation sensitive to a wide range of aggregate sizes. Less is known about Standards and Technology, Gaithersburg, MD, USA.

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We report ‘planar inverse anapole’ microresonators with high quality factors Sherbrooke, QC, Canada, 3Department of Chemistry, Dalhousie University, (Q-factors) and nanoliter active volumes, that increase both absolute sensitivity Halifax, NS, Canada. and concentration sensitivity of inductive-detection electron paramagnetic 19F nuclear magnetic resonance (NMR) spectroscopy provides highly sensi- resonance (EPR) spectroscopy at room temperature. Due to its inherently tive and practically background-free monitoring of pertubations to biomole- high sensitivity, ability to interrogate specific sites by site-directed spin-label- cules. This is being applied to the apelinergic system, which comprises a ing, and advances in pulsed measurements, EPR spectroscopy has gained class A G-protein-coupled receptor (GPCR), the apelin receptor, and two importance as a technique to elucidate protein structure and dynamics. Howev- families of peptide ligands, the apelin ligands (13-55 residue bioactive er, the limit of detection of state-of-the-art EPR spectrometers (approximately forms) and the apela (or ELABELA) ligands (11-32 residue bioactive forms). 50 uL of a 50 uM solution) is still too high compared to the amounts that are A ligand and its cognate receptor are being labelled with 19F probes orthog- easily obtainable for some biomacromolecules - for example, membrane pro- onal to each other with respect to 19F NMR chemical shift ranges. Specif- teins, which are notoriously difficult to express and purify in sufficient amounts ically, 13-residue long 19F-labelled apelin analogues containing a C- for macromolecular structural determination. To further decrease the detection terminal 2,4,5-trifluorophenylalanine residue are titrated with apelin receptor limit, an advantageous approach is to miniaturize the resonator, thereby segments biosynthetically-labelled with 4-, 5-, 6- and/or 7-fluorotryptophan. decreasing the interrogated sample volume. This miniaturization can be easily Interesting distinctions in ligand-receptor interaction are seen between the accomplished using standard photolithographic techniques to create resonant synthetic fluorophenylalanine-containing ligand, which has relatively high devices with micron-sized features and nanoliter active volumes.[1], [2] Howev- GPCR affinity, and longer (32- or 36-residue) natural ligands, which have er, such miniaturization also increases radiation losses from the resonators, re- lower affinity. Upon addition of high affinity apelin analogues, shorter apelin sulting in poor Q-factors and offsetting the gains in sensitivity achieved by receptor fragments (e.g., only transmembrane segment 1 þ the N-terminal decreasing the active volume. Here, we report an innovative planar microreso- tail) exhibit less specificity for extracellular domain perturbation than longer nator that is designed to have minimal radiation losses at room temperature,[3] fragments (transmembrane segments 1-3 þ the N-terminal tail), implying a thereby ensuring high Q-factors without the need to operate at cryogenic tem- more favourable extracellular domain-ligand interaction with the presence peratures. Using this combination of a small active volume and high Q-factor, of both N-terminal tail and extracellular loop 1. The apelin analogue, we project an increase in the sensitivity of room-temperature EPR spectroscopy notably, perturbs 19F NMR chemical shifts in the receptor fragments. This (and by extension, low-temperature EPR spectroscopy) of two orders of contrasts directly with longer natural ligands, where the extracellular region magnitude. of the same GPCR fragment exhibits ligand-dependent changes in 19FNMR References: lineshape, but not chemical shift perturbation. The C-terminal phenylalanine 1. Twig, Y., E. Dikarov, and A. Blank, Molecular Physics, 2013. 111(18-19): p. of the analogue also exhibits distinct behaviour as a function of 19F substi- 2674-2682. tution site, implying potential specificity in sidechain orientation with 2. Probst, S., et al., Applied Physics Letters, 2017. 111(20): p. 202604. respect to the receptor. As a whole, this strategy provides a sensitive means 3. Basharin, A.A., et al., Physical Review B, 2017. 95(3). of both site-specifically and species-specifically tracking both ligand and re- ceptor during a titration using relatively rapid one-dimensional NMR 1408-Pos experiments. Aortic Atherosclerosis with Consideration of the Anisotropic Properties of Lipids in MRI Erik N. Taylor1, Nasi Huang1, Matthew Diamse1, Farzad Mortazavi2, 1410-Pos 15 H 13 a Markus Bachschmid3, James A. Hamilton4. Accurate Measurement and Prediction of N and C Chemical Shift 1Physiology and Biophysics, Boston University, Boston, MA, USA, Tensors in Proteins 2Anatomy and Neurobiology, Boston University, Boston, MA, USA, Matthew Fritz1,2, Caitlin M. Quinn1, Mingzhang Wang1,2, Guangjin Hou1, 3Cardiology and Vascular Biology, Boston University, Boston, MA, USA, Xingyu Lu1, Leo Koharudin2,3, Jochem Struppe4, David A. Case5, 4 Angela M. Gronenborn2,3. Dept Biophysics, Boston Univ Sch Med, Boston, MA, USA. 1 Atherosclerosis is the major underlying cause of cardiovascular disease that Chemistry and Biochemistry, University of Delaware, Newark, DE, USA, 2 can lead to heart attack and stroke. The disease starts with deposition of lipo- Pittsburgh Center for HIV Protein Interactions, Pittsburgh, PA, USA, 3 proteins rich in cholesterol and cholesteryl esters (CE) from the blood into the Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, 4 5 intimal layer of the vessel walls, and then with inflammation and immune cell PA, USA, Bruker Biospin Corporation, Billerica, MA, USA, Chemistry and infiltration, can progress into advanced plaques. Structural cells that make up Chemical Biology, Rutgers University, Piscataway, NJ, USA. the majority of the mechanical properties of the vessel wall, particularly a The chemical shift tensor (CST) is a rich probe of structure and dynamics, thick layer of smooth muscle cells in the media, are also infiltrated by lipids dependent on electronic environment, local geometry, H-bonding, and dy- and acquire inflammatory-like cell features, with detrimental effects on their namics. Isotropic chemical shifts and chemical shift anisotropies can be em- structural properties. The objective of this study was to apply diffusion- ployed as structural restraints in determination, based molecular imaging of lipids (CE) with new considerations of supplementing distance restraints. To establish robust protocols for using vascular-tissue structural properties. These studies were performed in a rabbit chemical shifts in structure determination, accurate experimental measurement model of aortic atherosclerosis that resembles many of the stages and features approaches as well as computations of CST parameters need to be established for well-characterized proteins. Here, we present an analysis of experimental of human plaques, including heterogeneous lipids in advanced inflamed pla- 13 a 15 H ques and plaque rupture (thrombosis). Our MRI approach (demonstrated at and computed C and N chemical shift tensors using a 132-residue lectin 11.7T field strength ex vivo) elucidated the lipid contents of plaques in from Oscillatoria agardhii (OAA). OAA is an ideal benchmark system because slice-based MR imaging. Further details of anisotropic tissue properties it crystallizes readily and reproducibly, its crystal structure has been determined ˚ were distinguished with evaluation of three-dimensional lipid diffusion prop- at high (1.2 A) resolution, and it has been extensively characterized by solution erties. These properties give insights into the architecture of the vessel wall. NMR. Compared to healthy rabbit vessel, highly inflamed and rupture-prone plaques We explored a hybrid quantum mechanics/molecular mechanics (QM/MM) contained thick intimal lipid deposits. CE-rich plaques displayed anisotropic approach for calculation of chemical shift tensors and observed excellent agree- diffusion properties resulting from cellular and extra-cellular matrix constitu- ment between MAS and solution NMR isotropic shifts. We recorded residue- ents present in the adjacent vessel wall. Reconstruction of 3D fiber orientation specific CSA lineshapes using RN symmetry recoupling sequences and corre- demonstrated anisotropic properties with respect to vessel wall depth. These lated those to the computed tensors. We examined the influence of crystal con- anisotropic regions are indicative of normal vessel wall, lipid infiltrate, and tacts, H-bonding, ligand molecules, and dynamics on the accuracy of chemical shit tensor calculations. We show that 13Ca chemical shift tensors are primarily foam cell expansion. With further development our MRI approach could be 15 H used in vivo to expand characterization of atherosclerosis as a disease of determined by backbone dihedral angles and dynamics, while N tensors the vessel wall. mainly depend on local electrostatic contributions from solvation and hydrogen bonding. In addition, the influence of including crystallographic waters, the 1409-Pos molecular mechanics geometry optimization protocol, and the level of theory Orthogonal 19F-Labelling for Simultaneous Receptor and Ligand on the accuracy of the calculated chemical shift tensors is discussed. Specif- Tracking in Titrations ically, we showcase that these calculations can accurately predict the ring cur- 1 N Jeff Simmons1, Alexandre Murza2,E´ric Marsault2, Jan K. Rainey1,3. rent effects on the unusually up-field shifted H G26 and G93 resonances. The 1Department of Biochemistry & Molecular Biology, Dalhousie University, results of this study indicate the potential of this approach for structure Halifax, NS, Canada, 2Institut de Pharmacologie, Universite de Sherbrooke, refinement.

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Posters: Computational Methods and 1413-Pos Incorporating Proteins into Geometrically Complex, Cell-Scale Mem- Bioinformatics I brane Models for Molecular Dynamics Simulations Noah Trebesch1, Emad Tajkhorshid1,2. 1411-Pos 1Center for Biophysics and Quantitative Biology, Beckman Institute for Introducing a Novel Multi-Level Method for Simulating the pH Depen- Advanced Science & Technology, University of Illinois at Urbana- dence of Charge State Fluctuations and Conformational Ensembles of Champaign, Urbana, IL, USA, 2Department of Biochemistry, University of Intrinsically Disordered Proteins Illinois at Urbana-Champaign, Urbana, IL, USA. Martin J. Fossat, Ammon E. Posey, Rohit V. Pappu. The membranes that surround cells and organelles play a vital role in biological Biomedical Engineering, Washington Univ in Saint Louis, Saint Louis, MO, function, acting as semipermeable barriers that separate and protect cells and USA. organelles from their surroundings. Molecular dynamics (MD) simulations pro- Simulations of intrinsically disordered proteins (IDPs) use fixed charge vide the means to investigate the fundamental molecular mechanisms that give models where the charge states of ionizable residues are set using model rise to membranes’ biological functions, but it is exceedingly difficult to build compound pKa values. Recent experiments have questioned the validity of MD models that capture cellular membranes’ immense scales and complex ge- fixed charge models. For a 100-residue IDP without sequence biases there ometries. To address this challenge, we previously introduced software called 3 can be roughly 10 or more unique possible charge states. Accurate and fully xMAS (Experimentally-Derived Membranes of Arbitrary Shape) Builder, and ergodic simulations require converged conformational sampling of each of we used it to generate a 4.5 billion atom (1.9mm3) model of a piece of the the charge states. To facilitate this we have developed a multi-level Monte endoplasmic reticulum with a helicoidal shape called a Terasaki ramp. In build- Carlo and free energy based method that leverages the ABSINTH implicit ing this model, xMAS Builder utilized electron microscopy and chromatog- solvation model to enable efficient simulations of the conformational ensem- raphy data, respectively, for the shape and lipid composition of the model. In bles of all possible charge states for a given sequence. This is achieved by previous work, we also ran a long-term equilibrium MD simulation on a smaller grouping charge states based on their net charge and adding auxiliary Mar- test system to demonstrate that xMAS Builder produces stable, high quality kov chains that enable protonation/deprotonation of ionizable residues. At models. Used to develop and test the base capabilities of xMAS Builder, these a given pH, the populations of thermodynamically accessible charge states previous models were somewhat limited in that they were composed entirely of and conformations are calculated using the Bennett Acceptance Ratio lipids. We now introduce an updated xMAS Builder capable of incorporating method and data from simulations obtained for all possible charge states. proteins into its membrane models. In xMAS Builder’s approach, proteins Key features of the ABSINTH model allow reductions in the number of rep- are automatically placed on a mesh representing the surface of the membrane, licas needed for free energy calculations. We also estimate shifts in free en- lipids are carefully filled in around the proteins using coarse-grained simula- ergies upon protonation / deprotonation relative to model compounds. Using tion-based techniques, and a sophisticated restrained equilibration procedure these estimates, we quantify charge state fluctuations - known as charge is used to ensure the quality and stability of the final model. With this new capa- regulation - to estimate pKa shifts within IDPs and extract the pH depen- bility, xMAS Builder can now produce cellular membrane models that can be dencies of the populations of charge states and conformational ensembles. used to provide compelling insight into the complex molecular interactions be- In peptides excised from IDP sequences we find that local sequence contexts tween proteins, lipids, and the cellular environment which ultimately give rise cause shifts in pKa values that can be larger than one pH unit. The method, to membrane structure and biological function. with the incorporation of heuristics to enable increased efficiencies, is being deployed for simulations of charge states and conformational ensembles of a 1414-Pos spectrum of IDPs. Pytheas: A Software to Map RNA Modifications via Tandem Mass Spec- trometry 1412-Pos Luigi D’Ascenzo, Anna Popova, James R. Williamson. Ion-Hydroxyl Interactions: From High-Level Quantum Benchmarks to The Scripps Research Institute, La Jolla, CA, USA. Transferable Polarizable Force Fields Ribonucleic acids (RNA) such as ribosomal RNA (rRNA) and messenger RNA Vered Wineman-Fisher1, Yasmine Al-Hamdani2, Iqbal Adduo1, (mRNA) are decorated at the post-transcriptional level by more than 150 char- Alexandre Tkatchenko2, Sameer Varma1,3. acterized chemical modifications. These modifications range from positional 1Department of Cell Biology, Microbiology and Molecular Biology, isomerization to the addition of complex chemical groups to nucleosides. In University of South Florida, Tampa, FL, USA, 2Physics and Materials the last decades, RNA modifications have been shown to enhance RNAs struc- Science Research Unit, University of Luxembourg, Luxembourg City, tural diversity and their intramolecular interactions, as well as promoting RNA Luxembourg, 3Department of Physics, University of South Florida, Tampa, diverse functions within complex regulatory networks involved in cellular FL, USA. metabolism. To characterize and quantify RNA modifications, tandem Mass Ion descriptors in molecular mechanics models are calibrated against refer- Spectrometry (MS/MS) has been proven to be a powerful and flexible analyt- ence data on ion-water interactions. It is then typically assumed that these ical tool. We present here a novel software pipeline, Pytheas, to assist in the descriptors will also satisfactorily describe interactions of ions with other interpretation of MS/MS data with the goal to characterize modified RNAs. functional groups, such as those present in biomolecules. However, several Through an interactive multi-step process, Pytheas generates a theoretical data- studies now demonstrate that this transferability assumption produces, in base from arbitrary RNA sequences and the user-defined parameters such as many different cases, large errors. Here we address this issue in a represen- digestion enzymes, nucleotide chemical compositions and isotopic labeling. tative polarizable model and focus on transferability of cationic interactions Then it uses an empirical scoring function derived from the SEQUEST prote- across alcohols that, like water, also use hydroxyls for coordination. We omics platform to score measured MS/MS spectra vs the theoretical library, obtain gas phase reference data from vdW-corrected DFT after bench- adopting the target-decoy approach for the false discovery rate (FDR) estima- marking against ‘‘gold-standard’’ diffusion Monte Carlo and CCSD(T) tion. The final steps allow visualization of the scored spectra and map the mod- methods. Taken together with experimental condensed phase data, the ifications on the RNA sequence. Pytheas is freely available, easy to use and collected reference energies inform us that the original polarizable model highly customizable; at any step the user can set parameters relevant for their yields large water -> alcohol transferability errors - the RMS and maximum experiments, such as type and number of modifications or instrument-based un- errors are greater than 4.5 and 10 kcal/mol, respectively. These errors are, certainty windows on m/z measurements. The applicability of Pytheas has been nevertheless, systematic in that ion-alcohol interactions are over-stabilized, tested using the Liquid Chromatography MS/MS datasets acquired on different and systematic errors typically imply that some essential physics is either instruments, and was successful in mapping known and discover novel modifi- missing or misrepresented. A comprehensive analysis shows that when cations present in tRNAs and B. subtilis 16S and 23S RNA. both low and high-field responses of ligand dipole polarization are described accurately, then transferability improves significantly - the RMS and 1415-Pos maximum errors in the gas phase reduce, respectively, to 0.9 and 2.5 kcal/ MAINMAST-MELD-MDFF: Denovo Structure-Determination with Data- mol. Condensed phase transfer free energies also improve, as the RMS error Guided Molecular Dynamics decreases from 8.9 to 6.6 kcal/mol, however, the error still remains system- Alberto Perez1, Mrinal Shekhar2, Genki Terashi3, Daisuke Kihara3, atic, which we attribute to the parameterization in long range electrostatics. Ken A. Dill4, Emad Tajkhorshid5, Abhishek Singharoy6. 1 2 Overall, this work demonstrates a rational approach to boosting transfer- Chemistry, University of Florida, Gainesville, FL, USA, Dept Biophysics, 3 ability of ionic interactions that will be applicable broadly to improving other University of Illinois, Urbana, IL, USA, Biol Scis, Purdue Univ, West 4 polarizable and non-polarizable models. Lafayette, IN, USA, Laufer Ctr Phys & Quant Bio, Stony Brook University,

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Stony Brook, NY, USA, 5Biochemistry, University of Illinois, Urbana, IL, high-throughput structural refinement, a necessary step in building large- USA, 6Biodesign Institute, Arizona State University, Tempe, AZ, USA. protein complexes. The past three decades of hybrid modeling has seen molecular dynamics (MD) simulations leverage NMR, X-ray crystallography and cryo EM data 1418-Pos for the determination of medium- to high-resolution structures atomic struc- From FRET Measurements to Database Deposition of Integrative Struc- tures. However, all these approaches, including our popular Molecular Dy- tural Models namics Flexible Fitting (MDFF), and its various extensions work under the Christian A. Hanke1, Hayk Vardanyan2, Mykola Dimura1, conventional molecular replacement paradigm, whereby any initial search Claus A.M. Seidel3. model is morphed to satiate the data-imposed constraints. As a natural conse- 1Heinrich Heine Univ Duesseldorf, Duesseldorf, Germany, 2Dept Molec/ quence, quality of the determined model remains heavily biased by choices of Phys Chem, Heinrich Heine Univ, Duesseldorf, Germany, 3Chemistry, the initial model. Here, we deliver a novel modeling pipeline that iteratively Heinrich Heine University, Duesseldorf, Germany. combines minimum spanning tree-based backbone tracing tool (MAIN- Fo¨rster Resonance Energy Transfer (FRET) experiments provide valuable in- MAST), Bayesian-likelihood based protein-folding methodology (MELD), formation on the dynamics and the tertiary and super-tertiary structure of dy- and a resolution exchange-based fitting protocol (ReMDFF). Starting from namic biomolecules. Previously we demonstrated that FRET experiments only sequence information, the algorithm places C-alpha atoms into the den- together with computational methods, combined in hybrid approaches, can sity, fits a random coil to this C-alpha trace, generates protein secondary struc- be employed for computational structural modeling of biomolecules with tures on-the-fly, and exhaustively samples the backbone and side chain high accuracy and precision. geometries to deliver a refined model. Overcoming limitations of traditional Here, we present structural models for an RNA four-way junction, which we approaches, the need for an initial model or homology information is obtained by combining a large number of high-precision single-molecule completely subsided, and de novo modeling is now made feasible even at FRET measurements with FRET-restrained structural modeling, molecular low resolutions. Available on cloud computing resources, the method is simulations and SAXS measurements in a hybrid approach. This approach al- equally applicable for determining globular and transmembrane protein struc- lowed us to obtain structural models for three co-existing conformers with a tures, as demonstrated for ubiquitin, TRPV1 and Magnesium-channel CorA precision between 1.3 and 3.0 A˚ . The software used in this workflow is avail- (all in the 3-5 A˚ resolution range), and the de novo atomic structure of a li- able on our webpage (http://www.mpc.hhu.de/software.html).As a next step poprotein flp3. after generating the structural models, we prepared the deposition of these models to PDB-Dev (https://pdb-dev.wwpdb.org/). In order to deposit the 1416-Pos generated integrative structural models to PDB-Dev, we extended the Transfer Learning for Efficient Segmentation of Subcellular Structures in hybrid/integrative modeling (IHM) dictionary by definitions and requirements 3-D Electron Microscopy for fluorescence-based integrative structural models. This includes the Matthew D. Guay1, Zeyad A. Emam2, Adam B. Anderson3, description of samples, fluorescent probes, analysis, structural modeling pro- Richard D. Leapman1. cedure and the resulting structural models. The developed descriptions and 1NIBIB, National Institutes of Health, Bethesda, MD, USA, 2Applied definitions for fluorescence data can serve as starting points for other label- Mathematics, University of Maryland, College Park, College Park, MD, based techniques. USA, 3Mathematics, University of Maryland, College Park, College Park, MD, USA. 1419-Pos Automating the segmentation of biological structures at the cell and organ- FISIK: Framework for the Inference of In Situ Interaction Kinetics from elle level is essential for making efficient use of 3-D scanning electron mi- Single-Molecule Imaging Data croscopy (SEM) technologies, such as serial block-face (SBF)-SEM and Luciana R. de Oliveira, Robel Yirdaw, Khuloud Jaqaman. focused ion beam (FIB)-SEM. Currently, automated segmentation using con- Biophysics, UT Southwestern Medical Ctr, Dallas, TX, USA. volutional neural networks (CNNs) typically addresses a specific biological Receptor interactions in the plasma membrane are critical for transmembrane system, and it is necessary to train a CNN for each new problem. However, signal transduction. Using live-cell single-molecule imaging it is possible to addressing each problem individually imposes impractical labor require- monitor inter-receptor interactions with high spatiotemporal resolution in their ments for machine learning developers, and requires large amounts of anno- native environment, although only for the labeled subset of receptors, which tated training data, which are costly to produce. Here, we present our work tends to be a small fraction (often <10% of the full population). As a result, on reducing this burden by applying transfer learning techniques to neural it has remained a challenge to calculate receptor interaction kinetics, especially network architectures to solve instance segmentation and semantic segmen- association rates, from single-molecule data. To overcome this challenge, we tation problems for multiple biological datasets imaged via 3-D SEM. We developed a mathematical modeling-based Framework for the Inference of in are able to train instance segmentation networks to detect and localize ob- Situ Interaction Kinetics from single-molecule imaging data (termed ‘‘FI- jects across different cells and tissues. By sharing semantic segmentation SIK’’). FISIK consists of (1) devising a mathematical model of receptor dy- network pathways between biological systems, we are able to reduce the namics and interactions, mimicking the biological system and data amount of training data required for effective segmentation of new EM acquisition setup, and (2) estimating the unknown model parameters, including datasets. receptor association and dissociation rates, by fitting the model to experimental single-molecule data. Due to the stochastic nature of model and data, we adapt- 1417-Pos ed the method of indirect inference for model calibration. To validate FISIK, High-Throughput Refinement of CryoEM-Based Structures we simulated trajectories of diffusing receptors that interact with each other, Chaoyi Xu, Alexander J. Bryer, Juan R. Perilla. considering a wide range of model parameters, and including tracking errors Department of Chemistry and Biochemistry, University of Delaware, that result in noisy data for model calibration. Our tests demonstrated that FI- Newark, DE, USA. SIK has the sensitivity to determine association and dissociation rates; its accu- Cryo-electron microscopy (CryoEM) has become a routine tool to determine racy depends on the labeled fraction of receptors as well as the extent of the structure of protein complexes, alongside X-ray crystallography (XRD) tracking errors. For conditions where the labeled fraction was relatively low and Nuclear magnetic resonance spectroscopy (NMR). However, several (e.g. to afford accurate tracking), we found that combining dynamic single- structures determined employing CryoEM and deposited to the protein data- molecule imaging data with static localization microscopy data improves FI- bank present clashes and other structural problems, which can be readily de- SIK’s performance. All in all, FISIK is a promising novel approach for the deri- tected using structure validation tools such as Molprobity. Recently, we devel- vation of receptor interaction kinetics in their native plasma membrane from oped an iterative protein refinement protocol based on CryoEM at near-atomic single-molecule data. resolution and the Molprobity score. Our method can successfully refine inter- atomic clashes, torsion angles, bond lengths and bond angles. Structural out- 1420-Pos liers are optimized employing structural restraints which are applied using Can Hydrogen-Deuterium Exchange Rates at Single Residue Level Be custom-made RosettaCM scripts. The process is iteratively performed until Obtained from HDX-MS Data? all structural issues found in a complex have been corrected. To validate Emanuele Paci1, Roman Tuma2, Simon Skinner1, our protocol, we present the unsupervised refinement of hundreds of Jeanine J. Houwing-Duistermaat3. CryoEM-derived structures. The overall scores of the refined models show 1Fac Biol Sciences, Univ Leeds, Leeds, United Kingdom, 2Astbury Ctr, Univ that our method can correct structural issues found in many CryoEM- Leeds, Leeds, United Kingdom, 3School of Mathematics, Univ Leeds, Leeds, derived structures. Furthermore, we highlight the use of our method for United Kingdom.

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Hydrogen/deuterium exchange probed by mass spectrometry (HDX-MS) is a and amino acid atoms, can be applied to filter multiple structural character- promising technique for fingerprinting structure and dynamics of proteins. istics and has been used to identify novel folding motifs in nucleic acid- The deuterium uptake of any fragment of the polypeptide chain depends protein complexes. The web server is available at http://web.x3dna-dssr. uniquely on the rate of exchange of its amide hydrogens but determining the org/. latter from the former is in general not possible. If time-resolved measurements are available for a number of overlapping peptides that cover the whole 1423-Pos sequence, rate constants for each amide hydrogen exchange (or equivalently, GDash: A Genomics Dashboard Integrating Modeling and Informatics their protection factors) can be predicted. In most cases, the solution may not Zilong Li, Ran Sun, Thomas Connor Bishop. be unique, yet well-defined. The example presented shows how such informa- Louisiana Tech Univ, Ruston, LA, USA. tion can be refined, and valuable even when not sufficient to determine the Genomics is a sequence based informatics science and a structure based mo- structure of proteins. lecular science. There are few tools available that unite these approaches in a scientifically robust manner. Here we introduce the concept of a genomics 1421-Pos dashboard as a tool specifically designed to integrate informatics and phys- Charmm-Gui NMR Structure Calculator: A Web-Based Tool for Calcu- ical modeling approaches for the study of chromatin. GDash is a prototype lating Biomolecular NMR Structures genome dashboard that integrates our Interactive Chromatin Modeling(ICM) Jumin Lee1, Yuanpeng J. Huang2, Gaetano T. Montelione2, Wonpil Im1. tools with the Dalliance genome browser. ICM is an interactive tool that al- 1Departments of Biological Sciences and Bioengineering, Lehigh University, lows users to rapidly fold any sequence of DNA into atomic or coarse- Bethlehem, PA, USA, 2Center for Advanced Biotechnology and Medicine, grained models of chromatin by combining the material properties of Rutgers University, Piscataway, NJ, USA. DNA and nucleosomes with nucleosome positioning data. GDash extends Calculating a NMR structure can be laborious as it requires multiple valida- ICM’s physical modeling capabilities by uniting modeling with public or tion steps to extract accurate distance restraints from the NOESY data. In private informatics data, e.g. experimentally or theoretically determined this work, we describe CHARMM-GUI NMR Structure Calculator (http:// nucleosome positions. GDash currently allows users to specify nucleosome www.charmm-gui.org/input/nmrstructure), a web-based biomolecular positions from various experimental or theoretical sources, interactively NMR structure calculation module available in CHARMM-GUI. By manipulate nucleosomes(add, delete and move) and assign different confor- providing web-based graphical user interface (GUI), NMR Structure mational states to each nucleosome (e.g. tetrasome, octasome, chromato- Calculator makes it straightforward to assign NOESY cross peaks, create some). GDash displays 3D structures using JSmol and data associated distance restraints, generate 3D structures from these data, and validate with the structure as tracks in a genome browser (e.g. Roll, Slide, or the resulting ensemble of NMR structures against the primary NOESY Twist). The exchange of data is bi-directional so any genome track can be data and NMR restraints. NMR Structure Calculator operates in the mapped onto a molecular structure (e.g. color by function). A coarse- following four steps. STEP1 includes i) reading of the sequence/structural grained model of chromatin is implemented in LAMMPS to optimize and information and the NOESY peak list data or NMR restraints, ii) their con- sample 3D structures in real time. As a sample application, GDash identifies version to CHARMM format, and iii) generation of CHARMM structure steric clashes and DNA knotting associated with a model of the CHA1 pro- calculation scripts. In the case when a user uploads raw NOESY data as moter based on experimentally determined nucleosome positions. Our an input, NMR Structure Calculator automatically converts it to NMR re- coarse-grained model can resolve these problems without significant varia- straints using the automate NOESY peak assignment program ASDP. In tion in structure, yet other questions arise. GDash is a novel tool for inves- STEP2, jobs are submitted to a local cluster for structure calculations using tigating structure-function relationships for regions of the genome ranging torsion angle molecular dynamics with NMR restraints. STEP3 is refinement from kilobases to megabases and for generating, validating and testing of the structures in an implicit solvent model. Finally, in STEP4, the models mechanistic hypotheses. are ranked based on the restraint violation, Ramachandran conformational statistics, and other structure quality assessment metrics, and an ensemble 1424-Pos of structures that best fit the NMR restraints are provided to users. This auto- On the Use of Short Reseeding Trajectories to Sample Markov State mated NMR structure calculation protocol has been successfully tested for a Models set of biomolecules and their complexes (protein-nucleic acid and protein- Hongbin Wan, Vincent Voelz. protein). We expect NMR Structure Calculator to be a useful tool to the Chemistry, Temple Univ, Philadelphia, PA, USA. NMR community for biomolecular NMR structure calculations in a user- In the last decade, advances in molecular dynamics (MD) and Markov State friendly manner. Model (MSM) methodologies have made possible accurate and efficient esti- mation of kinetic rates and reactive pathways for complex biomolecular dy- 1422-Pos namics occurring on slow timescales. A promising approach to an enhanced A New Web Server for the Identification of Novel Nucleic Acid Structural sampling of MSMs is to use so-called ‘‘adaptive’’ methods, in which new Motifs and their Interactions with Proteins MD trajectories are ‘‘seeded’’ preferentially from previously identified states. Shuxiang Li1, Xiang-Jun Lu2, Wilma K. Olson1. Here, we investigate the performance of various MSM estimators on reseeding 1Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, trajectory, for both a simple 1D free energy landscape and for mini-protein USA, 2Department of Biological Sciences, Columbia University, New York, folding MSMs of WW domain and NTL9(1-39). Our results reveal the practical NY, USA. challenges of reseeding simulations, and suggest a simple way to better esti- The diversity of nucleic acid structural motifs and their interactions with pro- mate both thermodynamic and kinetic information. teins are of fundamental importance to crucial cellular processes, such as gene expression and regulation. The identification of new motifs has been 1425-Pos and continues to be of great interest to structural biology and bioinformatics. Automatic Assignment of Bonded Force Field Parameters for Small We have developed a novel web server and tools for the characterization and Molecules Using Machine Learning visualization of 3D structural motifs in RNA and DNA molecules. The web Praveer Narwelkar1, Hui Sun Lee2, Sihong Xie1, Wonpil Im2. server combines the DSSR software for analyzing and annotating nucleic 1Computer Science and Engineering, Lehigh University, Bethlehem, PA, acid tertiary structures with 3D and secondary structure molecular viewers, USA, 2Department of Molecular Biosciences and Center for Bioinformatics, a MySQL database, and a set of interactive tables. The web server takes The University of Kansas, Lawrence, KS, USA. atomic coordinates as input and produces lists of modified nucleotides, ca- Reliable force field parameters for small molecules are an essential component nonical and noncanonical base pairs, rigid-body parameters, higher-order of molecular mechanics and molecular dynamics studies for biological macro- co-planar base associations, various helices and loops, and torsion angles molecular systems in complex with ligands. However, it is unrealistic for of each nucleotide. The structural characteristics can be viewed on the currently available computational tools to fully cover the bonded parameters web through interactive 3D images of the molecule and Grid-View tables, of every possible combination of atom types. Machine learning has become which enable the efficient recognition of 3D motifs and their interactions a great tool for many areas of research, but there are very few works which uti- with the surrounding environment. The tables of selected parameters can lize this technology for prediction of force field parameters. To make progress also be downloaded for statistical analysis. Notably, the web server is able in this field, we have developed a computational method to automatically to simultaneously display the 1D sequence, 2D secondary structure, and generate bonded force field parameters for small molecules using machine 3D folding of nucleic acids. The web server, together with in-house tools learning. We defined 26 descriptors representing the microenvironment of and the SNAP software for detecting the interactions between nucleotide each atom type in the CHARMM General Force Field (CGenFF) and generated

BPJ 9378_9380 290a Monday, March 4, 2019 machine learning models optimally predicting specific parameters (e.g., force assembly in vivo, which may operate quite differently from purified in vitro constant for bonds, valence angle parameter for angles). The pre-trained ma- models due to such effects as spatial confinement, macromolecular crowding, chine learning models are used for making predictions for any given combina- and influences of extrinsic cellular factors. Simulation has thus proven a valu- tions of atom types that are unavailable in the CGenFF. We perform benchmark able adjunct to experimental methods, facilitating interpretation of experi- performance evaluation to examine the reliability of the bonded parameters mental data and inferences about experimentally unobservable aspects of predicted using the machine learning models. self-assembly. A variety of simulation methodologies have been used for modeling self-assembly dynamics — including approaches based on Differen- 1426-Pos tial Equations (DE), Gillespie Algorithm (SSA), Brownian Dynamics (BD), Defining Conformational States of Proteins using Dimensionality Reduc- Green’s Function Reaction Dynamics (GFRD), and variations thereon — tion and Clustering Algorithms each offering tradeoffs with respect to the ranges of phenomena they can Eugene Klyshko1, Sarah Rauscher1,2. model, their computational tractability, and the difficulty of fitting them to 1Physics, University of Toronto, Toronto, ON, Canada, 2Chemical and experimental measurements. Here, we explore a multiscale approach Physical Sciences, University of Toronto Mississauga, Mississauga, ON, combining efficient SSA-like sampling suitable for well-mixed systems with Canada. the slower but space-aware GFRD model, assuming as with GFRD that Molecular dynamics (MD) simulations of proteins produce large data sets - reactions occur in a spatially heterogeneous environment that must be explic- long trajectories of atomic coordinates - and provide a representation of the itly modeled. We generalize the GFRD model to more efficiently handle sampling of a given molecule’s structural ensemble. A deep quantitative anal- bimolecular association reactions fundamental to self-assembly, using a ysis using advanced machine learning techniques is a means to interpret MD rejection method for exact and efficient sampling of reaction locations in trajectories. To visualize the conformational space of the molecule and prop- spatially heterogeneous regions. To further accelerate models with minimal erly identify conformational states, we suggest combining clustering methods loss of accuracy, we are exploring regression-based methods for approximate and dimensionality reduction algorithms. We investigate different choices of integration. While the resulting model remains a theoretical abstraction, future features to represent individual structures, clustering algorithms, similarity work will explore its ability to capture an important subset of cellular self- metric, and methods to assign the number of clusters. assembly for which classic SSA is too simplified but GFRD and BD too inef- ficient to capture accurately the phenomena in realistic biological parameter 1427-Pos ranges. An Efficient Algorithm to Calculate the Common Solvent Accessible Volume 1430-Pos In Jung Kim. Electrostatic Force Driven Molecular Dynamics Simulations Computer Science, Pennsylvania State University Capital College, Yunhui Peng, Mahesh Koirala, Emil Alexov. Middletown, PA, USA. Dept Physics, Clemson University, Clemson, SC, USA. Protein-protein interactions are often mediated by solvent molecules. These Electrostatic interactions play crucial role at various levels of biological sys- solvent molecules often build a bridge between the proteins sharing hydration tems. Being a long-range force, electrostatic force is known to be the major shells, and as a result, the proteins are constrained to be correlated in their mo- driving force for biomacromolecules interactions at long distance. Here, we tions. Efficiently evaluating the common volume of these hydration shells may developed a new molecular dynamics protocol named Electrostatic Force lead to a better understanding of protein-protein interactions. In this work, we driven Molecular Dynamics (EFMD) simulation, which utilizes DelPhi- propose a new algorithm that calculates the common solvent accessible volume Force along with NAMD molecular dynamics (MD) package. In EFMD (SAV) between any two (charged) atoms in proteins, which will be used to es- the calculated electrostatic forces at each atom of the target are used in timate the strength of the bridge between the two atoms in proteins in our future steered MD simulations. This protocol was applied to study the binding of work. The proposed algorithm utilizes a well-known sweep-line algorithm to substrates to protein receptor and to model protein-protein recognition. It efficiently find all intersection points between each sphere of protein atoms is shown that EFMD greatly facilitates the success rate and reduces compu- with van der Waals and SAV radii, from which the common SAVs are calcu- tational time. lated. We apply 10 proteins, whose sizes vary from 15 to 8015 residues, to vali- date the accuracy and to evaluate the computational speedup of our algorithm 1431-Pos compared to the benchmark cube-based method. The proposed method may be A New Open Source Toolkit for Segmenting 3D Intracellular Structures in used to develop a new implicit solvent model that involves the solvent mediated Microscopy Images atom-atom interactions in the future. Matheus Palhares Viana, Susanne Rafelski. Cell Science, Allen Institute, Seattle, WA, USA. 1428-Pos The Allen Institute for Cell Science is developing a state space of stem cell CHARMM-GUI Multicomponent Assembler for Modeling and Simulation structural signatures to understand the principles by which cells reorganize of Complex Heterogeneous Biomolecular Systems during the cell cycle and differentiation. We have developed a pipeline that Nathan R. Kern. generates high-replicate, dynamic image data on cell organization and activ- Computer Science & Engineering, Lehigh University, Bethlehem, PA, USA. ities using endogenous fluorescently tagged human induced pluripotent stem Manually creating complex molecular models for simulation is tedious and cell (hiPSC) lines (www.allencell.org). Each line expresses a monoallelic often quite difficult. In CHARMM-GUI, there are already several tools for EGFP-tagged protein that represents a particular cellular structure. For each automating this process. Most notably, Solution Simulator prepares a model structure, we take advantage of thousands of replicate high-resolution 3D im- of a single solvated structure of interest, such as a protein. Similarly, Mem- ages to develop quantitative image-based assays, analyses, and computational brane Builder models a single structure embedded in a heterogeneous biolog- models. An important step in extracting information from the image data is to ical membrane with surrounding water. Both of these tools work by accurately segment each cellular structure in 3D. However, there remains a combining one PDB structure with a well-defined environment, such as water significant gap between state-of-the-art 3D computer vision algorithms and or a membrane. While these are useful for studying the isolated behavior of a the openly accessible tools that biologists without computer vision expertise single structure of interest in its typical environment, many important molec- can easily use to accurately segment their 3D image data. To address this, ular behaviors involve multiple such structures. Here we describe a more gen- we developed a Python-based open source toolkit for 3D segmentation that eral method for creating solvated models with an arbitrary number of unique combines traditional segmentation algorithms with an iterative deep learning molecular structures in any proportion in any solvent and membrane workflow. The general workflow is based on a minimal number of selectable environment. algorithms and tunable parameters. We successfully applied this workflow to over 30 different intracellular structures, and our results can be used like a 1429-Pos ‘‘lookup table’’ starting point for new applications. We implemented an iter- A Stochastic Spatial Simulation Method for Self-Assembly Reactions ative deep learning workflow that uses the results of the traditional segmenta- Marcus Thomas, Russell S. Schwartz. tion together with minor manual inspection to generate a ground truth for Computational Biology, Carnegie Mellon University, Pittsburgh, PA, USA. training deep learning models. This approach improves the accuracy and The ability of collections of molecules to spontaneously assemble into large robustness of those segmentation challenges that are not satisfactorily solved functional complexes is central to all cellular processes. Yet the fine details with standard algorithms. This open source segmentation toolkit aims to facil- of complex self-assembly processes normally cannot be directly experimen- itate quantitative cell biology, conveniently leveraging state-of-the-art algo- tally observed due to their small scale and rapid dynamics. Intractability of rithms in computer vision for specific image segmentation needs and experimental approaches is particularly acute for understanding self- challenges.

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1432-Pos lenging. Moreover, preparations of systems containing combinatorial ligand A Systematic Approach to Understanding Macromolecular Crowding structures are nontrivial and time consuming due to the alchemical hybridiza- Effects on Biomolecular Interactions through Computational ‘‘Toy’’ tion of topologies and structures among the various derivatives. Here, we Models describe CHARMM-GUI Ligand Binder to help users to prepare relative Rachel Kim1, Mala L. Radhakrishnan2. free energy calculations by providing necessary input files and force field pa- 1Wellesley College, Wellesley, MA, USA, 2Chemistry, Wellesley College, rameters for various simulation programs and free energy calculation Wellesley, MA, USA. methods. The cellular environment is crowded, with macromolecules occupying up to 40% of the cell’s volume. These ‘‘crowders’’ have been shown to have sig- 1435-Pos nificant impacts on biomolecular recognition in the cell. Thus, it is important One Nanometer Precision by Bayesian Grouping of Localizations to realistically account for these effects when studying biological com- Mohamadreza Fazel1, Bernd Rieger2, Ralf Jungmann3, Keith A. Lidke1. plexes. Rather than approaching an understanding of crowding effects 1Physics and Astronomy, Univ New Mexico, Albuquerque, NM, USA, 2Delft through a specific system, this study uses theoretical ‘‘toy’’ models to inves- Univ Tech, Delft, Netherlands, 3Dept Physics, Tech Univ Munich, Garching, tigate the impact of macromolecular crowding on binding energetics of a Germany. complex as a function of the intrinsic properties of the binding partners Single-molecule-localization-based super-resolution methods such as DNA- and the crowders, focusing on electrostatic interactions and solvation prop- PAINT and (d)STORM result in multiple observed localizations from erties. The use of toy model molecules allows for controlled, systematic each dye or binding site that are not a priori assigned to the specific variation of their physical properties, such as charge distribution, size, and dyes or binding sites. We describe a Bayesian method of combining local- shape. Using a continuum electrostatics model, binding partners and izations from multiple blinking/binding events that improve localization randomly-placed crowders are represented as low-dielectric regions. By precision to around 1 nm. The known statistical distribution of the studying the trends of crowding effects on both binding affinity and speci- number of binding/blinking events per dye/docking strand along with ficity through the lens of the intrinsic, physical properties of the binding localization precision of each localization event are used to estimate the partners and the crowders, we hope to achieve a unified, general framework true numbers and locations of emitters in closely spaced clusters. The for understanding and predicting how macromolecular crowding affects bio- algorithm uses Reversible Jump Markov Chain Monte Carlo to find both molecular interactions that will help guide future crowding studies, both the number of dyes/docking strands and their locations as well as their un- computational and experimental. certainties. The output is the posterior distribution, which is a weighted average over all possible numbers of emitters and their locations. 1433-Pos The most likely model can also be extracted from the posterior. We demon- Calculation of Protein-Protein Binding Free Energies using Umbrella strate the algorithm performance using DNA-Origami and membrane pro- Sampling with Dual Resolution Water Models tein data. Jagdish Suresh Patel1, F. Marty Ytreberg2. 1Center for Modeling Complex Interaction, University of Idaho, Moscow, ID, 1436-Pos USA, 2Department of Physics, Center for Modeling Complex Interactions, Implicit Solvent Calculations at Large-Scale Virus-Level Poisson-Boltz- University of Idaho, Moscow, ID, USA. mann and Multiscale Simulations for Electrostatics The binding free energy between two proteins is a measure of how strongly Matias Martinez1, Horacio Vargas-Guzman2, Christopher D. Cooper1. they will interact under physiological conditions. All-atom statistical-me- 1Dept. Mechanical Eng, Univ Santa Maria, Valparaiso, Chile, 2Max Planck chanics-based free energy calculation methods have the potential to accurately Institute for Polymer Research, Mainz, Germany. calculate binding free energy but are completely unfeasible for large-scale In molecular simulations, continuum electrostatic theory leads to a coupled sys- applications due to their high computational cost. This motivates the develop- tem of partial differential equations on two domains: the ‘solvent’ and ‘mole- ment of efficient and accurate free energy calculation approaches. Dual- cule’ regions, interfaced by the molecular surface. From a continuum approach, resolution molecular simulation approach allows one to simulate at the atomic the solvent consists of water with salt, yielding the Poisson-Boltzmann equa- level in the regions of interest and at a coarser level, but much faster, in less tion, and in the ‘molecule’ domain the potential is modeled with the Poisson relevant parts of the biomolecular systems. This hybrid approach has shown a equation. This approach is widely used for biomolecules, and in this work substantial reduction of computational cost without compromising accuracy in we take it to a larger scale to study the electrostatic potential of full virions, a classical molecular dynamics simulations. Here, we investigate whether through a shared-memory parallelized version of the Poisson-Boltzmann solver combining the statistical-mechanics-based free energy calculation method, PyGBe. PyGBe solves the system of partial differential equations with a umbrella sampling, with dual resolution water models provides a good multipole-accelerated boundary integral approach, to compute the solvation trade-off between speed and accuracy. We chose Barnase-Barstar protein- energy. protein complex as a test system since it is relatively small to allow conver- We will show results where we systematically vary the ionic concentration in gence of the simulations, has a known structure, and empirical relative the solvent, to analyze dissolution and rupture mechanisms of the viral capsids, binding free energy difference values (DDG) of their observed mutations in particular, for Dengue and Zika. Also, we aim to study the effect of the ionic are reported in the literature. We selected eight different mutations occurring concentration gradient in mechanical stability, as the solvent inside and outside at the different sites with broadly varying empirical DDG values. We then the viral capsid may have a different amount of salt. calculated DDG values for each mutation using umbrella sampling simula- These results aim to contribute towards more detailed computational multiscale tions, where we treated protein-protein complex and one surrounding water molecular modeling, i.e. at the protein-capsomer level. Hence, we will layer at an atomic level and the bulk of the water using WT4 coarse- conclude on how computationally effective would be to bridge the multiscale grained water model. We obtained a high correlation between experimental and continuum approaches. and calculated DDG values. This dual resolution free energy calculation approach has the potential for future high-throughput studies of protein- 1437-Pos protein binding. Unsupervised Learning of Conformational States Present in Molecular Dynamics Simulation Data for Summarization of Equilibrium Conforma- 1434-Pos tional Dynamics Charmm-Gui Ligand Binder for Relative Binding Free Energy Calcula- Kazi Lutful Kabir, Nasrin Akhter, Amarda Shehu. tions Department of Computer Science, George Mason University, Fairfax, VA, Seonghoon Kim, Wonpil Im. USA. Bioscience and Bioengineering, Lehigh Univ, Bethlehem, PA, USA. Markov State Model (MSM)-based analysis of Molecular Dynamics (MD) The relative binding free energy calculation methods have become an impor- conformational data has become very popular in molecular modeling as a tant class of techniques for obtaining thermodynamic predictions in the field way of summarizing the conformational dynamics present in the simulated of drug discovery, ligand and peptide binding prediction, and solvation free MD trajectories. The availability of hallmark software, such as EMMA and energies. The major methods are based on the free energy perturbation, ther- MSMBuilder, has democratized such analysis among computational biolo- modynamic integration, and lambda dynamics, which have different advan- gists. However, such analysis proceeds in a formulaic manner, first preparing tages and disadvantages in terms of calculation speed and accuracy. Each conformational data for a clustering-based analysis, carrying out the analysis method is embedded in different simulation programs, so that preparations and denoting detected clusters as conformational states, utilizing such states to of initial modeling and necessary input files are quite different and chal- build the MSM, and then analyzing the model’s quality and interpreting

BPJ 9378_9380 292a Monday, March 4, 2019 it. The identification of states as clusters relies on standard clustering algo- Posters: Micro- and Nanotechnology I rithms that ignore conformational energy and instead use similarity over low-dimensional representations (a process referred to as featurization in cur- 1440-Pos rent MSM analysis) of conformations. In this work, we propose to leverage Soft Material Programming through the Spatiotemporal Release of the fact that the conformations sampled in simulation populate an unknown Oligonucleotides energy landscape. The neighborhood structure of the landscape can be ex- Moshe Rubanov, Phillip Dorsey, Dominic Scalise, Wenlu Wang, tracted via spatial statistical analysis that does not ignore energy but instead Rebecca Schulman. uses it to identify basins and basin-separating saddles in the landscape. We Chemical and Biomolecular Engineering, Johns Hopkins University, utilize this information to identify states and show that such a definition of Baltimore, MD, USA. states is more robust than that obtained via clustering. We proceed to analyze The programmable spatiotemporal release of molecular outputs is a prerequi- in this manner various MD trajectories and summarize the conformational dy- site for creating new classes of stimuli responsive biomaterials capable of namics in a comparative manner, showing that a landscape-based identifica- executing sensing and computational programs. Here we demonstrate the abil- tion of conformational states is promising for detecting and summarizing ity to fabricate heterogeneous micromaterials that implement sequentially conformational dynamics. activated molecular cascades with programmed timescales of activation and release from a hydrogel. These materials use enzyme-free DNA-based 1438-Pos strand-displacement to direct the sequential release of short oligonucleotides Genetic Mutation Classification using Machine Learning from spatial domains. Maskless photolithography enables the spatial sequestra- Hammad Farooq, Naeem Rehmat, Sanjay Kumar, Hammad Naveed. tion of acrylate-modified short oligonucleotides within hydrogels at sizes of Computer Science, National University of Computer and Emerging Sciences, tens of microns. To control temporal release, a DNA reaction-cascade that re- Islamabad, Pakistan. lies on toehold mediated strand displacement was implemented, allowing for DNA-Sequencing of tumor cells has revealed thousands of genetic mutations the controlled release of oligonucleotides at 8-hour intervals. The tuning of but cancer is caused by some of them. The challenge is to identify the muta- reactant concentrations and toehold sizes allows for the temporal control of tions that contribute to tumor growth which is currently being done manually. DNA release. The programmed release of DNA oligonucleotides from hydro- A clinical pathologist manually reviews and classifies each genetic mutation gel substrates enables the scalable development of DNA-based reaction-diffu- based on evidence from clinical literature. This process is very cumbersome sion systems that regulate the availability of oligonucleotides at different points and expensive in terms of money and time. We have used different Machine in space and time. Learning (ML) classifiers (Random Forest, k-Nearest Neighbors, Naive Bayes, Support Vector Machine and Gradient Boosting etc) for multiclass 1441-Pos classification of cancer mutations. We used OncoKB dataset which has three Detection and Mapping of dsDNA Breaks using Graphene Nanopore features (Gene, Mutation/Variation, Clinical literature). Gene and Variation Transistor were encoded to numerical features from categorical features using one-hot Nagendra Athreya, Olgica Milenkovic, Jean-Pierre Leburton. encoding. Clinical literature was converted to numerical vectors of fixed Electrical and Computer Engineering, University of Illinois at Urbana- length using tf-idf, word2vec and doc2vec and then fed into ML models. Champaign, Urbana, IL, USA. We achieved 0.9 logloss and 68% accuracy by XGBClassifier, while the Single-strand DNA (ssDNA) breaks that are often converted into DNA random model gives 2.68 logloss and 12% accuracy. Our model performs double-strand (dsDNA) breaks make up a vast majority of lesions a normal significantly better than random model (3x on logloss and 5.67 times on ac- human cell undergoes everyday. If the repair mechanism fails, the dsDNA curacy). This work will assist the pathologist in classifying the genetic muta- break can cause chromosomal instability leading to tumorigenesis. Addition- tion in less time and save the patient’s life and time by reducing the ally, a single break in a critical gene can cause the cell to undergo apoptosis. misdiagnosis rate. Moreover accurate treatment will reduce toxicity in cancer Existing genome sequencing techniques are not suitable for detecting such patients. changes directly. As an alternative methodology, we propose to employ graphene-Quantum Point Contact nanopore transistor to detect and map 1439-Pos defects in the DNA backbone, as miniscule as ssDNA breaks, efficiently using Characterization of Spectral Feature Upon Cellular Morphodynamics and electronic sheet currents obtained across the transistor membrane. For this its Application purpose, we use large-scale comprehensive all-atom molecular dynamics Xiao Ma1, Ellen O’Shaughnessy2, Klaus M. Hahn2, Gaudenz Danuser1. simulation techniques accompanied with electronic transport calculations, 1 Dept of Bioinformatics, University of Texas Southwestern Medical Center, data denoising and signal detection. In all our simulations, we observe 2 Dallas, TX, USA, Dept of Pharmacology, University of North Carolina at the molecule sticking in the pore at the nicked site due to strong hydrophobic Chapel Hill, Chapel Hill, NC, USA. attraction between the graphene membrane and the damaged-backbone. Cellular morphodynamics is a phenotypic outcome of numerous cellular While the ionic currents calculated for the translocation of 20 base-pair processes, including migration, proliferation, and differentiation. It is dsDNA strand with a break in the backbone does not show any distinct commonly utilized as an indicator of physiological and pathological state signature from the nicked-site in the signal, a clear dip is seen in the trans- of cells. Therefore, quantitative characterization on morphodynamic feature verse sheet current signal corresponding to the location of the breakage, of cells is essential for evaluating cell state and understanding the effect of thereby enabling us to detect and map these damages, electronically. We underlying molecular pathway that governing the morphogenetic process. validated our methodology by detecting other sequence specific dsDNA By employing Hilbert-Huang transform (HHT) based spectral decomposi- breaks along randomly sequenced strands. We strongly believe such a detec- tion method, we decomposed the convoluted cell edge movement time series tion mechanism enables the development of versatile semiconductor elec- into several analyzable functions with instantaneous frequency and magni- tronics for early cancer detection caused by structural modification of the tude characterized at each location, and formed the frequency spectra to genome. represent the signaling topology of individual cell. We then applied the fre- quency spectra of cell migration to evaluate the performance of several 1442-Pos FRET based biosensors expressing GTPases RhoA, Rac1 and Cdc42. The Autochemophoretic DNA Motors Generate 100D Piconewton Forces intrinsic and inter-group variation of the spectral features among control, Aaron Blanchard1, Khalid Salaita2. wildtype and mutated biosensors were compared, and the dependence of 1Biomedical Engineering, Emory University, Atlanta, GA, USA, 2Chemistry, morphodynamic variation on the expression level of biosensors were quan- Emory University, Atlanta, GA, USA. titatively estimated. We discovered consistent frequency spectra with DNA walkers, which ‘‘walk’’ along linear or planar tracks via burnt-bridge considerable heterogeneity upon amplitude spectra within each biosensor hybridization interactions, are promising synthetic analogs of motor proteins condition, while evidently distinguishable spectral patterns between such as kinesin, myosin, and dynein. DNA walkers can precisely transport different conditions. One type of mutated biosensor (TQ10) is found to pre- nanoscale cargo but cannot generate piconewton-scale force. This primary sent much more stable performance with less adverse perturbation of cells function of motor proteins is necessary for countless processes including mus- over a range of expression levels compared to the control cells tagged cle contraction, clotting, immunosensing, embryogenesis, and mechanosensa- with membrane marker CAAX. The proposed spectral profiling method, tion. We present progress towards the design of force-generating with high sensitivity, consistency and efficiency, could have promising nanomachines by showing that highly polyvalent DNA motors (HPDMs) application prospect in biosensor development, and provide incisive infor- generate 100þ piconewtons of force via a novel mechanism that we term au- mation upon the correlation between cellular morphogenesis and underlying tochemophoresis. HPDMs are DNA-coated microparticles that connect to molecular regulation pathways. planar RNA-functionalized surfaces via DNA-RNA hybridization (Yehl &

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Salaita et. al., Nature Nanotechnology 2016). Ribonuclease H enables translo- and excluded volume interactions, under constant voltage bias. So, we inves- cation by cleaving the RNA-DNA duplexes, resulting in an RNA depletion tigated the conformational variations of this polymer evaluating the dwell track in the HPDM’s wake. Translocating for hours at micron/minute speeds, time in such nanocavities and the DNA speed when passing from one cavity HPDMs are the fastest, most processive DNA-based motors reported to date. to another one. These studies are important for advances in DNA barcoding To test HPDMs’ force generation capability we designed a single molecule technology. fluorescence microscopy experiment which enables direct visualization of mechanically-ruptured molecular bonds. Surprisingly, we found that HPDMs 1445-Pos generate forces that mechanically rupture 25 basepair DNA duplexes and Biomimetic, Voltage-Sensitive Nanopores with Local Control over Pore biotin-streptavidin bonds (the strongest noncovalent bonds found in nature Position, Size and Surface Chemistry with 100þ pN force thresholds). HPDMs lack directed tracks and conforma- Cody Combs1, Nick Teslich2, Elif T. Acar1, Francesco Fornasiero2, tional switching such as ATP-fueled powerstrokes, thus underscoring the nov- Zuzanna S. Siwy1, Steven F. Buchsbaum2. elty of this result. To study this fundamental mechanism of force-generation, 1Dept Phys/Astron, Univ Calif Irvine, Irvine, CA, USA, 2Dept Phys & Life we developed a simulation method that accurately reproduces most properties Scis, Lawrence Livermore Natl Lab, Livermore, CA, USA. of HPDM motion via direct modeling of the distance-dependant biophysics of Excitable ion channels play an essential role in biology by regulating ion DNA-RNA interactions. These simulations highlight the mechanism of transport across cell membranes. A key characteristic of these nanoscale pores HPDM force generation and demonstrate that motion is driven by autochemo- is the ability to respond to environmental stimuli such as changes in the local phoresis, which has been observed in biological systems (Sugawara & Ka- membrane voltage or ion concentration. The replication of this behavior in neko, Biophysics 2011). Our work suggests that autochemophoresis may be synthetic nanopore platforms has the potential to enable new responsive ma- a third fundamental method of force generation in molecular motors and terials useful in applications such as biomimetic componentry, drug delivery living systems. and filtration technologies. While voltage gating has been demonstrated before in synthetic nanopores, previous fabrication methods make it difficult 1443-Pos to incorporate such pores into more complex systems due to lack of control Optically Assisted Localization of Solid-State Nanopore during Controlled over key attributes such as pore location. Here, we present the fabrication Breakdown Fabrication of a voltage and ion concentration responsive nanochannel with local control Kamyar Akbari Roshan, Weihua Guan. over the pore location, size, shape and surface chemistry. This is achieved us- Electrical Engineering, Pennsylvania State Univeristy, State College, PA, ing focused ion beam (FIB) to nanomachine a pore in a SiNx membrane fol- USA. lowed by ion-beam-controlled deposition of SiOx around the pore entrance. A number of nanopore-based applications such as tunneling current sensing and Chemistry selective for SiOx is then used to graft single stranded DNA plasmonic nanopores would require the nanopore to be specifically localized. (ssDNA) only to the regions where the local deposition was performed. Re- So far, CBD-based nanopore localizing efforts often require additional lithog- corded voltage-sensitive transport is complex and dictated by both the charge raphy patterning process, thus limiting its flexibility and tunability. Optical and conformational changes of ssDNA chains in confinement. For small beams can be manipulated to tightly focus into any arbitrary spot, offering enough pore diameters, interplay between the applied voltage and local ion an alternative approach for solid-state nanopore localization. It has been shown concentrations at the pore opening results in three pore states, each with an enhanced electric field by pre-patterned plasmonic structure could lead to different conductance behavior. Hysteresis was also observed, which is attrib- nanopore fabricated in desirable locations. In addition, high laser power was uted to steric barriers to ssDNA conformational changes in a confined space. shown to directly etch (drill) the nanopore on silicon nitride (SiNx) membranes This successful demonstration of biomimetic nanopores with local control of through heat effect. While both the localized electric field enhancement and the multiple chemical and geometric properties is an important step towards the heat effects were demonstrated for nanopore localization in previous studies, it integration of nanopore technology into more complex multi-component remains unclear if a non-enhanced and non-etching optical beam could assist systems. the nanopore localization during the controlled breakdown fabrication. To this end, we studied the nanopore formation characteristics under the influence 1446-Pos of various beam intensities and wavelengths. We demonstrated nanopore local- A Robust Mechanism to Render Artificial Nanopores Potassium Ion ization within the submicron range of the optically exposed area. We anticipate Selective the optically assisted nanopore localization could enable broader access to the Elif T. Acar1, Steven Buchsbaum2, Cody Combs3, Francesco Fornasiero2, solid state nanopore-based sensing applications. Zuzanna S. Siwy1. 1Dept Phys/Astron, Univ Calif Irvine, Irvine, CA, USA, 2Lawrence 1444-Pos Livermore Natl Lab, Livermore, CA, USA, 3Univ Calif Irvine, Irvine, CA, Nanofluidic Chips for DNA and Nanoparticles Detection and Manipulation USA. Denise Pezzuoli, Elena Angeli, Diego Repetto, Giuseppe Firpo, Biological channels in a cell membrane have inspired scientists to create bio- Patrizia Guida, Roberto Lo Savio, Luca Repetto, Ugo Valbusa. mimetic nanopores for biosensing, separation, and design of ionic circuits. Physics, Universita` degli studi di Genova, Genova, Italy. One of the key properties of biological channels is the ability to transport Fluidic transport through nanostructures allows to probe fundamental phe- one type of ion, for example potassium ion, but prevent the passage of other nomena at nanoscale and develop tools for DNA and nanoparticles’ (NPs) ions of the same valence, like sodium ions. This ability has been attributed sensing and manipulation. Moreover, it allows to handle small sample quan- to the sub-nanometer dimension of biological channels’ opening combined tities for fast, high-resolution and low-cost analysis [Huh et al Nature Mate- with a pore wall chemistry tuned with atomistic precision. While crucial in rials, 2007]. many physiological processes including nerve signaling, this exquisite selec- Here, we used different planar elastomeric nanofluidic devices, fabricated first tivity is very difficult to reproduce in artificial nanopore systems. We have using Focused Ion Beam (FIB) milling technique and then a Poly(DiMethylSi- developed a strategy to create potassium ion selective channels, which is appli- loxane) (PDMS) based REplica Molding (REM) process [Fanzio et al Lab on cable to solid-state nanopores machined in essentially any material. As proof of chip, 2011], that allows to control the geometry of nanostructures. In particular, concept, we used a model system consisting of a single nanopore formed in a 30 we have optimized this manufacturing process using hard-PDMS (h-PDMS) nm thick silicon nitride membrane by the process of dielectric breakdown. The that, having a higher Young’s modulus, allows to avoid the structural collapse nanopore was subsequently subjected to chemical modification, which resulted of nanostructures, during the sealing process with a coverslip, which typically in a monolayer of 4’-aminobenzo-18-crown-6 ether on the pore walls and on occurs when standard PDMS is used because of surface interactions between one membrane surface. The other membrane surface was decorated with replica and glass. We have used these versatile devices both for counting and ssDNA. Ion selectivity was quantified as the ratio of ionic currents measured sizing NPs and biomolecules, and for studying DNA stretching dynamics. in KCl and NaCl. Nanopores with diameters below 2 nm exhibited currents On one side, combining the optimized fabrication process with Resistive Pulse in KCl that were up to 80 times larger than currents measured in NaCl, even Sensing (RPS) technique [Angeli et al Nano Letters, 2015], we have analyzed at a salt concentration of 1 M. Experimental data and modeling provide evi- translocation processes and the NPs/DNA motion inside the nanochannel as dence that the potassium selectivity occurs via facilitated transport of potas- transient variation in ionic current during translocation events, allowing a sium ions, which undergo binding/unbinding to crown ethers on the pore label-free sensing that is a crucial application for many fields such as medi- walls. Transport of Naþ is limited by the small opening, which hinders any cine, environment and public health. While, on the other side we have studied non-selective passage. Our fabrication strategy of ion selective pores opens DNA stretching dynamics into nanostructures provided of several cavities. We new avenues toward advanced separation processes, biosensing technologies observed how DNA stretches and recoil, due to the balance between entropic and biomimetic nanopore systems.

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1447-Pos demonstrate the formation of a planar lipid bilayer coating on a SiN pore us- Comparing Ubiquitin and Insulin Translocation Dynamics through a ing a photothermal heat shock. We first demonstrate that 100-nm-diameter li- Nanopore in an Electrically Biased Solid-State Membrane posomes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) can Craig C. Wells, Dmitriy V. Melnikov, Maria E. Gracheva. be electroosmotically driven to a SiN aperture by showing their translocation Physics, Clarkson University, Potsdam, NY, USA. through a 300 nm diameter pore. We then use this electroosmotic flow to The ability to separate proteins is desirable for many fields of study and nano- reversibly trap/dock liposomes at the entrance of a 50 nm pore under applied porous membranes may offer a method for rapid protein filtration at high voltage, as evidenced by a reduced ion current through the pore. Once a lipo- throughput volume provided there is an understanding of the protein dynamics some is trapped, we use a laser pulse to rapidly heat the SiN pore, which involved. We use Brownian dynamics simulations to model the motion of causes a thermal shock that ruptures the liposome, forming a planar lipid coarse-grained proteins insulin and ubiquitin in an electrically biased mem- bilayer with low leakage. We demonstrate that the resulting ruptured vesicle brane under zero electrolyte bias. The time each protein takes to find a cylin- is a bilayer by testing its voltage stability, as well as by demonstrating DNA drical nanopore embedded in a thin silicon dioxide membrane (waiting time) translocations through a a-hemolysin loaded liposome that was ruptured on and successfully translocate it in a single attempt (translocation time) are calcu- the SiN aperture. Our lipid coating using SiN photothermal heating aids lated. In our model, each protein is subjected to various biases applied to the high throughput and highly stable lipid bilayers for studies of single- silicon membrane equipped with a nanopore of different radii. We observe in- molecule transport and sensing. sulin finding the nanopore and translocating it faster than the electrically neutral ubiquitin due insulin’s slightly smaller size and net negative charge. 1451-Pos While ubiquitin’s dynamics is also affected by the size of the pore, surprisingly, Highly-Stable Bio-Inspired Peptide/MoS2 Membranes for Efficient Water its translocation process is noticably changed by the membrane bias. By inves- Desalination tigating the protein’s dipole and quadrupole moments, we demonstrate that its Bedanga Sapkota, Laxmi Pandey, Abdelkrim Benabbas, Meni Wanunu. waiting and translocation time dependency on the membrane bias is due to the Physics, Northeastern Univ, Boston, MA, USA. protein’s charge distribution. We report here on bio-inspired integrated membranes that comprise a multilay- ered atomically-thin ‘holey’ molybdenum disulfide (MoS2) laminate structure, 1448-Pos intercalated with a self-assembled layer of charged peptide molecules. We use Brownian Dynamics with Self-Consistent Force Calculations for a Neutral these membranes to filter monovalent and divalent salts, as well as a range of Nanoparticle Translocating through a Nanopore organic dyes (common industrial dyes used for coloring cotton, silk, and wool), Zachery K. Hulings1, Dmitriy V. Melnikov2, Maria E. Gracheva1. 1 2 and observe significant improvement in filter durability (month-scale) and salt Department of Physics, Clarkson University, Potsdam, NY, USA, Clarkson rejection performance, as compared to membranes without peptides. We find a University, Potsdam, NY, USA. high degree of selectivity to the commonly found ions in sea water (for We present a novel computational approach to self-consistently describe a example, >99% rejection of 0.5 M NaCl), a steady performance of membranes nanoparticle moving through a nanopore embedded in a solid-state membrane. for over a month, and a low degree of impact of fouling to proteinaceous fou- We calculate the electrostatic and hydrodynamic forces a particle experiences lant. The performance of our membranes outrivals commercial as well as state- at various locations within a nanopore and incorporate these forces into a of-the-art membrane materials (with hour-scale performance) reported in the Brownian dynamics model at each time step. The value of the ionic current literature. We attribute the high performance of our membranes to the synergis- through the pore is determined based on the particle’s location, and in tic effect between charge, sub-nm porosity, and presence of intersheet peptide conjunction with our Brownian dynamics model, a realistic ionic current trace molecules. Our bio-inspired integrated membrane is low-cost, non-toxic, and is obtained due to the motion of a nanoparticle through a nanopore. We find environmentally friendly. Overall, this work represents the effectiveness and that in addition to the usual geometric blockade, the variations of the current potential of bio-inspired nanoporous transition metal dichalcogenide for along the axis of the pore are largely caused by a concentration polarization membrane-based water separation technology. induced by the presence of the translocating nanoparticle in the nanopore while the current changes in the radial (perpendicular to the axis) direction 1452-Pos occur because of the local buildup of the ionic charge between the particle Array of Freestanding Planar Lipid Bilayers for Parallel Optical and and the nanopore surface. Electrical Recordings 1 1 1 2 1449-Pos Gerhard Baaken , Ekaterina Zaitseva , Soenke Petersen , Taras Sych , 3 4,5 Variations in Electroosmotic Flow Outside Glass Nanopores with Species Kubick Stefan , Jan C. Behrends . 1Ionera Technologies GmbH, Freiburg, Germany, 2Institute for Biology II, of Monovalent Cation 3 Jeffrey Mc Hugh1, Kurt Andresen2, Ulrich F. Keyser1. BIOSS Center, University of Freiburg, Freiburg, Germany, Fraunhofer 1Physics, University of Cambridge, Cambridge, United Kingdom, 2Physics, Institute for Cell Therapy and Immunology (IZI-BB), Potsdam, Germany, 4Department of Physiology, Faculty of Medicine, University of Freiburg, Gettysburg College, Gettysburg, PA, USA. 5 Nanopores are an important technology in molecular sensing systems. Their Freiburg, Germany, Freiburg Centre for Interactive Materials and behaviour is strongly affected by flow characteristics that are not well under- Bioinspired Technologies, University of Freiburg, Freiburg, Germany. stood. Utilising a novel 3 dimensional combination optical tweezers and capil- Here we report on the design and validation of an array platform for parallel lary nanopore measurement system, we have measured flow behaviour outside high-resolution electrophysiological and optical recordings. The recording m conical glass nanopores as a function of species of group 1 cation. We present a chip is based on a SU-8 coated glass slide (ca. 200 m thickness) containing full 3 dimensional flow field about the nanopore. We use this 3 dimensional 4 cavities with individual ring-shaped Ag/AgCl-microelectrodes as well as a measurement system to find the flow force as a function of distance from the common ground electrode. Four suspended lipid bilayers can be self- pore, allowing us to determine the momentum flux for each salt condition. assembled on the chip surface over the cavities. The electrodes are connected We find strong salt dependence on the strength of momentum flux from the to a multichannel amplifier capable of simultaneous electrical recording with < nanopore. Additionally we find previously reported voltage dependent flow high-bandwidth and low noise ( 1pA rms@10kHz). The ring shape of the elec- reversal to be dictated by salt species. For a subset of species we observe trodes creates an optical window allowing access with a high-NA oil or water- EOF in the expected direction as predicted by classical electrokinetic theory, immersion objective on an inverted microscope. This allows for e.g. single- while for others the direction of the flow is reversed. We present a model ex- molecule fluorescence measurements from one of the four bilayers using plaining the observed behaviour. These data and corresponding model are time-correlated-single-photon-counting (TCSPC). key to understanding ion transport, fluid flow and translocation dynamics in Fluorescently labeled alpha-hemolysin for optical and electrical measurements voltage driven nanopore systems. was expressed in-vitro and purified. BODIPY at position 131 was incorporated during the translation in a site-directed manner via stop or four-base codon sup- 1450-Pos pression using pre-charged tRNAs. Photothermally-Assisted Lipid Bilayer Coating on a Sin Nanopore for A series of characterization and validation experiments performed with the sys- High-Throughput Protein Channel Formation tem include: (1)Studies of diffusion rates of fluorescently labelled Hemolysin Hirohito Yamazaki1, Yinghua Qiu1, Xinqi Kang2, Meni Wanunu1. and lipid molecules in the bilayer using fluorescence correlation spectroscopy 1Department of Physics, Northeastern Univ, Boston, MA, USA, 2Department (FCS); (2)Monitoring of alpha-hemolysin membrane insertion using fluores- of Bioengineering, Northeastern Univ, Boston, MA, USA. cence lifetime imaging (FLIM) and (3)Visualization of fusion of labeled pro- In our recent study, we found that irradiation of a visible laser beam on a sil- teoliposomes resulting in membrane channel insertion. In the two latter icon nitride (SiN) membrane results in localized heating, which allows control cases, changes in fluorescence intensity were monitored simultaneously with over the temperature at a nanopore sensor with ms-scale kinetics. Here we alterations in membrane conductance.

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This system allows for rapid membrane array formation and by providing ready (IRI) polymers, inspired by extremophile species which survive freeze-thaw cy- combination of fluorescence microscopy with high-resolution electrophysi- cles, to enable solvent-free cryostorage of bacteria. Our group is developing syn- ology, enables correlated optical and electrical characterization and monitoring thetic polymer mimics of antifreeze (glyco)proteins (AFPs) with the aim of of the interactions of channel forming proteins in artificial lipid bilayers on a reproducing their ice recrystallization inhibition (IRI) activity. Here, we use single-molecule level. The technology can be further implemented for studies poly(vinyl alcohol) as an ice growth inhibiting polymer in combination with of membrane protein folding and mobility as well as of membrane transport. poly(ethylene glycol) as a hydrophilic bulking agent. A key benefit of polymeric formulations is that they are low cost and highly tuneable in terms of structure 1453-Pos and hence function. We have previously shown that addition of these polymers pH Responsive Morphological Change in Polymer Nanostructures to DMSO mediated cell cryopreservation can dramatically improve the post- Ryan L. Hamblin1, Stacy M. Copp2, Gabriel A. Montan˜o3. 1 thaw viability. In this work, we have developed an IRI active polymer formula- Physics and Astronomy, University of New Mexico, Albuquerque, NM, tion to investigate the cryopreservation of bacteria. The unique polymer formu- USA, 2Center for Integrated Nanotechnologies, Los Alamos National 3 lation was observed to provide a synergistic effect for all strains, giving equal or Laboratory, Los Alamos, NM, USA, Northern Arizona University, Flagstaff, better levels of recovery than . The formulation results in a 4-fold in- AZ, USA. crease in Escherichia coli yield post-thaw at 1.1 weight percent compared to Amphiphilic block copolymers (ABCs) can self-assemble into membranes with the customarily used 25 weight percent of glycerol. These results expose the ver- properties that mimic biological membranes. ABC membranes are attractive as satile nature of this polymer formulation, as the method is appropriate for Gram surrogates for lipid membranes in various applications due to their tunable negative, Gram positive and Mycobacteria strains. structural characteristics, such as membrane thickness, stability, and fluidity, corresponding to the adjustable parameters of ABC chain length, molecular 1456-Pos weight, and block chemical type. Self-assembled ABC nanostructures have An Algorithm to Construct Biologically Relevant Chondroitin Sulfate been used to study the dynamics of membranes and membrane proteins, as Biopolymer Models at Atomic Resolution well as applied to the targeted delivery of hydrophilic drugs. Some ABCs Elizabeth K. Whitmore, Gabriel Vesenka, Hanna Sihler, Olgun Guvench. also have potential for the creation of stimuli-responsive materials. Here we Pharmaceutical Sciences, University of New England College of Pharmacy, study micelle structures of one such ABC, polybutadiene-b-polyacrylic acid Portland, ME, USA. (pBDpAA), whose acrylic acid (pAA) block expands with increasing pH. Un- One of the most abundant proteoglycan (PG) biopolymers in the derstanding such expansion on the nanoscale is important for potential applica- body is the linear glycosaminoglycan (GAG) chondroitin sulfate (CS). CS con- tions. To investigate pH-driven pAA expansion, we monitor morphological sists of repeating disaccharide units of b-D-glucuronic acid (GlcA) and b-N- changes of pBDpAA micelles using Fo¨rster resonance energy transfer acetylgalactosamine (GalNAc). A single CS GAG may contain hundreds of (FRET). An amphiphilic donor fluorophore is embedded in the micelle core, disaccharide units. Atomic-resolution simulations of large systems are compu- and acceptor fluorophores float freely in solution. Dynamic light scattering tationally taxing so we aimed to find a more efficient method of generating bio- (DLS) shows that micelle diameter expands monotonically as pH increases, logically relevant CS GAGs atomic resolution. We therefore developed an yet FRET efficiency does not concomitantly monotonically decrease, as would algorithm that applies glycosidic linkage phi/psi dihedral angle probabilities be expected if micelle expansion increases the average donor-acceptor separa- from unbiased all-atom explicit-solvent molecular dynamics (MD) simulations tion. Further experiments suggest that acceptor fluorophores have unexpected of non-sulfated CS 20-mers to rapidly construct CS biopolymers of arbitrary and complex pH-dependent interactions with the pAA corona of the micelles. length. Our algorithm was used to construct non-sulfated CS 20-mer conforma- These results underscore the necessity of in-depth characterization of pAA- tional ensembles and was validated by comparing end-to-end distances in the based nanostructures, which is key to understanding their potential behavior constructed ensemble with the MD ensemble. We noticed a consistent, though in applications or biological contexts. subtle, difference in end-to-end distances in MD-generated versus constructed 20-mer ensembles: the constructed ensemble tended toward slightly higher 1454-Pos end-to-end distances indicating less compact polymer configurations. In an Characterization of Biocompatible Nanoparticles for Biophysical and effort to explain the difference, we analyzed the dihedral angles of each mono- Biomedical Applications saccharide ring in the 20-mer. Although these rings did not undergo any major Vincent J. Altimari, Louis Remy, Stephen C. Hickey, Cara Mawson, conformational changes during simulation, we found that ring flexibility con- Hannah M. Work, Charles D. Hughes, Nathaniel V. Nucci. tributes to end-to-end distance. By incorporating glycosidic linkage and mono- Department of Physics & Astronomy, Rowan University, Glassboro, NJ, saccharide ring dihedral conformational probabilities from the simulated 20- USA. mer ensemble, our algorithm is able to construct biologically relevant CS poly- Reverse micelles are soft nanoparticles created when amphiphilic molecules mer models. surround small volumes of water in a bulk nonpolar solvent. Typically these mixtures spontaneously organize into roughly spherical complexes. RM parti- 1457-Pos cles are quite stable, and can be used to encapsulate small hydrophilic molecules. Lignin-Cellulose Binding Affinity Dependence on Cellulose Face and Under optimal conditions, they can also be used to encapsulate macromolecules Lignin Composition without perturbing their structures. Recently, a mixture composed of decylmo- Josh V. Vermaas1, Gregg T. Beckham2, Michael F. Crowley1. noacyl glycerol (10MAG) and lauryldimethylamine-N-oxide (LDAO) has been 1BioScis Ctr, Natl Renewable Energy Lab, Golden, CO, USA, 2Natl shown to encapsulated a wide array of proteins and nucleic acids. In addition to Bioenergy Ctr, Natl Renewable Energy Lab, Golden, CO, USA. its versatility, this mixture also has the benefit of biocompatibility. The Nucci lab The interaction between lignin and cellulose is essential to the structural integ- is exploring the utility of this mixture for a variety of biomedical and nanotech- rity of plant cell walls, and these strong interactions are key to biomass recal- nology applications. The purpose of the present study is to thoroughly charac- citrance. Previous studies have indicated that the hydrophobic face of cellulose terize the shape and dispersity of 10MAG/LDAO RMs in the presence and is the primary face of interaction between cellulose and lignin. However, due to absence of macromolecular cargo. Dynamic light scattering is a primary tool the structural inhomogeneity of lignin and the small lengthscales that would for this endeavor and is augmented by electron microscopy and nuclear magnetic need to be probed, the relative binding affinities for lignin to different cellulose resonance. This characterization informs further application of this mixture as a faces has not been previously determined. Through the creation of idealized in- biotechnological development platform. finite cellulose faces for the (200), (110), (1-10) and, (010) planes, we deter- mine through computation the relative binding affinities for selected lignin Posters: Biomaterials compounds to these different faces of cellulose. The preference for individual lignin monomers to bind to hydrophobic faces of cellulose is found to be 1455-Pos remarkably weak. We develop a mathematical model to determine the struc- Solvent-Free Cryostorage of Microorganisms using Ice Growth Inhibiting tural features of lignin have the strongest relationship with the computed bind- Polymers ing affinities for large lignin polymers. Muhammad Hasan, Alice Fayter, Matthew I. Gibson. Chemistry, University of Warwick, Coventry, United Kingdom. 1458-Pos In the fields of micro- and molecular biology it is vital to successfully store, bank Flow Imaging Microscopy of a Self-Assembling Protein Polymer Material and transport micro-organisms with maintenance of membrane integrity and Eva Rose M. Balog. viability. Glycerol is currently the gold standard cryoprotectant, however it is Chemistry & Physics, University of New England, Biddeford, ME, USA. intrinsically toxic to many micro-organisms and can be a problem in biotechno- Biological and bioinspired polymer microparticles have broad biomedical and logical applications. Here we introduce the use of ice recrystallization inhibiting industrial applications, including drug delivery, tissue engineering, surface

BPJ 9378_9380 296a Monday, March 4, 2019 modification, environmental remediation, imaging, and sensing. Full realiza- nm diameter carbon nanotube porin (CNTPs) channels in an amphiphilic tion of the potential of biopolymer microparticles will require methods for block-copolymer matrix. We demonstrate that CNTPs maintain high proton rigorous characterization of particle sizes, morphologies, and dynamics, so and water permeability in these membranes. Our experiments also show that researchers may correlate particle characteristics with synthesis methods that CNTPs can mimic the behavior of biological gap junctions by forming and desired functions. Toward this end, we evaluated biopolymer microparti- bridges between vesicular compartments that allow transport of small mole- cles using dynamic imaging particle analysis, also known as flow imaging. cules and Ca2þ ions. This technology is becoming more widely used in the biopharmaceutical indus- try but is not yet well-known among the biomaterials community. Our polymer, 1462-Pos a genetically engineered elastin-like polypeptide (ELP), self-assembles into Location-Location-Location: Designing Cationic Charge Placement on micron- and submicron-scale coacervates. We performed flow imaging of Lipid Vesicles Determines their Interactions with Living Cells ELP coacervates using two different instruments, one with a lower size limit Aprameya Ganesh Prasad, Dominick Salerno, Alaina K. Howe, of approximately 2 microns, the other with a lower size limit of approximately Omkar Mandar Bhatavdekar, Stavroula Sofou. 300 nanometers. We validated flow imaging results by comparison with dy- Chemical and Biomolecular Engineering, Johns Hopkins University, namic light scattering and atomic force microscopy analyses. We explored Baltimore, MD, USA. the effects of various solvent conditions on ELP coacervate size, morphology, Cationic lipid vesicles are extensively used in cell transfection due to their abil- and behavior, such as the dispersion of single particles versus aggregates. We ity to interact with the cell membrane and to deliver intracellularly their ther- found that flow imaging is a superior tool for thorough and statistically power- apeutic cargo. In these interactions the critical role of the cationic charge in ful particle analysis of ELP assemblies in solution. We anticipate that re- enabling close approximation of and lipid rearrangement/exchange between searchers studying many types of microscale protein or polymer assemblies the apposing lipid membranes has been extensively studied. There are instances will be interested in flow imaging as a tool for rapid, quantitative, solution- in drug delivery, however, where only the adhesion of lipid vesicles on cells is based characterization. desired and any form of internalization by cells must be avoided. To design lipid vesicles with these properties, we hypothesize and then demonstrate 1459-Pos that it is the proximity of the cationic charge to the lipid membrane of the Investigations into the Mechanism of Fibril Formation in a Peptide charge-bearing lipid vesicles that (following the initial adhesion) may be crit- Hydrogel ical in affecting the extent of fusion and/or endocytosis of the vesicles by living Gabriel A. Braun1, Sara S. Linse2, Karin Akerfeldt Akerfeldt1. cells. 1Chemistry, Haverford College, Haverford, PA, USA, 2Department of We designed lipid vesicles with grafted PEG-chains and varied the distance of Biochemistry and Structural Biology, Lund University, Lund, Sweden. the cationic charge relative to the plane of the vesicle headgroups. We The mechanism of self-assembly of a hydrogel-forming peptide was investi- compared vesicles with the cationic charge directly on the lipid headgroups gated, using kinetics assays (which relate the rate of aggregation to underlying or on the free PEG-chain ends. Lipid vesicles with the cationic charge directly physical processes) and disc centrifugation (which reports on the length of the on the lipid headgroups interact very differently with the apposing living cell peptide fibrils in the gel). The kinetics assays reveal that aggregation in this membranes from lipid vesicles with the cationic charge on the ends of PEG- system is consistent with a secondary mechanism of fibril formation. These chains and the same zeta potential. Additionally, on the drug delivery end, assays also show that the aggregations kinetics are concentration dependent we show that the primary effect of the cationic charge when on the end of un- only up to 50 mM, suggesting that one or more processes saturate above dulating PEG-chains is to obstruct cell internalization of lipid vesicles and to this concentration. The disc centrifugation data show an inverse relationship delay their clearance from solid tumors in vivo. between monomer concentration and fibril length, which could result from Overall, the location of electrostatic charges on lipid vesicles can be used as a the combination of a saturating fibril growth mechanism and a non- tool to precisely tune the interactions of lipid vesicles with living cells with im- saturating fibril formation mechanism. Future directions for this project plications in drug delivery and therapy. include seeded kinetics assays (in which pre-formed fibrils are used to accel- erate aggregation) and disc centrifugation studies which focus on fibril length 1463-Pos as a function of time, both of which would help determine between possible Effects of Aging on the Viscoelastic Properties of Tissues and Cancer Cell aggregation mechanisms. Behavior Seungman Park1, Jiaxiang Tao2, Li Sun1, Chen-Ming Fan2, Yun Chen1. 1 1460-Pos Mechanical Engineering, Johns Hopkins University, Baltimore, MD, USA, 2 Self-Healing DNA-Based Reaction-Diffusion Patterns Department of Embryology, Carnegie Institution for Science, Baltimore, Phillip J. Dorsey, Rebecca Schulman. MD, USA. Chemical and Biomolecular Engineering, Johns Hopkins University, Aging is accompanied by the declined biological functions of living organ- Baltimore, MD, USA. isms. Aging is a known risk factor for various diseases, including cancer. We have developed a system for fabricating and perturbing self-healing oligo- Though a vast body of studies have been reported regarding how aging- nucleotide-based patterns in photolithographically-patterned hydrogels using associated mutations lead to higher incidence of cancer development and electromagnetic radiation. Digital maskless photolithography combined with more aggressive tumor progression in elder patients, few studies, if any, microfluidic handling provides the ability to construct soft materials of arbi- address how changing characteristics of the microenvironment in aging tissues trary shape at biologically relevant length scales of tens to hundreds of microns might affect the course of cancer pathology. In addition, the systematic and in a matter of seconds. We use this approach to first form self-healing linear and quantitative comparison between young and aging tissues in humans or hill-shaped DNA-based gradients in poly(ethylene-glycol) diacrylate hydro- commonly used animal models is yet to be established. In this study, I gels. Undisturbed, these spatial patterns were stable over at least 24 hours. hypothesized that aging epidermis, exhibiting distinct mechanical characteris- When patterns were perturbed in particular areas they reformed. The ability tics, promotes the progression of skin cancer in elder patients via p53- to grow and perturb gradients of biomolecules such as DNA may facilitate mediated pathways. First, to prove my hypothesis, I developed a versatile the study of cell populations in dynamic microenvironments, providing exper- and economic device, coined indentation-based mechanical analyzer (IMA), imentalists control over where and when chemical cues are induced or inhibited to perform in situ and in vivo measurement of the viscoelastic properties at the microscale. of the human epidermis of different ages. We observed that both elastic moduli and viscosities of skin tissues increased with age using the old 1461-Pos mouse and young mouse. Second, to systematically investigate how the Carbon Nanotube Porins in Block Copolymers as Fully Synthetic Mimics altered viscoelasticity promotes tumor progression, I created an in vitro model of Biological Membranes system in mimicry of young and aging tissues. The in vitro model system Aleksandr Noy. will be used to evaluate the rate of tumor progression, drug responses, Physics and Life Sciences, Lawrence Livermore Natl Lab, Livermore, CA, p53 activity and protein degradation in skin cancer cells growing in the USA. varied viscoelastic environment. In summary, this study will advance the Biological membranes provide a fascinating example of a separation system understanding of the complex relationship between aging and cancer, from that is multifunctional, tunable, precise, and efficient. Biomimetic membranes, the perspective of altered tissue mechanics and its consequences manifested which mimic the architecture of cellular membranes, have the potential to in mechanosignaling-dependent regulations. Such understanding might deliver significant improvements in specificity and permeability. We report inspire a distinct and more effective treatment strategy for elder cancer a fully synthetic biomimetic membrane that incorporates ultra-efficient 1.5 patients.

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