12a Sunday, March 3, 2019

Symposium: Biological Systems Single Molecule encode hundreds of ribosomal DNA operons (rDNA) that exhibit extensive sequence variation with the rRNA components of the ribosome, which are at the Time both conserved and expressed in a tissue-specific fashion. Using E.coli as a genetically tractable model system, we now show that nutrient limitation- 65-Symp induced stress changes the relative expression of rDNA operons to alter the ri- The Mechanism of Dynein Directionality bosomal RNA (rRNA) composition within the actively translating ribosome Ahmet Yildiz. pool. The most upregulated operon encodes the unique 16S rRNA gene, Physics, Univ. California Berkeley, Berkeley, CA, USA. rrsH, distinguished by conserved sequence variation within the small The ability of cytoskeletal motors to move unidirectionally along filamentous ribosomal subunit. rrsH-bearing ribosomes alter the levels of the RpoS sigma tracks is central to their role in cargo transport, motility and cell division. factor, the master regulator of the general stress response, to affect the expres- While kinesin and myosin motor families have members that move in oppo- sion of functionally coherent gene sets. These impacts are associated with site directions, all dyneins studied to date exclusively move towards the phenotypic changes in antibiotic sensitivity, biofilm formation, and cell microtubule (MT) minus-end. In order to understand the mechanism of motility, and are regulated by ribosome-associated stress response proteins. dynein’s directionality, we sought to engineer a plus-end-directed dynein Specific aspects of these phenotypic differences could be reconstituted in vitro guided by cryo-electron microscopy and molecular dynamics simulations. using highly purified translation components. These findings establish that As shown by single-molecule assays, elongation or shortening of the endogenously encoded, naturally occurring rRNA sequence variation can coiled-coil stalk that connects the motor to the MT controls helical direction- modulate ribosome function, central aspects of gene expression regulation, ality of S. cerevisiae dynein around MTs. By changing the length and angle of and cellular physiology. the stalk, we successfully reversed dynein motility towards the MT plus-end. These modifications act by altering the direction dynein’s linker swings 68-Symp relative to the MT, not by reversing the asymmetric unbinding of the motor Probing the Dynamics and Interactions of Disordered Proteins with Single- from MT. Because the length and angle of dynein’s stalk are fully conserved Molecule Spectroscopy among species, our findings provide an explanation for why all dyneins move Ben Schuler. towards the MT minus-end. University of Zurich, Zurich, Switzerland. The functions of proteins have traditionally been linked to their well-defined 66-Symp three-dimensional, folded structures. It is becoming increasingly clear, how- In Situ Imaging of Transcriptome and Genome in Single Cells ever, that many proteins perform essential functions without being folded. Xiaowei Zhang. Single-molecule spectroscopy provides new opportunities for investigating Chemistry and Chemical Biology, Howard Hughes Medical Institute, the structure and dynamics of such unfolded or ‘intrinsically disordered’ pro- Harvard University, Cambridge, MA, USA. teins (IDPs). The combination of single-molecule Fo¨rster resonance energy In situ transcriptomic analysis of single cells promise to transform our under- transfer (FRET) with nanosecond correlation spectroscopy, microfluidic mix- standing in many areas of biology, such as regulation of gene expression, devel- ing, and other advanced methods can be used to probe intra- and intermolecular opment of cell fate, and organization of distinct cell types in complex tissues. distance distributions, reconfiguration dynamics, and interactions on a wide We developed a single-cell transcriptome imaging method, multiplexed error- range of timescales, and even in heterogeneous environments, including live robust fluorescent in situ hybridization (MERFISH), which uses combinatorial cells. labeling and sequential imaging to massively multiplex single-molecule FISH I will illustrate the use of single-molecule FRET in combination with other bio- measurements and error-robust encoding schemes to minimize measurement physical methods in the context of an unexpected interaction mechanism that error, enabling RNA imaging and profiling at the transcriptomic scale. By we recently discovered: The two intrinsically disordered human proteins his- allowing single-cell transcriptomic analysis in the native context of cells and tone H1 and its nuclear chaperone prothymosin a associate in a one-to-one tissues, MERFISH facilitates the delineation of gene regulatory networks, the complex with picomolar affinity, but they fully retain their structural disorder, mapping of RNA distributions inside cells, and the mapping of distinct cell long-range flexibility, and highly dynamic character. Based on the close inte- types in complex tissues. In this presentation, I will talk about our technology gration of single-molecule experiments, NMR, and molecular simulations, development and recently applications of MERFISH, with a focus on mapping we obtained a detailed picture of this complex that can be explained by the the organization of distinct cell types in the brain. large opposite net charge of the two proteins without requiring defined binding I will also talk about a multiplexed FISH method that we developed for imaging sites or interactions between specific individual residues. This type of interac- the 3D conformation of chromosomes in single cells. The spatial organization tion has interesting ramifications for kinetic mechanisms of binding and of genome plays an important role in many essential genome functions from cellular regulation. gene regulation to genome replication. However, many gaps remain in our un- derstanding of the 3D organization of chromatin in the nucleus, partly because of the lack of proper imaging tools. Our multiplex FISH method allows Symposium: Proton-coupling Bioenergetics numerous genomic loci to be imaged and localized. Using this approach, we traced the 3D conformation of chromatin in cell nucleus with high resolution, 69-Symp C and revealed novel spatial organization of chromatin domains and compart- The Proton/Electron Coupling Mechanism of Cytochrome Oxidase 1 2 ments in individual chromosomes. Peter R. Rich , Vivek Sharma . 1Structural and Molecular Biology, University College London, London, 67-Symp United Kingdom, 2Physics, University of Helsinki, Helsinki, Finland. Endogenously Encoded Ribosomal RNA Sequence Variation within the Mitochondrial cytochrome c oxidases (CcOs) are members of a large haem- Assemble Ribosome can Regulate Stress Response Gene Expression and copper oxidase superfamily. They fall within the A1 branch which includes Phenotype closely structurally-related bacterial CcOs and quinol oxidases. Three possible Scott C. Blanchard1, Chad M. Kurylo1, Matt M. Parks1, Manuel F. Juette1, networks of amino acids and waters have been identified in subunit I for intra- Boris Zinshteyn1, Roger B. Altman1, Theresa C. Vincent1, protein transfer of substrate and translocated protons. The D and K channels are Michael R. Wasserman2, Jose L. Alejo Amaya3, Daniel S. Terry1. very similar in all structures. The H channel was firstly described in bovine 1Physiology and Biophysics, Weill Cornell Medicine, New York, NY, USA, CcO, though several of its features are evident bacterial A1-type CcOs. In bac- 2Rockefeller Univ, New York, NY, USA, 3Weill Cornell Med Coll, New terial systems, there is substantial evidence that the D channel provides the York, NY, USA. route for pumped protons to a temporary proton trap site, though the exit route Prevailing dogma holds that ribosomes are uniform in composition and func- into the P phase remains uncertain. In contrast, in mammalian mitochondrial tion. Single-molecule investigations of translation reveal, however, that the CcO structural and functional data have suggested that the H channel may ribosome and translation factors visit metastable, transient intermediates that instead provide the route for pumped protons to a similar trap site location, can be highly sensitive to even modest perturbations, including small- and that its ‘top’ half provides the exit route from the trap into the P phase. molecule drugs and single-nucleotide substitutions in ribosomal RNA Direct measurements of coupling efficiencies of yeast mitochondrial CcO (rRNA). These insights suggest the possibility that subtle changes in the ribo- with mutations in these potential proton pathways showed that pumped protons some’s composition may have functional impacts. Recent evidence further im- are transferred through its D channel. Furthermore, MD simulations indicate plicates changes in the ribosome’s core protein composition with gene-specific that the ‘lower’ region of its H channel could not provide a transfer route changes in translational efficiency. In this light, we set out to examine the po- into the trap site. This is consistent with prior MD simulations of this region tential physiological impacts of endogenously encoded sequence variations in of the bovine CcO H channel which supported a dielectric well, but not a proton the rRNA components of the assembled ribosome. Mammalian genomes transfer, role. However, the ‘top’ part of the H channel may indeed form a facile

BPJ 9279_9291 Sunday, March 3, 2019 13a route for proton exit from the trap, possibly gated around the region of two ar- variation; responding to drug resistant somatic variation in targeted cancer ginines that are close to the haem propionates. Hence, some degree of conver- therapies and phenotypic differentiation of disease mechanisms. In cancer, gence of thought on the mechanism of the bacterial, yeast and bovine CcOs evolutionary mechanisms drive the emergence of drug resistant variation may be proposed. which may ultimately render a targeted therapy ineffective. Simulations of high affinity programmed death-ligand 1 (PD-L1) inhibitor interactions 70-Symp with variant specific protein structural templates predict certain drugs will How the C-Subunit Stoichiometry of F1F0 ATP Synthase Controls Bio- bind to certain variants with increased affinity. This insight can inform selec- Energetic Thermodynamic Efficiency tion of a second line therapy from a variety of approved interventions. Addi- Todd P. Silverstein. tionally, results of protein structure simulation of arrhythmia associated Chemistry, Willamette University, Salem, OR, USA. variants in calmodulin (CAM) reveal the potential to predict divergent disease The F0 portion of the bovine mitochondrial F1F0-ATP synthase was shown in mechanisms. Machine learning based on structural and energetic features of n 2010 to contain eight ‘c’ subunits ( = 8). This surprised many in the field, MD trajectories not only separate variants based on divergent phenotypic as previously, the only other mitochondrial F0 (for yeast) was shown to have effects, but also correlate with the severity of functional disruption. Overall, ten ‘c’ subunits. The metabolic implications of ‘c’ subunit copy number lead the creative exploitation of protein structure simulations has the potential to E. coli n to several surprising conclusions: (1) Aerobically respiring ( = 10) revolutionize clinical decision making and improve the successful administra- n and animal mitochondria ( = 8) both have very high F1F0 maximum thermo- tion of personalized medicine. dynamic efficiencies of z 90% under typical conditions, whereas maximum z n efficiency is only 65% for chloroplasts ( = 14). Reasons for this difference, 73-Plat 6c including the importance of transmembrane potential ( ) as a rotational cata- Mechanism of Passenger Cleavage in Autotransporter EspP Explored with lyst, as opposed to an energy source, are discussed. (2) Maximum theoretical QM/MM Molecular Dynamics Simulation n P/O ratios in animal mitochondria ( = 8) are calculated to be 2.73 ATP/ Anna Pavlova, James C. Gumpart. NADH and 1.64 ATP/FADH2, yielding 34.5 ATP/glucose (assuming NADH Dept Physics, Georgia Tech, Atlanta, GA, USA. import via the malate/aspartate shuttle). The experimentally measured values EspP is an extracellular serine protease and one of the major virulence factors 5 5 5 of 2.44 ( 0.15), 1.47 ( 0.13), and 31.3 ( 1.5), respectively, are only about expressed by E. coli. As in all autotransporters, it contains a C-terminal b-barrel 10% lower, suggesting very little energy depletion via transmembrane proton domain embedded in the outer membrane and an N-terminal extracellular leakage. (3) Finally, the maximum thermodynamic efficiency of oxidative passenger domain. EspP belongs to the family of serine protease autotransport- not phosphorylation is lower than that of substrate level phosphorylation, as ers of Enterobacteriaceae (SPATE), whose passengers can cleave various previously believed. The overall maximum thermodynamic efficiencies of mammalian host proteins. The sequence of SPATE proteins is highly z oxidative phosphorylation, glycolysis, and the citric acid cycle are 80% in conserved, particularly around the passenger cleavage site. It has been shown all three processes. that cleavage occurs through asparagine cyclization and that several amino 71-Symp acids in the vicinity of the reacting asparagine are essential for the reaction. Adaptive Immunity Shaped by Large Multiprotein Membrane Complexes Surprisingly, presence of the protein-cleaving serine protease domain or other Robert Tampe. serines and histidines, commonly employed in enzymatic protein cleavage re- Dept. of Biochemistry, Goethe University Frankfurt, Frankfurt, Germany. actions was not required for cleavage to occur. Therefore, mechanisms Identifying and eliminating infected or malignantly transformed cells are involving several aspartate residues and a water molecule have been proposed, fundamental tasks of our adaptive immune system. For immune surveillance, although the plausibility of these mechanisms have not been previously the cell’s metastable proteome is displayed as broken bits (peptides) on major explored by computational methods. Here, we employ state of the art hybrid histocompatibility complex class I (MHC I) molecules to cytotoxic T-lympho- quantum mechanical/molecular mechanical (QM/MM) molecular dynamics cytes. Our knowledge about the track from the cellular proteome to the presen- simulations and free energy methods to investigate several paths for asparagine tation of peptides has greatly expanded, leading to a quite comprehensive cyclization and the consequent cleavage of the passenger domain in EspP. It is understanding of the antigen processing pathway. The seminar will report on shown that the reaction involves several consecutive proton transfers and that the mechanism of antigen translocation, chaperoning, editing, and ER quality presence of a water molecule and multiple amino acids at the catalytic site control. Following on an integrative approach, the contribution of individual greatly reduces the reaction barrier. Our results can be generalized for other proteins as well as the architecture of the MHC I peptide-loading complex SPATE proteins and facilitate development of SPATE inhibitors in order to (PLC) and other MHC I chaperone complexes, also in the context of viral im- combat bacterial infections. mune evasion, will be addressed. The work provides the framework for under- standing the quality control of antigen selection and unveils the molecular 74-Plat details underlying the onset of an adaptive immune response. Uncovering the Molecular Basis for the Clinical N642H Mutation in STAT5B Using Atomistic Molecular Simulations Deniz Meneksedag-Erol1,2, Elvin D. de Araujo1,3, Fettah Erdogan1,3, Platform: Molecular Dynamics I Hyuk-Soo Seo4,5, Sirano Dhe-Paganon4,5, Patrick T. Gunning1,3, Sarah Rauscher1,2. 72-Plat 1Department of Chemical and Physical Sciences, University of Toronto Translational Applications of Protein Structure Simulation: Predicting Mississauga, Mississauga, ON, Canada, 2Department of Physics, University Phenotype of Missense Variants of Toronto, Toronto, ON, Canada, 3Department of Chemistry, University of Matthew D. McCoy1, Subha Madhavan1, Sridhar Nimmagadda2, Toronto, Toronto, ON, Canada, 4Department of Cancer Biology, Department Dmitri Klimov3, Mohsin S. Jafri4. of Biological Chemistry & Molecular Pharmacology, Harvard Medical 1Oncology, Innovation Center for Biomedical Informatics, Georgetown School, Boston, MA, USA, 5Dana-Farber Cancer Institute, Harvard Medical University Medical Center, Washington, DC, USA, 2Radiology and School, Boston, MA, USA. Radiological Science, Johns Hopkins University, Baltimore, MD, USA, The cytosolic protein STAT5 (signal transducer and activator of transcrip- 3Dept Bioinform/Comput, George Mason Univ, Manassas, VA, USA, 4Sch tion 5) is involved in cell proliferation and differentiation. Hyperactivation Syst Biol, George Mason Univ, Fairfax, VA, USA. of STAT5 occurs in leukemia and lymphoma; a clinical mutation within Simulating the atomic scale biophysical properties of protein structures pro- STAT5 has been linked to increased drug resistance. Despite the importance vides insight into functional interactions and dynamics of complex molecular of STAT5 as a therapeutic target, drug design efforts have been limited due systems. For example, flexible docking algorithms predict molecular confor- to lack of structural and dynamic information. Here, we use molecular dy- mations which optimize energetic interactions and molecular dynamics (MD) namics simulations to elucidate the dynamics of the wild type and oncogenic simulations can survey the global conformational landscape of protein N642H mutant of human STAT5B dimers, and to provide a molecular basis motion using experimentally parameterize force fields. Defining variant spe- for the increased oncogenicity of the STAT5BN642H. We carried out three cific changes to simulated structural and energetic features allows for the independent, atomistic simulations for the STAT5B and STAT5BN642H variant divergence from the wildtype system to be quantified and creative dimers, and the STAT5B monomer. The wild type STAT5B dimer exhibited analysis has enormous translational potential. In particular, understanding marked instability and dissociated rapidly in all the tested systems. In the specific contribution of changes to the protein sequence can inform ther- contrast, STAT5BN642H dimer remained intact during 1 ms-long simulations. apeutic interventions in personalized medicine. Here we demonstrate two ap- The STAT5BN642H dimer was observed to be highly flexible, populating plications where results from simulations of protein structure can be used to different dimer interfaces. Additional inter-chain contacts formed in the address the challenge of interpreting the functional significance of sequence STAT5BN642H compared to the wild type may account for the enhanced

BPJ 9279_9291 14a Sunday, March 3, 2019 dimer stability of STAT5BN642H. Notably, these contacts are not close to the 77-Plat N642H mutation site, suggesting a possible allosteric communication Molecular Mechanism of Potent Capsid-Targeting Antiretroviral Drugs pathway between the interface domains and the SH2 domain bearing the Sruthi Murlidaran1, Juan R. Perilla2. N642H mutation. Ongoing structural and dynamic analysis will reveal the 1University of Delaware, Newark, DE, USA, 2Beckman Inst, Univ Delaware, intra- and inter-chain contacts that provide enhanced stability to the onco- Newark, DE, USA. genic STAT5BN642H dimer. Human immunodeficiency virus type 1 (HIV-1) adapts quickly to antiviral drugs, requiring new strategies for thwarting the virus be continuously sought. 75-Plat At the core of HIV-1 lies a key determinant of infection, the capsid, a special- Modeling Vibrational Stark Effects using Polarizable Force Fields: KSI as ized protein shell that encases the genetic material of viral pathogens. The an Exemplar capsid is made up of 1,500 capsid proteins(CA) that organize themselves Jonathan W. Essex1, Richard T. Bradshaw1, Stephen D. Fried2. into hexamers and pentamers to form a conical shell. Each CA protein sub- 1School of Chemistry, University of Southampton, Southampton, United units consists of two domains (NTD and CTD) connected by a flexible linker. Kingdom, 2Department of Chemistry, Johns Hopkins University, Baltimore, In the past decade, a number of highly potent CA-targeting HIV inhibitors MD, USA. have been identified like GS-CA1 and BI compounds. They are known to Vibrational Stark effect spectroscopy can provide a direct link between the bind to a broadly conserved site, lying at the NTD-CTD intersubunit interface frequency of a vibrational probe and the external electric field it experi- within CA-hexamers and disrupt the capsid assembly and disassembly and ences. It has become increasingly widely used to investigate electrostatics therefore prevent the delivery of the viral genetic material. These inhibitors in buried biomolecular environments and to shed light on the nature of are also known to compete with capsid-binding host proteins that determine intermolecular interactions, protein structure, and ultimately function. A both the efficiency of nuclear entry, thereby interfering with infection. There- notable example is the case of ketosteroid isomerase (KSI), where very large fore the exact molecular mechanism by which these drugs inhibit capsid func- electric fields have been reported in the binding site (143 MV cm1)with tion is unknown. Here, we have derived a set of atomistic force field implications for the catalytic mechanism. However, the combination of parameters to establish the dynamics of the inhibitor bound CA-hexamers. computer simulation with these experimental measurements, to rationalise By employing a divide and conquer strategy, quantum mechanical calcula- effects on enzyme structure and function at an atomistic level, is still tions were carried out on the drug fragments to derive molecular mechanics uncommon. parameters. To analyze the effect of the inhibitor binding to the HIV-1 capsid, In this presentation, we report the analysis of molecular dynamics simulations collective motions of inhibitor-bound CA-hexamers were derived from all- of KSI, performed using both conventional fixed-charge force fields, and the atom molecular dynamics simulations. Importantly, in comparison with the most theoretically sophisticated AMOEBA polarizable force field. We find a wild-type, the collective motions reveal altered dynamics and translocation number of binding geometries for the ligand, only one of which (with two pro- rates of charged ions in the inhibitor bound CA-hexamers. Altogether, our re- tein hydrogen bonds to the steroid carbonyl group) has the very high electric sults reveal changes in the physical properties of CA caused by the binding of fields reported by experiment, and only when analyzed using AMOEBA. the inhibitors. Fixed-charge force fields are unable to quantitatively replicate the experimen- tally observed electric fields. To address the issue of whether simulation and 78-Plat experiment are consistent, we calculate the vibrational spectral lineshapes Molecular Dynamics Simulations of an Entire HIV Virion directly from the MD. We find that the simulations are generally consistent Tyler Reddy1, Juan R. Perilla2. with the experimentally observed vibrational spectra, and predict how each 1Chemistry and Biochemistry, Los A´ lamos National Laboratory, ligand binding mode affects lineshape position and broadening. The theoret- Albuquerque, NM, USA, 2Chemistry and Biochemistry, University of ical observation of multiple short-lived ligand binding modes is consistent Delaware, Newark, DE, USA. with the experimental vibrational Stark effect data. This combination of simu- The HIV viral particle contains all the necessary components to infect a hu- lation and experiment potentially allows for simulation models to be tested man cell. The so-called virion is made of glycoproteins, lipids, the Gag and validated, and for structural interpretations of the experimental data to polyprotein, viral RNA and other essential proteins. After budding, be derived. several biological process occur inside the virion, including a major re- arrangement of the virion’s cargo commonly referred as maturation. Here, 76-Plat we present the steps performed towards the construction of an atomistic Conformational Flexibility of the HIV VIF Protein Complex model of a mature and immature virion. Our model includes all major com- 1 1 2 1 K. Aurelia Ball , Lieza M. Chan , David Stanley , Elise Tierney , ponents, including glycosylated proteins, twenty-four lipid types and capsid 1 2 2 2 Sampriti Thapa , Hai M. Ta , Lily Burton , Jennifer M. Binning , protein. Our effort constitutes one of the major efforts to construct a realistic 3 3 Matthew P. Jacobson , John D. Gross . HIV virion at atomic resolution. In addition, we discuss the techniques 1Chemistry Dept, Skidmore College, Saratoga Springs, NY, USA, 2Univ 3 developed to prepare the system and the steps required to simulate and Calif San Francisco, San Francisco, CA, USA, Dept Pharm Che, Univ Calif analyze our atomistic HIV virion model, which contains over 800 million San Francisco, San Francisco, CA, USA. atoms. HIV-1 viral infectivity factor (Vif) is an intrinsically disordered protein respon- sible for the ubiquitination of the APOBEC3 family antiviral proteins. Vif directly binds the Cullin-RING E3 ligase (CRL), consisting of Elongin-B Platform: Voltage-gated K Channels (EloB), Elongin-C (EloC), CBF-b, and Cullin5 (CUL5), creating the Vif- CBFb-EloB-EloC-CUL5 (VCBC-CUL5) complex, which has a published crys- 79-Plat tal structure. Vif also recruits an APOBEC3 by directly binding to it, but the Atomic-Level Characterization of C-Type Inactivation for Voltage-Gated structure of the VCBC-APOBEC3 complex has not yet been solved. We Potassium Channels Shaker and hERG have used Molecular Dynamics (MD) simulations to characterize the dynamics Jing Li, Young Hoon Koh, Ahmed Rohaim, Eduardo Perozo, of the VCBC complex with and without CUL5 bound. We find that the VCBC Benoit Roux. complex undergoes global dynamics and samples alternate conformational Dept Biochem & Molec Biol, Univ Chicago, Chicago, IL, USA. states from the crystal structure, while VCBC-CUL5 is more static and main- C-type inactivation is a time-dependent process observed in many Kþ tains a conformation similar to the crystal structure. This observation from channels whereby prolonged activation by an external stimulus leads to a MD is supported by methyl-transverse relaxation optimized spectroscopy reduction in ionic conduction. While C-type inactivation has been suggested (TROSY) NMR data, which indicates that the VCBC complex without as a constriction of the selectivity filter in pH-activated bacterial potassium CUL5 is dynamic, while the VCBC complex bound to APOBEC3F (A3F) is channel KcsA, the structural basis of the C-type inactivation in voltage-gated more conformationally restricted. Although there is no structure of VCBC- potassium channels remains a long-standing question. Our recent computa- A3F from which to initiate simulations, we find that MD simulations initiated tional study on KcsA revealed that the full opening of the intracellular from a modeled VCBC-A3F structure are less dynamic than the VCBC simu- gate could trigger a spontaneous and rapid conductive-to-constricted transi- lations. Key to the global dynamics observed, Vif contains a flexible linker re- tion of the selectivity filter through an allosteric coupling. Following this gion located at the hinge of the VCBC complex, which is involved in finding, we captured the first stable constricted (inactive) structure in the conformational changes of the complex. The conformational changes of the voltage-gated potassium channel Shaker by inducing further opening of VCBC complex and quenching of dynamics resulting from binding additional the intracellular gate in molecular dynamics simulations. The computational partner proteins may have implications for HIV-1 Vif function and therapeutic model displays several unique features different from the known constricted targeting. X-ray structures of the KcsA channel, which is also confirmed by a new

BPJ 9279_9291 Sunday, March 3, 2019 15a crystallographic data for a shaker-like KcsA mutant from our lab. The 82-Plat conductive-to-constricted transition of the selectivity filter in simulation Modulation of KV10.1 Potassium Channel Function by Intracellular Heme also reveals the underlying molecular mechanism of several key hydrogen Nirakar Sahoo1,2, Ina Coburger2, Kefan Yang2, Sandip M Swain2, bonds working as timer for C-type inactivation. Meanwhile, an asymmetrical Guido Gessner2, Reinhard Kappl3, Diana Imhof4, Toshinori Hoshi5, constricted-like conformation is identified in our extended MD simulations Roland Schoenherr2, Stefan H. Heinemann2. for another voltage-gated potassium channel hERG. The constricted 1Department of Biological Science/Department of Chemistry, University of hERG filter involves a considerable reorientation of phenylalanine at Texas Rio Grande Valley, Edinburg, TX, USA, 2Center of Molecular position 627 along the selectivity filter to dock into the binding pocket occu- Biomedicine, Department of Biophysics, Friedrich Schiller University Jena pied by the inactivating water molecules in the constricted KcsA structure. & Jena University Hospital, Jena, Germany, 3Institute of Biophysics, All of these results support the notion that constriction of the filter alloste- Saarland University, Homburg, Germany, 4Pharmaceutical Chemistry I, rically controlled by intracellular gate is a general molecular basis of Institute of Pharmacy, University of Bonn, Bonn, Germany, 5Dept of C-type inactivation in a variety of potassium channels, but with alternative Physiology, University of Pennsylvania, Philadelphia, PA, USA. structural factors stabilizing the constricted conformation. Heme, an iron-protoporphyrin-IX complex, is a cofactor bound to various he- moproteins and promotes a broad range of functions, such as electron transfer, 80-Plat oxygen transport, signal transduction, and drug metabolism. In recent years, there has been growing recognition of heme as a non-genomic modulator of C-Type Inactivation in KV2.1 Channels Carlos A. Villalba-Galea1, Takeharu Kawano2, Diomedes E. Logothetis2. ion channel functions. Here, we show that intracellular free heme/hemin mod- 1Physiology and Pharmacology, Univ the Pacific, Stockton, CA, USA, ulates human ether a` go-go (hEAG1, Kv10.1) voltage-gated potassium chan- 2Pharmaceutical Sciences, Northeastern University, Boston, MA, USA. nels. We used an integrative approach that combines electrophysiology and biochemical techniques to dissect the impact of heme on human Kv10.1 The voltage-gated channel KV2.1 displays voltage-dependent inactivation that occurs prior to pore opening. K 2.1 channels also display a partial recovery channels. Electrophysiology experiments were conducted in the inside-out V patch-clamp configuration on Xenopus oocytes. Intracellular application of he- from inactivation when applying high positive potentials. This phenomenon 3þ has been referred to as ‘‘U-shaped’’ inactivation. The molecular mechanism min (Fe -protoporphyrin-IX) potently inhibited Kv10.1 channels with an apparent IC50 of 4.250.3 nM (oxidizing condition) and 63.354.5 nM for this process remains unclear. Here we hypothesize that ‘‘U-shaped’’ inac- þ tivation is simply a combination of C-type inactivation occurring at negative (reducing condition). Hemin did not affect other delayed rectifier K channels potentials and channel activation happening at more positive ones. In testing such as Kv1.1, Kv1.5, and Kv1.6. The onset of the hemin inhibitory effect on the current followed an exponential time course and was very slowly this hypothesis, we reasoned that, if C-type inactivation occurs through a 2þ similar mechanism than in other potassium channels, then conserved residues reversible upon washout. Protoporphyrin-IX and Fe were ineffective on Kv10.1 channel currents. Hemin acts as a gating modifier by reducing the involved in C-type inactivation may play similar roles in KV2.1 inactivation. Following this rationale, we identified two key residues in the S6 transmem- probability of channel opening in a voltage-dependent manner. Studies on re- brane segment of rat Kv2.1. One of these residues is I405. This is homologous combinant channel fragments encompassing the C terminus of Kv10.1 (M478- to residues I470 in Shaker and F103 in KcsA. Like in these latter channels, N673) revealed a heme-responsive binding site in the C-linker region I405 is critically involved in inactivation. Mutating this residue to a Phenyl- involving histidine 543 and 552. Heme binding to the C-linker possibly in- alanine (I405F) renders a channel that displays faster inactivation. Since the duces a conformational constraint that interferes with channel opening. Over- mutation F103A abolished inactivation in KcsA, we then tested the mutation all, this study suggested that free heme is a physiologically relevant modulator of Kv10.1 channels activity and neuronal excitability. I405A, finding that it abrogated inactivation in KV2.1. These observations suggested that C-type inactivation may underlie U-shaped inactivation. The second residue was I401. This neighboring residue was also critically 83-Plat involved in inactivation. We showed that the I401F mutation seems to favor Structural Basis for Lipid-Dependent Gating of a Voltage-Gated Potassium the transition into closed-inactivated states, decreasing the rate of channel Channel opening as a function of the holding potential. The range of holding potentials Gaya P. Yadav1, Mahesh Chandak1, Liang Shi2, Hui Zheng3, that changed the activation kinetics was around 50 mV more negative than the Qiu-Xing Jiang1. half-maximum potential for activation. This indicates that inactivation occurs 1Dept Microbiology and Cell Science, University of Florida, Gainesville, FL, well before activation takes place, with a voltage-dependence like that of the USA, 2Dept of Cell Biology, UT Southwestern Medical Center, Dallas, TX, wild-type. Our findings are consistent with our initial hypothesis, strongly USA, 3Dept Neuroscience, UT Southwestern Medical Center, Dallas, TX, suggesting that both U-shaped inactivation and C-type inactivation share a USA. common mechanism. Protein-lipid interactions are important for a large array of eukaryotic mem- brane proteins. Studies from our lab and others have shown that nonphospho- 81-Plat lipids can change the energetics underlying the conformation of the voltage Structural Basis for Electromechanical Coupling in a Hyperpolarization- sensing domains (VSDs) of voltage-gated ion channels. Lipid-dependent Activated Ion Channel gating of the voltage-gated ion channels was proposed to account for a Michael D. Clark, Gustavo Contreras, Rong Shen, Eduardo Perozo. more general phenomenon that phospholipids favor the active conformation Dept Biochem & Mol Biophys, Univ Chicago, Chicago, IL, USA. of a voltage sensor domain whereas nonphospholipids favor it in a resting Hyperpolarization-activated ion channels serve as unique model systems for state. It suggests that a voltage-gated ion channel and the annular lipids studies of electromechanical coupling due to their reversed gating polarity: around it form a functional unit and both specific and non-specific interactions they activate upon membrane hyperpolarization, the opposite behavior as contribute to the energetic differences between different gating states of the the classic depolarization-activated Shaker channel. We present the cryoEM VSDs and in turn the channel. Recent findings from primary neurons showed structure of a hyperpolarization-activated ion channel at 3.5ang resolution, that lipid-dependent gating happens in vivo, supporting the proposition that it as well as associated functional interrogation of VSD-pore coupling. Strik- may be relevant to a larger group of voltage-gated ion channels. To analyses ingly, the channel forms a dimer of non-domain-swapped tetramers, with the conformational changes induced by nonphospholipids, we used the high- two channels stacking back-to-back via their cytoplasmic domains. The throughput screen to retrieve peptides that recognize the non-phospholipids- structure offers a glimpse of a voltage sensor in the ‘up’ conformation stabilized resting state of a canonical voltage-gated potassium (Kv) channel coupled to a tightly-closed pore, as well as two major interfaces that, we (KvAP). We found that these peptides can keep the channel in a resting state hypothesize, mediate VSD-pore coupling. The first interface is formed by when they were fused in right positions to the C-terminus of the channel. We the extracellular ends of the S1,S4 and S5 helices, and based on mutagen- determined the structure of the peptide-fusion protein with cryoEM and esis is critical for channel structure. The second interface is formed by the observed an inward-facing conformation of the voltage sensor domain. As a intracellular ends of the S4 and S5 helices, as well as the C-linker hairpin control, the wild-type channel without the peptide was studied to reveal its of the adjacent protomer. Based on mutagenesis, this interface is essential active conformation. The new peptide-stabilized resting state appears for VSD-pore coupling. Taken together, our structural and functional different from the mechanism of helix-sliding inside a four helix-bundle data suggest a model in which the ‘default’ state of this particular model, and allows the S4 to gain more peripheral access, and face the annular channel’s pore is closed, and the VSD does work to open the pore at nega- lipids. These changes appear to offer a mechanistic view of lipid-dependent tive potentials. gating.

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84-Plat movement. Supported by 1150273 to R.L. The Centro Interdisciplinario de Molecular Determinants of Gating Polarity in Hyperpolarization- Neurociencia de Valparaı´so is a Millennium Institute (P029-022-F). Activated HCN Channels John Cowgill1, Vadim Klenchin2, Claudia P. Alvarez Baron1, Debanjan Tewari1, Baron Chanda1. Platform: Protein Structure and Conformation I 1Neuroscience, University of Wisconsin Madison, Madison, WI, USA, 2Univ Wisconsin, Madison, WI, USA. 87-Plat Hyperpolarization-activated channels, cyclic nucleotide-gated (HCN) channels Conformational Bias in Unfolded Proteins Studied by Sequence Reversal show an inverted voltage response compared to virtually all other voltage-gated Steven T. Whitten, Lance R. English. ion channels (VGICs), opening on hyperpolarization rather than depolarization. Dept Chem/Biochem, Texas State Univ, San Marcos, TX, USA. The basic motions of voltage sensing and pore gating are thought to be Sequence patterns of charge, hydrophobicity, hydrogen bonding, and other conserved, implying that these domains are inversely coupled in HCN chan- amino acid physicochemical properties contribute to mechanisms of protein nels. Although the structure of the HCN1 channel was recently solved, the folding, but how sequence composition and patterns influence the conforma- structural element(s) responsible for the inverted gating polarity of HCN are tional dynamics of the denatured state ensemble is not fully understood. To not known. We used a structure-guided protein engineering approach to sys- investigate structure-sequence relationships in the denatured state, we reversed tematically assembled an array of mosaic channels between HCN1 and the the sequence of staphylococcal nuclease and characterized its structure, thermo- depolarization-activated EAG that display the full complement of voltage- dynamic character, and hydrodynamic radius using circular dichroism spectros- activation phenotypes observed in the VGIC superfamily. Our studies reveal copy, dynamic light scattering, analytical ultracentrifugation, and size-exclusion that the voltage-sensing domain of the HCN channel has an intrinsic ability chromatography as a function of temperature. The macromolecular size of to drive pore opening in either direction. Specific contacts at the voltage ‘‘Retro-nuclease’’ was found to be highly expanded in solution with character- sensor-pore interface and unique interactions near the pore gate force the istics similar to biological intrinsically disordered proteins. In contradistinction HCN channel into a hERG-like inactivated state, thereby obscuring their open- to a disordered state, Retro-nuclease exhibits a broad sigmoid transition of its hy- ing upon depolarization. Our findings reveal an unexpected common principle drodynamic dimensions as temperature is increased, indicating a thermodynam- underpinning voltage gating in the VGIC superfamily and identify the essential ically controlled compaction. Counterintuitively, the magnitude of these determinants of gating polarity. temperature-induced hydrodynamic changes exceed that observed from thermal denaturation of folded unaltered staphylococcal nuclease. Undetectable by calo- 85-Plat rimetry and intrinsic tryptophan fluorescence, the lack of heat capacity or fluo- Molecular Simulations of Ion Permeation, Gating and Selectivity in KD rescence changes throughout the thermal transition indicate canonical Channels hydrophobic collapse did not drive the Retro-nuclease structural transitions. Wojciech Kopec, Bert L. de Groot. Instead, temperature-dependent circular dichroism spectroscopy and computer simulation correlate the temperature sensitivity to the intrinsic sampling of back- Computational Biomolecular Dynamics Group, Max Planck Institute for a Biophysical Chemistry, Go¨ttingen, Germany. bone conformations for polyproline II and -helix. Using analytical techniques Potassium channels play a pivotal role in many biological functions, such as that relate backbone conformational bias to hydrodynamic size, the observed formation of the membrane potential and mediating electrical signals in excit- changes in Retro-nuclease structure with temperature indicate backbone confor- able cells (e.g. neurons). Structural and functional studies revealed main mational propensities and coil transition enthalpies that are in good agreement features of these channels, including rapid and selective Kþ ion permeation with calorimetric and spectroscopic studies of short peptides. through a narrow selectivity filter (SF), channel gating at the helix bundle 88-Plat crossing (activation gate), and distinct gating at the SF, termed C-type inac- Time-Resolved Crystallography Measurements Elucidating the Mechanism tivation. Despite such insights, the molecular mechanisms of permeation and of Bacterial HMG-CoA Reductase gating phenomena remain largely unknown, and are further complicated by Vatsal Purohit1, Calvin Steussy1, Tim Schmidt1, Chandra J. Critchelow1, differences exhibited in numerous, structurally distinct members of the potas- Tony Rosales2, Cynthia Stauffacher3,4, Paul Helquist2, Olaf Weist2. sium channel family. Nowadays, Molecular Dynamics (MD) simulations 1Biology, Purdue University, West Lafayette, IN, USA, 2Department of allow studying thousands of individual ion permeation events on the atom- Chemistry and Biochemistry, University of Notre Dame, South Bend, IN, istic scale, that directly correspond to experimentally measured macroscopic USA, 3Purdue Insitute of Inflation, Immunology and Infectious Diseases, currents. Here, we will show simulations of several potassium channels West Lafayette, IN, USA, 4Purdue Center for Cancer Research, West (KcsA, MthK, NaK2K, Kv1.2 and TRAAK), all sharing nearly identical Lafayette, IN, USA. SFs. Our simulations show that all studied Kþ channels achieve their high þ HMG-CoA reductase (HMGR) is the rate limiting enzyme in the Mevalonate conductance rates by permeation of ‘naked’ K ions through the SF. Simul- pathway that converts 3-hydroxy-3-methylglutarylCoA (HMG-CoA), an inter- taneously, the interactions of fully desolvated potassium ions in the SF effi- mediate in the metabolic pathway, into Mevalonate which is a precursor com- ciently exclude any sodium ions from permeation, due to their higher pound used in the formation of steroids and cholesterol. Hence, it is the target of dehydration penalty. Moreover, we will present simulation data that link cholesterol-lowering drugs, commonly known as statins, that are used to treat channel and SF conformations with the ion permeation rates, thus explaining patients suffering from hypercholesterolemia. Since the mevalonate pathway channel gating at the molecular level. is also critical for the survival of multiple gram-positive bacteria, HMG-CoA reductase can also act as a target for novel antibacterials. The mechanism of 86-Plat the bacterial enzyme in Pseudomonas mevalonii has previously been studied Voltage-Sensing Residues in the Voltage Sensor of the BK Channel and characterized by Steussy et. al (2013) and Haines et. al (2012) resulting Willy R. Carrasquel-Ursulaez, Ignacio Segura, Yenisleidy Lorenzo, in the hypothesis that the substrate, HMG-CoA undergoes conversion into Dario Basaez, Ramon Latorre. two intermediates, Mevaldyl-CoA and Mevaldehyde, before it is converted Centro Interdisciplinario de Neurociencia de Valparaı´so, Universidad de into the product, Mevalonate. However, the presence of only one of the inter- Valparaı´so, Chile. 2þ þ mediates, Mevaldyl-CoA, has been structurally observed by Steussy et. al Voltage- and Ca -activated K (BK) channels are modular proteins with allo- (2013). Understanding the structural changes that the bacterial enzyme un- steric gating. BK channels damp excitatory stimuli mediated by voltage- 2þ dergoes at the catalytic site during the conversion of substrate to product can dependent Ca channels, and they are involved in modulating physiological help us better understand its mechanism in order to design inhibitors for processes such as muscle contraction and neuronal excitability. To identify HMGR. Current work on HMGR focuses on identifying conditions that are the voltage-sensing residues that contribute to the gating charge movement is suitable for running the enzymatic reaction in the crystal and also altering crucial to understand the mechanism of BK channel voltage-dependent activa- the rate of the reaction within the HMGR crystal to trap and observe the inter- tion at the molecular level. Here, to determine which charged amino acids in mediates formed during catalysis. transmembrane segments S1-S4 contribute to the voltage sensitivity of the BK channel, we have measured gating currents in BK channels containing 89-Plat neutralization mutations. Only neutralization of basic residues contained in Understanding the Molecular Underpinnings of Collagen-Protein S4 can decrease the voltage dependence of the BK channel voltage sensor. Interactions in Healthy and Pathological States Neutralization of other charged residues in the voltage sensor domain changed Cody L. Hoop, Jie Zhu, Allysa Kemraj, David A. Case, Jean Baum. the equilibrium of the active-resting equilibrium, revealing a modulating role in Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ, USA. the free energy necessary to activate the voltage sensor. The results reveal that The extracellular matrix (ECM) protein collagen is extremely biologically in BK channels the S4 segment is solely responsible for the gating charge active, interacting with numerous receptors and matrix molecules to achieve

BPJ 9279_9291 Sunday, March 3, 2019 17a critical cellular functions including platelet aggregation, cell development, and of forces. We aim to understand how the tip-link responds to varying amount differentiation. Collagen I assembles into highly ordered fibrils, in which the of forces and withstand high forces and enable proper hearing. We also tried receptor binding sites are often buried away from the interaction surface, yet to understand how disease causing point mutation in these two proteins these essential cellular processes do occur. The mechanisms by which collagen change the force sensing behavior of the tip-link. Towards, this we first ligands recognize their binding sites in the collagen fibril are very poorly under- attempt to understand the unbinding kinetics of the tip-link complex against stood. In particular, integrin binding sites, required for platelet aggregation and force using single molecule force spectroscopy mimicking in-vivo inner ear hemostasis, are buried within the collagen fibril and seemingly inaccessible. environment. We have found different energy landscape of unbinding of the Through a multifaceted approach integrating nuclear magnetic resonance spec- wild-type and mutant complex which may arises from the conformational troscopy, computational methods, biological assays, and other biophysical difference of mutant complexes as revealed by single molecule FRET study. tools, we investigate the molecular mechanism of weak collagen–integrin I Structural mutation leads to broadening of the distance to the transition state domain (aI) interactions in healthy and pathological states both with the in energy landscape during unbinding which lead to greater tilting of the po- collagen fibril and the triple helix using collagen model peptides (CMPs). tential energy curve upon application of force compared to wild-type com- We show, through an all-atom molecular dynamics simulation of a fully sol- plex implying low force bearing property of the mutant complex. Further, vated collagen I fibril model with an explicit interaction surface, that the fibril we measured catch bond characteristics of the tip-link complex where surface is not static but rather has nanosecond timescale dynamics that allow it bond-lifetime increases upto a certain force and decline thereafter using to sample conformations not observed in static models. These fluctuations force clamp spectroscopy which explains the higher force-tolerance behavior expose a major ligand binding region within the fibril that includes a cryptic of the tip-link complex. Mutant complexs show different force responsive integrin binding site. We propose that the dynamics of the triple helices within behaviour which may lead to the molecular mechanisms of deafness associ- the fibril play an important role in facilitating binding processes. Further work ated with these mutations. on CMPs that model both healthy and pathological states show that a glycine mutation within a critical aI binding site reduces aI adhesion, destabilizes 92-Plat hydrogen bonding, and enhances mobility local to the mutation. Our novel Structure Determination of Active Full Length Human Taspase1: Towards model of the dynamic collagen fibril provides a fundamentally new framework Novel Anti-Cancer Therapeutics 1 1 1 for thinking about collagen–receptor interactions that are critical for cellular Jose M. Garcia , Nirupa Nagaratnam , Rebecca Jernigan , 1 2 2 3 function. Gihan Ketawala , Silvia Delker , Thomas Edwards , Derek Mendez , Chufeng Li3, Nadia Zatsepin1, Raimund Fromme1, Liang Tong4, 90-Plat Joel Schneider5, James Hsieh6, Andrew Flint7, Petra Fromme1. Structural Insights into Mdn1, an 540 kDa AAA Protein Required for 1Center for Applied Structural Discovery, Arizona State University, Tempe, Ribosome Biogenesis AZ, USA, 2Beryllium Discovery Corporation, Bainbridge Island, WA, USA, Zhen Chen1, Hiroshi Suzuki2, Yuki Kobayashi3, Ashley C. Wang1, 3Department of Physics, Arizona State University, Tempe, AZ, USA, Frank DiMaio4, Shigehiro A. Kawashima3, Thomas Walz2, 4Department of Biological Sciences, Columbia University, New York, NY, Tarun M. Kapoor1. USA, 5Center for Cancer Research, National Cancer Institute, National 1Laboratory of Chemistry and Cell Biology, The Rockefeller University, Institute of Health, St. Louis, WA, USA, 6Department of Medicine, New York, NY, USA, 2Laboratory of Molecular Electron Microscopy, The Washington University, St. Louis, MO, USA, 7Fredrick National Lab for Rockefeller University, New York, NY, USA, 3Graduate School of Cancer Research, National Cancer Institute, National Institute of Health, Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan, Frederick, MD, USA. 4Department of Biochemistry, University of Washington, Seattle, WA, USA. Taspase1 (threonine aspartase 1), an endopeptidase is a potent anticancer drug Ribosome biogenesis in eukaryotes is a dynamic and multistep process that target. Loss of Taspase1 activity disrupts proliferation of human cancer cells involves more than 200 assembly factors. Specific energy-harnessing en- in vitro and in mouse tumor xenograft models of glioblastoma. Therefore, zymes, such as ATPases or GTPases, are required to remodel the precursors Taspase1 has been included in the drug discovery and development pipeline of ribosomes at fast time scales. Mdn1 is an essential dynein-like AAA pro- of the Chemical Biology Consortium (CBC) of the US National Cancer Insti- tein (ATPases Associated with various Activities) that releases specific assem- tute (NCI). Several promising Taspase1 inhibitors have already been identified bly factors from the precursors of the 60S subunit of ribosomes (pre-60S). in high throughput screens. A high-resolution structure of the enzymatically However, how Mdn1 performs its remodeling function remains poorly under- active full-length Taspase1 and Taspase1 in complex with substrate and stood due to its large size (5000 aa in a single polypeptide) and limited ho- inhibitors would greatly enable rational design and optimization of pharmaco- mology to other well-studied proteins, including dyneins. In particular, it is logically potent Taspase1 inhibitors. Until recently, no structure of active, puzzling how conformational changes of the N-terminal AAA ATPase do- full-length Taspase1 nor complexes of the catalytically active enzyme with in- mains couple to its C-terminal pre-60S-interacting MIDAS (Metal Ion- hibitors existed. We have grown the first crystals of full-length Taspase1 and Dependent Adhesion Site) domain, as they are separated by more than 2000 tested at APS synchrotron facility. These crystals diffracted to 3.5 A˚ but aa without known functions. Here, we solved three cryo-EM structures of show strong diffraction anisotropy. Therefore, Taspase1 micro-crystals were full-length or truncated Mdn1 (resolution up to 4.0 A˚ ) that provided the first tested at LCLS and PAL XFEL facilities for serial femtosecond crystallog- pseudo-atomic models for Mdn1 at two distinct nucleotide states. Interest- raphy (SFX) to determine the first high-resolution structure of the full length ingly, we observed intramolecular docking of the C-terminal MIDAS domain Taspase 1 and complexes with inhibitors. Here we present the first crystallo- onto the N-terminal AAA ring in a nucleotide state-specific manner. These graphic structure of the full length active human Taspase1 to 2.7A˚ resolution. data indicate that conformational changes in the AAA ring can be directly The results reported here will open new insights in the enzymatic mechanisms transmitted to the MIDAS domain. Together, the structural studies of Mdn1 of Taspsae1 and help design novel drugs that inhibit the function of Taspase1 reveal how a previously named dynein-like AAA protein performs its me- enzyme. chanical function by a mechanism unlike dyneins or other AAA proteins. These conformational dynamics also provide insights into how Mdn1 could 93-Plat drive the selective release of assembly factors from the pre-60S particles Isotopically Edited Vibrational Spectra and Dynamics for Three-Strand and contribute to ribosome biogenesis. B-Sheet Peptides. DFT Spectral and MD Dynamics Simulations Timothy A. Keiderling1, Heng Chi2, Dan McElheny1, David Scheerer3, 91-Plat Mohammad Shahid Islam1, Karin Hauser1. Deciphering the Mechanism of Force Dissemination through Tip-Links in 1Dept Chemistry, Univ Illinois Chicago, Chicago, IL, USA, 2Jiangsu Food Hearing and Pharmaceutical Science College, Huai’an, China, 3Dept Chemistry, Jagadish P. Hazra1, Nisha Arora1, Sabyasachi Rakshit2. Universitat Konstanz, Konstanz, Germany. 1Chemical Science, Indian Institute of Science Education and Research Peptide b-sheets are difficult for theoretical simulation, due to multiple forms Mohali, Mohali, India, 2Department of Chemical Science, Indian Institute of and aggregation, but multi-strand hairpin models can have monomer structures Science Education and Research Mohali, Mohali, India. with defined cross-strand anti-parallel H-bonding. Hairpins can be partially sta- Cadherin mediated tip-links (Cadherin 23 in alliance with protocadheirn 15) bilized with selected turn sequences (e.g. DPro-Gly or Aib-Gly) or by incorpo- connecting two stereocilia on top of hair cell in inner ear serves as gating rating aromatic cross-strand interactions. In three-stranded b-sheet models, the springs for mechanoresponsive ion channels which open beyond a threshold central strand is H-bonded to two others, possibly having a complex folding force and convert sound induced mechanical force into electrical signal mechanism, depending on the balance of cross-strand and turn effects. We stud- which our brain can decode. Our hearing range is huge (20 Hz-20kHz and ied a series of 23-residue peptides having two turns composed of either DPro- 0 dB120 dB) and different intensities of sound exert different magnitude Gly or Aib-Gly sequences and various cross-strand aromatic couplings. IR and

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VCD thermal equilibrium spectroscopic studies are supported by NMR-based advantages over filopodia on soft substrates: 1) parallel focal adhesion com- structures and DFT modeling. MD dynamics simulations are correlated with plexes are able to compensate for the mechanical failure of neighboring IR-detected temperature-jump kinetics. These all have b-sheet structures, but focal adhesions, which allows for prolonged cell-substrate interactions, differ in stabilities for different turns and aromatic contacts. DFT computations and 2) lamellipodia spatially distribute the cell generated forces over a wider for an Ala-based structure with NMR constrained f,c angles yield IR and VCD area, allowing for greater traction build up at the cell-substrate interface. simulations consistent with experimental data. The structures are sharply Importantly, the model recapitulates in vitro findings on stiff substrates, twisted with the first hairpin being better formed. Aromatic stabilization was where similar mechanical abilities are evident in filopodia and lamellipodia. energetically effective for Trp-Tyr on strands 1-2, less on strands 2-3, but a Finally, it also provides a potential explanation for the observation that focal similar sequence with no aromatic interactions formed better sheets. MD adhesions have a larger mean size in filopodia than in lamellipodia. In silico studies show bistable fluctuation in the DPro-Gly turns from Type 1’ to 2’, results show that lamellipodia with a larger integrin count have shorter cell- but Aib-Gly turns, with less torsional barrier, sampled more forms. These be- substrate interactions with a higher frequency of mechanical failure, while haviors were evident in site-selected IR-detected T-jump relaxation kinetics. the inverse is true for filopodia. This implies that more frequent and smaller Isotopic labeling allows selected monitoring of turns and strands, and Aib- focal adhesions are mechanically advantageous in lamellipodia, while larger Gly have less spectral interference than DPro-Gly turns. Equilibrium IR as and less frequent focal adhesions are advantageous in filopodia. These re- well as T-jump dynamics reflect higher stabilities for strand 1-2 interactions. sults have implications for the broader field of cell-substrate interactions during cell motility. Platform: Cell Mechanics, Mechanosensing, and 96-Plat Motility Numerical Investigation of Leukocyte Rolling, Adhesion and Bond Formation on Surface Coated with Varying P-Selectin Density 94-Plat Grishma S. Prabhukhot1, Rohan Banton2, Charles D. Eggleton3. Microtubule Function in the Mechanosensitive Regulation of Cell 1UMBC, Baltimore, MD, USA, 2US Army Research Laboratory, Aberdeen, Migration MD, USA, 3Dept Mech Eng, Univ Maryland Baltimore, Baltimore, MD, Shailaja Seetharaman, Bertille Bance, Sandrine Etienne-Manneville. USA. Cell Polarity Migration and Cancer, CNRS-Institut Pasteur, Paris cedex 15, Leukocyte attraction towards the site of inflammation, is modulated by selec- France. tins expressed on activated endothelial cells. Scientists have studied the role Cell migration requires a dramatic and coordinated reorganization of the of P-selectin in the progression of systemic disease in mice; the experimental different elements of the cytoplasmic cytoskeleton, composed of three types results have shown that, deficiency of P-selectin results in failure to respond of filamentous structures: actin microfilaments, microtubules and intermedi- to an inflammation. P-selectin molecules have been shown to be a key contrib- ate filaments. Mesenchymal cells strongly interact with and exert traction utor of leukocytes recruitment to sites of inflammation. forces on the extracellular matrix through integrin-based focal adhesions to To date, there are limited theoretical investigations of the effects of changes in move forward. Cell migration is mechanosensitive cellular function during P-selectin density on leukocytes rolling, tethering and adhesion. To address which the traction forces exerted by the cell on the substrate are adapted to this, we have employed a 3D numerical model of an elastic capsule coated the substrate rigidity. Microtubules have been shown to play a key role in with P-selectin glycoprotein-1 (PSGL-1) molecules (representative of a the control of focal adhesion dynamics and distribution and in focal adhesion leukocyte) to simulate its motion on a surface decorated with P-selectins un- associated stress fibers. To ensure the coordinated regulation of focal adhe- der linear shear flow (Jadhav et. al, 2005). We utilized an immersed boundary sions as the cell moves forward microtubule activity must be differentially method to solve the Stokes equation governing the motion of an elastic controlled between the front and the rear of polarized migrating cells. Micro- capsule near a plane wall. The capsule membrane is modeled using tubule regulation involves the binding of microtubule associated proteins and Mooney-Rivlin constitutive equation. The stochastic nature of the bond for- post-translational modifications of tubulins including detyrosination and acet- mation and breakage between P-selectin receptors and PSGL-1 ligands is ylation. On investigating the role of these microtubule modifications during simulated using Monte Carlo method, and the binding kinetics from the directed migration, we showed that microtubule acetylation, but not microtu- Bell model (Bell 1978). bule detyrosination, controls the turnover focal adhesions by modulating We have examined the effects of changes in P-selectin density (NR) on the microtubule anchoring and vesicular fusion near focal adhesions. We hypoth- leukocyte rolling velocity, average force per bond, leukocyte footprints on esize that microtubule functions may be influenced by substrate rigidity to the substrate by varying NR from 30-150 mm2 on the substrate. The results participate in the mechanosensitive regulation of focal adhesions. We found indicate that the average rolling velocity of a leukocyte decreased from 2.6 that microtubule acetylation, but not detyrosination, is promoted by b1 integ- to 2.1 mm/s and the average force per bond decreased from 11.3 to 2.35 pN rin signaling and increases with substrate rigidity. Microtubule acetylation al- with increase in NR values. Correspondingly elevated stresses are shown by ters the distribution of focal adhesion, increases the generation of traction the leukocyte footprint at lower NR values. We are currently simulating forces and promotes cell migration on stiff substrate. Preventing acetylation PMN rolling on substrates with P-selectin patterns. leads to a cell migratory behavior similar to that observed on soft substrate. Altogether our results point to microtubule acetylation as a key element of 97-Plat the feedback loop mechanism contributing to the mechanosensitive regulation Inferring Cell Colony Forces across Time from Tight Junction Intersec- of focal adhesions and migration. tions in Human Induced Pluripotent Stem Cells Ritvik Vasan1, C David Williams2, Mary M. Maleckar3, 95-Plat Padmini Rangamani4. Cell Migration on Compliant Substrates Requires Actin Polymerization by 1Dept Mech, Univ Calif San Diego, San Diego, CA, USA, 2Allen Inst for Cell the Arp2/3 Complex Sci, Seattle, WA, USA, 3Modeling, Allen Inst for Cell Sci, Seattle, WA, Devin B. Mair1, Matthew Perrone1, Jin Zhu2, Ceylin Elmasli3, USA, 4Univ Calif San Diego, La Jolla, CA, USA. Seth H. Weinberg4, Rong Li2,3. Cell mechanics is thought to play a key role in the dynamical rearrangement of 1Dept Biomed Eng, Johns Hopkins Univ, Baltimore, MD, USA, 2Department cells in a colony. Several studies have thus proposed methods to infer the dis- of Cell Biology, Johns Hopkins University, Baltimore, MD, USA, tribution of force from shape. However, a tool for inferring the dynamical evo- 3Department of Chemical and Biomolecular Engineering, Johns Hopkins lution of cell force from change in colony shape remains open to interpretation. Univ, Baltimore, MD, USA, 4Department of Biomedical Engineering, In this study, we build upon previous work to devise a tool that can predict col- Virginia Commonwealth University, Richmond, VA, USA. ony tensions and pressures across time given the evolution of cell shape. We The brain is among the most compliant tissues in the body, which makes cell validate this method by comparison to both prior work in the field and to finite migration in the brain mechanically challenging. Previous work has shown element simulations of force development in synthetic colonies. We show that that migration of neural progenitor cells lacking the Arp2/3 complex, which by conditioning the solution with information from a previous time step, we can polymerizes branched actin cytoskeletal structures such as lamellipodia, are obtain solutions that are smoother across time and are less sensitive to noise. unable to migrate effectively on compliant substrates. However, they are still Using this method, we predict tensions and pressures in a skeletonized time- able to migrate on stiffer substrates. Here, we show that Arp2/3 knockout series of mEGFP tagged tight junction protein ZO-1 in human induced plurip- glioblastoma cells present a similarly deficient migratory ability. To deter- otent stem cells (hiPS) observed under a spinning confocal-disk microscope. mine the underlying mechanistic reasoning for this, an elastic stochastic Our results suggest that major topological changes, such as a mitotic event, model of lamellipodia versus filopodia cell-substrate interactions was devel- lead to large stochasticity (i.e sudden large change) in tension and that tension oped. The model indicates that lamellipodia provide two primary mechanical changes play a key role during differentiation.

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98-Plat synthase (cGAS), reveals a steady LBR gap at rupture site colocalized with Relationship between Cell Force, Shape, and Motion in Collective Cell cGAS binding DNA in tens of seconds. Mislocalization is greatly enhanced Migration by lamin A depletion, requires hours for nuclear reentry, and correlates Aashrith Saraswathibhatla, Jacob Notbohm. with an increase in pan-nucleoplasmic foci of the DNA damage marker Engineering Physics, U. Wisconsin-Madison, Madison, WI, USA. gH2AX. Excess DNA damage is rescued in ruptured nuclei by cooverexpres- Cells migrate collectively to form tissues, to heal wounds, and, in cancer, to sion of multiple DNA repair factors as well as by soft matrix or inhibition of metastasize. During these biological processes, the collective migration ex- actomyosin tension. Increased contractility has the opposite effect, and stiff hibits a transition from a solid-like state, wherein cell positions remain fixed, tumors with low lamin A indeed exhibit increased nuclear curvature, more to a fluid-like state, wherein cells flow freely and rearrange their positions frequent nuclear rupture, and excess DNA damage. Additional stresses likely with their neighbors. Recent theoretical models and experiments have play a role, but the data suggest high curvature promotes nuclear rupture, demonstrated that this transition can be predicted by average cell shape, which compromises retention of DNA repair factors and favors sustained with cells having more elongated shapes and greater perimeters more easily damage. sliding past their neighbors. At each cell-cell interface, it has been proposed that active actomyosin contraction generated within the cell cortex acts as an effective surface tension tending to reduce each cell’s perimeter. Cell-cell Platform: Membrane Physical Chemistry I adhesions have the opposite effect, tending to reduce the surface tension, thereby increasing the perimeter. It is unclear how cells regulate these 101-Plat competing factors in a confluent monolayer. Here, we investigate the factors Tuning Length Scales of a Modulated Phase in Model and Cell-Derived affecting cell surface tension and cell perimeter, and we quantify the corre- Membranes sponding effects on collective migration. For this, we perturb actin and Caitlin E. Cornell1, Allison D. Skinkle2, Shushan He1, Ilya Levental3, myosin-II using pharmacological treatments in a monolayer of Madin- Kandice R. Levental3, Sarah L. Keller1. 1Dept Chemistry, University of Washington, Seattle, WA, USA, 2Rice Darby Canine Kidney cells and study the effects of the treatments on force, 3 shape, and motion. To quantify the cells’ active contractile forces, we University, Houston, TX, USA, Univ Texas Houston, Houston, TX, USA. L L employ traction force microscopy and monolayer stress microscopy; to mea- Micron-scale, coexisting liquid-ordered ( o) and liquid-disordered ( d) sure cell perimeters and to quantify the relative amounts of cortical actin and phases are well characterized in giant unilamellar vesicles (GUV) composed adhesion molecules at cell-cell interfaces, we use fluorescent imaging; to of ternary lipid mixtures. Experimentally, uniform membranes undergo characterize the cell migration, we analyze cell trajectories. By combining demixing when temperature is decreased: domains subsequently nucleate, these experimental measurements, our study provides experimental testing diffuse, collide, and coalesce until only one domain of each phase remains. of the theoretical models and establishes new principles relating cell force, The sizes of these two domains are limited only by the size of the system. shape, and motion. Under different conditions, vesicles exhibit smaller-scale domains of fixed sizes, leading to the question of what sets the length-scale. In membranes 99-Plat with excess area, small domains are expected when coarsening is hindered Mechanics of Cell Sheet Folding - Embryonic Inversion in the Green Algae or a microemulsion or modulated phase arises. Here, we test theoretical pre- Volvox dictions of how the size, morphology, and fluorescence levels of small and Stephanie S.M.H. Hoehn1, Pierre A. Haas1, Aurelia R. Honerkamp-Smith2, ordered domains vary with the membrane’s temperature, tension, and Raymond E. Goldstein1. composition. Using GUVs and cell-derived giant plasma membrane vesicles 1DAMTP, Univ Cambridge, Cambridge, United Kingdom, 2Dept of Physics, (GPMVs), we find that: (1) the characteristic size of domains decreases Lehigh Univ, Bethlehem, PA, USA. when temperature is increased, or membrane tension is decreased, (2) stripes Cell sheet folding is an essential process during multi-cellular development, ex- are favored over circular domains for lipid compositions with low energy amples including gastrulation, neurulation and organogenesis. To understand per unit interface, and (3) fluorescence levels are consistent with domain these morphogenetic processes it is essential to analyse how local cellular registration across both monolayer leaflets of the bilayer. Our experimental changes translate into global deformations at the tissue level. The so-called results are consistent with several elements of current theories for microe- inversion process, during which the spherical embryos of the green alga Volvox mulsions and modulated phases, and inconsistent with others, suggesting a literally turn themselves inside-out, serves as a simple model for cell sheet reason to reexamine current theories. folding. Different types of inversion - that have evolved in different Volvox species - 102-Plat are associated with different physical constraints. A combination of light Twisting of a Mechanosensitive Molecular Probe Detects Lipid Order in sheet fluorescence microscopy and mathematical modelling was used to Membranes reveal that equatorial invagination during type B inversion has to be comple- Giuseppe Licari1,2, Emad Tajkhorshid2,3. 1Center for Biophysics and Quantitative Biology, University of Illinois at mented by active contraction and expansion in the posterior and anterior 2 hemisphere, respectively. Laser ablation experiments are used to map the ten- Urbana-Champaign, Urbana, IL, USA, NIH Center for Macromolecular Modeling and Bioinformatics, Beckman Institute, University of Illinois at sion and stresses that emerge during the morphological changes of inverting 3 embryos. While inversion is achieved by a single wave of cell-wedging in Urbana-Champaign, Urbana, IL, USA, Department of Biochemistry, Center some species, in others the anterior cell sheet is pulled over an inflection point for Biophysics and Quantitative Biology, University of Illinois at Urbana- by contraction rather than bending. Computational modelling yields a spatio- Champaign, Urbana, IL, USA. in situ temporal map of local deformations explaining the global dynamic shape The lack of an experimental technique for detecting phase order in changes of the cell sheet which may impact on future engineering of self- cell membranes has limited accurate studies of important processes in living folding tissues. organisms, such as activation of mechanosensitive pores, cell shape changes or formation of lipid domains. Dithienothiophene-based planarizable push- 100-Plat pull probes have been recently introduced as innovative mechanosensors Nuclear Rupture at Sites of High Curvature Compromises Retention of that can detect and respond to the lateral forces experienced within biolog- DNA Repair Factors ical membranes. The planarization in liquid- or solid-ordered lipid bilayers Irena L. Ivanovska, Yuntao Xia, Kuangzheng Zhu, Lucas Smith, results in a large red shift in excitation spectra and an increased fluorescence. Cory Alvey, Jerome Irianto, Charlotte Pfeifer, Jiazheng Ji, Dazhen Liu, Several spectroscopic and imaging techniques have proven the applicability Sangkyun Cho, Rachel Bennett, Andrea Liu, Roger A. Greenberg, of these probes in vesicles and cells. However, the microscopic interactions Dennis E. Discher. involved in mechanosensing cannot be directly studied by these experiments. Univ Pennsylvania, Philadelphia, PA, USA. In this work, we elucidate the operative mechanism performing extensive The nucleus is physically linked to the cytoskeleton, adhesions, and extracel- all-atoms molecular dynamics simulations for hundreds of nanoseconds. lular matrix - all of which sustain forces, but their relationships to DNA dam- Model membranes in the same conditions as in experiments were used, age are obscure. We show that nuclear rupture with cytoplasmic namely pure DOPC or DPPC bilayers, which give access to disordered mislocalization of multiple DNA repair factors correlates with high nuclear and ordered phases, respectively. The probe was subjected to highly accurate curvature imposed by an external probe or by cell attachment to either aligned parameterization using quantum mechanical calculations. The molecular dy- collagen fibers or stiff matrix. We imaged lamin B receptor (GFP-LBR), an namics simulations were rigorously compared to the experimental outcomes integral membrane protein that binds lamin B and mirrors lamin B meshwork in order to validate the computational approach and the simulated absorption rupture under AFM tip. Short time kinetics of LBR and cyclic GMP-AMP spectra resulted in agreement with previously published data. The study

BPJ 9279_9291 20a Sunday, March 3, 2019 unveils the atomistic details of the probe planarization upon ordering of the 105-Plat bilayers and provides critical features of the mechanosensitivity, such as the Characteristic Conformations of Pseudomonas Quinolone Signal Interact- difference in twisting angle distributions for unary, binary and ternary mem- ing with Bacterial Outer Membrane brane mixtures. The acquired knowledge will promote further developments Ao Li1, Jeffrey W. Schertzer2, Xin Yong1. of more efficient and selective probes, e.g., targeting diversified membranes 1Mechanical Eng, Binghamton Univ at SUNY, Binghamton, NY, USA, in the cell. 2Biological Sciences, Binghamton Univ at SUNY, Binghamton, NY, USA. With the role of packing cargos such as genomes, virulence factors and cell-to- 103-Plat cell communication signals, outer membrane vesicles (OMVs) of Gram- Homeoviscous Adaptation in Mammalian Cell Membranes in Response to negative bacteria have great importance in many disease-related processes. Dietary Lipid Perturbations In contrast to their versatile functions, our understanding about OMV formation Kandice R. Levental, Ilya Levental. remains limited. Recent studies have shown a strong link between the quorum UT Health Science Center at Houston, Houston, TX, USA. sensing molecule, Pseudomonas Quinolone Signal (PQS), and OMV biogenesis Membrane functionality requires maintenance of a narrow range of physical in Pseudomonas aeruginosa as well as other neighboring species. Here we con- properties which are constantly challenged by dietary sources with poten- ducted all-atom molecular dynamics (MD) simulations to elucidate the specific tially profound physiological consequences. We report that differentiation interaction between PQS and an asymmetric lipid membrane physiologically of human mesenchymal stem cells (MSCs) into osteoblasts or adipocytes relevant to the one of P. aeruginosa. We discovered two characteristic states results in extensive remodeling of the plasma membrane (PM), producing of PQS interacting with the membrane, namely attachment on the membrane cell-specific membrane compositions and biophysical properties. Supple- surface and insertion into the Lipid A leaflet. Dominant intermolecular mentation with docosahexaenoic acid, a lipid component characteristic of hydrogen bonds between PQS and Lipid A phosphates in the two states were osteoblast membranes, induced broad lipidomic remodeling in MSCs and identified respectively. The time-resolved position of PQS and the angle be- potentiated osteogenic differentiation. In addition to these effects on differ- tween its heterocyclic ring and alkyl chain reveal a four-staged dynamical pro- entiation, we observed that lipidomic remodeling comprises a homeostatic cess: flotation, attachment, folding, and insertion. Remarkably, PQS bends its response to dietary lipid challenge of membrane physical properties. Obser- hydrophobic chain into a closed conformation to lower the energy barrier for vations in prokaryotes and other non-thermoregulatory organisms have es- penetration through the hydrophilic Lipid A head-group zone, which was tablished that proper membrane functionality requires maintenance of a confirmed by the potential of mean force (PMF) measurements. Simulation narrow range of physical properties under challenge from external inputs. with multiple PQS exhibits significant aggregation of these amphiphilic mole- Homeostatic adaptation of membrane properties to temperature variation is cules in the surrounding aqueous phase. Yet, both attached and inserted states a fundamental and ubiquitous design feature in ectothermic organisms; how- were simultaneously observed even in the presence of PQS aggregation. These ever, such responsive membrane adaptation has not been observed in mam- findings present direct evidence of the PQS insertion into bacterial outer mem- mals. Here, we show that polyunsaturated fatty acids are rapidly and brane and provide critical insight into OMV biogenesis. extensively incorporated into membrane lipids and induce a reduction in membrane packing. Both in vitro and in vivo, we observe a rapid compensa- 106-Plat tion by upregulation of saturated lipids and cholesterol and suppression of Measuring the Interaction of Polyglutamine Peptides with Lipid Mem- other polyunsaturated lipids. These responses are mediated by activation branes of the classical mammalian sterol regulatory machinery, and ultimately Warren A. Campbell1, Maxmore Chaibva2, Xiang Gao2, Ziliang Zhao3, result in recovery of membrane fluidity. Inhibition of membrane remodeling Justin Legleiter2, Shelli L. Frey1. results in decreased cellular fitness when membrane homeostasis is chal- 1Dept Chemistry, Gettysburg College, Gettysburg, PA, USA, 2Dept lenged by dietary lipids. This suggests that mammals have maintained an Chemistry, West Virginia University, Morgantown, WV, USA, 3Max Planck essential mechanism for membrane homeostasis, analogous to homeoviscous Institute of Colloids and Interfaces, Potsdam, Germany. adaptation; however, mammalian cells remodel their membrane lipidomes in Huntington’s disease is an inherited neurodegenerative disorder associated with response to dietary lipid inputs, rather than temperature, in order to maintain motor and cognitive decline, caused by a mutation in the poly-glutamine functional membrane phenotypes. (polyQ) region near the N-terminus of the huntingtin (htt) protein. Expansion of the polyQ region results in the disease that is characterized by oligomeric 104-Plat and fibrillar aggregates of mutated protein. The polyQ expansion in htt is Liquid-Crystal Phase Transitions in Cellular Lipid Droplets flanked by a 17 amino acid N-terminal sequence (Nt17) and a proline-rich Julia Mahamid. (polyP) region. Patients with Huntington’s disease have reduced ganglioside Structural and Computational Biology, European Molecular Biology GM1 levels and restoring these levels in mouse models improves motor func- Laboratory, Heidelberg, Germany. tion, suggesting a neuroprotective effect, but with an unknown mechanism. To Lipid droplets (LDs) are dynamic organelles preserved in all eukaryotic explore the role of GM1 in the cell membrane environment without down- cells. They consist of a neutral lipid core (predominantly TAGs and CEs) stream metabolic effectors, total brain lipid extract (TBLE) model membranes surrounded by a single monolayer of phospholipids. LDs attracted recent in- enriched with either ganglioside GM1 or sphingomyelin (SM) were exposed to terest due to their emerging role as central hubs for lipid flux utilized for either a synthetic polyQ peptide or a full-length htt-exon1 recombinant protein. storage, energy generation or membrane synthesis. Current understanding The interactions between htt and lipid membranes were measured with a com- of their structure and function relies heavily on microscopies that lack spatial bination of Langmuir trough monolayer techniques, vesicle permeability and resolution and/or the ability to preserve native lipid and protein composi- binding assays, and in situ atomic force microscopy. The addition of either tions. Our data constitute the first systematic and quantitative molecular- SM or GM1 decreased htt insertion into lipid monolayers. However, TBLE ves- resolution structural description of LDs in their native state and within the icles with increased SM content were more susceptible to htt-induced permea- context of their unperturbed cellular environment, enabled by state-of-the- bilization, while GM1 had no effect on permeablization. Pure TBLE bilayers art cellular cryo-electron microscopy and tomography. We show that while and TBLE bilayers enriched with GM1 developed regions of roughened, gran- LDs in control HeLa cells are typically amorphous, LDs in mitotic arrest and ular morphologies upon exposure to htt-exon1, but plateau-like domains with a starvation exhibit internal order with a spacing of 3.4 nm, that is distinctly smoother appearance formed in bilayers enriched with SM. Oligomeric aggre- different from stacked phospholipid bilayers observed in the same speci- gates were observed on all bilayer systems regardless of induced morphology. mens. We suggest that the crystalline organization of LDs is a result of a Collectively, these observations suggest that lipid composition strongly influ- biologically-relevant increased local concentration of CE molecules and a ences htt binding and aggregation on lipid membranes. Additionally, results consequent phase separation, leading to their structural transition into will be presented on how the addition of the N-terminal peptide fragment af- a smectic liquid-crystal phase. The structural transition correlates with fects the material properties of the membrane. cellular stress and metabolic scenarios, as well as association with other cellular organelles, and is reversed by treatments beyond the phase transition 107-Plat temperature of CE. The structural phase transition may have a major impact A Link between Peptide Lipidation and Membrane Curvature Modulus on the accessibility of LD lipids to specific binding to enzymes or lipid trans- John M. Sanderson, Hannah M. Britt, Jackie A. Mosely. porters, that may become restricted when the LDs are crystalline and inhibit Dept Chemistry, Durham Univ, Durham, United Kingdom. the exchange of lipids with surrounding membranes. Therefore, the compo- Peptides and drugs are subject to lipidation reactions in membranes, and in some sition, and in turn the resulting internal and surface biophysical properties of cases appear to additionally facilitate lipid hydrolysis. Lipidation reactions LDs is expected to play a key role in their cellular function as a central hub involve acyl transfer from membrane lipids to reactive sites on a bound peptide of lipid fluxes. or drug. For peptides, an amphiphilic structure, typical of peptides such as

BPJ 9279_9291 Sunday, March 3, 2019 21a melittin, appears to favor lipidation, where as for drugs, the depth of bilayer par- and refined structural models of Msh4-Msh5 interacting with HJ and duplex titioning appears to be a crucial factor. In membranes with saturated lipids such DNA using homology modeling and molecular dynamics simulations. Impor- as DPPC or DMPC, melittin undergoes little or no lipidation, whereas with un- tantly, these modeled structures reveal a putative DNA-binding region (DBR), saturated lipids such as POPC, modest lipidation is found. Cholesterol incorpo- in which the protein makes asymmetric contacts with the junction. We further ration into all membrane types leads to striking increases in lipidation activity, identified DNA bases and protein residues that are potentially important for bind- alongside changes in the chemical selectivity of the transfer process. For some ing and recognition. Mutation of these residues or deletion of the DBR results in POPC/cholesterol mixtures, less than 50% of the original unmodified melittin is reduced affinity for HJ and dsDNA. Furthermore, DNA bases predicted to present after 24 hours. The level of activity does not correlate with the affinity of interact with the protein exhibit changes in dynamic motion upon binding that melittin for each membrane type. Rather the curvature modulus of the mem- are reduced with mutated protein. Taken together, these results provide signifi- brane is a better predictor of membrane activity. We hypothesise that the qual- cant insight into MutSg binding interactions and the structure-function relation- itative relationship between curvature modulus and lipidation activity is a ships of these complexes. consequence of changes in the penetration depth of melittin. Access of water to lipidation intermediates may also influence lipidation rates. 110-Plat Fluorescence Fluctuation Spectroscopy Reveals Double Strand Break 108-Plat Recruitment of 53BP1 Dimers and Assembly into Higher-Order Oligomers Intrinsically Disordered Proteins Sense Membrane Curvature at the DNA Repair Locus Wade F. Zeno1, Upayan Baul2, Wilton T. Snead1, Andre C.M. DeGroot1, Jieqiong Lou, Jee Khor, David Priest, Elizabeth Hinde. Liping Wang3, Eileen M. Lafer3, Dave Thirumalai4, Jeanne C. Stachowiak1. Biochemistry and Molecular Biology, University of Melbourne, Melbourne, 1Dept Biomed Eng, Univ Texas Austin, Austin, TX, USA, 2Physics, Albert Australia. Ludwig University of Freiburg, Freiburg, Germany, 3Department of The tumour suppressor 53-binding protein 1 (53BP1) is critical for double- Biochemistry and Structural Biology, Univ Texas Health Science Center San strand break (DSB) repair by non-homologous end joining. While biochemical Antonio, San Antonio, TX, USA, 4Chemistry, Univ Texas Austin, Austin, insight has been gained into the molecular mechanism by which 53BP1 recog- TX, USA. nises a DSB site, the spatiotemporal dynamics that underlie 53BP1 recruitment From endocytic vesicles to cytoskeletal protrusions, the ability of proteins to to a DSB remains unclear. Previous size exclusion chromatography studies sense membrane curvature is essential for efficient initiation and assembly of have revealed that 53BP1 forms dimers and oligomers in vitro. Here, by per- curved membrane structures. To date, all known mechanisms rely on protein forming fluorescence fluctuation spectroscopy in a genetically inducible DSB domains with specific structural features such as wedge-like amphipathic heli- cellular system (called DiVa) that is expressing eGFP-53BP1, we investigate ces and crescent-shaped BAR domains. Many proteins that contain these struc- how 53BP1 oligomerisation facilitates DSB detection and repair. In particular, tured domains also contain large intrinsically disordered regions. Nonetheless from application of Number and Brightness (N&B) analysis alongside a 2D studies of curvature sensing have focused on structured domains in isolation, cross pair correlation analysis (pCF), we demonstrate in live cells that a pre- based on the assumption that curvature sensing requires structural order. In existing pool of 53BP1 dimer is recruited to DSBs and assembled into a high contrast, here we report that disordered domains are themselves potent sensors order oligomer at the DNA repair locus. of membrane curvature. Comparison of Monte Carlo simulations with quanti- tative in vitro and live-cell measurements demonstrates that the polymer-like 111-Plat behavior of disordered domains found in endocytic proteins, including Replication Origins Exposed on the Surface of a Replication Domain by AP180, Epsin1, and Amphiphysin1, drives them to partition preferentially to Transcription Elongation are Preferentially Fired for DNA Replication convex membrane surfaces, which place fewer geometric constraints on their Yongzheng Li. conformational entropy. For highly charged disordered proteins, electrostatic Biomedical Pioneering Innovation Center, Peking University, Beijing, China. effects stiffen the peptide chain, reducing entropic curvature sensing. However, In mammals, DNA replication is initiated at discrete loci known as replication in these cases, increased repulsion between the disordered protein and the origins. Among totally 250,000 potential replication origins, only a small membrane surface give rise to an alternative, electrostatically derived mecha- subset is activated and used for complete replication of the whole genome nism of curvature sensing. Finally, full-length endocytic proteins, which during any S phase, but the selection mechanism remains elusive. Using Sto- contain both structured curvature sensors and disordered regions, are more chastic Optical Reconstruction Microscopy (STORM) super-resolution imag- than twice as curvature sensitive as their respective structured domains alone. ing, we mapped the spatial distribution of replication origins relative to These findings demonstrate curvature sensing mechanisms that are independent individual replication domains (RDs). Metabolic pause labeling revealed of protein structure and illustrate how structured and disordered domains can that DNA replication mainly initiated at the spatial boundary of replication collaborate to synergistically enhance curvature sensitivity. domains. Further, we choose two adjacent RDs and used fluorescence in situ hybridization (FISH) to fluorescently label the RDs and replication origins of them. Interestingly, we found that predominant origins of the early S phase Platform: DNA Structure, Dynamics, and replication domain (ESRD) which were actually inside of the RD became exposed on the surface of the RD from middle G1 phase to the beginning Function of S phase (G1/S phase), not the case with dormant origins of ESRD or pre- dominant origins of the late S phase replication domain (LSRD). To under- 109-Plat stand what drives ESRD predominant origins out, we inhibited transcription Msh4-Msh5 Induced DNA Conformational Changes Provide Insights into elongation or knocked down CTCF or cohesin and got spatial redistribution Its Role in Meiotic Recombination of predominant origins disappeared. To know why origins who were located 1 1 1 1 Sudipta Lahiri , Bharat Lakhani , Yan Li , Manju M. Hingorani , outside could be fired, we labeled proliferating cell nuclear antigen (PCNA), David L. Beveridge2, Ishita Mukerji1. 1 minichromosome maintenance complex component 2 (MCM2) and CCCTC- Dept Molec Biol/Biochem, Wesleyan Univ, Middletown, CT, USA, binding factor (CTCF) in G1 phase and found PCNA clusters distributed 2Chemistry, Wesleyan Univ, Middletown, CT, USA. g outside of replication domains while MCM2 or CTCF distributed randomly. MutS plays a role in meiotic recombination facilitating crossover formation be- Taken together, our STORM images reveal that DNA replication initiates at tween homologous chromosomes. Failure to form crossovers leads to improper origins exposed on the surface of replication domains where replication pro- segregation of chromosomes and aneuploidy, which in humans results in infer- teins distribute. Replication Origins selection may be a result of their spatial tility and birth defects. To improve current understanding of MutSg function, we g redistribution mediated by cooperation of transcription elongation and CTCF- investigated the binding affinities and structures of MutS in complex with DNA cohesin formed loops. substrates that model homologous recombination intermediates. Our findings demonstrate that Sc Msh4-Msh5 binds Holliday Junction-like substrates, 3’ 112-Plat overhangs, single stranded (ss) forks and the D-loop with nanomolar affinity. En- Elucidating Complimentary Base Specificity of Thymine DNA Glycosylase ergy transfer experiments further demonstrate that DNA structure is modulated via Potential of Mean Force Molecular Dynamics Simulations by the binding interaction with the largest changes associated with substrates Ozge Yoluk1, Alexander C. Drohat2, Alexander D. MacKerell1. containing a ss end. For junction-like intermediates, Msh4- Msh5 binding either 1Pharmaceutical Sciences, Univ. of Maryland, Baltimore, Baltimore, MD, stabilizes the existing stacked structure or induces formation of the stacked X USA, 2Biochemistry and Molecular Biology, Univ. of Maryland, Baltimore, conformation. Significantly, we find that upon binding Msh4-Msh5 stacks an Baltimore, MD, USA. open junction construct to the same extent as the standard junction. These results Base excision repair (BER) is a conserved repair pathway that removes suggest that MutSg stabilizes the stacked X junction conformation, which is re- damaged or chemically modified bases from DNA. Defects in BER lead to fractory to branch migration, possibly until resolution by MutLg. We developed elevated mutation rates, eventually pushing the cell to a cancerous state.

BPJ 9279_9291 22a Sunday, March 3, 2019

Thymine DNA glycosylase (TDG) is an essential component of BER that re- The depletion of CTCF slightly affect Cohesin association rate, confirming the moves thymine from mutagenic G,T mispairs that arise via deamination of hypothesis that CTCF is needed to position Cohesin on chromatin but not for its 5-methylcytosine (G,mC to G,T). Notably, TDG also excises two modified loading. No significant effect is observed on Cohesin dissociation rate in the forms of mC, 5-formylcytosine (fC) and 5-carboxylcytosine (caC). Considering absence of CTCF. This result support a scenario in which Cohesin is stalled the 17,000-fold faster excision of thymine from G,T relative to A,T pairs, the at CTCF sites but not actively stabilized there. complementary base is strongly indicated to drive specificity in TDG. This To further characterize Cohesin Dynamics in presence/absence of CTCF, we specificity is critical for avoiding activity on the vast background of A,T pairs, analyzed the features of long trajectories of Cohesins. For each trajectory we but its mechanism remains unknown for TDG and other G,T glycosylases. Us- compute the mean-square displacement and extrapolate the apparent diffusion ing molecular dynamics simulations with replica exchange umbrella sampling, coefficient (Dapp) and the anomalous exponent (alpha). As a reference, we also we investigated the origins of this base specificity by focusing on G,T, H,T track genomic loci. When depleting CTCF, a subpopulation of Cohesins show a and A,T pairs (H: hypoxanthine). Potential of mean force (PMF) calculations more dynamic behavior. targeted the T base flipping in DNA alone and the enzyme-DNA complexes. Given their values of Dapp and alpha, these mobile molecules are most likely Results show that formation of the enzyme-DNA complex in G,T pair leads bound to chromatin, suggesting the existence of a non-stalled fraction of Co- to destabilization of the Watson-Crick base-pair state and the presence of a hesins. We are currently testing this hypothesis by comparing our experi- local minimum of the flipped base corresponding to binding in the enzyme cat- mental data to computational predictions in the context of the loop alytic site. In an A,T pair, a partially flipped state is found to be more energet- extrusion model. ically favorable then the fully rotated state. The specificity of TDG for G,T over A,T pair is indicated to be due to both a lowering of the barrier to flipping 115-Plat of the T base and stabilization of the T base in the enzyme active site. The Three-Dimensional Architecture of the Human Genome: It’s Nuclear Physics! 113-Plat Michele Di Pierro. RNAP as a Moving Barrier to Loop Extrusion CTBP, Rice Univ, Houston, TX, USA. Aafke A. van den Berg1, Gordana Wutz2, Roman R. Stocsits2, The human genome is composed of 46 DNA molecules — the chromosomes Hugo Brandao3, Georg Busslinger4, Jan-Michael Peters2, Leonid Mirny1. — with a combined length of about 2 meters. Chromosomes are stored in the 1Massachusetts Institute of Technology, Cambridge, MA, USA, 2Research cell nucleus in a very organized fashion that is specific to the cell type; this Institute of Molecular Pathology, Vienna, Austria, 3Dept Biophysics, Harvard three-dimensional architecture is a key element of transcriptional regulation University, Cambridge, MA, USA, 4Hubrecht Institute, Utrecht, Netherlands. and its disruption often leads to disease. What is the physical mechanism Recent theoretical and experimental studies indicate that continuous loop leading to genome architecture? If the DNA contained in every human cell extrusion is one of the mechanisms underlying chromatin organization during is identical, where is the information — the blueprint — of such architecture interphase. According to the model for loop extrusion, a cohesin complex is stored? In a series of works, we were able to demonstrate that the architecture loaded onto chromatin and extrudes loops until it dissociates or encounters of interphase chromosomes is encoded in the one-dimensional sequence of an obstacle such as CTCF. Though cohesin is important for gene regulation, in- epigenetic markings much as three-dimensional protein structures are deter- teractions of cohesin with genes remain poorly understood. mined by their one-dimensional sequence of amino acids. In contrast to the Cohesin-gene interactions can best be studied when CTCF boundaries are situation for proteins, however, the sequence code provided by the epigenetic removed and cohesin’s lifetime on chromatin is extended. In mammalian cells, marks decorating the chromatin fiber is not fixed but is dynamically rewritten cohesin accumulates between sites of convergent transcription under these con- during cell differentiation, modulating both the three-dimensional structure ditions (Busslinger Nature 2017). Further, we observed that cohesin positioning and gene expression in different cell types. In vivo, segments of chromatin on genes correlates with RNAP positioning. Since positioning of cohesin characterized by homogeneous epigenetic markings undergo a process directly influences the 3D structure of chromatin, we asked: how do these similar to phase separation under the action of the proteome present in the cohesin-gene interactions shape the 3D genome around genes and what is the nucleus. This process forms liquid droplets, which rearrange dynamically underlying mechanism? by splitting and fusing, thereby modulating DNA distal interactions and To answer these questions, we studied patterns in Hi-C around actively tran- generating the genomic compartments characteristic of chromosomal scribed genes in the CTCF-Wapl KO and other mutants. We find that both tran- architecture. Our theory — together with our computational tools — allows scription start and termination sites form CTCF-independent, insulating predicting and studying the spatial conformation of genomes with unprece- boundaries and that the strength of the insulation depends on transcription dented accuracy and specificity, thus opening the way to the study of the levels and, surprisingly, on gene length. To reconcile these observations, we functional aspects of genome architecture. propose a model where cohesin cannot bypass PolII. In this case cohesin traces PolII at its low speed in the direction of transcription, while a cohesin approach- 116-Plat ing PolII in the opposite direction is shuffled back to the end of the gene. This Measuring the Physical Properties of DNA on a Genomic Scale 1 2 3 3 model can faithfully reproduce both ChIP-seq patterns of cohesin accumulation Aakash Basu , Tunc Kayikcioglu , Thuy Ngo , Quicen Zhang , 1 3 3 3 and patterns in Hi-C around active genes. Interestingly, the model also provides Basilio Cieza Huaman , Miroslav Hejna , Tomas Rube , Jun Song , 1 a mechanism for dynamic enhancer-PolII tracking during transcription elonga- Taekjip Ha . in vitro 1Dept Biophysics, Johns Hopkins University, Baltimore, MD, USA, 2Johns tion (Lee Gen & Dev 2015). Future experiments and live-cell imaging 3 can further test the predictions of this model. Hopkins Univ, Baltimore, MD, USA, University of Illinois Urbana Champaign, Urbana, IL, USA. 114-Plat The physical properties of DNA have been extensively measured. However, Single Molecule Imaging of CTCF and Cohesin. Dissecting the Dynamic relatively little is known on how DNA sequence modulates DNA flexibility, Interplay between Chromatin Loop Regulators and how sequence mutations affect downstream biological processes by Laura Caccianini1, Elphege P. Nora2, Johannes Nuebler3, Agnes LesSaux1, altering DNA flexibility. To address these questions, we have developed Edith Heard1, Leonid Mirny4, Benoit Bruneau2, Maxime Dahan5. an assay to create a genome-wide map of DNA flexibility. An input library 1Curie Institut, Paris, France, 2Gladstone Institut, San Francisco, CA, USA, of thousands of short DNA molecules representing nucleosomal sequences 3Massachusetts Inst of Tech, Cambridge, MA, USA, 4Dept Hlth Sci, from S. Cerevisiae is end modified to permit each molecule to form a Massachusetts Inst Tech, Cambridge, MA, USA, 5Lab Phys/Chem, Curie loop. A selection condition is imposed whereby stiffer molecules that fail Institut, Paris, France. to loop after various amounts of time are digested. The surviving molecules Recently proposed models on the 3D dynamical structure of chromatin mainly are sequenced at various time points, and individual kinetic curves for rely on population assays in fixed cells. We here integrate the results of these DNA cyclization is constructed for each sequence. Our measurements static assays with the information that can be inferred from live microscopy at indicate that þ1 nucleosomes have asymmetric cyclization rates, with the the single cell level. We present a single molecule (SM) tracking study of promoter proximal half more cyclizable than the distal half. We compare CTCF and Cohesin, central players in the formation of Topologically Associ- our data with existing biochemical and genome-wide NET-seq data to un- ating Domains (TADs). To study the interplay between the two proteins we derstand how nucleosomal flexibility asymmetry affects polymerase translo- perform SM tracking of Cohesin in the absence of endogenous CTCF, cation through the nucleosome. We also find that CpG and TpA content depleted via the auxine-inducible degron system. With SM tracking, we deter- have a marked effect on DNA cyclization rate. Our assay provides a plat- mine two parameters, the bound fraction of a nuclear factor and its residence form for understanding how sequence features modulate DNA flexibility, time on chromatin, from which the association and dissociation rate can be and more broadly, how physical forces may have shaped the evolution of inferred. genomes.

BPJ 9279_9291 Sunday, March 3, 2019 23a

Symposium: Proteins: Exploring Sequence Space rational design efforts. HT-MEK is applicable to enzyme function and stability, provides data that rivals or exceeds the precision typically obtained from stan- via Computation and Experiment dard assays, and reduces the time, effort, and cost to quantitatively profile the effects of individual substitutions on enzyme function by orders of magnitude, 117-Symp providing a technology to access previously inaccessible questions across mul- Engineering and Evolution of Allosteric Communication tiple systems and disciplines. Kimberly A. Reynolds. Green Center for Systems Biology and the Dept. of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA. Symposium: Glutamate Receptors Allosteric regulation permits the modulation of protein activity at a site distinct from the protein active site. This regulation can be mediated by 120-Symp protein-protein interactions, small molecule binding, or post-translational Optical Control and Report of AMPA Receptor Activation modification - but in all cases the critical feature is that thermodynamic inter- Andrew Plested. actions between spatially distributed amino acids serve to couple perturba- FMP-Berlin & Humbolt University Berlin, Berlin, Germany. tions at the allosteric site to the active site. Understanding which amino AMPA receptors are essential and ubiquitous in vertebrate nervous systems, acids provide the basis for allostery is necessary to predict regulatory sites, where they respond to neurotransmitter on the millisecond timescale. We engineer regulation, and understand how allostery might evolve. We show have adapted several approaches to allow regulation and observation of that analysis of amino acid sequence co-evolution can provide a practical AMPA receptor activity by light. Incorporation of optically-active unnatural strategy for engineering new regulation and predicting functional sites. More- amino acids, under genetic means, provides an unbiased approach to introduce over, the observation of sparse co-evolving networks inside of proteins im- a timed, progressive perturbation of receptor structure. Using this method, plies that allostery can evolve readily at a diversity of latent allosteric coupled to fast perfusion electrophysiology, we could identify gating modules surfaces, and suggests a strategy for examining communication not only in the channel domain of the receptor. In the process, we generated receptors within, but also between proteins. that behave identically to wild-type channels, but upon UV illumination are either inhibited or potentiated. To observe the activity of receptors and forma- 118-Symp tion of complexes between AMPA receptor subunits with auxiliary proteins, we Hypervariable Proteins in Microbes have fused fluorescent proteins to both parties and generated FRET pairs. Eugene Koonin. FLIM-FRET and patch clamp fluorimetry reveal specific signals dependent National Center for Biotechnology Information, National Institutes of Health, on the activation state of the complex. Full genetic encoding of these probes Bethesda, MD, USA. paves the way for their deployment in neurons to study synaptic transmission. Numerous proteins involved in essential cellular functions are highly conserved 121-Symp in evolution. On the opposite end of the evolutionary spectrum are proteins that The Eukaryotic Specific M4 Segments are Allosteric Conduits for NMDA function in defensive and ‘‘offensive’’ roles. These proteins evolve extremely Receptor Signaling fast, often under the pressure of positive selection, producing enormous diver- Lonnie Wollmuth. sity. We investigated in detail different classes of hypervariable proteins in Neurobiology & Behavior and Biochemistry & Cell Biology, Stony Brook archaea and bacteria including anti-CRISPR proteins, polymorphic toxins University, Stony Brook, NY, USA. and antitoxin immunity proteins. Using dedicated computational pipelines The regulation of protein function by allostery - coupling of binding at one and machine learning methods, we predicted thousands of proteins in each of site to affecting activity at another often remote site - is central to biology. these functional groups that are encoded in a broad diversity of bacteria and NMDA and AMPA receptors (NMDARs and AMPARs) are glutamate- archaea as well as viruses and integrated elements. Most of these proteins gated ion channels ubiquitous in the human brain and contribute to all brain have not been studied experimentally and are currently unannotated or misan- functions including perception, motor control, learning and memory. High- notated in genomic sequences. These hypervariable proteins were classified lighting their central role are de novo and inherited missense mutations in into families using sensitive methods for sequence analysis, their evolutionary NMDAR and AMPAR subunits that are associated with epilepsy, autism, in- relationships were investigated and specific functions predicted wherever tellectual disability, movement disorders, and schizophrenia. Missense muta- possible. tions often occur at key points in allosteric pathways. NMDARs and AMPARs are composed of 4 highly modular, layered domains: Extracellular amino- 119-Symp terminal (ATD) and ligand-binding (LBD) domains, a transmembrane domain Bringing Enzymology into the Genomic Era: Developing and Deploying (TMD) forming the ion channel, and an intracellular C-terminal domain New Tools to Quantitatively Map Functional Connections Throughout (CTD). In homology to Kþ channels, the core of the ion channel and lining an Enzyme the permeation pathway is the pore domain: M1 and M3 transmembrane he- Craig Markin1, Daniel Mokhtari1, Fanny Sunden1, Dan Herschlag1, lices and an M2 pore loop. Surrounding this core gating domain are eukaryotic Polly M. Fordyce2. 1 2 specific M4 transmembrane helices. These M4 segments are central allosteric Biochemistry, Stanford University, Stanford, CA, USA, Genetics and conduits of the distinct physiology of NMDARs and AMPARs, integrating in- Bioengineering, Stanford University, Stanford, CA, USA. formation within and across domains. Within the TMD, the M4 segments Despite enormous advances in our understanding of enzymes and their mech- regulate the dynamics of the bundle helical crossing and the M2 pore loop, anisms over the past decades, how they achieve their enormous rate enhance- impacting the rates of activation, deactivation, and desensitization, and the ments and exquisite specificities remains elusive, as evidenced by our limited magnitude of Ca2þ influx. The M4 segments are connected to the extracellular ability to engineer new enzymes. A fundamental reason for this lack of under- domains via the D2 lobe and the intracellular regulatory CTD. These connec- standing is the large number of interconnected residues and long-range interac- tions allow the M4 segments to integrate information across remote domains. tions that cannot be revealed via traditional, low-throughput quantitative The M4 segments, while a recent evolutionary addition to ionotropic gluta- assays. While high-throughput screens and selections can identify enzymes mate receptors, represent a key allosteric pathway to increase the structural with desired changes, the improvements obtained are typically modest and and functional diversity of NMDARs. the qualitative or limited quantitative data obtainable by these approaches is insufficient to reveal functional interconnections between remote and active 122-Symp site residues. To meet this unmet need, we have developed a new technology, After the Structure Comes the Dynamics: Molecular Modeling of Gluta- HT-MEK (High-throughput Microfluidic Enzyme Kinetics), that allows mea- mate Receptors Reveals Long-Range Allosteric Coupling between Ligand surement of myriad quantitative biochemical parameters for 1000 rationally Binding Site and Channel Gate chosen enzyme variants in a single experiment. Using this platform, we carried Maria G. Kurnikova. out deep functional analysis of multiple substitutions at each position within a Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA. single enzyme, the Alkaline Phosphatase superfamily member PafA. For each Ionotropic glutamate receptors (iGluR) are ligand-gated channels found in the substitution, we measured multiple biochemical parameters, including postsynaptic membranes of excitatory synapses. These receptors mediate a ma- Michaelis-Menten constants for cognate and non-cognate substrates and inhibi- jority of excitatory neurotransmission, and their dysfunction is associated with tion constants for multiple inhibitors and transition state analogs. The results numerous neurological diseases. Hence it is important to understand regulation provide a detailed functional signature for each position within PafA. We of the receptor functional states and the mechanisms of interconversion be- observe distinct physically contiguous ‘functional units’ throughout the tween them. AMPA type iGluRs exhibit the fastest gating, and desensitization enzyme that tune specific aspects of function, yielding blueprints for future kinetics among several families of the ligand-gated ionotropic receptors,

BPJ 9279_9291 24a Sunday, March 3, 2019 making them especially attractive for molecular modeling. Computationally LS). Scanning the line Bessel sheet across the cell allows us to create fast intensive molecular modeling technology has seen a steady improvement in 3D, multi-color movies of live nuclei during deformation. We investigate nu- recent years, in part due to new computer architectures for high performance clear deformation with actin depolymerization, lamin A/C knock down, and computing and in part from developments of better simulation algorithms chromatin (de)compaction to decouple each structure’s role in regulating nu- and molecular models. Simulations of protein systems are now capable of clear mechanics. We observe similar elastic behavior during short time-scale reaching the biologically relevant time-scales. We present extensive molecular deformations (10 s), yet lamin A/C KD nuclei show varied local deformations modeling of the smallest functional glutamate receptor: a homotetrameric around the AFM tip and increased chromatin flow during compression. We are modified GluA2 receptor consisting of the ligand binding domains (LBD) progressing towards mapping chromatin strain to determine how force is and the transmembrane domains (TMD). Thus truncated protein remains a distributed in each intervention and understanding how nuclear volume, shape, functional glutamate receptor with somewhat altered kinetics of gating and and internal chromatin organization are changed during and after compression. desensitization. All simulations were performed with a protein structure embedded in lipid bilayer and water and in presence of permeant monovalent 125-Plat ions. Our simulations reveal for the first time allosteric coupling between qua- Multi-Modal Fluorescence Characterization of Cell Cycle Progression and ternary, tertiary and secondary structures of the LBD and TMD thus shedding Cytokinesis light on the mechanisms of sub-conductance levels as well as receptor activa- Rachel Cinco, Per Niklas Hedde, Leonel Malacrida, Michelle A. Digman, tion and inactivation. We have also characterized how a set of novel non- Enrico Gratton. competitive inhibitor molecules interact with the binding site at the membrane Biomedical Eng, UC Irvine, Irvine, CA, USA. protein interface created by the pre-M1, M3 and M4 helices. The role of TMD- Cell division is central to both the creation of life and the propagation of dis- LBD linkers will be extensively analyzed. eases such as cancer and neurodegenerative disorders. While many of the genes and enzymes responsible for regulating cell cycle progression have been exten- sively characterized, much of the cellular bioenergetics responsible for fueling Platform: Optical Microscopy and cell cycle progression and biophysics of cell division is poorly defined. Here, we characterize cell cycle progression in human HeLa cells using Fluorescent Superresolution Imaging I Ubiquitination-based Cell Cycle Indicator (FUCCI) with a variety of advanced fluorescence optical approaches. Application of NADH phasor Fluorescence 123-Plat Lifetime Imaging Microscopy (FLIM) identified significant changes both in DNA Intercalators Tilt, Wobble and Twirl; Elucidating the Structure of NADH concentration and free/bound NADH between several stages of the S-DNA cell cycle defined by FUCCI. We also present detailed timelapse images of hu- 1 2 2 2 Adam Backer , Andreas S. Biebricher , Graeme A. King , Gijs J.L. Wuite , man HeLa cells undergoing cytokinesis using NADH Phasor FLIM, Laurdan Iddo Heller2, Erwin J.G. Peterman2. 1 2 Phasor FLIM, and timelapse 740nm 2-Photon spectral phasor to spatially char- Sandia National Labs, Albuquerque, NM, USA, Vrije Universiteit acterize and quantify metabolism, membrane fluidity, and unlabeled fluores- Amsterdam, Amsterdam, Netherlands. cence emission during the completion of cell division. We also present DNA structural transitions drive life processes and are key to the development timelapse pair correlation function of Laurdan labeled cells undergoing cytoki- of DNA-based biotechnology. Accordingly, we seek to quantify how DNA de- nesis to investigate membrane trafficking during the end of cell division. [Work forms under mechanical tension. A major obstacle that impedes the investiga- supported by NIH P41GM103540]. tion of elongated DNA conformations is the narrow width (2 nm) of the DNA molecule. Due to optical diffraction, the structural changes that result from an 126-Plat applied stretching force cannot be visualized using light microscopy. To Eliminating Background Noise for STED Super-Resolution Microscopy address this challenge, we employ concurrent single-molecule polarization im- Using Polarization Switching aging and DNA manipulation to probe the orientations and rotational dynamics Jong-Chan Lee1,2,YeMa2, Kyu Young Han3, Taekjip Ha2,4. of DNA-intercalated dyes—small fluorescent molecules that bind between 1DGIST, Dalseong-gun, Republic of Korea, 2Johns Hopkins University, adjacent DNA base pairs. Our method uses optical tweezers to precisely extend, Baltimore, MD, USA, 3CREOL, Orlando, FL, USA, 4Howard Hughes align, and (re)orient a single DNA molecule within the image plane of a fluo- Medical Institute, Baltimore, MD, USA. rescence microscope. Optical polarization reveals the nanoscale molecular re- Stimulated emission depletion (STED) super-resolution microscopy (or ordering that occurs as a single DNA molecule is stretched. Our data shows that nanoscopy) offers significant enhancement of optical resolution compared at extensions beyond the so-called ‘‘overstretching transition’’ intercalators to conventional microscopy. To achieve resolution beyond the diffraction- adopt a dramatically tilted orientation relative to the DNA-axis (approximately limit, STED nanoscopy uses orders of magnitude (roughly 10^5) more pho- 54 degrees), distinct from the perpendicular orientation (approximately 90 de- tons than the conventional confocal microscopy. Those additional ‘STED’ grees) normally observed at lower extensions. Strikingly, by imaging single photons, which are designed to deplete the fluorescence at the periphery of intercalated dye molecules with polarized illumination, we also demonstrate focus, can induce unintended background noise. Increased low spatial fre- that intercalators rapidly rotate (i. e. ‘‘twirl’’) about the DNA-axis, revealing quency background noise decreases the signal-to-background ratio (SBR) underlying Brownian twisting dynamics of the DNA substrate. Taken together, and deteriorates the image quality by masking the high spatial frequency, these results shed new insight on S-DNA: a DNA conformation that forms un- super-resolved signal. Here, we report a simple and easy-to-implement der tension that, at present, is not well understood. method, which we call polarization switching STED (psSTED), that can effi- ciently eliminate the low spatial frequency background appearing in STED 124-Plat images. In psSTED, we switch the STED beam polarization between two Nuclear Deformation with Combined AFM and 3D Multi-Color Live-Cell different circularly polarized states to record a regular STED image and a Line Bessel Sheet Imaging background noise image. A simple, unambiguous subtraction process be- 1 1 1 2 Chad Hobson , Evan F. Nelsen , Joe Hsiao , Andrew Stephens , tween these two images accomplishes a background-free super-resolved 1 1 1 E. Timothy O’Brien , Michael R. Falvo , Richard Superfine . image. With both simulation and experimentation, we demonstrate psSTED 1University of North Carolina - Chapel Hill, Chapel Hill, NC, USA, 2 works universally for different STED conditions. Finally, we compare the Northwestern University, Evanston, IL, USA. performance of psSTED with other state-of-the-art background subtraction The nucleus has increasingly been shown to be a mechanosensor that dynam- methods and highlight its capability of efficient background suppression ically responds to external stimuli. Transcription upregulation has been corre- with a much simpler hardware implementation. lated to external force-induced chromatin stretching, and cell migration through tight constrictions has been shown to rupture nuclei causing DNA damage. 127-Plat Fully understanding the mechanics of nuclear mechanotransduction requires The Nucluear Pore Complex as Intrinsic Reporter for Isotropic Expansion detailed mapping of how stress is distributed and determination of which Microscopy cellular structures give rise to these distributions. Here, we investigate the me- Paolo Bianchini1, Luca Pesce1,2, Marco Cozzolino1,2, Luca Lanzano’1, chanical properties of the nucleus and how they depend on the surrounding Alberto Diaspro1,2. structures of the cell using precise control of applied force and high spatio- 1Nanoscopy and NIC, Istituto Italiano di Tecnologia, Genoa, Italy, temporal resolution fluorescence imaging of the nucleus and chromatin. By 2Department of Physics, University of Genoa, Genoa, Italy. combining an atomic force microscope (AFM) with a versatile optics system, Expansion microscopy (ExM) is a super-resolution imaging method that does we can carefully measure and apply forces (10 pN resolution) and monitor not require any special optical microscope(1). The key point resides on the the behavior of the nucleus in the plane of applied force through use of Pathway possibility of uniformly chemically expanding a sample, thus increasing Rotated Imaging for Sideways Microscopy with vertical lightsheet (PRISM- the relative distances among objects of interest as fluorescent molecules

BPJ 9279_9291 Sunday, March 3, 2019 25a labeling specific components. ExM is highly invasive; it involves gelation Molecular Physiology & Biological Physics, Univ Virginia, Charlottesville, and digestion steps that could introduce artifacts and heterogeneities in the VA, USA, 4Dept Biomedical Engineering, Univ Virginia, Charlottesville, relative spatial distribution of complex proteins in the cells. The possibility VA, USA. to combine STED(2) and ExM (ExSTED)(3) allows not only an unprece- Tissue-like 3-dimensional (3D) microbial communities called biofilms colo- dented resolution but also a higher sensitivity to discover possible pitfalls. nize a wide variety of biotic and abiotic surfaces and, in aggregate, consti- The present study aims to determine the robustness of such a technique, quan- tute a major component of bacterial biomass on earth. As such, biofilms have tifying the expansion parameters, i.e., scale factor, isotropy, uniformity. Our a tremendous impact on the biogeochemistry of our planet and the biochem- focus is on the nuclear pore complex (NPC)(4). In particular, we show that istry of higher living organisms. However, the spatial distributions of Nup153, a filamentous subunit localized in the nuclear pore basket(5), is an different genotypes or phenotypes that shape the emergent properties and ca- excellent reporter to address the isotropy of the expansion process quantita- pabilities of biofilms remain largely unknown. A critical barrier is that con- tively. The quantitative analysis carried out on NPCs, at different spatial ventional imaging modalities are not able to resolve individual cells within scales, allows concluding that expansion microscopy can be used at the nano- thick 3D biofilms in a non-invasive manner. Lattice light-sheet microscopy scale with consistent accuracy in the range of 20 nm. It is an excellent method is a new imaging technology that effectively combines low photo-toxicity for structural studies of macromolecular complexes. 1. Chozinski, T.J., A.R. and high spatiotemporal resolution making it a promising tool for live-cell Halpern, H. Okawa, H.-J. Kim, G.J. Tremel, R.O.L. Wong, and J.C. Vaughan. biofilm research. We use lattice light-sheet microscopy to image the initial 2016. Nat Meth. 13: 485-488. 2. Vicidomini, G., P. Bianchini, and A. Dia- phases of surface colonialization and biofilm formation by the exoelectro- spro. 2018. Nat Meth. 15: 173-182. 3. Gao, M., R. Maraspini, O. Beutel, A. genic bacterium Shewanella oneidensis MR-1 and the motility of the preda- Zehtabian, B. Eickholt, A. Honigmann, and H. Ewers. 2018. ACS Nano. : tory bacterium Myxococcus xanthus in all three spatial dimensions. To acsnano.8b00776-8. 4. Szymborska, A., A. de Marco, N. Daigle, V.C. measure biofilm remodeling due to cell growth, division, motility, and Cordes, J.A.G. Briggs, and J. Ellenberg. 2013. Science. 341: 655-658. dispersal, we present progress towards automated cell segmentation algo- 5. Walther, T.C., M. Fornerod, H. Pickersgill, M. Goldberg, T.D. Allen, rithms that enable quantitative tracking of individual cells in developing bio- and I.W. Mattaj. 2001. EMBO J. 20: 5703-5714 films. Resolving cellular level details in biofilms may provide crucial information to guide the development of predictive computational models 128-Plat of biofilm growth and reveal new strategies to control biofilms in natural Multicolor Single-Particle Reconstruction of Protein Complexes and artificial environments. Christian Sieben1, Niccolo´ Banterle2, Kyle M. Douglass1, Pierre Go¨nczy2, Suliana Manley1. Platform: Membrane Proteins I 1School of Basic Sciences, EPFL, Lausanne, Switzerland, 2School of Life Sciences, EPFL, Lausanne, Switzerland. 131-Plat Single-particle reconstruction from electron microscopy (EM) images is a valu- Investigating How Membrane Elasticity Impacts Membrane Protein able and well established tool in structural biology, today able to reach atomic Folding resolution. However, since the image contrast in EM is dependent upon the Miranda L. Jacobs1, Neha P. Kamat2. local electron density within biological structures, the resulting image lacks 1Interdisciplinary Biological Sciences Graduate Program, Northwestern direct information on protein identity. To address this limitation, we developed University, Evanston, IL, USA, 2Biomedical Engineering, Northwestern, a computational and analytical framework that reconstructs and co-aligns mul- Evanston, IL, USA. tiple proteins from 2D super-resolution fluorescence images. We demonstrate Membrane protein expression and insertion into vesicle membranes is impor- our method by generating multicolor 3D reconstructions of several proteins tant for developing functional model cellular systems, bioreactors, and arti- within the human centriole, revealing their relative locations, dimensions and ficial cells. Many membrane proteins have been successfully incorporated orientations. Our method offers great flexibility and can be applied to other into artificial membranes, however, the effects of membrane properties on structures in cell and infection biology. protein insertion is not well understood. The incorporation of non-natural 129-Plat amphiphiles, such as diblock copolymers, into artificial membranes allows Intracellular Analysis of Individual Cells and Organelles for Both Oxygen for fine tuning of membrane physical properties, which has not been possible Concentration/Consumption and NADH Free/Bound Redox State using using natural biological molecules alone. Here, we use a cell free protein Fluorescence Lifetime Imaging expression system to characterize how membrane properties impact mem- Rozhin Penjweini1, Alessio Andreoni1, Anahit Gevorgyan1, Dan L. Sackett2, brane protein folding using a model mechanosensitive channel protein, the Jay R. Knutson1. mechanosensitive channel of large conductance (MscL). Folding studies 1NHLBI, NIH, Bethesda, MD, USA, 2NICHD NIH, Bethesda, MD, USA. were conducted by expressing MscL in the presence of small unilamellar Measuring the oxygen consumption and linked metabolic pathways in cancer phospholipid vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocho- cells, especially their covariant and/or compensatory interplay, is important line (DOPC). Protein folding efficiency and total production were measured for understanding transformation mechanisms, mechanisms of malignant by monitoring the fluorescence from a GFP C-terminal fusion protein and growth, cancer cell drug resistance, and for discovering potential drugs quantitative western blots. We first observed that increasing the total concen- specific for cancer cells. Fluorescence lifetime imaging (FLIM) already of- tration of vesicles available in a cell-free reaction increased the efficiency fers a sensitive and non-invasive probe of the metabolically critical free/ and yield of protein folding. We then blended the highly elastic diblock enzyme-bound states of intracellular NADH and FAD. FLIM can also be copolymer, poly(ethylene oxide)-b-poly(butadiene), into phospholipid mem- used in combination with our novel Fo¨rster resonance energy transfer branes and observed the folding and total production of MscL improved (FRET)-based oxygen sensing probe, Myoglobin-mCherry (Myo-mCherry). beyond folding observed in pure phospholipid membranes. Finally, we This allows real-time, non-invasive monitoring of the intracellular (or demonstrated this effect is reproducible with a different membrane composi- tion, by incorporating an alternate molecule that has been known to make even organelle-specific) oxygen concentration ([O2]). Herein, we demon- strate that the response of NADH to the metabolic transition from normoxia membranes more elastic and observing enhanced protein folding with respect to hypoxia is more complex than a simple increase in NADH concentration to pure lipid membranes. Overall, these results indicate that membrane phys- ical properties - such as membrane surface area and elasticity - play a role in ([NADH]). These contemporaneous intracellular measurements of both [O2] and the changes in metabolic redox ratio ([NADH]/[FAD]), via NADH the spontaneous folding and expression of MscL in a cell free reaction. conformation and environment, provides a foundation for quantitative func- Moreover, our results introduce how non-natural amphiphiles may be used tional imaging of cancer energy metabolism and can shed light on the mech- to uncover the impact of physical properties of membranes on membrane anisms of adaptive responses to hypoxia - or some common physiological protein behaviors. and disease states. 132-Plat 130-Plat An Inner Activation Gate Controls TMEM16F Phospholipids Scrambling 3D Imaging of Single Cells in Bacterial Biofilms using Lattice Light-Sheet Trieu Le1, ZhiGuang Jia2, Yang Zhang1, Son C. Le1, Jianhan Chen3, Microscopy Huanghe Yang1. Mingxing Zhang1, Ji Zhang1, Jie Wang2, Alecia M. Achimovich3, 1Biochemistry, Duke University, Durham, NC, USA, 2Chemistry, Univ Arslan A. Aziz4, Jacqueline Corbitt1, Scott T. Acton2,4, Massachusetts Amherst, Amherst, MA, USA, 3Chemistry/Biochem & Andreas Gahlmann1,3. Molecular Biology, Univ Massachusetts, Amherst, MA, USA. þ 1Dept Chemistry, Univ Virginia, Charlottesville, VA, USA, 2Dept Electrical TMEM16F is an enigmatic Ca2 -activated phospholipid scramblase (CaPL- and Computer Engineering, Univ Virginia, Charlottesville, VA, USA, 3Dept Sase) that passively transports phospholipids down their chemical gradients

BPJ 9279_9291 26a Sunday, March 3, 2019 and mediates blood coagulation, bone development and viral infection. Despite 135-Plat D the recent advances in understanding the structure-function of the fungal Cryo-EM Structures Reveal Bilayer Remodeling during Ca2 Activation TMEM16-CaPLSases, how mammalian CaPLSases open and close, or gate of a TMEM16 Scramblase their phospholipid permeation pathways remains unclear. Through functional Maria Falzone1, Jan Rheinberger1, Byoung-Cheol Lee1, Thasin Peyear1, and computational approaches, here we identified TMEM16F-CaPLSase’s in- Linda Sasset1, Ashleigh Raczkowski2, Edward Eng2, Annarita Di Lorenzo1, ner activation gate. Disrupting the inner gate residues dramatically altered Olaf Anderson1, Crina Nimigean1, Alessio Accardi1. TMEM16F phospholipid permeation. Interestingly, a gain-of-function muta- 1Weill Cornell Medicine, New York, NY, USA, 2Simons Electron tion at the inner activation gate not only made TMEM16F CaPLSase constitu- Microscopy Center, New York, NY, USA. tively open, but also converted TMEM16A Ca2þ-activated Cl- channel (CaCC) The plasma membranes of eukaryotic cells are organized asymmetrically to a CaPLSase. A gating model is proposed to describe the Ca2þ-dependent for cellular function. At rest, polar and charged lipids are sequestered to gating mechanism of TMEM16 CaPLSases. the inner leaflet by the activity of ATP-driven pumps. Activation of a specialized class of membrane proteins – phospholipid scramblases – 133-Plat causes rapid collapse of this asymmetry and externalization of negatively Prediction of the Closed Conformation and Insights into the Mechanism of charged phosphatidylserine molecules. As a result, extracellular signaling the Membrane Enzyme LpxR networks, controlling processes such as apoptosis, blood coagulation, mem- Graham M. Saunders, Hannah E. Bruce Macdonald, Jonathan W. Essex, brane fusion and repair, are activated. The TMEM16 family of membrane - Syma Khalid. proteins includes phospholipid scramblases and Cl channels, all of which 2þ Chemistry, University of Southampton, Southampton, United Kingdom. are Ca -dependent. Prior structural and functional analyses of the fungal Covalent modification of outer membrane lipids of Gram-negative bacteria can TMEM16 scramblase from Nectria haematococca identified a membrane- impact the ability of the bacterium to develop resistance to antibiotics as well as exposed hydrophilic groove that serves as the lipid translocation pathway. modulating the immune response of the host. The enzyme LpxR from Salmo- In the TMEM16A channel, this pathway is sealed from the membrane, thus nella typhimurium is known to deacylate lipopolysaccharide molecules of the preventing lipid access and enabling ion permeation. However, the mecha- 2þ outer membrane, however the mechanism of action is unknown. Here we nisms underlying Ca -dependent gating of TMEM16 scramblases, and employ molecular dynamics and Monte Carlo simulations to study the confor- their effects on the surrounding lipid bilayer, remain poorly understood. mational dynamics and substrate binding of LpxR in representative outer mem- Here we describe three high-resolution cryo-electron microscopy structures brane models and also detergent micelles. We examine the roles of conserved of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconsti- 2þ 2þ residues and provide an understanding of how LpxR binds its substrate. Our tuted in lipid nanodiscs. Differences between the Ca -free and Ca - 2þ simulations predict that the catalytic H122 must be N -protonated3 for a single bound states reveal that Ca binding induces a global rearrangement of water molecule to occupy the space between it and the scissile bond, with a free the transmembrane and cytosolic regions, which causes the lipid pathway binding energy of 8.5 kcal mol1. Furthermore, simulations of the protein to open. Further, comparison of these structures and a third in the presence within a micelle enable us to predict the structure of the putative ‘closed’ pro- of a long-chain inhibitory lipid, together with functional experiments, tein. Our results highlight the need for including dynamics, a representative reveal that scramblases cause profound remodeling of the surrounding environment and the consideration of multiple tautomeric and rotameric states membrane. Specifically, scramblase activation causes the bilayer to thin of key residues in mechanistic studies; static structures alone do not tell the full at the open lipid pathway and the outer leaflet to bend towards the center story. of the bilayer at the dimer interface. We propose a model in which trans- bilayer lipid movement is enabled by these membrane and protein rear- 134-Plat rangements and provide a general mechanistic framework for phospholipid Studying Conformation of the Voltage-Sensor Domain (VSD) of the scrambling. Human KCNQ1 Potassium Ion Channel in Proteoliposomes using EPR Spectroscopy 136-Plat Indra D. Sahu1, Gunjan Dixit2, Warren Reynolds1, Ben Harding1, Uncovering Eukaryotic Glycosylation Mechanism by Cryo-EM Colleen Jaycox1, Fathima Dilhani Mohammed Faleel1, Lin Bai, Huilin Li. Robert M. McCarrick1, Charles R. Sanders3, Gary A. Lorigan1. Van Andel Res Inst, Grand Rapids, MI, USA. 1Dept Chem/Biochem, Miami Univ, Oxford, OH, USA, 2Miami University, Glycosylation is the most dominant modification of eukaryotic secretory and Oxford, OH, USA, 3Department of Biochemistry and Center for Structural membrane-bound proteins. It mainly occur in endoplasmic reticulum (ER) Biology, Vanderbilt University, Nashville, TN, USA. and Golgi. Glycosylation plays important role in many essential biological KCNQ1 is a voltage-gated potassium channel modulated by members of the processes, such as intercellular recognition, protein folding and stability, KCNE protein family. KCNQ1 is involved in the cardiac repolarization phase protein-protein or protein-ligand interactions, and many enzyme activities. of the heart beat and Kþ homeostasis in the inner ear. Dysfunction of KCNQ1 We investigated the structural mechanism of eukaryotic glycosylation in causes several diseases including several cardiac arrhythmias, congenital ER using cryo-EM. One example is the oligosaccharyltransferase complex, deafness, and type II diabetes mellitus. Human KCNQ1 is a 676-residue pro- OST, which is the key enzyme in N-glycosylation. We report a 3.5-A˚ reso- tein consisting of 100-residue N-terminal cytosolic domain, followed by an lution cryo-EM structure of the Saccharomyces cerevisiae OST, revealing 260-residue channel domain containing six transmembrane (TMD) helices, the assembly of its eight subunits. We found that seven phospholipids and a 300-residue cytosolic C-terminus. The first four TMD helices (S1-S4) mediate many of the inter-subunit interactions, and an Stt3 N-glycan medi- form the voltage-sensor domain (Q1-VSD) that is linked to the pore domain ates interactions with Wbp1 and Swp1 in the lumen. Ost3 was found to (helices S5 and S6). Roughly 40% of the >200 reported disease-related mu- mediate the OST-Sec61 translocon interface. Our studies provide novel in- tations in the KCNQ1 gene result in amino acid substitutions in the VSD, sights into co-translational protein N-glycosylation and some other functions making structural and dynamic studies of this domain important in unraveling of glycosylation in eukaryotic cell. molecular mechanisms in human pathophysiology. Q1-VSD is an indepen- dent structural and functional unit maintaining the structural conformation 137-Plat and functional properties in a similar manner to the full-length channel Folding Mechanism of b-Helical Passenger Domains from a Bacterial (VSD þ pore domain). In this study, CW-EPR and DEER distance measure- Autotransporter ments were performed on several dual spin labeled Q1-VSD constructs in Anthony Hazel, Yui Tik Pang, James C. Gumbart. several membrane environments (micelles, bicelles, liposomes, and lipodisq School of Physics, Georgia Institute of Technology, Atlanta, GA, USA. nanoparticles) to compare structural and conformational dynamics of Q1- Virulence factors, small peptides attached to larger autotransporter proteins, are VSD. Our data indicated that the secondary structure of Q1-VSD is similar a common source of virulence in infectious, Gram-negative bacteria and a new in micelle and lipid bilayers, while tertiary folding of Q1-VSD changes in focus for antimicrobial drug development. While the transportation of the viru- lipid bilayers when compared to micelles. The DEER distance restraints lence factor across the inner membrane is well-studied, the mechanism of how were further utilized to obtain a refined structural model of Q1-VSD in lipid these peptides are secreted through the outer membrane remains unclear due to bilayers using simulated annealing molecular dynamics simulation. Our re- the absence of traditional energy sources, such as ATP or ion gradients, at the sults are consistent with the previously published EM and NMR studies. outer membrane. Interestingly, many of these proteins come attached to large This study will provide important mechanistic information on the Q1-VSD b-helical passenger domains, which do not appear to be participating in any channel upon binding with KCNE1. of their virulence behavior. In addition, folding of the b-helix occurs

BPJ 9279_9291 Sunday, March 3, 2019 27a significantly faster in vivo than in vitro, so the passenger domain likely folds 140-Plat along a faster, vectorial pathway as its being secreted through the outer mem- Atomic Level Characterization of an Ensemble of Amyloid Beta Oligomers brane. Experimental results show that the relative stability at the N- and C-ter- Cecily K. Campbell-Bezat1, Albert C. Pan1, Daniel Jacobson1, minus of the passenger domain largely affects the secretion rate of these Shivam Verma1, David E. Shaw1,2. proteins, suggesting that the folding of the b-helix structure plays an important 1D E Shaw Research, New York, NY, USA, 2Biochemistry and Molecular role in efficient secretion. In this study, we used three different simulation Biophysics, Columbia University, New York, NY, USA. methods to investigate the folding mechanism and kinetics of the passenger Many neurodegenerative diseases, such as Alzheimer’s, are associated with domain of Pertactin, an autotransporter from Bordetella pertussis. Multidimen- the cytotoxic effects of the low molecular weight oligomers and fibrillar ag- sional replica-exchange umbrella sampling simulations reveal how cooperative gregates of amyloid proteins within the central nervous system. While recent folding beginning at the C-terminus enhances the kinetics of folding, while experimental work has afforded a detailed characterization of the structures steered molecular dynamics simulations show differences in mechanical re- and growth mechanisms of fibrils, a similarly detailed characterization of sponses at the N- and C-terminus. Lastly, we use Markov state modelling to the formation of amyloid oligomers has not been possible due to their tran- find intermediate folding states as well as possible misfolded structures, which sient and heterogeneous nature. Here, using long timescale molecular dy- may act as kinetic traps. namics simulations with simulated tempering, we have observed the reversible formation of an ensemble of oligomeric complexes comprised of 138-Plat a C-terminal segment of amyloid beta (Ab), the amyloid protein associated Monitoring Rotation Dynamics of Membrane Protein in Live Cells with Alzheimer’s disease. The most stable oligomeric complex corresponds Youngchan Park1, Sangwon Shin2, Hyeonggyu Jin2, Jiseong Park2, closely to the hexameric cylindrin conformation seen in a recent crystal struc- Yeonki Hong2, Hyunjoon Song1, Daeha Seo2. ture of a toxic ab crystallin (ABC) amyloid oligomer. Similar to the ABC cy- 1Chemistry, Korea Advanced Institute of Science and Technology, Daejeon, b 2 lindrin, the A cylindrin structures observed in our simulations, composed Republic of Korea, Daegu Gyeongbuk Institute of Science and Technology, primarily of anti-parallel b-sheets, are rich in dangling hydrogen bonds, a Daegu, Republic of Korea. feature associated with toxicity in amyloid oligomers. The simulations addi- Dynamic behavior of membrane protein provides critical information in molec- tionally suggest that the energy landscape of the Ab cylindrin is characterized ular and cellular mechanisms. To have access to the mobility of a membrane by deep, metastable wells. Our results are consistent with experimental data, protein, single-particle tracking has been advanced for the microscopic mech- including Ab hexamer cross section measurements determined by ion anism understandings. Among various molecular motions, however, only the mobility mass spectrometry. lateral motion of the protein has been monitored due to the lack of in situ im- aging tool enabling observation for rotation and vibration. Here, we developed 141-Plat plasmonic nanoparticles which can monitor rotational diffusion dynamics as Ordered and Disordered Segments of Amyloid Beta Drive Sequential Steps well as lateral motion. This nanoparticle probe allows direct evidence and of the Toxic Pathway quantitative analysis of rotation dynamics, and furthermore, observation of Barun K. Maity1, Anand Kant Das2, Simli Dey1, Ullhas Kaarthi Moorthi3, conformation changes of proteins and the protein-protein interactions in live Amandeep Kaur3, Dayana Surendran1, Rucha Pandit4, Mamata Kallianpur1, cells. This study provides an insight into the molecular mechanism regarding Bappaditya Chandra5, Murlidharan Chandrakesan1, Senthil Arumugam6, the intracellular signaling process. Sudipta Maiti1. 1Department of Chemical Science, Tata Institute of Fundamental Research, Mumbai, India, 2Institute for Applied Physics - Biophysics Group, Vienna Platform: Intrinsically Disordered Proteins (IDP) University of Technology, Vienna, Austria, 3EMBL Australia Node in and Aggregates I Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, Australia, 4Clem Jones Centre for Ageing 139-Plat Dementia Research, Queensland Brain Institute, Queensland, Proximity Rulers in Amyloids and Liquid Droplets of Intrinsically Australia, 5St. Jude Children’s Research Hospital, Memphis, Disordered Proteins TN, USA, 6ARC Centre of Excellence in Advanced Molecular Anupa Majumdar1, Debapriya Das2, Priyanka Dogra2, Shiny Maity2, Imaging, Sydney, Australia. Samrat Mukhopadhyay3. The mechanisms of toxicity of disease-causing disordered peptides, such as 1Department of Biological Sciences, Indian Institute of Science Education Alzheimer’s disease (AD) associated amyloid beta (Ab), remain poorly un- and Research (IISER) Mohali, Mohali, India, 2Department of Chemical derstood. Ab oligomers are highly toxic partially structured peptide assem- Sciences, Indian Institute of Science Education and Research (IISER) blies with a distinct ordered region (residues 10-40) and a shorter Mohali, Mohali, India, 3Department of Biological and Chemical Sciences, disordered region (residues 1-9), which offer an opportunity to probe the Indian Institute of Science Education and Research (IISER) Mohali, Mohali, role of disorder in toxicity. We probe the interactions of an array of Ab frag- India. ments with lipid membranes and cultured neurons, using the techniques of Pathological amyloid deposits of two intrinsically disordered proteins (IDPs) confocal imaging, lattice light sheet imaging, fluorescence lifetime imaging, namely, a-synuclein (a-syn) and tau, are found in the intracellular Lewy and fluorescence correlation spectroscopy (FCS). Remarkably, we find that bodies in Parkinson’s disease and neurofibrillary tangles in Alzheimer’s dis- neither part (Ab10-40 or Ab1-9) is toxic by itself. The ordered part (Ab10-40) ease, respectively. The molecular mechanism underlying the transition of is the major determinant of how Ab attaches to lipid bilayers, enters neuronal these two IDPs from their soluble monomeric to their aggregated amyloid cells, and localizes in the late endosomal compartments. The disordered N- forms remains elusive. Here, we embarked upon studies aimed at gaining in- terminal part plays a minor role, but it helps increase cellular entry and sights into the proximity between different residue positions within the am- slightly reduces the level of endosomal localization. However once it enters yloid fibrils of full-length human a-syn. Our findings revealed that the the cell, the disordered part (only when it is a part of the full length peptide) decrease in the steady-state fluorescence anisotropy values associated with has a strong interaction with an unknown cellular component. This interaction these amyloid fibrils could be correlated well to the extent of site-specific appears to divert Ab to the toxic pathway. Our findings correlate well with energy migration via homo-Fo¨rster resonance energy transfer between the Ab sites of familial AD mutations. We conclude that while the required steps proximal residues bearing identical fluorophores. Furthermore, site-specific of membrane attachment and cellular entry are dictated by the ordered region, picosecond time-resolved fluorescence anisotropy measurements allowed the key to toxicity lies in the intracellular interactions of the disordered N-ter- us to quantify the rate of this energy migration, which in turn revealed the minal of Ab. intermolecular proximity within the amyloid architecture. We utilized this distance-dependent readout to construct the site-specific proximity map 142-Plat that illuminates the structural details of the amyloid fibril core of a-syn. I Understanding the Molecular Parameters Determining the Pathological will also discuss our recent results on liquid droplets of tau, which has Properties of Amyloid Fibrils recently been found to phase separate into protein-rich liquid-like droplets Harish Kumar, Jayant B. Udgaonkar. that can mediate the formation of amyloid aggregates. Our preliminary re- National Centre for Biological Sciences, Tata Institute of Fundamental sults revealed the changes in the conformational dynamics as well as in Research, Bangalore, India. the inter-residue proximities within the polypeptide backbone of tau Transmission of amyloid fibrils in the brain via a prion-like mechanism is a ma- upon the transition of monomeric protein into phase-separated liquid-like jor feature of various neurodegenerative diseases. In this context, it is crucial to droplets. understand the mechanism by which amyloid fibrils of tau can act to seed the

BPJ 9279_9291 28a Sunday, March 3, 2019 growth of fibrils from native tau. During fibril growth native protein is con- regions (IDRs). Acidic IDR has ten phosphorylation sites, and they are fully verted into multiple misfolded aggregated conformations, each being a strain phosphorylated when FACT expressed in the cell. The hyperphosphorylated that propagates faithfully and which is linked to a specific disease. An under- FACT does not bind to the nucleosome, while the non-phosphorylated form standing of the molecular mechanism of template-driven conversion of native shows the binding. FACT binding ability recovers in a sigmoidal manner ac- soluble protein into structurally different type of fibrils will lead to a better un- cording to the number of phosphates in the acidic IDR, which resembles the derstanding in general of molecular parameters which determine the patholog- ‘ultrasensitive’ response previously found for the phosphorylation-dependent ical properties of amyloid fibrils in various neurodegenerative diseases, and change in the interaction between Cdc4 and Sic1 (Mittag et al. PNAS will help in development of therapeutics for various protein aggregation dis- (2008)). eases. In this study, the molecular mechanism of template-driven growth of This work demonstrates the ultrasensitive response of FACT occurs the tau fibrils is shown to be describable by a simple Michaelis-Menten-like through the intramolecular interaction between the acidic and basic IDRs two-step model, in which monomers act like the substrate, and fibrils act like in the DNA binding domain of FACT. The ultrasensitive response of the enzyme. In the first step, monomer attaches to the ends of fibrils, and in FACT, however, does not rely on a large ensemble of the IDR-containing the second rate limiting step, the conformational transition takes place. complex as in the Cdc4-Sic1 interaction, but it requires rather restricted Modeling tau fibril growth to such a simple model provides a mechanistic ratio- conformational dynamics in the interaction between acidic and basic nale for the asymmetric seeding barrier which exists between two isoforms of IDRs. With the finding, we propose the other mechanism for the IDR- the tau protein. Importantly, tau-4R and 3R are found to form structurally mediating ultrasensitivity, as an additional function associated explicitly different fibrils: the structural core is more ordered in tau-3R fibrils than in with the IDR. tau-4R fibrils. These results provide a structural rationale forthe asymmetric seeding barrier. Hence, the current study provides a detailed understanding 145-Plat of the molecular parameters which determine the pathological properties of am- Targeting the Formation of Amyloid Oligomers using Rationally Designed yloid fibrils. Antibodies 1 1 1 2 143-Plat Francesco A. Aprile , Pietro Sormanni , Michele Perni , Paolo Arosio , 3 4 4 Redox Kinetics of the Amyloid-Beta-Copper Complex and Its Biological Sara Linse , Tuomas P. Knowles , Christopher M. Dobson , Michele Vendruscolo1. Implications 1 1 2 2 3 Dept Chemistry, Univ Cambridge, Cambridge, United Kingdom, Paul Girvan , Xiangyu Teng , Nicholas J. Brooks , Geoffrey S. Baldwin , 2 3 1 Chemistry and Applied Bioscience, ETH Zurich, Zurich, Switzerland, Dept Liming Ying . 4 1National Heart and Lung Institute, Imperial College London, London, Biophys Chem, Lund Univ, Lund, Sweden, Dept of Chemistry, Univ United Kingdom, 2Department of Chemistry, Imperial College London, Cambridge, Cambridge, United Kingdom. London, United Kingdom, 3Department of Life Sciences, Imperial College Antibodies are extensively used in biomedical research, as a result of their London, London, United Kingdom. strong binding affinities and high specificity. Our goal is to develop anti- Since the redox cycling of the Ab-Cu complex is implicated in the progres- bodies as research and diagnostic tools, and therapeutic molecules against sion of AD, the mechanism and kinetics of the reduction and oxidation steps pathological protein aggregation. In particular, the aggregation in the brain are of particular interest. Due to the differences between the major Cu(I/II) of normally soluble proteins into amyloid fibrils is often associated with neu- species coordinating environments, direct electron transfer between them re- rodegeneration. During this process, small soluble and highly neurotoxic quires a significant ligand rearrangement. Electrochemical studies have oligomers are formed. These oligomers play a major role in the onset and demonstrated that this is unfeasible, and instead proposed that the redox progression of the disease but they are also highly instable and difficult to goes through a ‘catalytic in-between state’ where the ligands are better ar- isolate with standard approaches. For this reason, a comprehensive under- ranged to facilitate fast redox cycling. We have previously reported the use standing of their mechanisms of formation is still missing. To address this of a fluorescence-based kinetic approach for studying the interactions of Ab issue, we developed a sequence-activity relationship approach to identify re- and Cu(II), which is highly sensitive to the copper environment. Now we gions of amyloidogenic proteins, which are important for the formation of describe the use of our kinetic approach to verify the presence of the lowly oligomers. This approach relies on an innovative antibody-discovery method populated, reactive intermediate state via a completely orthogonal technique. called ‘antibody scanning’ to readily produce a small panel of computation- Finally, we quantify the kinetics of the redox system to address whether Ab- ally designed antibodies that ‘scan’ a complete series of epitopes along a Cu redox occurs on a biologically relevant timescale, therefore being relevant target protein to obtain the full coverage of its sequence. We applied this b b to the progression of AD - an issue which has currently received little method to generate a pool of five antibodies against the amyloid- (A )pep- attention. tide, whose aggregation into amyloid fibrils is a hallmark of Alzheimer’s dis- ease (AD). Then, we screened these antibodies for their ability to inhibit the 144-Plat microscopic steps associated with the formation of oligomers and identified Multiple-Phosphorylation to IDR in the Chromatin Remodeler Fact Shows one affecting early oligomer formation (epitope 18-25) and another affecting an ‘Ultrasensitive’ Response in Its Nucleosome Binding oligomer propagation (epitope 29-36). Both of these antibodies were able to Shin-ichi Tate. rescue a C. elegans model of AD overexpressing Ab. Our results provide a Dept Math/Life Sci, Hiroshima Univ, Higashi Hiroshima, Japan. first step forward towards the development of a methodology to identify pro- The DNA binding domain of the chromatin remodeler FACT (FAcilitate tein regions, which are relevant for pathological processes, such as the pro- Chromatin Transcription) comprises acidic and basic intrinsically disordered duction of oligomers.

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Platform: Cardiac Muscle Mechanics, Structure, tropomyosin are dominated by its dimeric coiled-coil region and that the main function of overlapping domain is to sponsor tropomyosin polymeriza- and Regulation I tion and cable formation.

146-Plat 148-Plat Advanced Morpho-Functional Analysis on Ventricular and Atrial Tissue Protein Kinase C-Mediated Cardiac Troponin I S43/45 Phosphorylation Reveals Cross-Bridge Kinetics Alterations and Sarcomere Energetic Causes Contractile Dysfunction in Human Heart Failure and in Rodents Impairment in Hcm Patients Vani S. Ravichandran1, Tabea M. Schatz1, Margaret V. Westfall2. 1 Giulia Vitale1, Erica Lazzeri2, Irene Costantini2, Francesco Giardini2, Cellular and Molecular Biology, The University of Michigan, Ann Arbor, 2 Giacomo Mazzamuto2, Claudia Crocini2, Nicoletta Piroddi1, MI, USA, Dept Cardiac Surg, Univ Michigan, Ann Arbor, MI, USA. Beatrice Scellini1, Manuel J. Pioner1, Cecilia Ferrantini1, Chiara Tesi1, Cardiac dysfunction is linked to increased protein kinase C (PKC) expression, Francesco S. Pavone2, Leonardo Sacconi2, Corrado Poggesi1. but the role of downstream myofilament phosphorylation is debated. The cur- 1Univ Florence, Florence, Italy, 2LENS, Sesto Fiorentino, Italy. rent experiments evaluate the contribution of PKC-mediated cardiac troponin Mutations in cardiac myosin-binding protein-C (cMyBP-C), are the most com- I (cTnI) S43/45 phosphorylation (p-S43/45) to contractile dysfunction. West- mon cause of Hypertrophic CardioMyopathy (HCM). The E258K-cMyBP-C ern analysis showed increased cTnI p-S43 and PKC expression in failing hu- is a penetrant missense mutation with poorly understood molecular mecha- man myocardium, and hearts from a rat pressure overload (PO) model. nisms. Mechanics and kinetics of contraction as well the energy cost of ten- Myocyte contractile dysfunction was also present in failing (F) versus non- sion generation were investigated using left ventricular (LV) and atrial failing (NF) human myocytes, and in PO versus sham rat myocytes. Contrac- tissue from E258K HCM patients and donor hearts. Kinetics of tension gener- tile function and cTnI p-S43 levels were partially restored in F human ation and relaxation were measured in LV and atrial myofibrils while ATPase myocytes after ventricular assist device support, and gene transfer of domi- and isometric active tension were simultaneously measured in permeabilized nant negative PKC-alpha. Based on these findings, transgenic mice were LV and atrial strips. The rate of tension generation following maximal generated with a range of cardiac-specific phospho-mimetic cTnI S43/45D Ca2þ-activation was faster in both LV and atrial E258K myofibrils compared (SD) replacing endogenous cTnI, to directly test the contribution of p-S43/ to donors. The rate of isometric relaxation was also faster in E258K myofi- 45 to cardiac dysfunction and remodeling. In young adults (4 wks), high brils, suggesting faster cross-bridge detachment and increased energy cost (HE-), medium (ME-) and low (LE-) SD expression did not cause significant of tension generation. Direct measurements in skinned LV and atrial strips changes in cardiac morphology/remodeling compared to non-transgenic (ntg) confirmed that tension cost was higher in E258K preparations compared to littermates. By 8 wks, HE-SD mice developed increased heart weight/tibial controls. To check whether cardiomyocyte disarray, typical of HCM hearts, length ratio which continued to increase at 12 wks compared to LE-, ME- may have contributed to artificially increase the tension cost measured in SD and ntg mice. Echocardiography showed progressive increases in left ven- the HCM preparations, the strips used for mechanical investigations were clar- tricular systolic dimension (LVDs) and decreases in ejection fraction between ified, immunostained and imaged at mesoscale level. An advanced tissue 8 and 12 wks in ME- and HE-SD mice (8 wks: ntg EF% = 54.9þ1.7, n=4, clearing method in combination with two-photon microscopy was employed ME-SD = 38þ1.3*, n=3; HE-SD = 27.1þ 5.9* n=3; 12 wks: ntg EF% = to reconstruct the 3D image of the strips at sub-micrometer spatial resolution. 52.8þ4.6, n=3; ME-SD = 35.1þ3.3*, n=5; HE-SD = 16.7þ6.2*, n=3; A 3D cytoarchitecture analysis tool based on 3D Fourier Transform was *p<0.05 by ANOVA), and 95% of HE-SD mice died before 20 wks of developed and applied to determine cardiomyocyte orientation across and age. Taken together, the results show that chronic phosphorylation of cTnI along the strips. Both global and local statistics of spatial disarray were S43/45 is a significant contributor to progressive cardiac dysfunction and heart derived and correlated to mechanical and energetic data. The results did not failure. highlight structural differences between donor and HCM strips strengthening the conclusion that the E258K mutation primarily alters apparent cross-bridge 149-Plat kinetics and impairs sarcomere energetics. Acknowledgement: H2020EU Beta-Myosin Heavy Chain Post-Translational Modifications in Failing and SILICOFCM grant agreement 777204. Non-Failing Human Hearts Michelle S. Parvatiyar1, Rakesh K. Singh2, Elizabeth A. Brundage3, 147-Plat Bryan A. Whitson4, Paul M.L. Janssen3, Brandon J. Biesiadecki3, Tropomyosin Cable Formation and Its Influence on the Structural J. Renato Pinto2. Dynamics of Tropomyosin 1Dept. of Nutrition, Food and Exercise Sciences, The Florida State Farooq A. Kiani1, William Lehman1, Stefan Fischer2, University, Tallahassee, FL, USA, 2Dept. of Biomedical Sciences, College of Michael J. Rynkiewicz1. Medicine, The Florida State University, Tallahassee, FL, USA, 3Dept. of 1Dept Physiology & Biophysics, Boston University School Medicine, Physiology and Cell Biology, College of Medicine, The Ohio State Boston, MA, USA, 2Interdisciplinary Center for Scientific Computing (IWR), University, Columbus, OH, USA, 4Dept. of Surgery, College of Medicine, University Heidelberg, Heidelberg, Germany. The Ohio State University, Columbus, OH, USA. Head-to-tail association of coiled-coil tropomyosin dimers produce four-helix Very little is known about post-translational modifications (PTM) of myosin bundle ‘‘overlapping domains’’ connecting the dimers together. This polymer- heavy chain (MHC) in the human heart. Protein acetylation is a PTM, shown ization process yields elongated super-helical cables that interact continuously of instrumental importance as a modification of histone proteins. More recently with the surface of the actin filament. While considerable computationally it has been uncovered that the realm of lysine-acetylation extends beyond the generated information is available on the local and global interactions between nucleus. Protein phosphorylation is a well-known PTM found on several sar- isolated (unpolymerized) tropomyosin dimers and F-actin (Li et al., 2011), lit- comeric proteins in the heart and is crucial for fine-tuning myocardial contrac- tle is known about the influence of the overlapping domain on the structural- tility. To our knowledge, we were first to report alpha-MHC phosphorylation mechanics of tropomyosin and hence corresponding tropomyosin behavior on in murine hearts. Given the emergent importance of the broader lysine- actin filaments. In an effort to elucidate localized overlapping domain - actin acetylome and sarcomeric protein phosphorylation during heart failure, we interaction and ensuing regulatory translocation across actin, we compared examined PTMs in human heart beta-MHC. Here, using bottom-up prote- molecular dynamics simulations of models of single dimeric tropomyosin mol- omics, we identified six high-confidence lysine acetylation sites (three each ecules (as in Li et al., 2010) with new models of tropomyosin cables. The sim- in myosin-S1 and rod domains) and eight high-confidence phosphorylation ulations of tropomyosin cables were performed with the tropomyosin polymer sites (six in myosin-S1 and two in myosin rod regions). Furthermore, the acet- constrained to a native super-helical configuration as if linked to a ghost ylation and phosphorylation status of some of these sites changed in ischemic F-actin filament at a radius of 39 A˚ , thereby maximizing native flexural failing (IF) and non-ischemic failing (NIF) hearts compared to donor non- and twisting motions without being dampened by interaction with F-actin. failing human hearts. Using label-free quantification, we found reduced acety- We have recently shown that torsional motions and sequence-dependent lation of four lysine residues in IF and NIF human hearts. However, in IF twisting of tropomyosin is initiated and propagated from aspartate-137 and human hearts differential acetylation was apparent, with two lysine residues surrounding amino acids and is critical for tropomyosin function (Lehman et in myosin-S1 exhibiting enhanced acetylation. Analysis of the phosphorylation al., 2018). We now have shown that the sequence-specific twisting pattern sites is currently in progress. Additionally, we identified a region in the of tropomyosin is virtually unaffected by the additional presence of the over- myosin-S1 ATP-binding pocket that exhibits both acetylated and phosphory- lapping domain, while the flexural motions of tropomyosin are dampened as lated residues, suggesting the importance of specific changes in the amino expected. In addition, related MD shows that torsional (and flexural) mobility acid architecture in this region. It will be of interest to determine: (i) the func- of the overlapping domain itself appears to be unaffected by the presence of tional role of these PTM sites; and (ii) whether altered acetylation of beta- the remainder of the molecule. We conclude that the material properties of MHC aberrantly effects human heart function and in turn contributes to

BPJ 9292_9302 30a Sunday, March 3, 2019 impaired function and the progression to heart failure of both ischemic and 152-Plat non-ischemic origins. Leveraging Natural Cardiomyocyte Variability to Discover Novel Genes in Canonical Signaling Pathways 150-Plat Jeffery A. Clark, Jonathan D. Weiss, Stuart G. Campbell. A Familial Dilated Cardiomyopathy Mutation Decreases Myosin Gener- Biomedical Engineering, Yale University, New Haven, CT, USA. ated Tension at the Molecular Level and Alters Mechanosensing at the Biological samples are inherently variable, probably due to stochastic pro- Cellular Level cesses that drive diversity in gene expression. Typically, cell-to-cell variation Sarah R. Clippinger1, Paige E. Cloonan2, Lina Greenberg1, William Stump3, makes experiments more complex and difficult to interpret, leading most inves- Michael J. Greenberg3. tigators to average out phenotypic diversity by measuring responses in 1Biochemistry & Biophysics, Washington Univ, St. Louis, MO, USA, numerous cells. Having carefully documented the inherent heterogeneity in 2Boston University, Boston, MA, USA, 3Dept Biochemistry, Washington mechanical function of individual isolated adult rat ventricular cardiomyocytes Univ Med Sch, St Louis, MO, USA. in previous work, we have now developed a sampling technique that takes Familial dilated cardiomyopathy is a leading cause of sudden cardiac death and advantage of cellular heterogeneity rather than ignoring it. Within a naturally heart failure in young people. DCM, which is frequently caused by mutations of heterogeneous population, cardiomyocytes exhibit varied phenotypic response sarcomeric proteins, including troponin T, is characterized by dilation of the to drugs. Correlating these varied responses with gene expression within indi- left ventricular chamber. However, it is not well understood how molecular- vidual cells could uncover novel molecular participants in signaling pathways based changes lead to alterations in cellular organization and contractility. and lead new insights into cardiac regulatory mechanisms. Here, we present a To address this gap in our knowledge, we studied the molecular and cellular novel high-throughput, low-cost single cardiomyocyte device that is capable of consequences of a mutation in troponin T that leads to DCM in human patients, testing phenotypic drug response at the single cell level on adult rat cardiomyo- DK210, and applied computational modeling to link our molecular and cellular cytes. Each cell can then be captured for single-cell gene expression analysis results. Using in vitro biochemical assays, we determined the molecular mech- and related back to the diverse phenotypic responses. Our system uses a anism of the mutation, demonstrating how it affects activation of the thin fila- high-speed sarcomere length camera to capture sarcomere shortening kinetics ment. Using a computational model (Campbell et al., 2010) and our coupled with calcium measurements through excitation of Fura-2 AM loaded measurements, it was possible to recapitulate hypocontractility seen in our into the cells. Cells are localized into micro-patterned wells allowing for motility results and make testable predictions about the transient responses rapid measurement and consistent positioning for multiple measurements of mutant sarcomeres to changes in calcium. To examine the cellular conse- over time. After phenotyping, cells can be picked up individually with a quences of the mutation, we used CRISPR/Cas9 to generate human stem cell computerized single cell aspirator and be sequenced via single cell RNA-seq derived cardiomyocytes with the DK210 mutation as a model of the early dis- to detect sarcomeric gene expression. This new method provides a rapid, ease, and we examined their structural and contractile properties on substrates low-cost screening mechanism to optimize further testing in larger-scale with different mechanical cues. We found that the structural organization of models and for discovering regulatory targets that are involved in heteroge- DK210 cells is less sensitive to the mechanical environment than WT cells. neous drug response. Moreover, consistent with our computational modeling, traction force measure- ments of single cardiomyocytes reveal that the DK210 cells generate less force 153-Plat per sarcomere; however, the cells undergo compensatory hypertrophy to Recruitment from Myosin Off State Steepens ESPVR in Finite Element normalize their force and power production. Taken together, our approach Model of Left Ventricle 1 1 2 3 was successful in bridging the molecular and cellular phenotypes, and our re- Charles K. Mann , Zhanqui Liu , Xiaoyan Zhang , Kenneth Campbell , Jonathan Wenk1. sults implicate changes in mechanosensing as an important factor in the devel- 1 opment of DCM. Mechanical Engineering, University of Kentucky, Lexington, KY, USA, 2University of California, San Diego, CA, USA, 3Physiology, University of 151-Plat Kentucky, Lexington, KY, USA. Myocardial Slices - A Novel Platform for In Vitro Biomechanical Studies Finite element (FE) modeling is becoming increasingly prevalent in the world Fotios Pitoulis, Samuel A. Watson, Eef Dries, Ifigeneia Bardi, of cardiac mechanics, however many existing FE models are phenomenolog- Raquel Nunez-Toldra, Filippo Perbellini, Cesare M. Terracciano. ical and thus do not capture cellular level mechanics. This work implements Myocardial Function, NHLI, Imperial College London, London, United a cellular level contraction scheme into existing nonlinear FE code to model Kingdom. ventricular contraction. Specifically, this contraction model incorporates three Most adult cardiac models cannot be chronically studied in vitro due to myosin states: OFF, ON, and an attached force-generating state. It has been myocardial remodelling. Myocardial slices are a novel 300mm-thick living speculated that force dependent transitions from the OFF to ON state may adult multicellular cardiac preparation with preserved structure and function. contribute to length-dependent activation at the cellular level (PMID: Culturing slices with physiological preload, by application of unidirectional 30054031). This work investigates the contribution of this OFF state to ventric- stretch, diminishes this remodelling. However, static stretch does not recapit- ular level function, specifically the Frank-Starling relationship, as seen through ulate the dynamic mechanical events of the in vivo cardiac cycle. We hypoth- the end systolic pressure-volume relationship (ESPVR). Five FE models were esized that the electromechanical events of the cardiac cycle can be simulated constructed using geometries of rat left ventricles obtained via cardiac MRI. FE on slices. simulations were conducted to optimize parameters such that the FE predicted Left ventricular slices were prepared from adult male Sprague-Dawley rats and ventricular pressures for the models were within 5% of experimentally human heart failure tissue, mounted on a force-transducer/linear actuator biore- measured LV pressures. The models were validated by comparing FE predicted actor, stretched to 2.2mm sarcomere length, and electrically stimulated at 1- and strain to experimentally determined strain. Simulations mimicking vena cava 0.5Hz respectively. A quasi-physiological cardiac cycle algorithm consisting of occlusion generated descending pressure volume loops from which ESPVRs isometric and isotonic contraction (0-, 0.5-, 1.5-, 3-, and 4.5% of slice’s resting were calculated. In simulations with the inclusion of the OFF state using length shortening), isometric relaxation and diastolic re-stretching was simu- force-dependent transitions to the ON state, the ESPVR calculated was steeper lated at control and increased contractile state (200nM norepinephrine). than in simulations excluding the OFF state. This suggests that force-dependent Force-length loops were plotted, and twitch force, kinetics, and mechanical recruitment of thick filaments from the OFF state at the cellular level contrib- work measured at different % shortening. utes to the Frank-Starling relationship observed at the organ level. In rat slices, twitch force decreased from 6.252.4 and 10.953.4 mNmm2 at 0% to 4.451.9 and 8.952.7 mNmm 2 at 4.5% shortening with or without Platform: Excitation-Contraction Coupling/ norepinephrine respectively (n=3). In human slices twitch force decreased from 17.0 mNmm2 at 0% to 11.3 Cardiac and Skeletal Muscle Electrophysiology I mNmm2 at 4.5% shortening. Mechanical work increased with increased % shortening from 0.69 mNmm1 at 0.5% to 5.3 mNmm1 at 4.5% (n=1). 154-Plat We show for the first time that a biomimetic cardiac cycle can be simulated in Oligomerization of Micropeptides that Regulate SERCA slices using a computer-controlled motorised bioreactor. Additionally, we pro- Deo R. Singh1, Ellen Cho1, Michael Dalton1, Marsha Pribadi1, vide evidence that slices respond physiologically to dynamic mechanical stim- Catherine A. Makarewich2, Eric N. Olson2, Seth L. Robia3. ulation, suggesting their suitability for biomechanical studies. As myocardial 1Cell and Molecular Physiology, Stritch School of Medicine, Maywood, IL, slices can be cultured in vitro, future studies will address the role of chronic USA, 2Department of Molecular Biology, University of Texas Southwestern biodynamic mechanical stimulation on slice’s structural and functional Medical School, Dallas, TX, USA, 3Dept Physiology, Loyola Univ Chicago, remodelling. Maywood, IL, USA.

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Newly discovered single-span transmembrane proteins endoregulin (ELN), downregulation of KATP in vascular smooth muscle cells (VSMC) reverses dwarf open reading frame (DWORF), myoregulin (MLN), and another- cardiac remodeling. This indicates that the complex CV abnormalities observed regulin (ALN) are reported to bind to the SERCA calcium pump in a manner clinically arise secondary to smooth muscle KATP GOF and hypo-excitability. similar to that of known regulators of SERCA activity, phospholamban CS mice also exhibit systemic hypotension, associated with a compensatory up- (PLB) and sarcolipin (SLN). To determine how micropeptide assembly into regulation of renin-angiotensin signaling (RAS), which we propose as the oligomers determines the availability of the micropeptide to bind to SERCA driver of cardiac hypertrophy. Finally, chronic administration of glibenclamide in a regulatory complex, we used fluorescence resonance energy transfer by slow-release pellets implanted subcutaneously in Cantu´ mice results in (FRET) and co-immunoprecipitation to measure micropeptide oligomerization normalization of vascular consequences and reversal of cardiac hypertrophy affinity and SERCA-binding affinity. All micropeptides except DWORF over 3 weeks. These results demonstrate the in vivo efficacy of glibenclamide formed avid homo-oligomers with high-order stoichiometry (n>2 protomers in targeting cardiovascular KATP channels, suggesting that the drug can be per homo-oligomer), but it was the monomeric form of all micropeptides clinically repurposed for the treatment of CS specifically - and potentially for that interacted with SERCA. Interestingly, we did not see poor SERCA- diverse cardiovascular conditions arising from decreased smooth muscle excit- binding for the most avidly oligomeric species. Instead, we observed a positive ability in general. correlation between oligomerization affinity and SERCA-binding. The data suggest micropeptide structural determinants that support oligomerization are 157-Plat also important for binding to SERCA. Moreover, the unique oligomerization/ Optogenetic Currents in Myofibroblasts Produce Acute Changes in Elec- SERCA-binding profile of DWORF is in harmony with its distinct role as a trophysiology of Cocultured Cardiomyocytes competitive activator, in contrast to the inhibitory function of the other Geran Kostecki1, Yu Shi1, Dan Reich1, Emilia Entcheva2, Leslie Tung1. SERCA-binding micropeptides. 1Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA, 2Biomedical Engineering, George Washington University, Washington, 155-Plat DC, USA. The Effect of the SK Channel Inhibitor ICAGEN in Intact Atria and Atrial After myocardial infarction, the infarcted area exhibits conduction slowing and Cardiomyocytes ectopic activity, promoting arrhythmia. This may be partly due to myofibro- Sara Dobi1, Godfrey L. Smith2. blasts that electrically connect to cardiomyocytes and depolarize them, thereby 1Institute of Cardiovascular and Medical Sciences, Glasgow, United inactivating sodium channels. In this study, we used optogenetics to induce in- Kingdom, 2Dept Cardiovas Sci, Univ Glasgow, Glasgow, United Kingdom. ward currents in myofibroblasts and found they can cause conduction slowing Small-conductance calcium-activated potassium channels (SK channels) have and spontaneous beating, and used computational modeling to determine the been suggested to mediate abnormal atrial electrophysiology in heart failure minimum cardiomyocyte-myofibroblast conductivity consistent with our and sustained atrial fibrillation. However, a mechanistic explanation of the experiments. function of SK channels in health and disease is lacking. In this study we exam- We transduced neonatal rat cardiac fibroblasts with the depolarizing opsin ined the effects of a putatively specific SK channel blocker, ICAGEN on the ChR2, and then treated them with 5 ng/mL TGF-b for >48 h to differentiate electrophysiology of atria and ventricle of rabbit hearts. Experiments were per- them into myofibroblasts (MFs). We then plated 200,000 ChR2-MFs or non- formed in isolated atrial cardiomyocytes and intact atria and ventricle of iso- transduced control MFs onto 500,000 3-day-old neonatal rat ventricular cardi- lated rabbit hearts using fluorescence based methods. Isolated Langendorff omyocyte (CM) monolayers in a 24-well plate. The co-cultures were optically perfused hearts were perfused with Di-4-ANEPPS to allow measurements of mapped with voltage-sensitive dye on day 6-8 to assess conduction velocity and atrial and ventricular action potentials (APs) optically. Addition of ICAGEN spontaneous beating. (1mM) to the perfusate caused significant prolongation of atrial AP duration Continuous application of blue light activated inward ChR2 current in MFs and at 5mins, (APD75 19.52% change þ/- 2.4 and APD90 23.90% change þ/- induced spontaneous CM beating, which immediately stopped upon cessation 6.63 ,n=4), but no change in ventricular APD (APD75 3.03% change þ/- of light. Spontaneous rate increased with brightness. Cycle length of ChR2- 1.70, APD90 2.30% change þ/-0.84, n=4). This effect was confirmed with cor- MF/CM co-cultures under 0.057 mW/mm2 light was significantly less than responding, independent, experiments in isolated atrial and ventricular cells. To that of MF/CM cultures, paced at 500 ms CL (33859 vs. 50051 ms, n=9 test the dependence of the effect on intracellular calcium, the effect of the drug vs. 13 monolayers, P=0.001). At 0.017 mW/mm2, below the spontaneous in hearts perfused for 20mins in low (0.3mM) extracellular Ca2þ was exam- beating threshold, slowing occurred in ChR2-MF/CM but not MF/CM co- ined. This manoeuvre which was designed to lower intracellular Ca2þ did cultures in a dose-dependent manner (3.751.8 vs. 0.350.6 cm/s, n=5 vs. not alter the ICAGEN-induced prolongation of APD. Similar results were 14 monolayers, P=0.003). A computational model of a 1-D strand of ChR2- observed in isolated atrial myocytes. This apparent lack of sensitivity of the MF/CM cells showed that a CM-MF conductance of >40 mS/(CM mF) was ICAGEN effect to intracellular Ca2þ is counter to the supposed Ca2þ- depen- necessary for inward current in MFs to cause the conduction slowing and spon- dence of SK channels. Further studies are required to understand the mecha- taneous beating seen experimentally. These results demonstrate that the electri- nism underlying the electrophysiological effects of ICAGEN and the SK cal coupling of myofibroblasts to cardiomyocytes is sufficiently large for channel in rabbit atrial electrophysiology. inward myofibroblast currents to affect cardiac conduction at a tissue level.

156-Plat 158-Plat The Mechanism of Cardiovascular Pathophysiology in Cantu Syndrome Blood Flow Control by ATP-Sensitive Potassium Channel in Heart and Response to Glibenclamide in Novel KATP Channel Mutant Mouse Guiling Zhao, Humberto C. Joca, W. Jonathan Lederer. Models University of Maryland School of Medicine, Baltimore, MD, USA. Conor McClenaghan, Yan Huang, Carmen Halabi, Theresa Harter, Blood flow within the heart is tightly coupled to its metabolic needs. Despite its Robert P. Mecham, Maria S. Remedi, Colin G. Nichols. importance, little is known about the way the local blood flow is regulated at the Washington Univ, St Louis, MO, USA. molecular and cellular levels. The current study has investigated the molecular Characterized by diverse features including hypertrichosis, craniofacial dys- and cellular regulatory mechanisms that control blood flow in the mammalian morphology, and edema, Cantu´ syndrome (CS) arises from gain-of-function heart using a perfused mouse cardiac papillary muscle preparation. Addition- (GOF) mutations in the genes encoding the cardiovascular KATP channel sub- ally, primary co-cultures of ventricular myocytes and capillary endothelial cells units Kir6.1 and SUR2 (KCNJ8 and ABCC9, respectively). Multiple cardiovas- have been used. Functional changes of arterioles and capillaries in heart were cular abnormalities are also reported in CS including vascular dilation and examined using high-speed confocal microscopes with a combination of XY tortuosity, dramatic cardiomegaly, pulmonary hypertension and low systemic (Nipkow disk) and point-scanning imaging. The preparations were examined blood pressure. How KATP dysfunction results in these complex abnormalities using a temperature and flow control superfusion bath and patch clamp instru- þ is not fully understood and there are no known therapies for the disorder. Here, mentation. Our results suggest that ATP-sensitive K channel (KATP) in cardi- we use novel ‘‘Cantu´ mouse’’ models, in which disease-causing SUR2[A478V] omyocytes are responsible for pinacidil and diazoxide induced vasodilation in and Kir6.1[V65M] mutations were introduced into KCNJ8 and ABCC9 using papillary muscle. Additional evidence demonstrates that the hyperpolarizing CRISPR/Cas9 genome editing, to dissect the pathophysiological mechanisms current generated by cardiomyocyte KATP can be injected into the apposed underlying CV remodeling and to test the efficacy of the KATP inhibitor gli- capillary endothelial cells, and then into arterial smooth muscle cells, leading benclamide in reversing abnormalities. Cantu´ mice exhibit decreased systemic to vasodilation, and blood flow elevation. An additional contribution to blood þ þ blood pressures and cardiac hypertrophy - recapitulating clinical manifesta- flow elevation and vascular relaxation is attributed to extracellular K ([K ]o) þ tions. We generated Cantu´ mice which also inducibly express dominant- increases resulted from KATP (and other K channels) in cardiac myocytes that negative Kit6.1 subunits specifically in smooth muscle. Following induction, lead to hyperpolarization of capillary endothelial cells through activating

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inward rectifier Kþ channels (Kir). Thus, the activation of KATP in cardiac mechanism is consistent with the delayed onset of exercise-induced weakness myocytes caused by [ATP]i drop and/or metabolites accumulation links the that occurs with rest after vigorous activity. metabolism demanding from myocytes to the increased blood flow in microves- Supported by the NIH R01-AR063182, R37-AR42703 and the MDA sels together via electrical transmission of hyperpolarizing current through (RG381149) þ gap junctions and [K ]o-induced hyperpolarization of capillary endothelial cells. 161-Plat S-Nitrosylation of Cx43 Hemichannels Promotes Cardiac Arrhythmias in 159-Plat a Duchene Muscular Dystrophy Mouse Model 1 2 2 2 Vegf-Induced Vascular Leak Promotes Atrial Fibrillation by Disrupting Mauricio A. Lillo , Eric Himelman , Lai-Hua Xie , Diego Fraidenraich , 1 Intercalated Disc Nanodomains Jorge E. Contreras . 1Pharmacology, Physiology & Neuroscience, Rutgers University, Newark, Louisa Mezache, Heather Struckman, Amara Greer-Short, Anna Phillips, 2 Alex Martinson, Justin Thomas, Przemyslaw Radwanski, Thomas J. Hund, NJ, USA, Cell Biology, Rutgers University, Newark, NJ, USA. Rengasayee Veeraraghavan. Connexin43(Cx43) facilitates conduction necessary for contraction in heart. The Ohio State University, Columbus, OH, USA. Recently, we found that Cx43 is upregulated and mislocalized from intercalated Atrial fibrillation (AF) is the most common arrhythmia and is associated with disks to lateral regions in cardiomyocytes of humans with Duchene Muscular inflammation. AF patients have elevated levels of inflammatory cytokines Dystrophy(DMD) and in DMD mouse models. Lateralized Cx43 protein forms (vascular endothelial growth factor A; VEGF-A), which promotes vascular aberrantly active, unpaired hemichannels. Blockade and reduction of Cx43 hem- b leak. This leads to cardiac oedema, which disrupts intercalated disc (ID) nano- ichannels prevents arrhythmias in DMD mice upon -adrenergic stress. More- domains and precipitates arrhythmias in ventricles. Specifically, cardiac so- over, we observed that cardiac stress induced by isoproterenol treatment triggers high levels of S-nitrosylated Cx43 in DMD mouse hearts compared dium channel (NaV1.5) -rich ID nanodomains with close intermembrane spacing (<30nm) are important for cell-to-cell electrical communication. to wild-type(WT). We therefore aimed to determine if S-nitrosylation promotes Widening of these sites and Na 1.5 translocation have been implicated in hyperactivity of lateralized Cx43 hemichannels in DMD mice. First, we demon- V u pro-arrhythmic conduction slowing. How this mechanism contributes to the strated that N -Nitro-L-arginine methyl ester(L-NAME), a generic nitric oxi- progression of AF is unknown. We hypothesized that elevated VEGF-A levels de(NO) synthase(NOS) blocker, prevents isoproterenol-evoked arrhythmias in may increase vascular leak, rendering the heart oedematous. This fluid accu- DMD mice, confirming a role of NO signaling. Subsequently, we found that mulation disrupts ID nanodomains, which may slow conduction and promote both Cx43 hemichannel blockers and L-NAME reduce isoproterenol-evoked atrial arrhythmias. Stochastic optical reconstruction microscopy (STORM) spontaneous action potentials in isolated cardiomyocytes from DMD mice, therefore implicating both NO signaling and Cx43 activity in isoproterenol- and sub-diffraction confocal imaging (sDCI) revealed NaV1.5 enrichment at gap junction-adjacent perinexi and N-cadherin-rich sites consistent with adhe- induced arrhythmias. Finally, we asked whether NO directly opens Cx43 hemi- rens junctions. Myocardial water content, measured as the ratio of wet to dry channels. Using electrophysiological techniques to measure currents from weight, was not significantly altered in VEGF-A -treated mouse hearts relative Xenopus oocytes expressing WT or mutant Cx43-expressing oocytes, we to untreated controls. However, transmission electron microscopy (TEM) re- applied NO donors and evoked hemichannel currents at negative and positive vealed expansion of the perinexus (6459nm versus 1751nm), as well as sites voltages. Non-injected oocytes were used as controls. Whereas NO-donors next to adherens junctions (6354nm versus 2752nm) in VEGF-A -treated open WT Cx43 hemichannels, we observed that deletion of the Cx43 C-termi- D hearts relative to controls. Importantly, STORM revealed translocation of nus(CT, Cx43 CT) prevents hemichannel opening in response to NO donors. Mutagenesis of single cysteine residues(C260,C271 or C298) located within NaV1.5 from perinexi, indicating loss of functional sodium current at these sites. VEGF-A treatment slowed atrial conduction as assessed by optical the CT to serine identified C271 as the critical site of S-nistrosylation. While voltage mapping in Langendorff-perfused mouse hearts (1050.4cm/s versus C260S and C298S mutant hemichannels remained sensitive to NO donor appli- 2151cm/s at baseline). AF was inducible by burst pacing in 4 of 5 hearts cation, NO-induced currents were not detected in C271S mutants. Thus, we have tested following VEGF-A treatment but not before. These results suggest identified a mechanism by which S-nitrosylation of lateralized Cx43 hemichan- that VEGF-A-induced vascular leak may acutely elevate AF risk by disrupting nels plays a key role in the development of arrhythmias in DMD mice. ID nanodomains and slowing conduction. These data highlight vascular bar- rier function and ID nanodomains as potential targets for anti-arrhythmic therapy. Platform: Micro- and Nanotechnology 162-Plat 160-Plat Porous Zero-Mode Waveguides for Picogram-Level DNA Sequencing Recovery from Intracellular Acidosis Triggers Loss of Force in Hypokale- Vivek S. Jadhav1, David P. Hoogerheide2, Jonas Korlach3, Meni Wanunu4. mic Periodic Paralysis 1Physics, Northeastern Univ, Boston, MA, USA, 2National Institute of 1 2 2 2 Wentao Mi , Fenfen Wu , Marbella Quinonez , Marino DiFranco , Standards and Tech, Gaithersburg, MD, USA, 3Pacific BioSci, Menlo Park, Steve C. Cannon2. 4 1 2 CA, USA, Dept Phys/Chem, Northeastern Univ, Boston, MA, USA. Neurology, UT Southwestern Medical Center, Dallas, TX, USA, Dept We have recently shown that nanopore zero-mode waveguides are effective Physiology, Univ Calif Los Angeles DGSOM, Los Angeles, CA, USA. tools for capturing picogram levels of long DNA fragments for single- Periodic paralysis is an ion channelopathy of skeletal muscle, with recurrent molecule DNA sequencing. Despite these key advantages, manufacturing of episodes of weakness or paralysis caused by a sustained depolarization of large arrays is not practical due to the need for serial nanopore fabrication. the resting potential. Episodes are often triggered by environmental stresses þ To overcome this challenge, we have developed an approach for wafer-scale such as shifts of extracellular K , cooling, or exercise. Rest after vigorous ex- fabrication of waveguide arrays on low-cost porous membranes, which are ercise is the most common trigger for weakness in periodic paralysis, and yet deposited using molecular layer deposition. The membrane at each waveguide the mechanism is unknown. Curiously, weakness does not occur during exer- base contains a network of serpentine pores that allows efficient electrophoretic cise and resuming light exercise can abort an impending attack. We used DNA capture at picogram levels while eliminating the need for prohibitive se- knock-in mutant mouse models of hypokalemic periodic paralysis (HypoPP; rial pore milling. Here, we show that loading efficiency of these porous wave- NaV1.4-R669H or CaV1.1-R528H) and hyperkalemic periodic paralysis guides is up to two orders of magnitude greater than their nanopore (HyperPP; NaV1.4-M1592V) to better define the coupling between pH and predecessors. This new device facilitates scaling up the process, greatly susceptibility to loss of muscle force, as a possible contributor to exercise- reducing the cost and effort of manufacturing. Further, the porous zero-mode induced weakness. Acidosis (pH 6.7 in 25% CO2) was mildly protective, waveguides can be usedfor applications which benefit from low-input single but unexpectedly a return to pH 7.4 (5% CO2) elicited a robust loss of force moleculereal-time (SMRT) sequencing. in HypoKPP but not HyperKPP muscle. A prolonged exposure to low pH (tens of minutes) was required to cause susceptibility to post-acidosis loss 163-Plat of force, and the force decrement was prevented by reducing Cl- entry via Nurturing Nature for Nanotechnology block of NKCC1 with bumetanide. Washout of acidosis restricted to the extra- Michael Zwolak. cellular space (HCl load) did not cause loss of force. Based on these data, we Biophysics Group, NIST, Gaithersburg, MD, USA. propose a mechanism for post-acidosis loss of force wherein the reduced Designing DNA origami folding pathways to enable rapid and high-yield ClC-1 conductance in acidosis leads to a slow accumulation of myoplasmic folding is one of the core challenges in the practical production of DNA- Cl-. A rapid recovery of pH, and of the ClC-1 conductance, in the setting based nanostructures. In DNA origami, many folds are necessary in order to of increased [Cl]in/[Cl]out favors the anomalously depolarized state of the reach a given target structure. Each fold will occur rapidly when the template bistable Vrest in HypoKPP muscle, which reduces fiber excitability. This has one, and only one, bound staple per fold, i.e., a ready-to-be-folded state.

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The formation of this ‘‘ready state’’ is orders of magnitude slower than the Bioengineering, UC San Diego, La Jolla, CA, USA, 7UC San Diego, San folding process itself under standard folding conditions. The actual folding Diego, CA, USA, 8Department of Anesthesiology, UC San Diego, San Diego, step, however, is a unimolecular reaction, i.e., its rate is independent of the CA, USA, 9Institute of Engineering in Medicine, UC San Diego, San Diego, overall concentration. These concepts are the basis for a theory that predicts CA, USA. that origami yield will decrease exponentially with the number of folds (e.g., Nanoparticles carrying therapeutics have emerged as promising candidates for with the size of the origami template). We will discuss this theory and related targeted drug delivery. These nanocarriers (NC) can initiate controlled and on- results on kinetics and fidelity. demand release at the target site. However, obtaining effective doses of NCs in diseased locations remains challenging. Magnetic NCs (MNCs) can be used as 164-Plat vectors to increase localized bioavailability of drug: external magnetic fields Measuring the Conformation of Single Stranded DNA using a DNA can accumulate MNCs at a target site and trigger controlled release of therapeu- Origami Nano-Structure tics in high concentrations. Additionally, dynamic magnetic field gradients can 1 1 2 2 Yuval Garini , Efrat Roth , Arkady Bitler , Olga Girshevitz . potentially guide MNCs through blood vessel networks, increasing the amount 1Physics Department & Institute for Nanotechnology, Bar Ilan University, 2 of therapeutic reaching the target site. Ramat Gan, Israel, Institute for Nanotechnology, Bar Ilan University, Ramat In our lab, we have designed two types of iron oxide- silica MNCs - magnetic Gan, Israel. gold nano-golf-balls (NGB) and magnetic nanobowls (NB). These MNCs were Measuring the mechanical properties of single-stranded DNA (ssDNA) is a functionalized for pH and temperature stimuli sensitive drug release. NGB and challenge. Here we describe an innovative method for studying the biophysical NB response to magnetic field was analyzed by microscopy in: (1) epithelial properties of ssDNA based on DNA origami. We synthesized a DNA origami HeLa cells, (2) neurons derived from induced pluripotent stem cells (iPSCs), structure that consists of two rigid rods with an ssDNA segment between them (3) porous hydrogels and (3) microfluidic systems. Magnetic fields increase up- and image it with atomic force microscopy. The rigid rods provide a means for take of NGBs in HeLa cells and neurons. Stimuli sensitive drug release was the exact identification of the ssDNA ends and we measure the end-to-end dis- demonstrated by encapsulating DOX in NGB. Magnetically actuated transport tance of the ssDNA and analyze it in various ways. By fitting the measured dis- in hydrogels suggests that these MNCs can be retained for long periods of time tribution to the ideal chain polymer model, we measured an effective in high elastic modulus tissues. Studying NBs in microfluidic systems revealed persistence length of 1.98 nm with a standard deviation of 0.72 nm. It also pro- characteristics of their response to magnetic field gradients in varied fluid flow vides information on the conformation and the loops-formation along the conditions. ssDNA. It allows to compare the data to previously-published models on the These findings enhance our understanding of factors affecting efficiency for loop formation of RNA and ssDNA structures.This method can be used for magnetically guided transport, namely, magnetic and hydrodynamic volume measuring stem loops, for determining the effect of repetitive nucleotide se- and magnetization of MNCs. Optimization of these factors in MNCs can reduce quences and environmental conditions on the mechanical properties of ssDNA, side-effects by enabling guided drug delivery through vasculature to a target and the effect of interacting proteins with ssDNA. site. 165-Plat Nanopore Detection of Surprising Molarity Dependence of DNA Knot 167-Plat Complexity Quantifying the Influence of Nanoparticle Polydispersity on Cellular Rajesh K. Sharma1, Liang Dai2, Ishita Agrawal3, Patrick S. Doyle4, Delivered Dose Slaven Garaj5. Stuart Johnston, Matthew Faria, Edmund Crampin. 1Singapore MIT Alliance for Research and Technology, National University The University of Melbourne, Parkville, Australia. of Singapore, Singapore, Singapore, 2Singapore MIT Alliance for Research Nanoparticles provide a promising approach for the targeted delivery of ther- and Technology, Singapore, Singapore, 3Biomedical Engineering, National apeutic, diagnostic and imaging agents in the body. However, it is not yet fully University of Singapore, Singapore, Singapore, 4Chemical Engineering, understood how the physico-chemical properties of the nanoparticles influence Massachusetts Institute of Technology, Cambridge, MA, USA, 5Physics, cellular association and uptake. Cellular association experiments are routinely Biomedical Engineering, National University of Singapore, Singapore, performed in an effort to determine how nanoparticle properties impact the Singapore. rate of nanoparticle-cell association. To compare experiments in a meaningful DNA knots are self-entanglements of long DNA molecules, crucial in key bio- manner, the association data must be normalized by the amount of nanopar- logical processes such as viral DNA packaging, DNA replication, etc. Nano- ticles that arrive at the cells, a measure referred to as the delivered dose. pore nanoscopy is a promising tool that allows for the large-scale sampling The delivered dose is calculated from a model of nanoparticle transport of single DNA molecules in solutions. Recently, we demonstrated the co- through fluid. A standard assumption is that all nanoparticles within the pop- existence of simple and complex knots with statistical significance using nano- ulation are monodisperse, namely the nanoparticles have the same physico- pore sensors. Here, using our large sampling of single-molecule events in chemical properties. We present a semi-analytic solution to a modified model concert with parameterized simulations, we investigated the complexity of of nanoparticle transport that allows for the nanoparticle population to be DNA knots and their dependence on several experimental parameters such as polydisperse. Combining characterization data obtained from a range of ionic concentration, nanopore diameter, etc. Our results showed a surprising in- commonly used nanoparticles and our model, we find that the delivered crease in the probability and complexity of knotting with increasing ionic con- dose changes by more than a factor of 2 if realistic amounts of polydispersity centration, even beyond the theoretical saturation point of persistence length of are considered. DNA. In addition, we probed the localization of knots and various modes of translocation as a function of ionic concentration. Our results reveal that equi- 168-Plat librium distributions of knots could be obtained via controlling the sliding of Revealing the Dynamics of Single-Molecule Reactions in a Single-Molecule knots by tuning the experimental parameters such as nanopore diameter. These Nanoreactor results are useful in understanding the knotting phenomena in elongated poly- Kaipei Qiu, Bo Yuan, Yi-Tao Long. mers as well as in the nanopore based next generation genome sequencing ap- East China University of Science and Technology, Shanghai, China. plications. Keywords: Solid-state nanopores, DNA knots, polymers, DNA The dynamics of bond breaking and formation at the single-molecule level are sequencing, topology Acknowledgement: This work was supported by National of both fundamental importance and practical significance. Tremendous ef- Research Foundation, Singapore Grant (Grant No. NRF-NRFF2012-09) and forts have been made over the last decade toward the visualization of such Singapore-MIT Alliance for Research and Technology Centre. transient phenomenon, with quite a few amazing single-molecule techniques being designed and developed. However, it remains a major challenge to 166-Plat realize the truly single-molecule (cascade) reactions largely due to the sto- Nanocarriers for Magnetically Actuated Targeted Drug Delivery chastic nature of, e.g. molecular motions. Inspired by the enzymatic biosyn- Vrinda Sant1, Liping Wang2, Grace Jang3, Deependra Ban3, Jay Seth4, thesis, the authors consider that a nanoscale confined space, or nanoreactor, Sami Kazmi5, Nirav R. Patel6, Qingqing Yang7, Joon Lee7, must be essential to the highly ordered single-molecule reactions. Moreover, Woraphong Janetanakit4, Shanshan Wang8, Brian Head8, Gennadi Glinsky9, an ideal nanoreactor also needs to possess a well defined structure that is of Ratnesh Lal3. comparable size to the individual reactant molecules. Hence, the rationally- 1Materials Science and Engineering, UC San Diego, San Diego, CA, USA, mutated biological nanopores may be applied as a new brand of single- 2Shanghai Jiaotong Univerity, Shanghai, China, 3Mechanical and Aerospace molecule nanoreactors, where the entry of reactants and exit of products are Engineering, UC San Diego, San Diego, CA, USA, 4Department of voltage-driven. Nanoengineering, UC San Diego, San Diego, CA, USA, 5Department of Herein, a mutant areolysin was adopted to study the unique confinement induced Chemical Engineering, UC San Diego, San Diego, CA, USA, 6Department of selectivity of nanopore reactor. In addition to regio- or stereo-selectivity, it

BPJ 9292_9302 34a Sunday, March 3, 2019 is hypothesized that the mismatch between the bond formation kinetics of Recent breakthroughs in experimental technologies and in high-performance reactant molecules and their translocation speeds can offer an extra level computing have enabled unprecedented measurements and simulations of of control on reaction selectivity. To prove this concept, the reaction dynamics complex chemical systems such as macromolecules. However, experiments of three constitutional isomers, 2-, 3-, and 4-mercaptobenzoic acid, at seven provide only a partial view of macromolecular processes and are limited identical reaction sites (i.e. at the same position on each monomer) were in their temporal and spatial resolution. On the other hand, atomistic simu- examined in this work. Surprisingly, those tiny structural variations of three lations are still not able to sample the conformation space of large com- isomers could be easily distinguished by not only the different current plexes, thus leaving significant gaps in our ability to study molecular blockades caused by each tethered molecule, but also the most frequent and pro- processes at a biologically relevant scale. We present our efforts to bridge longed current levels as well. This observation shed light on the transient dy- these gaps, by using experimental data as a starting point in a computational namics of single-molecule reactions within a single-molecule nanoreactor, modeling approach. We use models at different resolutions and ‘‘anchor’’ providing a crucial first step to the ultimate objective of 100% yield of targeted them to experimental measurements, to provide quantitatively accurate product. representations of systems of interest, and address open biophysical questions. 169-Plat Development of a New Antimicrobial Photosensitizer from Brominated 172-Symp Carbon Dots: Metal-Enhanced Phosphorescence and Singlet Oxygen Dealing with Dynamics and Disorder by Combining Simulation and Generation Experiment Rachael Knoblauch, Christopher D. Geddes. Gerhard Hummer. Department of Chemistry and Biochemistry, The Institute of Fluorescence, Department of Theoretical Biophysics, Max Planck Institute of Biophysics, University of Maryland Baltimore County, Baltimore, MD, USA. Frankfurt, Germany. The ever-growing wave of antibiotic resistance in bacterial strains has placed Many biomolecular systems are highly dynamic. In mechanistic studies using pressure on the scientific community to develop new, diverse methods for the tools of structural biology, dynamics and disorder pose major challenges. In fighting deadly microbials. One such method investigated is antimicrobial my talk, I will show how the combination of a range of experiments with struc- photodynamic inactivation (aPDI), whereby photosensitizing compounds or tural modeling and molecular simulations can address these challenges. I will particles that commonly occupy a triplet excited state (T1) may generate show how results from disparate experiments and computational approaches toxic species, such as reactive oxygen, when irradiated. Subsequently, these can be combined within a Bayesian formulation of ensemble refinement. On short-lived reactive agents inflict critical damage on nearby microbials. this basis, I will describe approaches to deal with dynamic systems in cryo- Although numerous photosensitizers have been developed and implemented electron microscopy, and on sampling and analyzing the conformation space for this purpose, the need remains to develop more photostable and dynamic and dynamics of disordered proteins and nucleic acids using NMR, SAXS, photosensitizers. Carbon nanodots are well known to exhibit strong photo- and fluorescence data. stability and biocompatibility, are easily synthesized, and may undergo sim- ple structural modification. We report a new singlet oxygen photosensitizer from a carbon nanodot scaffold, termed ‘‘Heavy Carbon Nanodots.’’ These 173-Symp luminescent, quasi-spherical nanoparticles are composed of brominated Towards Simulating Bacterial and Eukaryotic Cells: Integration of Exper- sheets of oxidized graphene that exhibit phosphorescence (T1/S0) when ox- iment and Theory ygen diffusion is slowed; however, in oxygenated, low viscosity solvents we Zaida Ann Luthey-Schulten. show singlet oxygen is readily generated by these particles. Further we Chemistry, University of Illinois, Urbana, IL, USA. explore the potential of silver-coated 96-well plates ‘‘Quanta PlatesÔ’’ to Ribosomes—the primary macromolecular machines responsible for trans- plasmon amplify both the phosphorescent signal and singlet oxygen genera- lating the genome into proteins—are complexes of precisely folded RNA tion, in processes known as metal-enhanced phosphorescence (MEP) and and proteins. The ways in which their production and assembly are managed 1 metal-enhanced generation of singlet oxygen (ME O2) respectively. We by the living cell is of deep biological importance. Our whole-cell model of report increased detection of triplet emission and increased generation of ribosome biogenesis in a slowing growing bacteria includes the effects of singlet oxygen by these metal-coated wells. These phenomena are also growth, DNA replication, transcription, translation, ribosome assembly, and observed to be pH-dependent. The data reported here provides the foundation cell division. All biological processes are described in terms of reaction- for future development of brominated carbon nanodots as novel agents for diffusion master equations (RDME) and solved stochastically using the antimicrobial photodynamic inactivation. GPU-based Lattice Microbes simulation software over a cell cycle. The repli- *all correspondence: [email protected] cation parameters are derived from single cell measurements of gene copy numbers in a series of E. coli strains with fluorescently labeled genes as a func- tion of cell length and time. The work on ribosome biogenesis can now be Symposium: Integrative Modeling from linked with the essential metabolism of a minimal bacterial cell, JCVI- Syn3A. To treat processes at biologically relevant length- (microns), time- Macromolecules to Cell (hours), and concentration- (nanomolar to millimolar) scales requires hybrid RDME simulations. I will report on our efforts to develop a complete kinetic 170-Symp model of a minimal bacterial cell and the challenges facing us to simulate eu- Mapping the Spatial Organization of Genomes through Data Integration karyotic cells. Frank Alber, Polles Guido, Hua Nan, Yildirim Asli, Zhan Yuxiang. Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los Angeles, CA, USA. Symposium: Cytoskeleton An accurate mapping of the 3D structure and dynamics of entire genomes re- quires a combination of complementary data and methods. As part of a joint 174-Symp analysis project we have developed computational methods for integrating var- Phase Separation of TPX2 Enhances and Spatially Biases Microtubule ied experimental data sources to produce quantitative models of the nuclear or- Nucleation ganization. Here, we will present an overview of our efforts and progress in Sabine Petry. performing a comparative structure analysis of genomes of three different hu- Princeton University, Princeton, NJ, USA. man cells. We will discuss cell-type specific structural features and analyze dif- Phase separation is an emerging paradigm in cellular physiology, yet its role ferences in the spatial organization of subcompartments between the cell types. for the cytoskeleton remains to be defined. Here, we demonstrate that the Our analysis allows insights into the guiding principles of genome organization microtubule nucleation effector, TPX2, forms a co-condensate with tubulin and its functional relevance. in vitro and in isolated cytosol. Co-condensates can nucleate microtubules and then localize to them. Importins regulate this process by inhibiting 171-Symp co-condensate formation. Most importantly, we demonstrate that TPX2’s Multiscale Modeling of Biomolecular Processes by Combining Experiment ability to form co-condensates with tubulin enhances the kinetic efficiency and Simulation of microtubule nucleation. We propose a model, in which importin- Cecilia Clementi. regulated phase separation of TPX2 and tubulin near chromatin creates a Chemistry, Rice University, Houston, TX, USA. co-condensate that initiates MT nucleation for spindle assembly. This

BPJ 9292_9302 Sunday, March 3, 2019 35a co-condensate, in turn, localizes to the initial microtubule to bias future 35% of all HCM mutations occur in the motor domain of human b-cardiac nucleation from it and autocatalytically generate spindle microtubules. myosin, while another 35% occur in myosin binding protein-C. Associated Our study demonstrates how phase separation enhances microtubule nucle- with HCM worldwide are heart failure, arrhythmias, and sudden cardiac ation while simultaneously conferring spatial control to allow for rapid and death at any age. We are using in vitro molecular studies of biochemically accurate spindle formation. reconstituted human sarcomeric protein complexes to elucidate the molecular basis of HCM-induced hypercontractility. We postulated in 2015 that a ma- 175-Symp jority of HCM mutations are likely to be shifting b-cardiac myosin heads Regulation of Bidirectional Motility of Kinesin-5 Motors from a sequestered off-state to an active on-state for interaction with actin, Leah Gheber. resulting in the hypercontractility seen clinically in HCM patients. This hy- Chemistry, Ben Gurion Univ, Be’er Sheva, Israel. pothesis is different from earlier prevailing views, and this viewing an old Mitosis is an essential process by which duplicated genetic information is disease in a new light is the basis of all of our current research. With a transmitted from mother to daughter cells. This process is mediated by the detailed molecular understanding of the resultant increase in power output mitotic spindle, a dynamic microtubule-based structure. The bipolar caused by HCM mutations, one should be able to design appropriate small kinesin-5 motors perform essential functions in mitotic spindle dynamics molecule therapies, which are desperately needed for treatment of these by crosslinking and sliding apart antiparallel microtubules emanating from diseases. opposite poles. We have recently demonstrated that the S. cerevisiae kinesin-5 Cin8 and Kip1 are minus-end directed and can switch direction- ality in vitro under a number of conditions (1, 2). The mechanism of this Platform: Ligand-gated Channels directionality switch and its physiological significance remain unclear. We also demonstrated that Cin8 is differentially phosphorylated during late 178-Plat anaphase at three cyclin-dependent kinase 1 (Cdk1) specific sites located High Throughput Validation of Non Canonical Amino Acid Incorporation in its motor domain (3). This phosphorylation regulates Cin8 activity during into Acid Sensing Ion Channel 1A anaphase, but the mechanism remains unclear. To address this point, we Nina Braun1, Søren Friis2, Weihua Tian3, Eric P. Bennett3, Jacob Andersen4, examined their in vitro motile properties and in vivo functions by TIRF mi- Stephan A. Pless1. croscopy and live-cell imaging. We found that Cdk1 phosphorylation of Cin8 1Department of Drug Design and Pharmacology, University of Copenhagen, regulates its directionality. We also found that in vitro, Cin8 not only moves Copenhagen, Denmark, 2Nanion Technologies, Munich, Germany, 3Center to- but also clusters at the minus-end of the MTs. This clustering causes Cin8 for Glycomics, University of Copenhagen, Copenhagen, Denmark, 4Vipergen to reverse its directionality from fast minus- to slow plus-end directed ApS, Copenhagen, Denmark. motility. Based on these results, we propose a revised model for activity of Human acid-sensing ion channel 1a (ASIC1a) is a trimeric proton-sensitive Cin8 and Kip1 during mitosis and propose a physiological role for their cation channel expressed in the central nervous system, where it plays a minus-end directed and switchable motility. 1. Gerson-Gurwitz A,etal. crucial role in the initiation of neuropathic pain and acid-induced ischemia (2011) Directionality of individual kinesin-5 Cin8 motors is modulated by after stroke. In order to target these conditions therapeutically, the molecular loop 8, ionic strength and microtubule geometry. Embo J 30(24):4942- links between ASIC1a activity and its surrounding cellular network have to 4954. 2. Fridman V,etal.(2013) Kinesin-5 Kip1 is a bi-directional motor be identified. We are thus aiming to investigate protein-protein and protein- that stabilizes microtubules and tracks their plus-ends in vivo. JCellSci peptide interactions of ASIC1a with a combination of patch-clamp electro- 126(Pt 18):4147-4159. 3. Avunie-Masala R,etal.(2011) Phospho- physiology and UV-induced cross-linking. This will allow mapping of both regulation of kinesin-5 during anaphase spindle elongation. JCellSci intra- and extracellular interaction sites. For the purpose of cross-linking, 124(Pt 6):873-878. the non-canonical amino acids (ncAAs) AzF, Bpa and Se-AbK are site- specifically incorporated at selected positions in ASIC1a via the nonsense 176-Symp suppression method. Here we introduce a workflow to functionally assess Regulation of Myosin Motors - from Single Molecules to Functional site-specific incorporation of ncAAs into a library of 103 ASIC1a variants. Ensembles After removing endogenous ASIC1a from HEK293 cells using CRISPR- Claudia Veigel. Cas9, we transiently transfect the test constructs into these knock-out cells. Ludwig-Maximilians-Universit€at Munchen,€ Munich, Germany. Following transfection, cells are submitted to fluorescence-activated cell Many types of cellular motility are based on the myosin family of motor sorting, after which we perform high-throughput patch-clamp recordings proteins. There are now known to be at least 35 different classes of myosin, on a SyncroPatch 384PE. Incorporation of ncAAs is tolerated throughout involved in intracellular transport processes, cytokinesis, muscle contrac- all channel domains, with highest success rates for AzF (61%) and lowest tion, exo- and endocytosis or signal transduction in vision and hearing. for Se-AbK (44%). Bpa is preferably incorporated at positions of aromatic The ability to coordinate the timing of motor protein activation lies at the origin, while no comparable trend is observed for the other ncAAs. Interest- very centre of this wide range of cellular motile processes. Using a com- ingly, replacement of residues around the acidic pocket produces channels bined approach of recombinant protein expression, single molecule mechan- with significantly reduced proton affinity. In conclusion, this approach allows ical and fluorescence imaging techniques, and cargo binding assays we rapid identification of suitable positions for cross-linking experiments, as study the basic mechanisms of activation, force production and movement well as efficient biophysical characterization of ncAA-containing ASIC1a of these molecular machines at the single molecule and ensemble level. variants. We will report on our recent studies on myosins forming acto-myosin lattice structures and myosins forming functional ensembles interacting with lipids 179-Plat enabling them to transport cargo over micrometer distances along the Measuring Dynamics of the Acid-Sensing Ion Channel N-Terminus using actin cytoskeleton in the cell. Supported by DFG SFB 863 B6 and Baur Transition Metal Ion FRET Stiftung. Megan Cullinan, Prafulla Aryal, John Bankston. Department of Physiology and Biophysics, Univ Colorado Anschutz Med 177-Symp Ctr, Aurora, CO, USA. The Myosin Mesa and Hypertrophic Cardiomyopathy: Mutations to Acid-sensing ion channels (ASICs) are proton gated, voltage-independent Mechanisms to Therapies membrane proteins that are activated by changes in extracellular pH. Func- James Spudich. tional channels are composed of three subunits, each with intracellular n- Stanford University, Palo Alto, CA, USA. and c-termini, two transmembrane domains, and a large extracellular domain. After 40 years of developing and utilizing assays to understand the molecular While static structures of ASIC1a are known, none of these structures resolve basis of energy transduction by the myosin family of molecular motors, all the intracellular termini, which are important for channel gating, assembly and members of my laboratory are now focused on understanding the underlying selectivity. The lack of structural information about the intracellular termini biochemical and biophysical bases of human hypertrophic (HCM) and dilated leaves a void in understanding of the molecular mechanisms of channel (DCM) cardiomyopathies. Our primary focus is on HCM since these muta- activation. To date, it has been a challenge to make short range measure- tions cause the heart to be hypercontractile, and we hope to understand the ments of conformational dynamics of proteins. Using a combination of molecular basis of this increased power output. HCM is most often a result fluorescence and electrophysiological approaches, we sought to map the of single missense mutations in one of several sarcomeric proteins, the sarco- position of the n-terminus of ASIC1a and look for conformational changes mere being the fundamental contractile unit of the cardiomyocyte. More than that occur in this region in response to channel activation. Typical

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fluorescence approaches are ineffective at measuring small changes in struc- Immense progress has been made in recent years concerning the structure ture within proteins so we turned to a new approach called transition metal and function of iGluRs but central questions remain unanswered. For example, ion FRET (tmFRET) that uses a small transition metal ion as the FRET how do ions permeate through the channel and how are they discriminated by acceptor and has a working distance range of 8-20A˚ . To get specific labelling the selectivity filter? These questions persist for both selective and non- of the n-terminus of ASIC1a with a small fluorophore, we used the amber sup- selective cation channels in general and permeation mechanisms remain a sub- pression approach to incorporate the small unnatural amino acid 3-(6-acetyl- ject of intense debate. The non-selective cation channel NaK has been used naphthalen-2-ylamino)-2-aminopropanoic acid (L-Anap). At most labelled widely as a model channel for permeation studies. Following the hypothesis positions along the n-terminus, we can measure large current with normal that the critical events in ion selection take place at the selectivity filter, we functional properties. We then incorporate Cu2þ at various positions in used NaK as a scaffold to introduce selectivity filter residues of GluA2. Crystal the channel using a cysteine-reactive metal chelator called TETAC and mea- structures of a mutant obtained under Naþ,Kþ,Rbþ,Ba2þ and Ca2þ show a sure distances between the n-terminus and our metal binding site. These ex- degree of flexibility to accommodate ions of different size, as well as preferred periments lay the groundwork for future work mapping the position of the binding sites and the contribution of water for ion coordination. These scaffolds n-terminus of ASIC1a and measuring how the n-terminus changes conforma- are also conveniently compact for use in molecular dynamics simulations, tion during ASIC gating. where they behave well and readily support cation permeation. Overall, our work provides clues towards conserved mechanisms of monovalent and diva- 180-Plat lent ion permeation between non-selective prokaryotic channels and a verte- A Mechanism for Desensitization of all Three Functional Mammalian Acid brate neurotransmitter receptor. Sensing Ion Channels Yangyu Wu, Zhuyuan Chen, Cecilia Canessa. 183-Plat Basic Sciences Medical School, Tsinghua Univ, Beijing, China. Role of NMDAR-BK Complexes in the Integration of Synaptic Inputs of ASICs are proton-activated sodium channels expressed in most neurons of the Barrel Cortex Pyramidal Neurons central and peripheral nervous systems. ASICs exhibit proton-induced desensi- Ricardo Go´mez1,2, Laura E. Maglio1,2, Alberto J. Gonzalez-Hernandez1,2, tization from the open and the closed states (steady-state desensitization or Belinda Rivero-Perez1,2, Teresa Giraldez1,2. SSD). Atomic structures of chicken ASIC1a have shown a combination of fea- 1Dept. Ciencias Medicas Ba´sicas-Fisiologı´a, Facultad de Medicina, tures that together are distinctive of the desensitized state: 1) the gate of the Universidad de La Laguna, San Cristo´bal de La Laguna (Tenerife), Spain, pore is shut in the transmembrane domain; 2) the acidic pocket is collapsed, 2Instituto de Tecnologı´as Biomedicas (ITB), Centro de Investigaciones and 3) two residues L415 and N416 in the b11-b12 linker are rotated almost Biomedicas de Canarias (CIBICAN), San Cristo´bal de La Laguna (Tenerife), 180oin opposite directions. We asked whether the linker is important in desen- Spain. þ þ sitization and if so, how it participates in this process. Previous mutagenesis of Large conductance Ca2 - and voltage-gated K channels (BK) form com- þ þ L415 and N416 (human ASIC1a numbering) and modification of cysteines with plexes with Ca2 -permeant ion channels, coupling neuronal Ca2 to mem- methanethiosulfonate reagents decreased the rate of desensitization of the open brane voltage signals. Postsynaptically, functional association of BK with state and shifted the pH50of SSD toward more acidic values, supporting the N-methyl-D-aspartate receptors (NMDAR) has been shown in olfactory functional importance of the linker. Using electrophysiological recordings of bulb (Isaacson & Murphy, Neuron 2001;31:1027) and dentate gyrus (Zhang human ASIC1a currents, mutagenesis, induction and reduction of disulfide et al., PNAS 2018;115:E9006) granule cells. The existence of NMDAR-BK bonds, and molecular dynamic simulations we show dynamic coupling of res- complexes in other brain regions and their physiological relevance in neuronal idues in the b11-b12 linker and the palm domain and that such interaction traps function remain unknown. Here we studied the role of NMDAR-BK com- the linker in a compressed state that is unable to transduce conformational plexes in somatosensory barrel cortex layer 5 pyramidal neurons (BC- changes locking the channel in the desensitized state. Disruption of interactions L5PN), where two neuronal populations (A- or B-type) were characterized þ between the palm and the linker result in channels with no desensitization but according to the presence or absence of NMDAR-mediated Ca2 spikes still sensitive to activation by protons and with pore properties -selectivity and following the action potential (Maglio et al., Cereb Cortex 2018;28:1568). conductance- indistinguishable from those of wild-type channels. Furthermore, We hypothesized that this difference relays on the presence of NMDAR- we demonstrate that the residues identified in hASIC1a play the same role in BK complexes in B-type BC-L5PN. Additionally, we predicted that these mammalian ASIC2 and ASIC3 channels supporting the notion that the uncov- complexes would make B-type BC-L5PN less sensitive to afferent synaptic ered mechanism governs desensitization in all mammalian functional ASIC inputs and induction of synaptic plasticity. We combined electrophysiological, channels. immunochemistry, and imaging experiments to test these hypotheses. Local application of NMDA at basal dendrites induced a biphasic current response 181-Plat in 35% of BC-L5PN at depolarizing voltages, consisting on an inward current A Critical Mobile Divalent Cation Site in the ATP-Binding Pocket of P2X3 immediately followed by a long-lasting outward current. NMDAR antagonist Receptors that Controls Channel Gating AP5 and BK blocker paxilline abolished this response, as well as ifenprodil Mufeng Li1, Yao Wang2, Shai D. Silberberg3, Motoyuki Hattori2, (selective NMDAR-NR2B antagonist). Inside-out patch-clamp recordings Kenton Swartz1. from transfected HEK293 cells demonstrated that functional NMDAR-BK 1NINDS NIH, Bethesda, MD, USA, 2Department of Physiology and complexes are mainly formed by NR2B-containing NMDAR. Independent Biophysics, Fudan University, Shanghai, China, 3NIH, Bethesda, MD, USA. FRET and PLA experiments confirmed NMDAR-BK close proximity. Magnesium ions play fundamental roles in regulating the activity of many types Finally, BC-L5PN expressing NMDAR-BK complexes (B-type) are less sen- of ion channels, yet the mechanism is unclear in most cases. P2X3 receptors are sitive to the induction of long-term potentiation (LTP) when compared to ATP-activated channels that are involved in pain and other irritating sensations. A-type neurons. In summary, our results reveal that NMDAR(NR2B)-BK þ Here we show how Mg2 binds to a unique mobile site in the ATP-binding functional coupling in basal dendrites of BC-L5PN constitutes a mechanism pocket of P2X3 receptors to modulate channel gating. We obtained crystal to control synaptic excitability and plasticity in somatosensory pyramidal structures of Mg-ATP bound hP2X3 receptors, and surprisingly found that neurons. Mg2þ binding is different from Apo state. The structures suggest that Mg2þ can either be coordinated solely by acidic residues in the receptor, or simulta- 184-Plat neously interacts with the g-phosphate of ATP and receptor. This Mg2þ bind- Probing Structural States of a Pentameric Ligand-Gated Ion Channel with ing site stabilizes ATP on the receptor, slows recovery from desensitization and Small Angle Neutron Scattering is required for the physiologically abundant divalent-bound form of ATP to Marie Lycksell1, Nicolai T. Johansen2, Rebecca J. Howard1, Lise Arleth2, promote channel opening. Interestingly, Ca2þ can antagonize Mg2þ effect by Erik Lindahl1. 1 competing to bind at the same site. Dept Biochemistry & Biophysics, Stockholm University, Stockholm, Sweden, 2Niels Bohr Institute, University of Copenhagen, Copenhagen, 182-Plat Denmark. Structural Studies of Mutants of the NaK Channel Ligand-gated ion channels transform chemical signals into electrical ones, Sonja Minniberger1,2, Saeid Abdolvand1,2, Han Sun1, Andrew J. Plested1,2. which makes them important factors for a range of illnesses, and they are 1Leibniz-Forschungsinstitut fur€ Molekulare Pharmakologie, Berlin, critical drug targets in the nervous system. The bacterial pentameric Germany, 2Institut fur€ Biologie, Humboldt-Universit€at zu Berlin, Berlin, ligand-gated ion channel GLIC is a valuable model system for eukaryotic Germany. channels, particularly as structures are available from several distinct states. The family of ionotropic glutamate receptors (iGluRs) mediates the vast major- However, there are still questions regarding the states of these structures, and ity of fast excitatory synaptic transmission in the central nervous system. how representative they are of room-temperature channels. We sought to

BPJ 9292_9302 Sunday, March 3, 2019 37a validate structural predictions using solution-phase small angle neutron scat- 187-Plat tering (SANS). We have acquired SANS data from GLIC under non- Investigating the Generality and Biophysical Underpinnings of Consensus activating buffer conditions, hiding the detergent micelle by use of Protein Stability Enhancement deuteration. Using structures and molecular dynamics simulations of different Matthew Sternke, Katherine W. Tripp, Doug Barrick. states, the agreement with the SANS data has been evaluated. Although dif- Dept Biophysics, Johns Hopkins Univ, Baltimore, MD, USA. ferences in the fit of the predicted models to the experimental data were A promising approach many have applied to increase protein stability is small, our work shows that a resting state model is consistent with the consensus sequence design. ‘‘Consensus sequences’’ are composed of the room-temperature experimental data. This work shows the potential for using most frequent residue at each position in an alignment of extant sequences. SANS in conjunction with other methods in the study of membrane proteins, While there have been many successful applications of consensus design, while underlining the importance of high quality SANS data of multiple the generality and underlying mechanisms with which succeeds in producing states. folded, stable proteins are unclear. Here, we address these questions by designing consensus sequences for a set of seven globular protein families. 185-Plat We find that all seven consensus proteins adopt the archetypal folds of their Cryo-EM Reveals Two Distinct Serotonin-Bound Conformations of Full- respective protein families. Five of the seven consensus proteins show Length 5-HT3A Receptor enhanced thermodynamic stabilities compared to natural homologues, with Sandip Basak1, Yvonne W. Gicheru2, Shanlin Rao3, Mark S. Sansom3, the other two showing near-average and above-average stabilities. All Sudha Chakrapani1. consensus proteins assayed for function maintain biological activities, 1Dept Physiol & Biophys, Case Western Reserve Univ, Cleveland, OH, USA, showing variable effects on molecular recognition and steady-state catalysis 2Case Western Reserve Univ, Cleveland, OH, USA, 3Department of activities. Biochemistry, University of Oxford, Oxford, United Kingdom. Constructing multiple consensus sequences from diverse families of proteins Serotonin receptors (5-HT3AR) are cation-selective, neurotransmitter-gated, allows us to examine sequence features that impart unique properties, such pentameric ion channels that play a crucial role in regulating cellular excit- as high stability, on consensus proteins. Comparisons of the sequence features ability. 5-HT3AR activities directly regulate gut movement and therefore drugs of consensus proteins and the multiple sequence alignments from which they that inhibit 5-HT3AR function are used to control vomiting reflexes. Serotonin derive show striking increases in charged residues at weakly-conserved surface binding induces a global conformational change encompassing the ligand- positions. Currently, we are examining how these sequence features contribute binding extracellular domain (ECD), the transmembrane domain (TMD), and to the observed effects of consensus substitution on stability and function the intracellular domain (ICD), the molecular details of which are unclear. through site-directed mutagenesis, making chimeric proteins that introduce Here, we determined two serotonin-bound structures of the full-length specific features of consensus protein into extant proteins. We are also ˚ ˚ 5-HT3AR in distinct conformations at 3.32 A and 3.89 A resolutions, which comparing our consensus design strategy to other sequence-based strategies we referred as State 1 and State 2, respectively. The new structures reveal such as ancestral reconstruction and residue covariation-based approaches. the mechanism underlying channel activation. When compared to Apo-5- For the homeodomain, we find that the high stability of the consensus sequence HT3AR, serotonin-bound states underwent a large twisting motion in the results neither from sequence differences at conserved positions, nor from dif- ECD and TMD leading to the opening of a 165 A˚ long permeation pathway. ferences in charged residues. Rather, the large stability increase for the Notably, this motion results in creation of lateral portals for ion permeation consensus sequence seems to result from many small contributions from varied at the interface of the TMD and ICD. Combined with molecular dynamics sim- substitutions. ulations, these structures provide novel insights into conformational coupling across domains and functional modulation. 188-Plat Protein-Solvent Attractive Interactions Dominate the Inverse Tempera- ture Dependence of Polypeptide Hydration Free Energies Tomar S. Tomar1, Michael E. Paulaitis2,3, Lawrence R. Pratt4, Platform: Protein Folding, Pathways, and Dilip N. Asthagiri5. Stability 1Dept Chem/Biomolec Eng, Johns Hopkins University, Baltimore, MD, USA, 2Dept Chem/Biomolec Eng, Ohio State Univ, Columbus, OH, USA, 186-Plat 3Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD, USA, Characterization of Conformational Diversity, Stability, and Catalytic 4Chemical and Biomolecular Engineering, Tulane University, New Orleans, Activity of TcmN, an Enzyme Involved in Antibiotic Biosynthesis LA, USA, 5Dept Chem/Biomolec Eng, Rice Univ, Houston, TX, USA. Veronica S. Valadares1, Luan C. Martins1, Lara G.R.V.M. Tannus2, Simulations show that both the excess free energy and the excess enthalpy Adolfo H. Moraes3, Elio A. Cino1. of hydration of a deca-alanine peptide in the helix and extended (coil) 1Biochemistry and Immunology, Federal University of Minas Gerais, Belo states increases with increasing temperature: as expected of a hydrophobic Horizonte, Brazil, 2Chemical Engineering, Federal University of Minas solute, the excess entropy of hydration is negative and the excess heat Gerais, Belo Horizonte, Brazil, 3Chemistry, Federal University of Minas capacity of hydration is positive. A similar trend is also found for a Gerais, Belo Horizonte, Brazil. helix-pair, a simple model for a protein tertiary structure. However, the Tetracenomycin aromatase/cyclase (Tcm) is an enzyme derived from Strepto- temperature dependency of the excess enthalpy is dominated by different myces glaucescens involved in polyketide cyclization, aromatization, and physics for methane, the side-chain analog of alanine and a prototypical folding. Polyketides are a diverse class of secondary metabolites produced hydrophobe, and the peptide models. For methane, the temperature signa- by certain groups of bacteria, fungi and plants with pharmaceutical applica- tures arise due to changes in water reorganization, but for the peptide tions. Examples include antibiotics, such as tetracycline, and anticancer drugs, models, it is due to the weakening of the effective attractive solute- such as doxorubucine. The N-terminal of Tcm (TcmN) participates in the spe- solvent interactions, a major fraction of which arises from peptide cific cyclization of polyketides, and is structurally classified as belonging to the backbone-solvent interactions. Different temperature dependencies are Bet v 1-like or START domain superfamily. TcmN catalyzes the formation of also observed for the excess free energy of a cavity, an ideal hydrophobe: the first (C9-C14), and second (C7-C16) rings of polyketides in vivo and increasing with temperature for methane but decreasing for the protein. in vitro. TcmN was expressed in Escherichia coli BL21(DE3) and purified. The temperature dependency of the excess free energy for the protein Secondary structure and stability were assessed in different buffer conditions in both helix and coil states mainly reflects temperature dependence of by circular dichroism (CD) and fluorescence spectroscopy. Nuclear Magnetic attractive protein-solvent interactions. Further, as expected in protein un- Resonance (NMR) spectra were acquired at different field strengths (900, folding, we find a positive heat capacity change in the unpairing of a 800, and 400 MHz). Duplicate 1 ms Molecular Dynamics (MD) simulations helix dimer, but this feature again arises due to protein-solvent attractive in- of TcmN were performed in explicit solvent. CD indicated that TcmN was teractions, with backbone-solvent interactions playing a dominant role. more stable in sodium phosphate at pH 6.8 and 8.0, respectively, rather than These first direct, all-atom calculations of the thermodynamics of hydration in sodium acetate at pH 4-5. NMR data indicated that TcmN is correctly folded, of protein models shows that acknowledging the role of attractive interac- and resonance assignment is underway. Combining experimental and computa- tions and the expansion of the solvent matrix upon heating provides a tional data, we aim to investigate TcmN substrate specificity through mapping parsimonious explanation for the unusual temperature signatures that have of interactions between active site residues and a substrate analogue. The re- often been explained by invoking hydrophobic hydration. Implications of sults are expected to be useful for future for engineering polyketide bio- this work for understanding intrinsic disorder and aggregation will be synthesis. noted.

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189-Plat spectroscopy and molecular dynamic simulations, where the results of Multi-Scale Simulations Yield Insight into Protein Diffusion and Stability each approach drive experiment design of the other, we reveal the presence in Crowded Environments of a hidden unfolding intermediate in the second PDZ domain of PTP-BL. Stepan Timr1, Simone Melchionna2, Philippe Derreumaux1, We hypothesize that this partially folded intermediate is important to the Fabio Sterpone1. process of amyloid fibril formation in this PDZ domain and have used 1LBT/IBPC, CNRS, Paris, France, 2ISC-CNR, Rome, Italy. various computational and experimental methods to evaluate its role. The In living cells, proteins operate in a highly crowded environment where a merging of computational and experimental approaches used here offers op- large fraction of the volume is filled by macromolecules. Despite intensive portunities to obtain a more complete characterization of the folding land- research, the effects of macromolecular crowding on protein mobility, sta- scape and new insights into amyloid fibril formation mechanism of globular bility, and ultimately function have not yet been fully elucidated. The proteins. need for a detailed microscopic understanding calls for insights from com- puter simulations; however, the wide spread of time- and length scales 192-Plat involved in crowding poses a serious challenge for current in silico ap- Probing Pressure Effects on Core Packing of a Repeat Protein using 13C proaches. Our novel multi-scale scheme, combining lattice Boltzmann mo- NMR lecular dynamics (LBMD) with all-atom replica exchange simulations, Siwen Zhang1, Scott McCallum2, Catherine A. Royer2. bridges a gap between existing simulation techniques and provides a 1Chemistry, Rensselaer Polytechnic Institute, Troy, NY, USA, 2Dept Biol, computational framework for exploring protein diffusion and stability in Resselaer Polytechnic Inst, Troy, NY, USA. the intracellular environment. Our LBMD simulations, employing the Repeat proteins greatly simplify the identification of the determinants for pro- OPEP coarse-grain model, reproduce the experimentally observed diffusion tein folding cooperativity. We use the leucine rich repeat (LRR) domain of the slowdown in several crowded binary protein mixtures and allow us to char- tumor suppressor pp32 as a model protein. The pp32 protein is composed of 5 acterize the timescales of protein reshuffling as well as the typical geome- LLR with two capping domains on both of its termini. Based on previous tries of local packing around the proteins. Furthermore, we use our 15N-1H high pressure HSQC experiments, we know that intermediates are computational scheme to shed light on the unfolding of superoxide dismut- populated in the folding of pp32. In order to obtain more information about ase 1 (SOD1) in crowded conditions, a process implicated in amyotrophic these intermediates, in particular pressure dependent changes in the core lateral sclerosis (ALS). We show that the thermal stability of a SOD1 barrel packing, we collected 13C-1H HSQC spectra as a function of pressure on depends on its interactions with the surrounding proteins. As a consequence, cavity-containing variants of pp32. We focused on the region of the spectra cor- the different states of local packing result in crowding-induced heterogene- responding to side-chain methyl groups of the Valine, Leucine and Isoleucine ity of SOD1 thermal stabilities. Thus, our results highlight the importance of residues to reveal pressure-dependent changes in the packing of the hydropho- considering the local environment when investigating protein function in the bic core. For one variant, L139A, the previous pressure-dependent 15N-1H cytoplasm. HSQC showed a highly concerted unfolding transition for all backbone amides in the sequence, indicating even higher cooperativity for this variant than the 13 1 190-Plat WT protein. In contrast, pressure-dependent C- H HSQC peaks of the core Fast Pressure Jump Reveals Site-Specific Protein Dehydration-Folding side-chain methyl groups of this variant revealed considerable deviation from Dynamics two state behavior. These observations are interpreted in terms of the energy Maxim B. Prigozhin1, Yi Zhang2, Klaus J. Schulten3, Martin Gruebele4, landscape of pp32 and the effects of mutations thereon. Taras V. Pogorelov4. 1Physics, Stanford, Stanford, CA, USA, 2Biophysics, University of Illinois, 193-Plat Urbana, IL, USA, 3Physics, University of Illinois, Urbana, IL, USA, Chaperone-Guided Co-Translational Folding 4Chemistry, University of Illinois, Urbana, IL, USA. Kaixian Liu, Kevin Maciuba, Christian M. Kaiser. Protein dehydration is a major but often neglected contributor to protein Johns Hopkins Univ, Baltimore, MD, USA. folding. Dehydration upon folding can be characterized directly by all-atom Co-translational folding likely simplifies the conformational search problem simulations of fast pressure-drop experiments, which create desolvated for large proteins, but the events leading to correctly folded, functional struc- pockets inside the nascent hydrophobic core. Here we study pressure drop re- tures remain poorly characterized. Domain-wise folding and help from chaper- ones are particularly important for multi-domain proteins, which constitute a folding of three lambda repressor fragment (l6-85) mutants computationally and experimentally. These mutants report on tertiary structure formation large fraction of all proteomes. Using optical tweezers, we have dissected early via fluorescent helix-helix contact pairs. All-atom simulations of pressure folding events of nascent elongation factor G, a multi-domain protein that re- drops capture refolding of all three mutants by a similar mechanism, thus vali- quires chaperones for folding. Interactions with the ribosome reduce inter- dating the non-perturbative nature of the fluorescent contact probes. As an domain misfolding and, depending on nascent chain length, can either reduce experimental confirmation of computer simulations, fast pressure-drop relax- or increase folding rates of the N-terminal G-domain. Successful completion ation experiments capture refolding on a millisecond timescale. These of G-domain folding is crucial because it is a prerequisite for folding of the experiments reveal that contacts between contiguous alpha-helix pairs 1-2 next domain. Unexpectedly, co-translational folding does not proceed unidirec- and 2-3 rapidly form and dissolve, while contacts between alpha-helices 1 tionally: unfolded polypeptide emerging from the ribosome can denature an and 3 experience a slower association/dissociation rate. Analysis of simulated already folded domain. The chaperone trigger factor, but not the ribosome, pro- inter-probe distances also shows that alpha-helix pair 1-3 distance displays tects against denaturation, thus helping multi-domain proteins overcome the slowest characteristic timescale, so packing of alpah-helices 1 and 3 is inherent challenges during co-translational folding. Misfolding among more indeed the rate-limiting folding step. Unlike the case of the two-state folder C-terminal domains, which are energetically coupled in the native structure, poses an additional obstacle for EF-G folding. The second majoe nascent a3D, whose drying and core formation proceed in concert, l6-85 repeatedly dries and rewets local tertiary contacts before finally forming a solvent- chain-binding chaperone in bacteria, DnaK, acts on these longer nascent chains excluded core, explaining the multitude of states observed during refolding and changes the folding energy landscape in fashion distinct from trigger fac- in molecular dynamics simulations. Our work demonstrates that proteins tor. Our single-molecule experiments define the folding pathway of a complex can explore desolvated pockets numerous times prior to reaching the native multi-domain protein and show how the ribosome and two differentially acting conformation. chaperones together modulate nascent chain folding.

191-Plat Single-Molecule Force Spectroscopy and Molecular Dynamics Simulations Platform: Spectroscopy and Single-Molecule Reveals Complex Folding Landscape and its Potential Role in Amyloid Fluorescence Fibril Formation in a PDZ Domain Ha H. Truong, Susan Marqusee. 194-Plat Dept Molec Cell Bio, Univ Calif Berkeley, Berkeley, CA, USA. FRET-Filtered Spectroscopy to Simultaneously Probe Local and Global Molecular understanding of partially folded high-energy structures accessible Conformations of Nucleic Acids from the native state or during the folding process of a protein is imperative Julia R. Widom. for understanding of protein aggregation and therapeutic intervention, Dept Chemistry, Univ Oregon, Eugene, OR, USA. yet structural and energetic characterization of these species remains a RNA performs a diverse set of biological functions, acting in turn as a catalyst, challenge. By using a powerful combination of single-molecule force an information carrier, a regulator of gene expression and more. I will briefly

BPJ 9292_9302 Sunday, March 3, 2019 39a present work in which I investigated the folding of a bacterial riboswitch that 197-Plat regulates gene expression during the process of transcription. I will discuss DNA Base Damage and Consequential Point Mutation Controls Telomere some interesting questions that this work brought up about the mechanism Conformation and Elaborates Telomerase Extension Activity by which this RNA folds into its biologically active structure, and how those Hui-Ting Lee1,2, Tapas Paul1, Joshua Choe1, Samantha Sanford3, questions can be addressed using spectroscopic techniques that combine elec- Patricia L. Opresko3, Sua Myong1,4. tronic coupling and Fo¨rster resonance energy transfer (FRET) as probes of nu- 1Department of Biophysics, Johns Hopkins University, Baltimore, MD, USA, cleic acid structure on different length-scales. In a new family of techniques 2Chemistry, Loyola University of Maryland, Baltimore, MD, USA, that I call FRET-filtered spectroscopy (FFS), two closely spaced fluorophores 3Department of Environmental and Occupational Health, University of (such as fluorescent base analogues) are positioned at a location within the Pittsburgh, Pittsburgh, PA, USA, 4Center for Physics of Living Cells, RNA whose local conformation is of interest. Electronic coupling between University of Illinois, Urbana-champaign, IL, USA. these fluorophores gives rise to changes in absorbance, circular dichroism Telomeres are protein-DNA complexes that cap the ends of chromosomes and other spectra that can be interpreted quantitatively in terms of their and protect the chromosomes from deterioration. Stem cells and most cancer A˚ ngstrom length-scale distance and relative orientation. An additional fluoro- cells maintain telomere length by activating telomerase, a reverse transcrip- phore, chosen to be a FRET acceptor for the coupled dimer, is placed at a loca- tase that extends telomeric overhang by adding TTAGGG tandem repeats. tion in the RNA for which nanometer length-scale fluctuations are of interest. Telomere is considered a hot spot for oxidative damage due to its high gua- By recording any type of excitation spectrum of the coupled dimer while de- nine content. We used single-molecule FRET (smFRET) microscopy to test if tecting fluorescence either directly from it or from the acceptor, the spectra ob- different positions and types of oxidative damages impart unique effect in tained can be decomposed into the individual spectra of subpopulations with telomere structure and telomerase activity. Our recent work showed that different FRET efficiencies from one another. I will present results on two nu- the G-quadruplex (GQ) formed by telomeric overhang is destabilized by a cleic acid systems, one of which is designed as a spectroscopic model system single site damage, leading to an increased telomerase activity. Here, we with well-defined FRET efficiencies and electronic coupling strengths, and the compared two common oxidatively damaged bases, 8oxo-guanine and O6- other of which is designed to probe local and global conformational changes in methylguanine, in three terminal guanine positions of telomere, which is the riboswitch. the key access point for telomerase. Consistent with previous findings, any modification of the second position is most disruptive while the other two po- 195-Plat sitions are better tolerated. At all positions, the structural instability induced Tetherless, Precise and Extended Observation of Single-Molecule FRET in by oxidative damage, especially the O6-methylguanine, far exceeds the an Anti-Brownian Trap perturbation by base substitutions, rendering high accessibility to comple- Hugh Wilson, Quan Wang. mentary DNA strand. Telomerase binding and extension activity is dimin- Princeton University, Princeton, NJ, USA. ished greatly by the two consequential point mutations (T and A) at the A comprehensive understanding of biomolecules calls for the ability to terminal guanines regardless of the position or structural disruption, likely observe single-molecule dynamics at the nanometer scale without perturba- due to the base-paring dependent mode of binding. In contrast, point muta- tion or constraints. Single-molecule Fo¨rster resonance energy transfer tions in upstream guanines causes same level of telomerase binding as (smFRET) is a powerful tool for probing nanoscale dynamics, but existing oxidatively damaged bases at the terminal guanines. Our finding demon- modalities have limitations. Solution based confocal measurements are strates how a single base damage can drive the structural alteration and restricted by the short (1ms), diffusion limited observation time. Surface im- lengthening of telomere in a position dependent manner. Furthermore, this mobilized measurements can extend the observation window, but at the data suggests a long-term and inheritable effect of oxidative damage on telo- expense of the molecule’s translational and rotational degrees of freedom. mere lengthening. Moreover, potential perturbations to the biomolecule’s function introduced by immobilization or encapsulation are always a concern. We overcome these 198-Plat limitations by combining smFRET optics with the capability to isolate indi- G-Quadruplex-Helicase Interactions and the Impact of Small Molecules vidual molecules in solution using an Anti-Brownian ELectrokinetic Parastoo Maleki, Hamza Balci. (ABEL) trap. Our new platform, ABEL-FRET, enables photon-by-photon Physics, Kent State Univ, Kent, OH, USA. recording of smFRET trajectories over tens of seconds in solution, without One of the therapeutic targets that has attracted attention in targeted therapy is tethering the molecule to a surface. We first demonstrate ABEL-FRET using guanine quadruplex (GQ), which forms in guanine-rich nucleic acid sequences, short (10-30bp) DNA rulers and achieve near shot-noise limited precision of such as telomeres. Studies on cancer cells show that stabilizing the GQ struc- DE0.01 for 5,000 photons. This is 10 times better than burst-based tures in telomeres with small molecules reduces telomerase activity and prolif- confocal measurements and 2 times better than TIRF-based measurements. eration of cancer cells. Because of their role in biological processes and their We also demonstrate the capability to observe dynamic smFRET transitions potential use as therapeutic targets, GQ structures have been utilized in charac- using Holliday junctions and DNA-protein interactions with millisecond terizing the efficacy of small molecule anti-cancer drugs. time resolution. We envision ABEL-FRET will fill a critical gap in the mod- In this study, we performed single molecule measurements to study how stabil- ern single-molecule toolkit. ity of GQ against Bloom helicase (BLM) is impacted by its interactions with several prominent small molecules (L1H1-7OTD, PDS and PhenDC3). We 196-Plat considered two different approaches to quantify this effect: dynamic approach Bayesian Nonparametrics for Fluorescence Methods and steady state approach. DNA constructs were accordingly designed depend- Meysam Tavakoli1, Sina Jazani2, Ioannis Sgouralis2, Steve Presse2. ing on the question we addressed in each approach. The dynamic assay with 1Physics, Indiana University-Purdue University, Indianapolis, IN, USA, time resolution of 50 ms reveals 50%-60% reduction in the GQ unfolding 2Physics, Arizona State University, Tempe, AZ, USA. rate when a small molecule is present. On the other hand, the fraction of GQ Fluorescence methods are widely used across Biophysics. Fluorescence signals molecules that remain folded against BLM-mediated unfolding rose by a factor contain important information on biomolecular chemistry and dynamics. This of two in the presence of these small molecules. The similarity of the results information can be further applied to characterize and monitor complex biolog- attained from the two approaches illustrates a consistent picture in terms of ical processes. However, it is normally difficult to establish accurate parametric the impact of these molecules on BLM-GQ interactions. Moreover, we inves- models for biophysical systems on account of the noise inherent to single mole- tigated the impact of these molecules on folding dynamics of GQ. Even though cule methods. For this reason, we introduce Bayesian nonparametrics (BNPs) GQ folding was too fast to characterize the impact of small molecules, we that go beyond parametric modeling where models of single molecule dy- observed a significant variation in the folding conformation in the presence namics are assumed from the onset. BNP approaches allow us to introduce of small molecules. an explicit mathematical formulation that quantitatively describes the func- tional relation from the observation data. This method provides a purely empir- 199-Plat ical identification of sample fluorescence properties that can serve as input in Chromatin Remodeling Induced by the Invasion of Yeast Pioneer Tran- future coarse-grained modeling simulations. BNPs are poised to play an impor- scription Factor Rap1 Revealed by Single-Molecule FRET tant role in the analysis of fluorescence signals since so few model features- Anne-Marinette Cao1, Maxime Mivelaz1, Iuliia Boichenko1, Louise Bryan1, such as the number of states of a single molecule in any time trace-are known Slawomir Kubik2, David Shore2, Beat Fierz1. a priori. Moreover, BNPs provide full posteriors over models and parameters in 1Institute of Chemical Sciences and Engineering, Ecole Polytechnique comparison to existing Bayesian approaches that provide only distributions Federale de Lausanne, Lausanne, Switzerland, 2Institute of Genetics and over parameters. Genomics in Geneva, University of Geneva, Geneva, Switzerland.

BPJ 9292_9302 40a Sunday, March 3, 2019

Deciphering chromatin regulation at the molecular level is of fundamental the dynamics of influenza hemagglutinin (HA), the canonical class-I viral importance for an understanding of cellular physiological and pathological glycoprotein, and Ebola GP, which shares structural similarities with HA. processes. Chromatin is an extremely complex system due to its molecular or- This novel approach was facilitated by introduction of non-canonical amino ganization, heterogeneous structure and multiscale dynamics induced by post acids into HA, which were subsequently labeled with fluorophores. In contrast translational modifications on chromatin itself and other regulatory effectors to the current model of class-I-mediated membrane fusion, we observe including transcription factors (TFs). One key class of chromatin interacting spontaneous and reversible exchange between pre-fusion and intermediate proteins are pioneer transcription factors. The main characteristic that distin- conformations. Acidification of pH and interaction with receptors shift the dy- guishes pioneer transcription factors from other TFs is their ability to namic equilibrium, favoring progression toward membrane fusion. These specifically recognize their target DNA sequences in compacted chromatin observations are leading to a new understanding of viral membrane fusion, and consequently to trigger chromatin opening, thus enabling the cellular ma- and conserved and divergent features of distinct class-I viral envelope chinery to locally access the DNA. In the context of cell fate reprogramming, glycoproteins. this pioneer action is crucial, but its molecular mechanism is poorly under- stood. Known to be an essential protein involved in multiple steps of DNA regulation, Saccharomyces cerevisiae repressor-activator protein 1 (Rap1) is Platform: Protein-Lipid Interactions: Channels/ the pioneer transcription factor inspiring us to explore its pioneer role in Structures such complex and dynamic system as chromatin. We recently established a single-molecule Fo¨rster resonance energy transfer (FRET) method, using mi- 202-Plat cromirror total internal reflection fluorescence (mmTIRF) microscopy, dedi- Toxic Amyloid Tape: A Novel Mixed Antiparallel/Parallel Beta-Sheet cated to investigate structural dynamics of chromatin fibers. Combining Structure Formed by Abeta on GM1 Clusters single-molecule FRET and chemically defined synthetic chromatin segments, Katsumi Matsuzaki1, Yuki Okada1, Kaori Okubo1, Keisuke Ikeda2, we uncovered the interconversion kinetics of discrete tetranucleosome units Yoshiaki Yano1, Masaru Hoshino3, Yoshio Hayashi4, Yoshiaki Kiso5, and the impact of the post-translational modifications by ubiquitylation on Hikari Itoh-Watanabe6, Akira Naito7. chromatin structure. Here, we demonstrate that Rap1 invades compacted chro- 1Grad Sch Pharm Sci, Kyoto Univ, Kyoto, Japan, 2Grad Sch Med Pharm Sci, matin and exerts chromatin remodeling. These studies yield fundamental in- Univ Toyama, Toyama, Japan, 3Kyoto Univ, Kyoto, Japan, 4Tokyo Univ sights into the molecular mechanisms of gene regulation by molecular Pharm Life Sci, Hachioji, Japan, 5Nagahama Institute Bio-Sci Tech, interactions. Nagahama, Japan, 6Yokohama Naitional Univ, Yokohama, Japan, 7Dept Engineering, Yokohama Natl Univ, Yokohama, Japan. 200-Plat The abnormal aggregation of amyloid beta-protein (Abeta) is considered Single Molecule Measurements Reveal Conformational Transitions central in the pathogenesis of Alzheimer’s disease. We focused on ‘mem- During DNA Clamp Loading and Unloading brane-mediated’ amyloidogenesis and found that amyloid fibrils formed on SeungWon Lee, Eunjin Ryu, Sukhyun Kang, Hajin Kim. monosialoganglioside GM1 clusters were more toxic than those formed in UNIST, Ulsan, Republic of Korea. aqueous solution. In this study, we investigated the structure of the toxic fibrils Proliferation cell nuclear antigen (PCNA) is a DNA clamp, playing an impor- by Abeta-(1-40) in detail in comparison with less toxic fibrils formed in tant role of providing a ‘‘platform’’ for various enzymes during DNA repli- aqueous solution. The less toxic fibrils contain in-resister parallel beta-sheets, cation. The loading of the closed trimeric ring of PCNA into duplex DNA whereas the structure of the toxic fibrils is unknown. Atomic force microscopy requires the ATP-dependent activity of replication factor C (RFC) complex. revealed that the toxic fibrils had a flat, tape-like morphology composed of a The unloading of PCNA from chromatin is crucial for the regulation of single beta-sheet layer. Isotope-edited infrared spectroscopy indicated that replication process and maintaining genomic stability and it was recently almost the entire sequence of Abeta is included in the beta-sheet. Chemical found that ATAD5 protein is complexed with RFC-like complex (RLC) cross-linking experiments using Cys-substituted Abetas suggested that the fi- to get involved in the unloading of PCNA. However, the molecular mecha- brils mainly contained both in-resister parallel and 2-residue-shifted antipar- nisms of PCNA loading and unloading processes have remained poorly un- allel beta-sheet structures. Solid-state NMR experiments also supported this derstood. Here, we report direct observation of the loading and unloading conclusion. Thus, the toxic fibrils were found to possess a novel unique dynamics of human PCNA driven by RFC and ATAD5-RLC complexes, structure. respectively, by single molecule fluorescence resonance energy transfer measurements. Distinct conformational stages during PCNA loading were 203-Plat clearly detected that represent open and closed conformations of PCNA Recruitment of Actin-Binding Proteins on the Membrane Interface: trimer and another loading intermediate that possibly triggers the dissociation Effects of Cholesterol on Protein/PIP2 Interactions of RFC complex. The unloading of PCNA occurred upon binding ATAD5- Ololade Fatunmbi1, Ryan Bradley1, Robert Bucki2, Paul Janmey2, RLC complex through an intermediate conformation, but not requiring the Ravi Radhakrishnan1. hydrolysis of ATP, which is later employed to recycle PCNA and ATAD5- 1Chemical and Biomolecular Engineering, University of Pennsylvania, RLC complexes. Our findings present a mechanistic model of the clamp Philadelphia, PA, USA, 2Physiology, University of Pennsylvania, loading/unloading dynamics and bring implications on the mechanism Philadelphia, PA, USA. controlling the exclusive functions of RFC and ATAD5-RLC complexes in Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) only represents 1% of the loading and unloading processes, respectively. It further provides a plat- membrane phospholipids but is a key ligand in a myriad of important physi- form to study how PCNA controls the exchange of various replication ological events such as membrane-cytoskeleton interactions. PI(4,5)P2 acti- enzymes. vates various actin-binding proteins that regulate actin microfilament assembly which in turns plays an essential role in dynamic changes in the 201-Plat cytoskeleton. Understanding PI(4,5)P2’s ability to bind numerous actin- Conformational Dynamics Related to Membrane Fusion Observed in binding proteins that contain structurally divergent PI(4,5)P2 binding sites Single Viral Envelope Glycoproteins will provide invaluable insights linking molecular-level specificity to cyto- Dibyendu Kumar Das, Natasha Durham, Angela Howard, James B. Munro. skeletal assembly and dynamics. However, the underlying atomistic mecha- Dept Molec Biol & Microbiol, Tufts University, Boston, MA, USA. nisms by which PI(4,5)P2 interacts with several actin-binding proteins has All enveloped viruses enter cells by fusing the viral membrane with a cellular not been well investigated. Cholesterol’s role in mediating peptide and lipid membrane. This fusion event in promoted by the viral envelope glycoprotein. interactions is also not fully understood. Previously, we have shown both The current model of class-I viral membrane fusion describes a static ‘‘spring- computationally and experimentally that lipid composition, specifically loaded’’ fusion domain. Upon triggering by acidic pH or interaction with a re- PI(4,5)P2 concentration and cholesterol presence regulates the ability for ceptor, the fusion domain undergoes a singular, irreversible conformational PI(4,5)P2 to interact with an actin nucleating protein, Mdia2 (a formin pro- rearrangement. Atomic-resolution structural models of several envelope tein). In this current study, we employed several all-atom molecular dynamics glycoproteins describe the endpoints of this dynamic event. Yet no direct visu- (MD) simulations on three systems, namely, peptides derived from the formin alization of these conformational dynamics has ever been made, leaving the protein mDia2, neural wiskott-aldrich syndrome (N-Wasp), an actin nucle- current model of class-I-mediated membrane fusion unverified. Here, using ating protein, and actin filament severing protein, gelsolin. The peptides single-molecule Fo¨rster resonance energy transfer (smFRET) imaging we were adhered on lipid bilayers containing varying levels of PI(4,5)P2 and directly visualized the conformational changes of individual envelope glyco- cholesterol concentration. Remarkably, our results reveal electrostatic interac- protein trimers on the surface of viral particles. Thus far, we have visualized tions between the peptides and lipid bilayers are uniquely specific to structure

BPJ 9292_9302 Sunday, March 3, 2019 41a of the peptide, PI(4,5)P2 concentration, and cholesterol presence. Analysis of Cholesterol is a major component of cell membranes and is an important regu- the electrostatic interactions PI(4,5)P2 and formin and gelsolin peptide indi- lator of multiple types of ion channels. However, the dynamics of its interac- cate that the presence of cholesterol, increases PI(4,5)P2 recruitment. Dy- tions and the specific mechanisms through which it mediates ion channel namic light scattering experiments performed on the peptides and bilayers function are not well understood. One family of ion channels, inwardly recti- were found to be in agreement of our MD simulations. These results suggest fying potassium (Kir) channels, was shown to be sensitive to cholesterol a new understanding of the underlying mechanisms of peptide binding to the through direct interactions with ‘‘cholesterol-sensitive’’ regions on the channel membrane in a physiologically relevant bilayer environment mediated by protein. It is unclear though whether these regions correspond to a well- PI(4,5)P2 and cholesterol. localized binding site or are representative of a broad and shallow thermody- namic landscape. To investigate the interactions between cholesterol and 204-Plat Kir channels, we developed Martini coarse-grained simulations of Kir2.2 Molecular Mechanism of Voltage-Gated Ca2D Channel Regulation by embedded in a POPC model membrane bilayer with varying concentrations Membrane PIP2 of cholesterol. This approach allowed us to simulate the unbiased diffusion Cheon-Gyu Park, Byung C. Suh. of cholesterol from the bilayer environment to both the surface and recessed Dept of Brain & Cognitive Sci, DGIST, Daegu, Republic of Korea. pockets of the channel. We found that, unlike small drug-like molecules, which Voltage-gated calcium (CaV) channels play essential roles in adjusting calcium tightly bind to a single site, cholesterol interacts with the channel on a range of influx upon membrane depolarization. CaV2 (N-, P/Q- and R-type) channels different time scales, with multiple cholesterol molecules in contact with the are concentrated in the presynaptic nerve terminals and important for the channel simultaneously. Following this, we utilized principles from network neurotransmitter release. Adjusting the presynaptic calcium channel gating ex- theory to identify four discrete cholesterol binding sites on the channel, which erts potent influence on synaptic plasticity. CaV channels need auxiliary sub- exist depending on the conformational state of the channel. Furthermore, we units for proper trafficking to the plasma membrane and the channel gating. found that a twofold decrease in the bilayer concentration of cholesterol, which Especially CaVb subunit plays crucial roles in the surface expression of CaV was shown previously to increase Kir2 channel activity, leads to a decrease in channels and fine-tuning of channel gating. It has been known that CaV chan- occupancy in only a subset of identified binding sites. These results allow us to nels are modulated by membrane phosphatidylinositol 4,5-bisphosphate discriminate between functionally relevant and functionally non-relevant (PIP2). The binding affinity between ion channel and PIP2 is important for cholesterol interaction sites, and hypothesize a mechanism of cholesterol the channel gating normally, but molecular mechanism of PIP2 regulation re- regulation. mains unclear. It was recently reported that subcellular localization of b sub- unit is a key factor for the control of PIP2 sensitivity of CaV channels. Here we 207-Plat found that the intracellular movement of I-II linker in a1 subunit is important Diffusion in Nanopore Connected Bilayer Networks for determining the PIP2 sensitivity of CaV channels. When the I-II linker was Manon Valet,Lea-Laetitia Pontani, E´ lie Wandersman, Alexis Prevost. shifted to the plasma membrane, current inhibition by PIP2 depletion signifi- Laboratoire Jean Perrin, Sorbonne Universite - CNRS, Paris, France. cantly decreased as like the responses triggered by membrane-tethered b sub- Cell-cell communication in biological tissues depends partly on the passive unit. Consistently we also found that inserting a flexible linker between molecular transport through transmembrane channels. How does such diffusive membrane-tethered Lyn and GK domain of b subunit increased the PIP2 sensi- transport lead to specific patterns in multicellular assemblies? We have ad- tivity of CaV channels. Polybasic motif at the C-terminal end of the I-II linker dressed this question by studying the diffusive properties of fluorophores in bio- of CaV channels is a potential PIP2 interaction site. Neutralization of the poly- mimetic tissues. These tissues consist of monodisperse aqueous microdroplets basic motif of I-II linker abolished PIP2 sensitivity of CaV channels. Together, connected by synthetic membranes - known as Droplet Interface Bilayers our results indicate that the conformational shift of I-II linker to the plasma (DIB) - decorated with passive alpha-hemolysin pores. Microdroplets are pro- membrane is the key mechanism for decreasing the PIP2 sensitivity of CaV duced in a bath of oil and lipids using a novel technique we have recently devel- channels and this shift is mainly regulated by auxiliary CaVb subunit in phys- oped, relying on the interplay between interfacial forces at the tip of a capillary. iological condition. Combined with pre-patterned substrates, this technique has allowed us to engi- neer linear arrays of DIBs. 205-Plat We have measured the diffusion kinetics of molecular fluorophores from a pH Induced Switch between Different Modes of Cytochrome C Binding to source droplet to its connected neighbors and related its dependence to the Cardiolipin Containing Liposomes pore concentration in the DIBs. We have interpreted our results using a theo- Bridget Milorey, Reinhard Schweitzer-Stenner. retical frame based on continuous time random walks and mean first passage Drexel University, Philadelphia, PA, USA. times. The scaling between the diffusion timescale and the pore concentration Fluorescence, visible circular dichroism, absorption and resonance Raman allows us to infer the spatial arrangement of the nanopores within the spectroscopy were combined to explore structural changes of ferricytochrome membrane. c upon its binding to cardiolipin containing liposomes (20% 1,1’,1,2’-tetrao- leyolcardiolipin (TOCL) and 80% 1,2-deoleyol-sn-glycero-3-phosphocholine 208-Plat (DOPC)) at acidic pH (6.5). According to earlier work of Kawai et al., cyto- Molecular Dynamics Simulation and Photo-Crosslinking Reveal a Specific chrome c binding at this pH is governed by interactions between the Cholesterol Binding Site for the Metabotropic Glutamate Receptor 2 phosphate head groups of cardiolipin and amino acid side chains of the so- Markus Kurth1, Rainer Beck1, Camilo A. Aponte-Santamaria2, called L-site which contains the charged residues K22, K25, K27 and poten- Britta Bruegger1. tially H26 and H33. We found that L-site binding causes a conformational 1Heidelberg University Biochemistry Center, Heidelberg, Germany, 2Max change that involves a change of the protein’s ligation and spin state. We Planck Tandem Group in Computational Biophysics, Universidad de los measured spectroscopic responses to an increasing number of cardiolipin con- Andes, Bogota´, Colombia. taining liposomes in the absence and presence of NaCl. The latter was found The Metabotropic glutamate receptor 2 (mGluR2) belongs to the family of to mostly inhibit protein binding already with 50 mM concentration. The in- G-protein coupled receptors, a large class of transmembrane proteins hibition effect was quantitatively reproduced by applying the electrostatic the- whose function has been shown to be regulated by their surrounding lipid ory of Heimburg and Marsh. A comparison with corresponding spectroscopic environment, especially by the cholesterol content in the membrane. Here, response data obtained at pH 7.4 reveal major differences in that the latter in- we hypothesize that specific protein-lipid interactions contribute to the volves hydrophobic binding followed by a conformational change that is elec- dynamics and regulation of mGluR2. Both tryptophan and tyrosine have trostatically driven, which we identify with the earlier proposed A-site been found in specific protein-lipid binding motifs. In the search of a sphin- binding. golipid or cholesterol binding sites for this protein, an alanine mutagenesis screening was carried out, targeting tryptophan and tyrosine residues at 206-Plat the transmembrane surface of mGluR2. Surface biotinylation and co- Molecular Dynamics Simulations of Kir2.2 and Cholesterol Reveal State- immunoprecipitation showed that neither trafficking nor dimerization are and Concentration-Dependent Binding Sites disturbed by substitution of these aromatic residues. In contrast, cellular Nicolas Barbera1, Manuela A. Ayee2, Belinda S. Akpa3, Irena Levitan4. photo-crosslinking assays demonstrated that cholesterol binding is compro- 1Chemical Engineering, Univ Illinois Chicago, Chicago, IL, USA, 2Chemical mised if one tyrosine residue located at the helix five or another at the helix Engineering, Dordt College, Sioux Center, IA, USA, 3Chemical Engineering, six are replaced. Thus, these experiments suggest these two helices to contain North Carolina State University, Raleigh, NC, USA, 4Department of specific cholesterol binding sites. All-atom and coarse-grained molecular dy- Pulmonary, Univ Illinois Chicago, Chicago, IL, USA. namics (MD) simulations of the mGluR2 transmembrane domain confirmed

BPJ 9292_9302 42a Sunday, March 3, 2019 this observation, by revealing a highly-localized density of cholesterol near ever, irreversible inactivation was observed in 53% of single-channel record- these residues, which smeared out when they were changed to alanine ings (n=15). Combinations of complementary fragments of ATP (PiþADP in silico. The simulations also revealed an important collective motion of or PPiþAMP or PPPiþadenosine) could not reproduce the effects of ATP, the helices which seems to be correlated with the presence of cholesterol. however, prior treatment with adenosine protected against PPPi-induced inac- Notably of this motion was the tilt of helix six in the outer leaflet close to tivation. Therefore, while the RyR2-ATP binding site can accommodate a one of the cholesterol binding sites. Overall, our study combining functional wide variety of ligands, including PPPi, the most effective ligands have assays and multiscale MD simulations demonstrate the existence of a specific at least three phosphate groups that are guided into place by an adenine cholesterol binding site in mGluR2. It will be highly interesting to investigate nucleoside thus preventing destabilising interactions that lead to channel the functional implications of this newly-found specific protein-cholesterol inactivation. interaction. 211-Plat 209-Plat A Cryo-EM Based Study of a Mutant Cardiac Ryanodine Receptor (RyR2) Direct Lipid Binding in a Pentameric Ligand-Gated Ion Channel Assessed Kavita A. Iyer1, Ashok R. Nayak1, Takashi Murayama2, by Native Mass Spectrometry Nagomi Kurebayashi2, Montserrat Samso1. Wayland W.L. Cheng. 1Virginia Commonwealth University, Richmond, VA, USA, 2Juntendo Anesthesiology, Washington Univ Sch Med, St Louis, MO, USA. University, Tokyo, Japan. Pentameric ligand-gated ion channels (pLGICs) such as the nicotinic acetyl- Ryanodine receptors (RyRs) are large intracellular Ca2þ channels that act as choline receptor or the prokaryotic homologue, Erwinia ligand-gated ion chan- gatekeepers to Ca2þ stored in the sarcoplasmic reticulum (SR). RyR2, the car- nel (ELIC), require anionic phospholipids for agonist-induced channel diac isoform of RyR, releases Ca2þ by a mechanism termed as Ca2þ-induced- activation. Whether this requirement is mediated by direct lipid binding events Ca2þ-release (CICR). The large cytoplasmic domain of RyR is composed of a to specific sites is not known, and understanding this phenomenon is essential number of smaller domains that interact with each other as well as external for the development of lipophilic allosteric modulators of pLGICs. We used binding partners arranged in a complex manner to precisely control channel native mass spectrometry (MS) coupled with thermal stability and liposome gating. The published structures of porcine RyR2 in both the open and closed flux assays to examine direct binding of phospholipids to ELIC and the func- state reveal a ‘‘breathing’’ motion exhibited by the cytoplasmic domain during tional effects of binding. ELIC from E. coli membranes co-purify with bound channel opening. Using cryo-EM, we examined a NTD mutation of RyR2 to phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) lipids, showing study its effect on the channel’s conformation. It is long suspected that muta- selectivity for PG lipids. Native MS analysis of de-lipidated ELIC using exog- tions in RyR2 might render them more susceptible to opening, thereby enous phospholipids reveal that anionic phospholipids such as PG directly bind causing a leak of Ca2þ from the SR. These leaky RyR2 channels lead to to ELIC with higher affinity than PE or phosphatidylcholine (PC). By corre- arrhythmia and have been associated with disorders such as catecholaminergic lating the stoichiometry of binding with lipid concentration, we determine polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ven- that two bound PG lipids per subunit are required to stabilize ELIC from ther- tricular dysplasia type 2 (ARVD2). We have successfully resolved and mal denaturation. To identify the sites of PG binding, single and double muta- modeled a 3D map of the mutant and wild type mouse RyR2 at 4.86 and tions of five arginine residues at the transmembrane domain interfacial region 6.15 A˚ resolution respectively. Our 3D reconstructions display densities that were generated. Analysis of these mutants reveal two clusters of positively have hitherto been unresolved within RyR2. The mutant RyR2 presents a charged residues at both extracellular and intracellular leaflets that are essential number of subtle but significant differences in the relative orientations of mul- for PG binding and thermal stabilization. Ongoing single particle cryo-EM tiple cytoplasmic sub-domains, such as the NTDs, CD and HD2, when studies of ELIC reconstituted in nanodiscs of defined lipid composition is antic- compared to wild type RyR2. It appears that differences in the orientation ipated to corroborate these sites of lipid binding, and may reveal the structure of of the NTDs might be causing other domains of the cytoplasmic domain to uncoupled and activated/desensitized ELIC channels induced by these lipid- undergo conformational changes. With this structural study we hope to binding events. shed light on the effect of a NTD mutation on the leaky phenotype of RyR2. Supported by grants AHA 14GRNT19660003, MDA352845, NIH Platform: Intracellular Calcium Signaling, R01 AR068431, and NIH U24GM116789. Sparks and Waves 212-Plat Detecting RyR Clusters with CaCLEAN: Validation and Influence of 210-Plat Spatial Heterogeneity Modelling the ATP Binding Site of RyR2 to Rationalise Ligand-Induced David Ladd1, Agne Tilunaite2, H. Llewelyn Roderick3, Christian Soeller4, Gating Behaviour Edmund Crampin2, Vijay Rajagopal5. Chris Lindsay1,2, Mano Sitsapesan1, Wei Mun Chan1, Elisa Venturi1, 1Systems Biology Lab, Department of Biomedical Engineering, University of William Welch3, Maria Musgaard4, Rebecca M. Sitsapesan1. Melbourne, Melbourne, Australia, 2Systems Biology Lab, School of 1Dept of Pharmacology, University of Oxford, Oxford, United Kingdom, Mathematics and Statistics, University of Melbourne, Melbourne, Australia, 2Chemistry Research Laboratory, University of Oxford, Oxford, United 3Laboratory of Experimental Cardiology, Department of Cardiovascular Kingdom, 3Dept Biochem, Univ Nevada Reno, Reno, NV, USA, Sciences, KU Leuven, Leuven, Belgium, 4The Soeller Lab, Living Systems 4Department of Chemistry and Biomolecular Sciences, University of Ottawa, Institute, University of Exeter, Exeter, United Kingdom, 5Cell Structure and Ottawa, ON, Canada. Mechanobiology Group, Department of Biomedical Engineering, University The cardiac ryanodine receptor, RyR2, is a ligand-gated ion channel that is trig- of Melbourne, Melbourne, Australia. gered to open by rises in cytosolic [Ca2þ]. The changes in gating caused by In cardiomyocytes, the coordinated release of calcium ions from intracellular Ca2þ binding to RyR2, however, depend heavily on the coincident interaction stores through ryanodine receptor (RyR) clusters is key to the generation of a of RyR2 with other regulators. ATP is an important constitutive cellular regu- calcium transient and induction of contraction following depolarisation of lator of RyR2 and can fully open the channel in the presence of activating cyto- the sarcolemma. Recently, a deconvolution algorithm from radio astronomy solic Ca2þ. Since other physiologically relevant adenine nucleosides and ATP was adapted and applied to analysis of calcium fluorescence imaging of cardi- fragments can also activate RyR2, we have used published cryo-EM structures omyocytes. The algorithm (CaCLEAN) showed potential in revealing both the of both RyR2 (Peng et al. (2016) Science 354, 5324) and ATP-bound RyR1 spatial locations of RyR clusters and their functional response in living cells. (des Georges (2016) Cell 167, 145-157) to investigate the likely relationships However, whether the RyR clusters identified by CaCLEAN analysis of the im- between ligand binding in the RyR2-ATP site and the subsequent modulation aging data were true or false positives remained unvalidated in the absence of of channel gating. ground truth values. In this work, a finite element model was developed to RyR2 open probability and gating behaviour was monitored using [3H]ryano- simulate reaction-diffusion of calcium emanating from RyR clusters during dine binding and single-channel recording of RyR2 after incorporation of sheep the rising phase (first 30 ms) of the calcium transient. Cluster distributions cardiac SR into artificial membranes under voltage-clamp. Ligand docking into were analysed from immuno-labeled confocal images. These distributions the RyR2-ATP site was performed using AutoDock 4.2.3. were applied to admissible regions of an eight sarcomere finite element model We found that most fragments of ATP could activate RyR2 but exhibited at each z-disk using a spatial statistics reconstruction. The effect of two sets of reduced affinity or efficacy. Even triphosphate (PPPi) alone could activate factors were examined with the model: (1) the number and spacing of simulated RyR2 demonstrating that the adenine moiety is not essential for binding, how- RyR clusters and (2) the effect of mitochondria acting as barriers to diffusion.

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Confocal fluorescence microscopy images were simulated from the model re- distance to the closest membrane. We observed a site-specific RyR cluster den- sults and analysed using CaCLEAN. The performance of CaCLEAN was found sity as well as the setting of local phosphorylation gradients that evolve with to be sensitive to cluster spacing and distance from the imaging plane when stimulation parameters and over time. Together, these data suggest that simulating imaging in the longitudinal plane. The inclusion of mitochondria spatio-temporal patterns of phosphorylation contribute to the synchrony of cal- acting as diffusion barriers also tended to marginally increase the number of cium signals throughout the cell volume. detected false positives. 215-Plat 213-Plat Calcium Sensing and Conformational Rearrangements in STIM1, the ER FRET-Based Trilateration Resolves Distinct Structural States and Transi- Calcium Sensor tions of Calmodulin Bound to RyR Aparna Gudlur1, Ana E. Zeraik1, Nupura Hirve1, V. Rajanikanth1, Bengt Svensson1, Robyn T. Rebbeck1, Jingyan Zhang1, Donald M. Bers2, Andrey A. Bobkov2, Elizabeth A. Komives3, Patrick G. Hogan1. David D. Thomas1, Razvan L. Cornea1. 1Dept Signaling/Gene Exp, La Jolla Inst Allergy/Immunol, La Jolla, CA, 1Biochemistry, Molecular Biolology and Biophysics, Univ Minnesota, USA, 2Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, Minneapolis, MN, USA, 2Department of Pharmacology, University of USA, 3Dept Chemistry and Biochemistry, University of California-San California, Davis, CA, USA. Diego, La Jolla, CA, USA. We used time-resolved FRET (TR-FRET) and trilateration to map the location STIM1 senses the depletion of free ER calcium and undergoes a resting-to- of calmodulin (CaM) bound to skeletal and cardiac ryanodine receptor (RyR) active state transition to gate the plasma membrane calcium channel, ORAI1. Ca2þ channels at resting vs. peak contracting [Ca2þ] (nM vs. mM). Eight The prevailing view has been that STIM1 has a single EF-hand calcium-bind- single-Cys variants of the 12.6-kDa FK506-binding protein (FKBP) were ing site and the loss of bound calcium triggers a dramatic loss of secondary labeled with FRET donor (AlexaFluor 488), and targeted to RyR1 and structure of the STIM luminal domain. Surprisingly, our studies show that RyR2. Four single-Cys variants of WT-CaM or the Ca2þ-insensitive STIM1 luminal domain has 5-6 previously undetected calcium-binding sites CaM1234, two N-lobe and two C-lobe sites, were labelled with acceptor (Alex- that are energetically coupled to the EF-hand site, and set the threshold for aFluor 568). FRET from donor-FKBP donors to acceptor-CaM was measured the activation of STIM1. We further show that the loss of bound calcium causes via fluorescence lifetime detection and data were fitted to Gaussian distance only a limited structural rearrangement of the STIM1 luminal domain that distributions. Our trilateration approach accounts for distance distributions propagates to the cytoplasmic domain of STIM1 through a concerted intra- from multiple FRET pairs, and shows the location of the acceptor as a prob- dimer pairing of the transmembrane and juxtamembrane helices into an ability distribution in the format of density map. This map can be displayed extended coiled coil. This intradimer rearrangement overcomes the relatively using common molecular graphics software. The TR-FRET-based trilateration weak CC1-SOAR interactions that hold STIM in an inactive conformation, has allowed us to resolve the location of the N- and C-lobes of CaM within releasing the SOAR domain to activate ORAI channels. Our studies present RyR1 and RyR2 cryo-EM maps at 30 nM and 30 mMCa2þ. Results show new insights into the calcium binding and the structural rearrangements that that Ca2þ-CaM is bound in a more compact conformation than apo-CaM on lead to activation of STIM1. both RyR1 and RyR2. In RyR1, WT-CaM in 30 nM Ca2þ binds close to 2þ the CaM1234 location, suggesting CaM is in apo form in resting Ca .In 216-Plat RyR2, CaM binds close to, but distinctly from CaM1234, suggesting that Abnormal Calcium Leak from Cardiac Sarcoplasmic Reticulum: New þ þ CaM is partially Ca2 loaded in nM (diastolic) Ca2 . By integrating our Insights Offered by Statistical Physics TR-FRET system in a stopped-flow experiment, we resolved millisecond Anna Maltsev1, Michael D. Stern2, Victor A. Maltsev3. þ Ca2 -driven structural transitions of CaM-bound to RyR. Results provide 1Dept Mathematics, Queen Mary Univ London, London, United Kingdom, þ insight in the mechanism of CaM’s action as a Ca2 sensor in modulating 2NIA NIH, Baltimore, MD, USA, 3IRP, NIA NIH, Baltimore, MD, USA. RyR channel function during muscle contraction. This work was supported Heart muscle contraction is activated by a synchronized systolic Ca release by NIH grants R01HL92097 (RLC, DMB) and R37AG26160 (DDT), and from sarcoplasmic reticulum (SR) via Ca sparks. In disease, Ca sparks fail to by American Heart Association Postdoctoral Fellowship 16POST31010019 terminate, causing a diastolic Ca leak that decreases contraction amplitude (RTR). and increases the risk of arrhythmia. The mechanisms and treatment of the abnormal Ca leak remain unclear. We have recently shown that spark 214-Plat termination emerges as collective behavior (synchronized closings) of Ca 2DDT Imaging of Calcium Signaling Microdomains in Cardiac Myocytes release channels (RyRs) that is identical to synchronization of spin orienta- Mouna Abdesselem, Guillaume Gilbert, H. Llewellyn Roderick, tion in ferromagnets described by a phase transition in Ising model. We em- Karin R. Sipido. ployed the Ising approach to investigate and classify mechanisms of spark Cardiovascular Sciences, Experimental Cardiology, KU Leuven, Leuven, termination failure and Ca leak. Methods and Results: The key parameters Belgium. determining whether spark termination succeeds or fails are SR Ca and Myocyte contraction is coupled with electrical depolarization via the process of RyR opening/closing rates. They define analogues of magnetic field h and excitation contraction coupling (ECC). During ECC, CM depolarization results the inverse temperature (b) in Ising model. Sparks terminate via a phase in opening of voltage-gated Ca channels and calcium influx, which in turn trig- transition known as ‘‘h polarity reversal’’ and the leak emerges when h gers a larger calcium release via RyR calcium release channels on the sarco- fails to change its sign. This happens when the SR depletes insufficiently plasmic reticulum (SR) store. Central to this process is the close apposition and RyR openings remained partially synchronized via Ca-induced-Ca- of the SR localized RyRs and plasma membrane voltage-gated calcium chan- release, generating long-lasting sparks. Leak can also occur via b, known nels. This close coupling relies on membranous invaginations of the sarco- as Onsager’s ‘‘order-disorder’’ transition. This happens at low SR Ca, lemma termed t-tubules, which ensure homogenous calcium elevations reducing RyR current and RyRs interactions, resulting in independent RyR throughout the cytosol. In large mammals and in disease, t-tubules are not regu- openings. The disorder leak is distinguished from synchronized leak by larly distributed. Consequently, not all calcium release units (RyR clusters) are larger Peierls contour lengths reflecting degree of disorder. Abnormal leak directly engaged by calcium influx, leading to potential inhomogeneities in results from either a probability imbalance during synchronized firing cytosolic calcium. of RyRs or from disordered RyR firing. Each leak type requires different Here we optimized a multi-beam array confocal microscope to image and balanced treatment to shift RyR operation towards normal spark signaling microdomains at high spatial (215 nm pixel size) and temporal res- termination. olution (125 fps) in intact pig myocytes. We fitted pixel-by-pixel the time evo- lution of calcium transients, extracted local key functional parameters and 217-Plat registered these pixel-based calcium changes with WGA stained cell mem- Filopodia Dynamics are Affected by Cation Flux through TRP Channels branes using a descriptor-based algorithm. When correlating functional pa- Carol A. Heckman, Marilyn L. Cayer, Omolade M. Ademuwiya. rameters to the distance to closest membrane, we observed gradients in Dept Biol Sci, Bowling Green State Univ, Bowling Green, OH, USA. local calcium handling. Such differences were smaller during prolonged stim- Filopodia are the sensors of a cell, receiving signals from both the substrate and ulation, suggesting a potential role for functional modulation, possibly soluble ligands in their environment. In nerve cells, they respond to the fre- through phosphorylation. quency of calcium transients, but the intracellular concentration and calcium We complemented live cells analysis with immunofluorescence staining of gradient are also considered important. The purpose of this research was to cells that were fixed after varying the duration of stimulation. We examined learn how filopodia of epithelial cells are affected by calcium flux. We sub- location and distribution of RyR clusters, their phosphorylation state and their jected cells to ER (endoplasmic reticulum) stress by cyclopiazonic acid

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(CPA) and monitored the relative concentration of intracellular calcium using line linkers, truncations, and hybrid AMPs we were able to produce a variety calcium orange fluorescence. Filopodia prevalence was estimated by an assay of DesHDAP1-BF2 mutant hybrids. Their effectiveness and mechanism were described elsewhere (Heckman et al, 2017, Cytoskeleton 74: 297). Cytoplasmic evaluated via bacterial assays and confocal microscopy. These observations calcium continued to increase for at least 1 hour in CPA-treated cells in have emphasized that combining these approaches results in more active calcium-free media. However, filopodia increased for 10 minutes and then re- BF2-DesHDAP1 hybrid peptides. Future work will further probe the mecha- turned to the level of untreated cells. Restoration of extracellular calcium then nism of these constructs and will investigate whether this design approach caused a rapid increase in filopodia prevalence. The increase was inhibited by a can be generalized to a wider range of peptides. TRPC channel inhibitor, SKF96365, but not by nifedipine or an analog of cal- cium release-activated channel (CRAC) inhibitor 3,5-bis(trifluoromethyl)pyra- 220-Plat zole (BTP). As nifedipine and other dihydropyridines block L-type calcium High-Resolution Structures of Two Metallated Host Defense Peptides that channels, the positive effect was not attributable to calcium influx through these Feature Copper-Dependent Bactericidal and Chemotactic Effects: Impor- channels. We confirmed the presence of a TRPC channel on filopodia of epithe- tance of Histidine for Anti-Infective Action lial cells by immunofluorescence localization. Although TRP channels are not Myriam Cotten1, Alexander Greenwood1, Steven Paredes1, Yimin Miao1, activated by depolarization, they are nonselective for cations and can allow Yawei Xiong1, Ella Mihailescu2. membrane depolarization. Previous work suggested that membrane potential 1Applied Science, William and Mary, Williamsburg, VA, USA, 2University was essential for filopodia. To investigate such effects, we depolarized cells of Maryland, Rockville, MD, USA. with 25 mM KCl. The results resembled those caused by TRPC inhibition Piscidins are membrane-active host-defense peptides (HDPs) that are rather than TRPC activation. TRP channels may affect dynamic structural re- distinctively rich in histidines. Although histidines are pH-responsive in the organizations leading to filopodia initiation or, if persistent at the tips of the physiological range, piscidins are pH-resilient, making them attractive drug structure, TRP channels may affect filopodial persistence by permitting cation templates. Having a conserved histidine at position 3, they possess an amino- flux through the membrane. terminal Cu- and Ni-binding motif. We recently established that upon Cu(II)-coordination, the homologs P1 and P3 oxidize lipids in bacterial membranes and exhibit enhanced efficacy. P1 and P3 share three his- Platform: Membrane Active Peptides and Toxins tidines in their N-terminal domain but P1 has an additional histidine near its C-terminus (H17). Here, we deploy complementary techniques (e.g., 218-Plat NMR, CD, neutron diffraction, SPR, MD) to mechanistically investigate Synergism of Magainins is Not Coupled to the Formation of a Well-Defined their pH-resilient and Cu(II)-dependent membranolytic and anti-infective Peptide Pore properties. Michael Pachler1, Ivo Kabelka2, Regina Leber1, Ilse Letofsky-Papst3, First, we show that P3 is less membrane active and pH-resilient than P1 and Karl Lohner1, Robert Vacha2, Georg Pabst1. requires neutral histidines for membrane disruption. Furthermore, the histidines 1 Institute of Molecular Biosciences, Biophysics Division, University of Graz, of P1, especially H17, feature lower pKa values and deeper bilayer insertions Graz, Austria, 2Central European Institute of Technology, Masaryk than those of P3. At high peptide concentration, the N-terminus of P3 directs University, Brno, Czech Republic, 3Institute for Electron Microscopy and membrane insertion while P1 orients diagonally in the bilayer, burying its Nanoanalysis and Center for Electron Microscopy, Graz University of C-terminus and H17. Thus, the different histidine contents of P1 and P3 trans- Technology, Graz, Austria. late into contrasted directionality of membrane insertion, membrane activity, We have studied the synergistic activity of PGLa and MG2a, two antimicro- and pH-resiliency. bial peptides secreted by the African clawed frog, on phosphatidylethanol- Second, we solve the high-resolution structures and topologies of metallated amine/phosphatidylglycerol mimics of the inner membrane of Gram P1 and P3 bound to bacterial membrane mimics containing oxidized lipids. negative bacteria using an array of experimental and theoretical techniques. We use this crucial structural information to explain how oxidized lipids In particular, we correlated the peptides’ dye-releasing capabilities from large and Cu(II)-coordination enhance the membrane-disruptive effects of the unilamellar vesicles with the induced structural changes on the microscopic to peptides. nanoscopic length scales combining small-angle X-ray and neutron scattering Finally, we explore the effects of P1 and P3 on host immune cells. Employing with transmission electron microscopy and molecular dynamics simulations. neutrophils and transfected cells, we demonstrate that they exclusively use At low concentrations and in the absence of pronounced dye leakage we found formyl peptide receptors to induce chemotaxis and that Cu(II)-coordination that MG2a is aligned parallel to the membrane surface in the lipid’s headgroup modulates this effect. region and causes a relocation of PGLa from the backbone region toward the The structural determinants identified here could help design novel therapeutics polar surface. In the synergistic regime, i.e. at high concentrations, the pep- with versatile anti-infective properties. tides transformed the unilamellar vesicles into collapsed multilamellar lipo- somes with the peptides acting as spacers between the bilayers. In this case, 221-Plat the PGLa and MG2a do not exhibit strong cross-interactions and show the ten- Comparison of the Effects of Daptomycin on Bacterial and Model dency to form fiber-like structures. PGLa and MG2a synergism in phosphati- Membranes dylethanolamine/phosphatidylglycerol mixtures consequently does not involve Ming-Tao Lee1,2, Pei-Yin Yang3, Nicholas E. Charron3, the formation of a stable peptide pore within the bilayer as conceived Meng-Hsuan Hsieh4, Yu-Yung Chang1, Wei-Chin Hung5, Huey W. Huang3. previously. 1Life Sci Group, Natl Synchro Rad Res Ctr, Hsinchu, Taiwan, 2Department of Physics, National Central University, Jhongli, Taiwan, 3Dept Phys & 219-Plat Astronomy, Rice Univ, Houston, TX, USA, 4Institute of Biotechnology, Combining Design Strategies in Engineering More Active Hybrid Antimi- National Taiwan University, Taipei, Taiwan, 5Dept Physics, ROC Military crobial Peptides Acad, Fengshan City, Taiwan. Anne K. Buck1, Louise E.O. Darling2, Donald E. Elmore1. Daptomycin is a phosphatidylglycerol specific, calcium dependent membrane- 1Department of Chemistry and Biochemistry Program, Wellesley College, active antibiotic that has been approved for the treatment of Gram-positive in- Wellesley, MA, USA, 2Department of Biological Sciences and Biochemistry fections. A recent Bacillus subtilis study found that daptomycin clustered into Program, Wellesley College, Wellesley, MA, USA. fluid lipid domains of bacterial membranes and the membrane binding was The ever-increasing bacterial resistance to conventional antibiotics makes it correlated with dislocation of peripheral membrane proteins and depolariza- imperative to discover alternatives. Antimicrobial peptides (AMPs), which tion of membrane potential. In particular, the study disproved the existence are found in the innate immune response of many organisms, including hu- of daptomycin ion channels. Our purpose here is to study how daptomycin in- mans, are a possible alternative. Previous research focused on optimizing the teracts with lipid bilayers so as to understand the observed phenomena on bac- activity of known AMPs has found that increased arginine composition, the for- terial membranes. We performed new types of experiments using aspirated mation of hybrid peptides, and the presence of proline linkers can individually giant vesicles with ion leakage indicator, making comparisons between dapto- produce a more effective AMP. Other work has investigated the value of trun- mycin and ionomycin, performing vesicle-vesicle transfers and measuring cation in making shorter peptides that are more cost-effective to produce but daptomycin binding to fluid phase vs. gel phase bilayers and bilayers have similar, or even increased, activity. Here, we build upon these studies including cholesterol. Our findings are entirely consistent with the observa- in designing novel AMPs based on two well-studied peptides, buforin II tions on bacterial membranes. In addition, daptomycin is found to cause ion (BF2) and DesHDAP1. By integrating observations on arginine mutants, pro- leakage through the membrane only if its concentration in the membrane is

BPJ 9292_9302 Sunday, March 3, 2019 45a over a certain threshold. The ion leakage caused by daptomycin is transient. It icantly reduced for the modified lipopolysaccharides. Simulations suggest the occurs only when daptomycin binds the membrane for the first time; after- initial interaction between the peptide and the outer membrane are governed wards they cease to induce ion leakage. The ion leakage effect of daptomycin by hydrogen bonding to the outer sugar groups rather than displacement of is not transferrable from one membrane to another. The membrane binding of ions in the inner lipopolysaccharide core. Our findings suggest that the struc- daptomycin is reduced in the gel phase versus the fluid phase. Cholesterol also tural changes in LPS of Salmonella outer membrane induced by the PhoPQ weakens the membrane binding of daptomycin. The combination of mem- system alters both the affinity and mechanism of antimicrobial action by hu- brane concentration threshold and differential binding is significant. This man LL-37. could be a reason why daptomycin discriminates between eukaryotic and pro- karyotic cell membranes. 224-Plat Synchrotron X-Ray Scattering Studies to Determine Structure of Amyloid 222-Plat Beta Interactions with Lipid Membranes Life and Death in a Bacterial Biofilm Under Antibiotic Attack Character- Crystal M. Vander Zanden1, Jaroslaw P. Majewski2, Erik B. Watkins3, ized by Fluorescence and Atomic Force Microscopy Eva Y. Chi1. Catherine B. Volle1, Kanesha Overton1, Helen Greer1, Megan A. Ferguson2, 1Chemical and Biological Engineering, University of New Mexico, Eileen M. Spain3, Megan E. Nunez4. Albuquerque, NM, USA, 2Lujan Center for Neutron Scattering, Los Alamos 1Biology, Cottey College, Nevada, MO, USA, 2Dept Chem, State Univ New Natl Lab, Los Alamos, NM, USA, 3MPA-11: Materials Synthesis and York New Paltz, New Paltz, NY, USA, 3Dept Chemistry, Occidental College, Integrated Devices, Los Alamos Natl Lab, Los Alamos, NM, USA. Los Angeles, CA, USA, 4Dept Chemistry, Wellesley College, Wellesley, Alzheimer’s disease (AD) is the 6th leading cause of death in the United MA, USA. States with more than 5 million people currently diagnosed. The cause of With the increase in bacteria developing resistance to traditional antibiotics, AD remains debated, but one known factor is that the peptide amyloid there is a pressing need for new antibiotic compounds. Researchers are beta (Ab) is present at high concentrations in the extracellular space around currently exploring the use of antimicrobial peptides as a new class of anti- neuronal cells. Ab is known to directly interact with neuronal cell mem- biotic drugs. Antimicrobial peptides are typically small and occur naturally branes, resulting in deadly consequences for the cell such as membrane per- as components of many immune systems. These peptides kill bacteria in meabilization and disruption to native membrane behavior. Ab is most one of two mechanisms. In the first mechanism, multiple peptide subunits popularly associated with the fibrils that make up amyloid plaques, the clas- join together to form pores in the bacterial membranes. In the second, the sical hallmarks of AD, however the protein forms oligomeric intermediates peptide crosses the membrane and binds to some molecule in the cell, pre- that are now thought to be the main source of toxicity. The purpose of venting its action. Magainin II, first isolated from the skin of the African this project was to study how Ab oligomers interact with lipid membranes clawed toe frog Xenopus leavis, is a classic example of a pore-forming pep- and perturb membrane structure. To address these questions, synchrotron tide and has been investigated in lipid micelles, bacterial spheroplasts, and x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD) various bacteria. However, much of the research on magainin II and other were used to determine the structural consequences of Ab/membrane interac- antimicrobial peptides has focused on free-swimming, planktonic bacteria, tions. Results indicate that Ab monomers, fibrillar oligomers (FO), and pre- yet many bacteria live not as planktonic cells, but in an organized commu- fibrillar oligomers (PFO) all interact with a model lipid membrane, however nity called a biofilm. Due to their complex architecture and excreted exopo- the a-helix rich PFOs are most disruptive to lipid packing. Monomeric Ab lymeric substances (EPS), cells in the biofilm are notoriously hard to kill. uniquely formed a protein layer that extended into the aqueous subphase, Thus it is essential to evaluate the effectiveness and mechanism of action and was the quickest to form a b-sheet rich protein structure at the mem- of any new classes of antibiotics on both planktonic and biofilm cells. brane surface. By measuring protein adsorption to an air/water interface, Here we use fluorescence microscopy and atomic force microscopy (AFM) we determined that all Ab forms are equally surface active. Furthermore, to evaluate the changes that occur in both planktonic and biofilm Escherichia GIXD revealed the protein layer to be b-sheet rich in all cases, regardless coli cells when exposed to this antimicrobial peptide in native conditions. of initial protein structure. In conclusion, these results support the notion Notably, using AFM we find distinct changes in both cell stiffness and the that the PFO form of Ab is most disruptive to native membrane behavior, morphology of the outer membrane after treatment of both planktonic and while monomeric Ab or FO may be most effective at nucleating fibrillation biofilm cells, but we also observe that the EPS protects some cells in the on a membrane surface. biofilm. 225-Plat 223-Plat Conformational Disorder is Required for Toxin Secretion, Folding and Interaction of Antimicrobial Peptide Ll-37 with Lipopolysaccharides Cell Intoxication Michael Martynowycz1, Amy Rice1, Konstantin Andreev1, Darragh Patrick O’Brien1,2, Dominique Durand3, Sara Cannella1,2, Thatyane M. Nobre Pavinatto2, Jeff Wereszczynski1, David Gidalevitz1. Alexis Voegele2, Patrice Vachette3,Sebastien Brier2, Daniel Ladant2, 1Dept Physics, Illinois Inst Tech, Chicago, IL, USA, 2Departamento de Fı´sica Alexandre Chenal2. e Cieˆncias dos Materiais, Instituto de Fı´sica de Sa˜o Carlos, Sa˜o Carlos, 1Oxford University, Oxford, United Kingdom, 2Dept Structural Biology and Brazil. Chemistry, Institut Pasteur, Paris, France, 3IBBMC, Univ Paris-Sud, Orsay, The outermost surface of Gram-negative bacteria is almost entirely composed France. of lipopolysaccharides. These lipids are targeted by host defense peptides as Bordetella pertussis, the causative agent of whooping cough, secretes an ad- an innate immune response. Pathogenic serotypes of Salmonella enterica enylate cyclase toxin (CyaA, an RTX protein of 1706 residues) that plays an possess the two-component regulatory system, PhoPQ, that modifies lipopoly- essential role in the early stages of respiratory tract colonization. The cell saccharides in response to the presence of host defense peptides. Host defense intoxication process of CyaA is still poorly understood. After its secretion peptides have typically been thought to circumvent antimicrobial resistance through a type 1 secretion system, CyaA intoxicates human cells via a mechanisms by targeting properties of lipid membranes rather than specific direct translocation of its catalytic domain (AC) across the plasma membrane. protein receptors. However, resistance to some peptides has been found in Once in the cytosol, AC binds to calmodulin (CaM) and catalyzes high bacteria with constituted lipopolysaccharide modifications. We report a amounts of cAMP, leading to cell death. Our results, based on a combination two-fold increase in bacteriostatic concentrations of human antimicrobial of biophysical approaches, including SAXS, HDX-MS and SR-CD, illustrate cathelicidin LL-37 for Salmonella enterica with modified lipopolysaccharides how the structural flexibility of CyaA is essential for its secretion, its as compared to the wild type. Both modified and unmodified lipopolysaccha- folding, its translocation into target cells, and its activation by CaM. All of rides are extracted from bacteria and examined by grazing incidence X-ray these steps involve disorder-to-order structural transitions that are finely diffraction and specular X-ray reflectivity. Initial interactions between LL- tuned to the environmental conditions that CyaA successively experiences 37 and both forms of lipopolysaccharide are investigated using all-atom mo- along its journey from the bacterium to the eukaryotic cell cytoplasm. lecular dynamics simulations. Peptide interaction with lipopolysaccharide These data open up new avenues for both basic science, as well as biotechno- monolayers reduces the number of lateral crystalline domains, but is found logical applications of recombinant CyaA as an antigen delivery vehicle, to increase the typical size of domains that remain in both cases. Peptide and as a potential protective antigen in the next-generation of pertussis intercalation into lipopolysaccharide hydrophobic core is found to be signif- vaccines.

BPJ 9292_9302 46a Sunday, March 3, 2019

Posters 229-Pos Computational Investigation of the Effect of Backbone Chiral Inversions on Polypeptide Structure Posters: Protein Structure and Conformation I Gul€ H. Zerze1, Pablo Debenedetti2, Frank Stillinger2. 1Dept Chem Eng, Princeton University, Princeton, NJ, USA, 2Princeton 226-Pos University, Princeton, NJ, USA. A New Amino Acid Similarity Matrix Based on Sequence Correlations and Studying a helix-folding peptide and a b-hairpin peptide with systematically Structural Features Yields Complete Sequence-Structure Congruence inserted chiral inversions, we investigate quantitatively the effect of chiral Kejue Jia. perturbations on the structure of peptides, thereby assessing the extent to BBMB, Iowa State Univ, Ames, IA, USA. which the backbone structure is able to fold in the presence of systematic het- Protein sequence matching does not properly account for some well-known erochiral perturbations. For the helix-folding polyalanine peptide, we invert features of protein structures: surface residues being more variable than core the backbone chiralities of Ala residues along a specific perturbation pathway, residues, the high packing densities in globular proteins, and does not yield starting from the homochiral L-Ala20 peptide until reaching the homochiral D- good matches of sequences of many proteins known to be close structural Ala20 peptide. Analysis of the helical contents of the simulated structural en- relatives. There are now abundant protein sequences and structures to enable sembles shows that even a single inversion in the middle of the peptide major improvements to sequence matching. Here, we utilize structural frame- completely breaks the helical structure in its vicinity, and drastically reduces works to mount the observed correlated sequences to identify the most impor- the helical content of the peptide. Our results show a monotonic change in tant correlated parts. The rationale is that protein structures provide the structure content as a function of perturbation coordinate and provide a quan- important physical framework for improving sequence matching. Combining titative proof of that arbitrary heterochirality is not tolerated by the alpha- the sequence and structure data in this way enables the incorporation of allo- helical structure. steric information into sequence matching and transforms it effectively from GB1 hairpin, on the other hand, does not show a monotonic change in the struc- a 1-D to a 3-D procedure. Our results show that there are major gains in the ture content, i.e. b-sheet content, along the perturbation coordinate. Some in- specificity of sequence matching across diverse proteins. Specifically, all versions yield a similar or higher secondary structure content compared to known cases where protein structures match but sequences do not match the homochiral hairpin, in contrast to the helical peptide. However, similarity well are resolved. in secondary structure content does not imply fold similarity: we have, in fact, observed cases in which the peptide with inversions folds into clearly 227-Pos different hairpin structures with respect to the non-chirally-perturbed (native) Structural Modeling of the Reflectin Protein peptide, while sharing similar secondary structure content. In other words, Dillion Fox1, Loukas Petridis2,3, Jeremy Smith2,3, Alison Sweeney1. the chirally perturbed hairpin peptides fold into different unique structures 1Department of Physics and Astronomy, University of Pennsylvania, when they are still able to fold. Therefore, our main conclusion applies to Philadelphia, PA, USA, 2UT/ORNL Center for Molecular Biophysics, Oak both of the structurally distinct proteins we examined, namely, that arbitrary Ridge National Lab, Oak Ridge, TN, USA, 3Department of Biochemistry and heterochirality conspires against proper folding. Cellular and Molecular Biology, University of Tennessee, Knoxville, TN, 230-Pos USA. Molecular Dynamics Simulations of gp120 and gp41 of HIV Env Provide Cephalopod skin has the ability to manipulate incident light with reflective cells Insights into Strain Specificity and the Role of the Membrane Environment to camouflage with their environment. This remarkable capability derives from Louis R. Hollingsworth IV1, Justin A. Lemkul1, Richard D. Gandour2, structurally colored skin cells that form elaborate dielectric structures; a self- David R. Bevan1, Anne M. Brown1. assembling protein called reflectin acts as an intracellular high-index material 1Dept Biochemistry, Virginia Tech Univ, Blacksburg, VA, USA, 2Dept and the extracellular space acts as a low-index material. The highly evolution- Chemistry, Virginia Tech Univ, Blacksburg, VA, USA. arily conserved region of reflectin that is unique to cephalopods has proven nsight into structural dynamics of targetable HIV proteins is necessary for intractable to most standard biophysical characterizations including crystalliza- immunogen and small molecule design. The sole antigenic target on HIV is tion. Therefore, we modeled the structure of the highly conserved reflectin the trimeric envelope glycoprotein (Env), which is comprised of three hetero- motif using two methods: ab initio structure prediction and monte carlo folding dimeric subunits that include a surface glycoprotein (gp120) and a transmem- simulations of randomly generated conformations. Both methods produced brane glycoprotein (gp41). Therefore, targeting Env with small-molecule and compact conformations consistent with experimental data. We then explored antibody therapies requires an understanding of both gp120 and gp41 dy- the conformational landscape accessible to the conserved motif using replica namics, which is often challenging given difficulties in describing highly flex- exchange molecular dynamics simulations. This talk will discuss how the ible, strain-specific loop dynamics or structural properties in a native calculated structure of the motif and the corresponding conformational membrane. Here, atomistic molecular dynamics (MD) simulations were per- landscape we observed may inform our understanding of reflectin function in formed to better understand these strain-specific dynamics, small molecule living cells. binding events, and the influence of an asymmetric, viral membrane environ- ment on the transmembrane domain (TMD) of the prefusion Env. MD simula- 228-Pos tions and analysis of small molecule binding to the V3 loop region of gp120 Modeling Electrostatic Force in Protein-Protein Recognition show some strains sample a closed state more frequently, whereas other strains 1 2 Mihiri Shashikala Hewa Bosthanthirige , Arghya Chakravorty , sample more open, epitope-exposing conformations. Simulations of the TMD 2 Emil Alexov . in a lipid bilayer showed the influence of an arginine - water - anion network, 1Physics and Astronomy, Clemson University, Clemson, SC, USA, 2 which plays an important role in viral fusion with the host cell, in modulating Department of Physics, Clemson University, Clemson, SC, USA. protein conformational changes within the membrane. Entropic forces Electrostatic interactions are important for understanding inter-molecular inter- contribute to the tilting of the TMD in the membrane and highlight key residues actions, since they are long-range interactions that guide binding partners to for targeting. Collectively, MD simulations have expanded the knowledge of their correct binding positions. To investigate the role of electrostatic force conformational landscapes sampled by these targetable proteins and provide in molecular recognition, we calculate electrostatic force between partners viable insight into therapeutic design. separated at various distances. The investigation is done on a large set of 600 protein complexes using recently developed DelPhiForce tool. To accomplish 231-Pos the goal, we develop a method to find an appropriate direction to move one Atomic Simulations of Trp-Cage Folding by Umbrella Sampling using chain in protein complex away from its bound position. The results indicate Q Function as Reaction Coordinate that based on the electrostatics force profile, the protein complexes can be Hamed Meshkin, Fangqiang Zhu. grouped into four main categories: Soft landing, Minimum force at the bound Physics, IUPUI, Indianapolis, IN, USA. state, Attraction force, and Multi variation force. These four categories are Spontaneous transitions between different protein conformations, especially the discussed in terms of the net charge of the partners, interfacial charge comple- transition between native and non-native states, typically occur rarely in an unbi- mentarity and other biophysical characteristics. It is demonstrated that while ased molecular dynamics simulation. Recently, it was shown that the thermody- electrostatic force is almost always guiding the partners toward their binding namics of protein folding can be described by a Q function, which is the collective position when they are separated at large distance, the electrostatic force fraction of the native contacts of protein atoms. In this study, we implement Um- contribution to the physical binding depends on the balance between attractive brella Sampling with Hamiltonian replica exchange, using Q as the reaction pair-wise interactions and desolvation penalty. coordinate, to model the folding of Trp-Cage protein. The CHARMM force field

BPJ 9303_9305 Sunday, March 3, 2019 47a and the NAMD2 program were used in the molecular dynamics simulations. The 235-Pos simulations show a satisfactory convergence along Q. Besides the native struc- Molecular Basis of Ligand Selectivity in Aminoglycoside Acetyltrans- ture, multiple folded states can be observed in the reconstructed equilibrium ferases ensemble. Even without native contacts, some protein structures are stabilized Prashasti Kumar1, Matthew J. Cuneo2, Brinda Selvaraj3, Engin H. Serpersu4. by hydrogen bonds not present in the native state. Overall, our result showed 1Genome Sci & Tech, Univ Tennessee, Knoxville, TN, USA, 2Structural that although Q is a reasonably reliable parameter to analyze the simulations, it Biology, St Jude Children’s Hospital, Memphis, TN, USA, 3Neutron is not necessarily the best reaction coordinate for enhanced sampling. In partic- Sciences, Oak Ridge National Laboratories, Oak Ridge, TN, USA, 4Molec ular, the folding of the a-helix is a slow degree of freedom for Trp-Cage, and Biophys Cluster, Natl Sci Fndn, Alexandria, VA, USA. the reaction coordinate may probably be improved by incorporating parameters Resistant bacteria employ drug modifying enzymes like aminoglycoside acetyl- that describe the a-helix conformation as well. transferases (AACs) to evade the effects of aminoglycoside antibiotics. Even though the AACs share a high level of sequence and structural similarity, they 232-Pos exhibit a range of substrate profiles. Thus, these enzymes present an excellent op- Multiple-Walker Curvilinear-Path Umbrella Sampling Simulations: to portunity to study the molecular principles underlying substrate promiscuity and Trace Protein-Protein Dissociation Trajectories and Compute Potential selectivity. AAC-VIa and AAC-IIIb possess the same structural fold, catalyze of Mean Force the same modification reaction and utilize the same catalytic machinery to do Dhananjay C. Joshi1,2, Jung-Hsin Lin1. so. Despite these similarities, they exhibit quite different substrate profiles. 1Research Centre for Applied Science, Academia Sinica, Taipei 115, Taiwan, AAC-VIa, is a highly selective AAC, capable of modifying only five aminoglyco- 2Taiwan International Graduate Program (TIGP-CBMB) and National sides, limited to kanamycin class of drugs. AAC-IIIb, on the other hand, is a highly Taiwan University (NTU-IBS), Taipei 115, Taiwan. promiscuous acetyltransferase and can modify upto 15 different aminoglycosides, The protein-protein interaction energetic can be obtained by calculating the from both the neomycin and kanamycin classes. Structure based sequence align- potential of mean force (PMF) from umbrella sampling simulations, in which ment shows that AAC-VIa has a loop with a four-residue insertion, which is absent samplings are often enhanced along a predefined vector as the reaction coordi- in AAC-IIIb. Instead, this region in AAC-IIIb is occupied by a subsite of the bind- nate. However, any slight change in the vector may significantly vary the ing site, utilized to bind neomycin class of aminoglycosides, which are not calculated PMF, and therefore, the energetic using a random choice of vector catalyzed by AAC-VIa. Unlike AAC-IIIb, AAC-VIa is able to kinetically distin- is still a subtle issue. Alternatively, a non-predefined curve path-based sampling guish between its substrates. This is accomplished by more preferable ligands enhancement is a natural approach, but was relatively less explored for protein- binding in a different conformation compared to the lesser preferable ligands. protein systems. In this work, dissociation of the Barnase-Barstar complex is AAC-IIIb utilizes a larger and bipartite binding site, to allow the binding of struc- simulated by implementing non-predefined curvilinear pathways in umbrella turally varied substrates and catalyzes all of them with similar efficiency. These sampling simulations. Multiple-walker simulations are carried out and the tra- structures provide structural framework for the unusual thermodynamic properties jectories were analyzed. Two major dissociation trends in the curvilinear disso- of enzyme-ligand complexes of AAC-IIIb. Overall, these studies shed light on ciation trajectories are observed that include interactions with the RNA-binding molecular principles underlying ligand selection by AACs and would contribute loop and the interactions with Val 36 to Gly 40 loop. On the other hand, sur- towards the design and development of more effective antibacterial drugs. prisingly the PMF values significantly varied along different curved paths. We found that the prolonged interface interactions along the dissociation paths 236-Pos are associated with higher PMF. Therefore, a simple variational principle is Structural Basis of Endocrine FGF Recognition by Beta-Klotho applied to determine the lower-bound PMF from the constructed PMF profiles, Sangwon Lee. which is then used to derive the standard free energy of binding. The estimated Pharmacology, Yale University School of Medicine, New Haven, CT, USA. free energy of binding is in good agreement with the experimental values. Thus, Endocrine FGFs signal through FGFRs in a manner that requires Klothos, a highly generalized multiple-walker curvilinear-path umbrella sampling which are cell surface proteins with tandem glycosidase domains. Here we approach is studied for revealing protein-protein interaction energetic along present the crystal structures of ligand-bound b-Klotho extracellular regions, with the intermediate conformations. revealing the molecular mechanism underlying the specificities of endocrine FGF towards b-Klotho and demonstrating how FGFR is activated in a 233-Pos Klotho-dependent manner. b-Klotho serves as a primary ‘‘Zip code’’-like re- On Restraints in End-Point Protein-Ligand Binding Free Energy Calculations ceptor for endocrine FGFs with an FGFR functioning as a catalytic subunit William Menzer1, Bing Xie2, David D.L. Minh2. that mediates intracellular signaling. Our structures suggest that a sugar- 1Biology, Illinois Institute of Technology, Chicago, IL, USA, 2Chemistry, cutting enzyme has evolved to become a specific receptor for hormones that Illinois Institute of Technology, Chicago, IL, USA. regulate important metabolic processes, and provide insights offering develop- Harmonic restraints are often used to maintain a particular configuration in the ment of novel therapeutics for diseases linked to endocrine FGFs. molecular simulation. In the popular MM/PBSA end-point binding free energy method, they enable free energies to converge more quickly. However, the 237-Pos effects of the restraining potential have not been adequately accounted for. Structural Rearrangements in the C-Terminal Domain Homolog of Furthermore, the effects of restraints on configuration space sampling have Orange Carotenoid Protein are Crucial for Carotenoid Transfer not yet been quantified. We have performed simulations with different spring Dvir Harris1, Adjele Wilson2, Fernando Muzzopappa2, constants on datasets with at least seven protein-ligand complexes in each. Nikolai N. Sluchanko3, Thomas Friedrich4, Eugene G. Maksimov3, For most datasets, we find that levels of restraints commonly used in Diana Kirilovsky2, Noam Adir1. MM/PBSA, even 1kcal mol-1 Angstrom-2, significantly restrict the entropy of 1Chemistry/Energy, Technion - Israel Institute of Technology, Haifa, Israel, each system. Entropic restriction reduces the correlation between calculated 2Biology, Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, and measured binding free energies. We recommend performing simulations Universite Paris-Sud, Universite Paris-Saclay, Gif sur Yvette, France, with weaker restraints and accounting for the effect of restraints in free energy 3Department of Biophysics, Faculty of Biology, M.V. Lomonosov Moscow estimators. State University, Moscow, Russian Federation, 4Inst of Chemistry, Technical Univ Berlin, Berlin, Germany. 234-Pos A recently reported family of soluble cyanobacterial carotenoproteins, homologs Proteins that Tell Time of the C-terminal domain (CTDH) of the photoprotective Orange Carotenoid Andy LiWang. Protein, is suggested to mediate carotenoid transfer from the thylakoid membrane Dept Chem/Chem Bio, Univ Calif Merced, Merced, CA, USA. to the Helical Carotenoid Proteins, which are paralogs of the N-terminal domain Circadian clocks arose in organisms as an adaptation to the rotation of the earth. of the OCP. Here we present the three-dimensional structure of a carotenoid-free These clocks produce involuntary anticipation of sunrise and sunset by gener- CTDH variant from Anabaena (Nostoc) PCC 7120. This CTDH contains a ating a succession of biochemical phases. In this talk, the mechanism of a cysteine residue at position 103. Two dimer-forming interfaces were identified, model system, that of cyanobacteria, will be described. Briefly, it depends on one stabilized by a disulfide bond between monomers and the second between phosphorylation, long-range allostery, dynamics, and protein metamorphosis. each monomer’s b-sheets, both compatible with small-angle X-ray scattering Because a simple mixture of clock proteins and ATP generate a persistent data and likely representing intermediates of carotenoid transfer processes. The macroscopic rhythm, the mechanism of the clock can be studied in real time crystal structure revealed a major positional change of the C-terminal tail. Further as it ticks. Now, signal transduction pathways have been reconstituted with mutational analysis revealed the importance of the C-terminal tail in both the oscillator so that rhythmic transmission of clock signals can be studied carotenoid uptake and delivery. These results have allowed us to suggest a in vitro. detailed model for carotenoid transfer via these soluble proteins.

BPJ 9303_9305 48a Sunday, March 3, 2019

238-Pos 241-Pos A New Open Structure of the Insulin Degrading Enzyme Provides Insights Transient Structural Distortion and Oligomerization of the Capsid Form- into the Conformational Transition of the Molecule ing Protein Arc is Attenuated by Ligand Binding Nicolae Sapoval1, Esmael J. Haddadian2, Wei Jen Tang3. Lau Dalby Nielsen, Simon Erlendsson, Kaare Teilum. 1University of Chicago, Chicago, IL, USA, 2Biological Sciences Collegiate Department of Biology, University of Copenhagen, Copenhagen N, Denmark. Division, University of Chicago, Chicago, IL, USA, 3Ben-May Department The Activity-regulated cytoskeleton-associated protein, Arc, is highly for Cancer Research, University of Chicago, Chicago, IL, USA. expressed in neuronal dendrites. Arc, which is homologues to the capsid form- Insulin Degrading Enzyme (IDE) is involved in the deconstruction of insulin, ing Gag proteins from the retrotransposon Ty3/Gypsy family, was recently amylin, and glucagon peptides involved in controlling blood glucose level and shown capable to form capsids that can encapsulate Arc’s own mRNA and has also been found to break down amyloid-b, a peptide involved in Alz- transport it to neighbouring neurons for transcription. However, the molecular heimer’s disease. IDE has two homologous N-and C-terminal domains, events that lead to formation of Arc capsids and thus how the process is regu- IDE-N and IDE-C. For IDE to degrade its substrates, both domains must lated are not known. Here we determined the structure of the Arc CA-domain in come together for the catalytic cleft to assume closed conformation. New solution, which consists of two sub-domains (lobes). We show that the capsid Cryo-Electron-Microscopy (Cryo-EM) data suggests IDE passes through domain of Arc has a locked inter-lobe orientation in contrast to other Gag five conformations: completely closed with substrate bound, partially closed proteins in which inter-lobe mobility is important for the capsid formation. with substrate bound, partially open with substrate unbound, and open without In apo Arc, we find transient structural distortions in the N-terminal lobe and substrate. Details of how these conformations connect are unknown. We also that structural changes occur in the N-terminal lobe during oligomeriza- investigated the mechanism by running microsecond-long molecular dy- tion. Both the capsid formation and conformational exchange in apo Arc are namics simulations of IDE in closed conformation with and without insulin efficiently suppressed by interactions with ligands, suggesting that the capsid plus the newly captured open states of the molecule obtained by Cryo-EM. formation of Arc may be a regulated process. The dynamical network analysis of our simulations shows that insulin stabi- lizes the local structure of IDE. We find that the Cryo-EM captured open 242-Pos conformations of IDE, except one of them, converge back to the closed state The Effect of Lithium Binding on Secondary and Tertiary Structure, over the course of the simulations, indicative of their higher energy state Hydrophobicity, Thermodynamics, and Interactions with Interacting compared to the closed conformation. A potential explanation for this Partners of Dream behavior is the presence of FAB-molecules in the Cryo-EM models that might Samiol Azam, Jaroslava Miksovska. shift the energy-landscape. We observe a new conformational state of open- Dept Chem & Biochem, Florida Int Univ, Miami, FL, USA. Liþ is widely used for the treatment of manic-depressive illness. Studies on IDE that suggests an opening mechanism in which domains D2 and D3 þ remain unchanged (compared to their positions in experimentally available neuronal calcium sensor protein1 (NCS-1) have found that Li can inhibit the interaction between NCS-1 and inositol 1,4,5-triphosphate receptor protein, alle- closed conformation of IDE) while domains D1 and D4 separate from þ each other. Our simulation and analysis offer new insight into the effect of viating hyperarousal of insomnia patient. Li can have neuroprotective or neuro- toxic effects depending on its concentration. In a rat model, chronic exposure to substrate binding on the structural stability of the IDE and provide a new þ mechanism for the conformational transition between the closed and open Li has shown a significant decrease in membrane-associated protein kinase C states. (PKC) in the hippocampus, suggesting potential clinical relevance. Here, we show that Liþ binds to a neuronal calcium sensor protein named Downstream 239-Pos Element Antagonist Modulator (DREAM), a protein homologous to NCS-1 Structure of a Non-Canonical and Flexible Middle Domain in Inner-Ear and expressed in the hippocampus region of the brain. Liþ association results Protocadherin-15 in a decrease in the emission intensity of tryptophan, suggesting rearrangements Brandon L. Neel, Carissa F. Klanseck, Marcos Sotomayor. of the tertiary structure of the protein. Liþ binding also exposes hydrophobic cav- Dept Chem & Biochem, Ohio State University, Columbus, OH, USA. ity of the protein as evidenced by binding of the hydrophobic molecule 1,8-ANS. Protocadherin-15 (PCDH15), an atypical member of the cadherin superfam- CD data reveal that Liþ binding alters the protein secondary structure. Trypto- ily, is essential in conveying force to inner-ear hair-cell mechanotransduc- phan emission and CD data are further supported by steady-state lifetime data. tion channels for sensation of sound and head movements. PCDH15 is Thermodynamic parameters for Liþ association to DREAM have been obtained composed of 11 extracellular cadherin (EC1-11) repeats and a membrane through isothermal titration calorimetry (ITC) measurements. Liþ-bound adjacent domain (MAD12). The EC repeats are similar in structure but DREAM interactions with binding partners have been probed by titrating not in sequence, with mechanical strength often provided by conserved FITC-tagged presenilin-1 and potassium channel against Liþ-bound DREAM. calcium-binding linker regions between successive ECs. Here we present Findings of this project suggest potentials for the Liþ-based compounds in the an- three crystal structures of PCDH15 fragments that show its middle domain imal model to investigate whether acute and chronic exposure to Liþ has any ef- comprised of EC4 through EC7. These structures, along with molecular fect on the expression level of DREAM and whether Liþ-based compounds dynamics simulations of a structural model of the full-length PCDH15, pro- influence interactions between DREAM and intracellular partners. vide insight into a novel calcium-binding linker region between EC5 and EC6, which supplies flexibility and elasticity needed for parallel homodime- 243-Pos rization of PCDH15 and tip-link assembly. These results provide a first view Investigating How Protein Mixtures Interact with Gold Nanoparticles of PCDH15’s middle domain and its elastic behavior essential for inner-ear Rebecca A. Hill, Katarina J. Boulet, Randika Perera, Mack B. Davidson, mechanotransduction. Nicholas C. Fitzkee. Chemistry, Mississippi State University, Mississippi State, MS, USA. 240-Pos Gold nanoparticles (AuNP) have numerous biological applications, including Selenium NMR Spectroscopy: A Versatile Probe for Biological Macromol- drug and gene delivery, chemo- and photo-therapy, biosensing, and bioimaging. ecules When exposed to biological fluids, AuNPs will interact with proteins in solution, Sharon Rozovsky. and these proteins will compete to bind to the AuNP surface. It remains impossible Department of Chemistry and Biochemistry, University of Delaware, to predict the outcome of competitive binding to AuNPs, but this knowledge could Newark, DE, USA. potentiallyaid scientists hoping to target nanoparticles to specific cells in the body. 77Se can be used to study sulfur-sites in proteins because the sensitivity of NMR has been used extensively to study interactions between biological mole- the only NMR-active isotope of sulfur is too low. Thus, 77Se NMR provides cules in solution. Here, we explore using 2D 1H-15N HSQC and CPMG NMR insights into the multifaceted roles of cysteine and methionine in enzymatic methods to study protein interaction kinetics with various nanoparticles. Specif- reactions, metal binding, and molecular recognition. However, it is yet not ically, we used a 1H-15N HSQC technique to quantify AuNP binding versus routinely employed for studies of biological systems because protocols to time for a mixture of GB3 and Ubiquitin, two small model proteins. GB3 and enrich proteins with 77Se were not available and the interpretation of the Ubiquitin signals were sampled at 15 min time points. An external standard was NMR data remains challenging. We describe our work on building the used to quantify absolute binding to the AuNPs. Binding was observed to be knowledge base necessary to interpret selenium NMR of biological sys- extremely fast, with a majority of binding occurring in the dead time (3 min) of tems. To that end, we recorded NMR parameters in model proteins as the experiment. We also used CPMG relaxation experiments to monitor the time- well as native selenoproteins. Through our work, we arrive at general scale of protein exchange with a pretreated, protein-functionalized AuNP surface. guidelines regarding optimal site selection, sensitivity, Se incorporation Our results suggest that competition between proteins can lead to increased sur- methods, and information content of solution and solid-state NMR experi- face exchange rates (ms). This differs slightly from a previous study, where ments. surface exchange was extremely slow (18 hr). Together, our results suggest a

BPJ 9303_9305 Sunday, March 3, 2019 49a mechanism by which nanoparticle surface character may change over time, and Human T-cell leukemia virus type 1 is the causative agent of serious diseases, this may be an important consideration in the design of nanoparticle-based such as adult T-cell leukemia and neuropathy myelopathy/tropical spastic para- therapeutics. paresis. In our efforts to understand critical viral mechanisms we study the regu- latory protein p13II, which interact with the inner mitochondrial membrane 244-Pos (IMM) where it is believed to self-associate into a Kþ channel. The resulting Ion-Dependent Binding-Site Configurations in Ef-Hand Proteins Measured channel activity affects the membrane permeability and potential, which results with Ultrafast Infrared Spectroscopy in modified mitochondrial homeostasis, possibly leading to cell apoptosis. How- 1 2 2 Sean Edington , Thomas R. Middendorf , David Brent Halling , ever, the structure and functional mechanism of this protein in IMM are poorly 3 1 Richard W. Aldrich , Carlos R. Baiz . understood. Here we report for the first time to the best of our knowledge the clon- 1Department of Chemistry, Univ Texas at Austin, Austin, TX, USA, 2Dept 3 ing, expression, and purification of large, milligram, quantities of highly pure Neurobiology, Univ Texas Austin, Austin, TX, USA, Dept NeuroSci, The full-length p13II. We also observed p13II self-association in lipid membrane Univ of Texas Austin, Austin, TX, USA. mimetics. The p13IIU fusion construct of wild type full-length p13II and an ubiq- Calmodulin (CaM) is essential for ion regulation in eukaryotic organisms and is uitin and His8 tag at p13II C-terminal was cloned in a pET15b vector and ex- one of the most well-studied calcium sensors. Four unique binding sites make pressed as a soluble protein in E. coli. The protein was purified by Ni-affinity CaM a sophisticated signal transducer that modulates hundreds of effector pro- 2þ and size-exclusion chromatography (SEC). The purified p13IIU is monomeric teins. The complex interactions between the Ca -binding sites make it difficult in solution based on the SEC results. Furthermore, double electron-electron reso- to measure individual occupations. Further, structural descriptions of ion- nance (DEER) spectroscopy, carried out on spin-labeled single cysteine mutant dependent binding configurations remain incomplete as experimental markers H63C of p13IIU,confirmed the monomer in solution. For the same p13IIU mutant are often unavailable or difficult to interpret. We examine changes in binding- self-association was detected by DEER when the protein was reconstituted into þ site configuration and site-disorder using Ca2 as well as lanthanide ions. Lan- LPPG or SDS micelles. The self-association persisted after cleaving the ubiqui- thanides are commonly used in biophysical studies of ion affinity due to their tin/His8 tag. DEER results were consistent for spin labels with different hydro- luminescence properties. We use ultrafast two-dimensional (2D IR) spectros- phobicity, namely iodoacetamido proxyl and spirocyclohexyl nitroxide labels. copy, the IR analog of 2D NMR, to measure frequencies and picosecond energy These results are relevant to the insertion and oligomerization of p13II in IMM. relaxation rates of the binding-site carboxylate modes. Our results are as follows: 1. Lanthanides eliminate bidentate configuration of Ca2þ-bound position 12 247-Pos glutamate residue and replace it with purely monodentate or pseudo-bridging High Concentration of Inert Solutes (Ficol 70 and Polyethelene Glycol configurations; Residue 12 is highly conserved across species and thought to 6000) Stabilize the Native form of Clarias Gariepianus GST (cgGST) be important for activation; 2. Lanthanide-bound sites exhibit greater structural Adedayo A. Fodeke1, Olusanjo I. Adewale2, Temidayo Ogumoyole2. disorder as evidenced by peak broadening and shorter vibrational lifetimes 1Department of Chemistry, Obafemi Awolowo University, Ile-Ife, Nigeria, compared to Ca2þ-bound CaM. 3. Amide-I to carboxylate cross peaks are 2Department of Biochemistry and Molecular Biology, Obafemi Awolowo well-defined in Ca2þ 2D IR spectra. These features indicate that the binding sites University, Ile-Ife, Nigeria. are more compact and rigid in the Ca2þ bound site. In general, the results show Unfolding studies of Clarias gariepinus glutathione transferase (cgGST) with that lanthanide ions significantly alter the geometries. Finally, we show that 0 - 2 mole.dm-3 GuHCl at 25oC were carried out fluorometrically in separate charge (not ionsize) constitutes the largest perturbation to the binding geometries. solutions of 0 - 100 mg/cm3 of each of Ficoll 70 and Polyethelene glycol Finally, we examine the effects of three binding-site mutations on the conforma- 6000 (PEG 6000) and in mixture of equal g/cm3 concentrations of both poly- tional landscape: one tryptophan insertion and two carboxylate deletions. mers pH 7.4. The data were analyzed based on model that assumes that the pro- tein dimer unfolds into two monomers. The standard free energy of unfolding 0 245-Pos (DG UN) increases with increasing concentration of each crowding agent in a Alkaline Confomer of Human Dimeric Cytochrome C manner which suggests that high concentration of PEG6000 and Ficoll 70 Haotian Lei, Bruce E. Bowler. favour the native cgGST relative to the unfolded form. Ficoll 70 stabilizes Dept Chem/Biochem, Univ Montana, Missoula, MT, USA. the native cgGST more than PEG 6000 at the same g/cm3 concentration. A The alkaline transition of monomeric cytochrome c (Cytc) has been studied for mixture of equal g/cm3 concentration of both crowding agents however stabi- decades and has been used as a model system to study protein conformational lizes the native form more effectively than either Ficoll 70 or PEG 6000 at change. More recently, it has been suggested that monomeric Cytc alkaline con- equivalent g/cm3 total concentration and is less sensitive to increase in concen- formers may play an important role in regulating initiate the intrinsic apoptotic tration. This is in strong agreement with the result of the folding studies, and pathway. Structural and biochemical studies have shown that an available coor- suggest that a mixture of crowder of widely different molecular weight might dination site on the heme iron of dimeric Cytc, leads to the enhancement of the exclude more volume than that predicted using hard particle model. This might peroxidase activity. We have recently solved a human dimeric Cytc alkaline be a useful phenomenon in regulating the effect of crowding in living cells. conformer crystal structure near pH 10. Unlike previously reported structures 248-Pos of either horse or yeast iso-1 Cytc dimers with H2O/OH bound to the heme, the human dimeric Cytc alkaline conformer contains Lys79-heme ligation. It Investigating the Function of Mutl Conformational Changes in Mismatch is known that strong Lys-heme ligation blocks peroxidase activity, indicating Repair using smFRET that the human dimeric Cytc alkaline conformer possesses significantly lower Sharonda J. LeBlanc1, Pengyu Hao2, Malikiya A. Hinds1, Andi N. Morgan1, peroxidase activity. We hypothesize that the conformation of dimeric Cytc is Korene Gbozah3, Keith R. Weninger2, Dorothy A. Erie1. 1 2 pH dependent conferring the ability to tune its peroxidase activity. To test this Chemistry, Univ of North Carolina, Chapel Hill, NC, USA, Physics, North 3 hypothesis, we are performing a variety of complementary biophysical methods Carolina State University, Raleigh, NC, USA, Biology, Univ of North to investigate the alkaline transition of dimeric Cytc. Using UV-Vis spectros- Carolina, Chapel Hill, NC, USA. copy, we are investigating the thermodynamics of the alkaline transition. Using Mismatch repair (MMR) is a highly conserved enzymatic system that corrects er- stopped-flow methods, we are investigating the kinetics of the alkaline transition rors made during DNA replication. MMR involves the coordinated stepwise action and the peroxidase activity. Using EPR spectroscopy, we are probing different of about ten different proteins on a DNA mismatch. In humans, mutations in the populations of high-spin and low-spin heme species across the pH range 6 to genes that code for mismatch repair proteins have been linked to hereditary non- 11. We observe that the alkaline transition of dimeric Cytc involves equilibria polyposis colorectal cancer (HNPCC), and some sporadic cancers. The connection among three species. As pH increases Met80 coordination is replaced with as between cancer and mutations in DNA repair proteins has been firmly established with the standard alkaline transition. However, a high-spin species, presumably with biochemical and in vivostudies,but we currently lacka molecular understand- ing of precisely how these mutations lead to tissue-specific tumorigenesis. H2O coordination, is also replaced by Lys coordination. The transition from the high-spin species to Lys coordination appears to be more facile than the transi- MutS recognizes a mismatch by binding to DNA, and then undergoes ATP- tion from Met80 to Lys coordination. dependent conformational changes to interact with MutL. This MutS-MutL com- plex then interacts with the processivity clamp in an ATP-dependent manner that 246-Pos activates MutL to nick the daughter strand. This nick serves as an entry point for Production and Characterization of Htlv-1-Encoded Mitochondrial exonucleases and polymerase to complete repair. MutL is the key player in the Protein P13II middle of the pathway that directs repair, likely acting as a switch to turn molec- Elka R. Georgieva1, Peter P. Borbat1, Shuyang Zhang2, Andrzej Rajca2, ular interactions of repair machinery on and off. Despite its importance in repair, Jack H. Freed1. the mechanism by which MutL carries out its functions is poorly understood. 1Department of Chemistry and Chemical Biology, Cornell University, Ithaca, Structural, biochemical, and single molecule experiments indicate that ATP bind- NY, USA, 2Department of Chemistry, University of Nebraska, Lincoln, NE, ing and hydrolysis regulate conformational changes in MutL to modulate tran- USA. sient interactions.

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We have used single molecule FRET to determine the key dynamic conforma- 252-Pos tional changes of MutL during its initial interactions with the MutS-mismatch Self-Assembly of the Tau Protein: Fibril Formation and Complex DNA recognition complex. We expect that conformational dynamics of MutL Coacervation are critical for MutL to activate downstream machinery in the MMR system Joan Emma Shea. and that defective interactions are presumably involved in the malfunction Univ Calif Santa Barbara, Santa Barbara, CA, USA. that leads to cancer. Tau is an intrinsically disordered peptide that plays an important role in the cell by binding to microtubules. Under pathological conditions, Tau can form 249-Pos fibrillar aggregates, a process that has been linked with Alzheimer’s disease. Inherent Flexibility and Oligomerization of CLIC5A and CLIC6 - A In addition to forming fibrils, the Tau protein can also phase separate into a Comparative Static and Dynamic Structural Study protein rich and a protein depleted phase, a process known as liquid-liquid Alisa Ferofontov, Milit Marom, Moshe Giladi, Yoni Haitin. phase separation (LLPS), or coacervation. I will present field theoretic simula- Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv tions that map out the phase diagram for Tau coacervation. The phase diagram University, Tel Aviv, Israel. is then used to predict the conditions under which Tau can be driven towards Chloride intracellular channels (CLICs) are a family of unique metamorphic pro- coacervation under live cell coculturing conditions. The theoretical predictions teins. While exhibiting both soluble and transmembrane states, the molecular basis are corroborated by experiments from the Han and Kosik groups at UCSB. of their inherent flexibility remains largely unknown. Here, we present the high- resolution crystal structures of the human CLIC5A and mouse CLIC6. The struc- 253-Pos tures reveal that both family members share monomeric arrangement in their Enhanced Sampling of Amyloid Beta-42 Dimer Ensemble; A Novel soluble state with a high degree of structural conservation with other CLICs. More- Approach with Conformational Symmetry over, Small Angle X-ray Scattering (SAXS) analysis under reducing conditions Levent Sari, Milo M. Lin. demonstrates monomeric distribution in solution, reminiscent of the crystallized Green Ctr for System Biology, University of Texas Southwestern Medical, form. Importantly, Ensemble Optimization Method (EOM) analysis of the Dallas, TX, USA. SAXS data revealed that both CLIC5A and CLIC6 can sample multiple conforma- Low molecular weight amyloid beta oligomers have been found to play key tions in solution, supporting the metamorphic classification of the family. CLICs roles in the pathology of Alzheimer’s disease (AD). Yet, unlike the fibril were previously shown toincorporate intomembranes and mediate ion conduction in vitro form, the molecular-level structure of any oligomeric form has resisted exper- , suggesting multimerization upon membrane insertion. Here, we provide imental efforts, and therefore no oligomeric structure has ever been resolved evidence for the ability of both CLIC5A and CLIC6 to undergo oxidation- even at the dimer level. On the other side, computational samplings in atomic dependent oligomerization in solution. However, comparative analyses of resolution face two well-known challenges; (i) inaccessibly large con- CLIC5A and CLIC6 revealed differential modes of oligomerization. While formational space and (ii) kinetic trapping in local minima. We have estab- CLIC5A shows both cysteine-dependent and independent oligomerization, lished a novel general approach to constrain multi-protein simulations to CLIC6 demonstratesonly weak assembly which iscysteine-independent and high- sampling the conformational symmetric subspace in which all copies of the ly enhanced by the presence of membrane mimetics. CLIC5A was suggested to protein adopt the same conformation, and have recently showed exponential play an important role in membrane remodeling processes. Interestingly, using speed-up of conformational sampling [(1)Lin M.M.,Chem. Phys. Letter., both dynamic light scattering and fluorescence measurements, we demonstrate in vitro Vol. 683, pp. 347-351 (2017); (2)Drombosky, Sari, Lin, et al., NSMB (sub- the ability of CLIC5A to mediate liposomal fusion , which is greatly mitted)]. To escape from local minima, we have integrated it with metady- enhanced by oxidative conditions. Together, these results provide insights into namics. Here, the combined method is applied on Ab42 dimer ensemble, the structural conservation and inherent flexibility of CLICs and shed light on their and is shown to produce convergent sampling overcoming both computational regulation by changes in cellular redox potential as well as the molecular mecha- challenges effectively. Simulations demonstrate that the dimer ensemble is nisms associated with their possible roles during membrane remodeling. populated by monomeric single and double hairpins, with a preference of 250-Pos dimeric face to face packing where intermolecular hydrophobic sidechain Integrative Methods for Protein Dynamics and Aggregation interactions stabilize the dimer unit. We have identified 3 main groups of Carlo Camilloni. dimer conformations; (1) dimers with compactly folded monomers with no Department of Biosciences, University of Milano, Milano, Italy. beta structure, (2) conformations with separated monomeric hairpins, (3) We investigate the relationship between protein structure, dynamics, stability compact dimers having monomeric hairpins stabilized by intermolecular and aggregation for well folded proteins associated to different form of systemic side chain interactions. amyloidosis like beta-2-microglobulin and light-chain antibodies. We employ a range of techniques from x-ray crystallography, solution and solid state NMR 254-Pos and molecular dynamics simulations coupled with experimental data through Identification and Study of Polymorphic Structures of Hierarchically inferential techniques. The systematic comparison of designed and natural se- Twisted Amyloid Fibrils by Atomic Force Microscope quences of b2m reveal how protein dynamics is key to interpret the aggregation Sergey K. Sekatskii, Jiangtao Zhou, Giovanni Dietler. properties of this protein. The same concepts are then applied to shed light on Inst Phys Sys Biol, Ecole Polytech Fed Lausanne, Lausanne, Switzerland. the aggregation properties of light chain antibodies associated with AL. Uncontrolled protein folding often results in amyloid fibrillogenesis associated with many neurodegenerative diseases. Nowadays the interest to this phenom- 251-Pos enon is driven also by the observation that some protein fibrils can be functional Investigation of Extracellular Gate Movement in a Glutamate Homologue in vivo, and by exciting perspectives to use them in (nano)technology and for Erika Riederer, Francis Valiyaveetil. new materials synthesis. At different conditions, numerous proteins might be Dept of Physiology and Pharmacology, Oregon Health and Science subject to aggregation, and rather different aggregation mechanisms and poly- University, Portland, OR, USA. morphic structures of aggregated proteins were reported. Well-characterized Glutamate transporters harness the ionic gradients across cell membranes to insulin molecules early emerged as a kind of a ‘‘model object’’ to study the pro- carry out the concentrative uptake of glutamate. The sodium coupled Asp sym- cess, and the conclusions obtained when investigating insulin fibrillization porter, GltPh is an archaeal homologue of glutamate transporters which has proved to be important for numerous other proteins. been extensively used to understand the transport mechanism. A critical aspect Here, we present the results of systematic Atomic Force Microscopy (AFM)- of the transport mechanism is the coupled binding of sodium and aspartate. The based studies of insulin molecules fibrillization. We fully exploited the ultahigh þ z Integrated crystal structures of GltPh shows that the Na ions do not form direct interac- -resolution of AFM, and introduce a new parameter, named tions with the substrate, suggesting that the binding of Naþ and substrate is Maximal Height (IMH), which is the sum of average height of a filament and coupled through an allosteric mechanism. Previous studies have suggested a half of its height fluctuation amplitude (i.e. the difference of maximal and critical role for hairpin-2 (HP2), the extracellular gate for the substrate binding minimal height), to classify the observed both twisted and ribbon-like fibril site, in coupling the binding of sodium and asparate to GltPh. We have devel- structures. The structures involving up to six mutually twisted protofilaments oped a fluorescence assay for monitoring HP2 movement by incorporating were observed and quantified. All possible arrangements were recorded for tryptophan and the unnatural amino acid, p-cyanophenylalanine into GltPh. those composed by up to four protofilaments, while ribbon-like structures We use the HP2 fluorescence assay in concert with traditional Naþ and Asp including five and six protofilaments were not observed, clearly indicating binding assays to investigate how key residues in the substrate binding sites that the formation of such ribbon-like structures are less favorite in our exper- modulate HP2 movement. Our studies reveal the intricate choreography of imental conditions. We discuss the possible relation between the interfilament side chain conformational changes linked to HP2 opening and closing that is forces and the peculiarities and statistics of the structures observed, and its þ the basis for the coupled binding of Na and Asp to GltPh. meaning for the general fibrillization mechanism.

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255-Pos Fluid(CSF) suggests that electric fieldcan be employed to control aggregation pro- Tetratricopeptides, a Versatile Protein Interaction Motif pensity of intrinsically disordered peptides. Furthermore, a cell-based primary Srihari Shankar, Jayaraman Sivaraman. cytotoxicity screening assay on two neuroblastoma cell lines (SH-SY5Y and Department of Biological Sciences, National University of Singapore, IMR-32) and human embryonic kidney cell line (HEK293) was performed using Singapore, Singapore. EF treated and untreated Ab1-42 samples. The EF treated peptides were 15-38% Kinesins are tetrameric proteins crucial for intracellular transport of large cargos to less toxic than the untreated ones under identical conditions. We propose the target sites within the cells. Tetratricopeptide repeats (TPR) are the versatile re- prototype of an electric field based device as a non-invasive therapeutic option gions present in the kinesins possessing the ability to distinctly bind to favorable for Alzheimer’s disease. cargos with acidic motifs. The mechanism of binding of TPR to these large protein cargos reveals the importance of acidic-motif interactions in the docking and lock- 258-Pos ing. Since cargo-carrying mechanisms are the crux of intra-cellular transport, it is Kinetic Characterization of a Novel Fosfomycin Resistance Enzyme from of wide importance and has its implications in normal cellular functioning, meta- Mycobacterium Bolletii bolic transporters, cancer cell progressions and pathway shuttling. The study in- Skye R. Travis, Madeline R. Shay, Matthew K. Thompson. volves elucidating this binding mechanism with well-known cargos and the Chemistryand Biochemistry, The University of Alabama, Tuscaloosa,AL, USA. identification of affinity calculations using biophysical methods. The cargos Fosfomycin is a small antibiotic that is effective against both Gram-positive used in the study are proteins involved in maintaining normal cell phenotype. Mu- and Gram-negative bacteria via inhibition of cell wall formation. Fosfomycin tationsinthese typesof cargos usually leadtodifferent neurodegenerative diseases, resistance enzymes have been observed in both Gram-positive and Gram- 2þ reduction in glucose pathway shuttling, disruptions in host-pathogen interactions negative bacteria: FosA, a M -dependent glutathione (GSH)-S-transferase 2þ þ and a host of other metabolic abnormalities. Many of these proteins are scaffolding found in Gram-negative species, FosB, a M - and K -dependent bacillithiol machinery thereby playing a role in multiple pathway mechanisms. The finding (BSH)-S-transferase found in Gram-positive species, and the evolutionary pre- 2þ suggests interaction between the proteins in-vitro. We aim to characterize the cursor FosX, a M -dependent hydrolase also found in Gram-negative species. 2þ 2þ structure-function relationship of this interaction to explore lock-dock mechanism Each of these enzymes is activated by Mn and inhibited by Zn . We have involved in the intracellular trafficking through biophysical methods. discovered, expressed, and purified a novel enzyme from Mycobacterium ab- scessus subspecies Bolletii that shares 55% sequence identity with FosX. Pre- 256-Pos liminary time trace kinetic data using 31P NMR suggest that this enzyme, which Biophysical Insights into the KRAS4b-FMe-Calmodulin Interaction we call FosM, is also a M2þ-dependent thiol transferase. Kinetic activity has Constance Agamasu1, Rodolfo Ghirlando2, Andrew Stephen1. been observed with both GSH and L-cysteine as thiol substrates. However, My- 1 2 FNLCR, Frederick, MD, USA, NIH/NIDDK, Bethesda, MD, USA. cobacteria do not produce GSH or BSH, and we believe FosM uses the natural Membrane localization of KRAS4b is an essential step for initiation of the thiol found in mycobacteria, mycothiol (MSH), as its substrate. Preliminary downstream signaling cascades that guide various cellular processes and dereg- data also suggest that FosM is activated by Mn2þ and inhibited by Zn2þ similar ulation of KRAS4b signaling pathway has been implicated in 30% of all can- to both FosA and FosB. Hydrolytic activity has also been observed, reinforcing cers. KRAS4b plasma membrane (PM) binding is mediated by insertion of a that FosM may be an evolutionary descendant of FosX. We are currently trying farnesyl moiety that is attached to the terminal carboxy-methylated cysteine, to obtain MSH to demonstrate that FosM is a new class of fosfomycin resis- in addition to electrostatic interactions of its positively charged hypervariable tance enzyme from Mycobacteria. region (HVR) with anionic lipids. Calmodulin (CaM) has been suggested to selectively bind KRAS4b to act as a negative regulator of the RAS/MAPK 259-Pos signaling pathway by displacing KRAS4b from the membrane. However, the Development of Fast Photochemical Oxidation of Proteins for In Vivo mechanism by which CaM can recognize and displace KRAS4b from the mem- Modification in Caenorhabditis elegans brane is not well understood. In this study, we employed biophysical and struc- Jessica A. Espino, Lisa M. Jones. tural techniques to characterize this detailed mechanism. We show that Pharmaceutical Sciences, University of Maryland, Baltimore, Baltimore, KRAS4b prenylation is required for binding to CaM and that the hydrophobic MD, USA. pockets of CaM can accommodate the entire farnesyl group, which might Caenorhabditis elegans, part of the nematode family, are used as model sys- represent a novel CaM binding motif. Remarkably, processed KRAS4b forms tems for many human diseases including those involving protein misfolding a 2:1 stoichiometric complex with CaM in a nucleotide-independent manner. such as Parkinson’s disease. Structural studies on C. elegans strains expressing The interaction between processed KRAS4b and CaM is enthalpically driven, the aggregate forms of the proteins involved in these diseases would shed light and electrostatic interactions also contribute to the formation of the complex. on how these aggregate structures lead to disease. The protein footprinting The KRAS4b terminal KSKTKC-FMe is sufficient for binding and defines method fast photochemical oxidation of proteins (FPOP) is especially suited the minimal CaM bind motif. This is the same region implicated in membrane for these in vivo studies because of the irreversible nature of the modification. and phosphodiesterase6-d (PDE6d) binding. Our data provide new insights into FPOP utilizes hydroxyl radicals to oxidatively modify solvent accessible sites the KRAS4b-CaM interaction and suggest a possible mechanism whereby CaM in proteins. Radicals are generated via hydrogen peroxide photolysis by a 248 can regulate KRAS4b membrane localization. nm excimer laser. Our lab has performed preliminary studies that indicate that FPOP can be used to study protein structure directly in C. elegans. We have 257-Pos designated this new iteration of the method in vivo FPOP (IV-FPOP). Here, Electric Field Mediated Disruption of Beta Amyloid; a Potential Non- we describe the use of penetration enhancers in order to increase the number Invasive Therapy for Alzheimer’s Disease of oxidatively modified proteins. C. elegans can uptake hydrogen peroxide Jahnu Saikia, Vibin Ramakrishnan. directly through their skin and the addition of penetration enhancers (i.e. Biosciences and Bioengineering, Indian Institute of Technology Guwahati, DMSO) will aid with hydrogen peroxide uptake. The viability of the worms Guwahati, India. in the presence of hydrogen peroxide and these enhancers were also tested. In proteins, folding and aggregation evolves along competing pathways. Misfold- LC-MS/MS analysis revealed and increase in oxidatively modified proteins ing and extracellular deposition of amyloid-beta (Ab) peptide, a fragment of by IV-FPOP when worms were treated with penetration enhancers. This b-amyloid precursor transmembrane protein (bAPP), is a key event in the forma- work further demonstrates the efficacy of IV-FPOP for studying proteins tive stages Alzheimer’s disease (AD). Previous studies reveal that promoting directly in an animal model. folding and thus inhibiting aggregation can potentially be an effective strategy against Ab-elicited toxicity. In particular, it is well established that the stabilization of the native state through mutations or solvent alterations are able to halt aggrega- Posters: Membrane Protein Structures tion. In this work, we experimentally explore the feasibility of using low strength electric field (EF) and magnetic field (MF) on the in-vitro fibrillogenesis of hydro- 260-Pos phobic core sequence Ab16-22 and its parent peptide Ab1-42 as aggregation inhibitor Application of Electron Paramagnetic Resonance Spectroscopy to Probe through the promotion of folding. Results from ThT fluorescence assay, static light the Structural Topology, Dynamics and Conformational Changes of S21 scattering, Circular Dichroism (CD) and Infrared (IR) spectroscopy, suggest that Pinholin Protein peptides are mostly unstructured in the absence of the field but experience a tran- Tanbir Ahammad1, Daniel L. Drew1, Sophia Rafferty1, Indra D. Sahu1, sition into a-helical states when the field is applied. Dynamic Light Scattering Robert McMarrick2, Gary A. Lorigan1. (DLS) and Transmission Electron Microscopic(TEM) studies confirmed the gen- 1Chemistry and Biochemistry, Miami University, Oxford, OH, USA, 2Miami eration of morphologically different aggregates in EF exposed Ab1-42.EffectofEF Univ Ohio, Oxford, OH, USA. on pre-formed aggregates show that the transition is accompanied by the disas- The Holin is a class of bacteriophage membrane proteins which are synthesized in sembly of pre-seeded stacked b-sheets. Experiments on human Cerebrospinal the infected bacterial cytoplasm. It is perhaps the simplest biological timing and

BPJ 9303_9305 52a Sunday, March 3, 2019 cell lytic system. These proteins are essential part of phage infection cycle and 263-Pos make hole in the cytoplasmic membrane to release the neonate virus. The Pinho- Mapping the Extracellular Loops of the Serotonin Transporter Using lin S21 (PHS21) is a type II Holin with two transmembrane domains (TMD), en- Crosslinking-Mass Spectrometry coded by lymbdoid Phage S21 gene. This dual start motif gene can express as 68 Elizabeth Castellano. residues active Holin or 71 residues Antiholin. In the active form, TMD1 can be Duquesne Univ, Pittsburgh, PA, USA. externalized from the membrane very quickly to oligomerize followed by very The serotonin transporter (SERT), a monoamine transporter family member, small hole formation, but in antiholin this externalization is slow which delayed controls the magnitude and duration of serotonergic neurotransmission by the oligomerization. We are using Electron Paramagnetic resonance (EPR) spec- facilitating reuptake of serotonin back into the pre-synaptic neuron. Selective troscopic techniques to probe the structural topology, dynamics and conforma- serotonin re-uptake inhibitors that prolong neurotransmitter signaling have tional changes of active and inactive form of PHS21. Fmoc-Solid Phase Peptide been developed to treat depression and other conditions. However, these Synthesis (SPPS) and site directed spin labeling was used to make EPR active drugs are not always effective and more specific and efficacious therapeutics samples in DMPC proteoliposomes. could result from a better understanding of the structure and mechanistic EPR Line shape analysis and EPR power saturation data indicate the external- function of SERT. The structures of SERT and related transporters have ization of TMD1, its orientation and interaction with lipid bilayer. DEER data been determined and serve as useful structural models; however they are and circular dichroism (CD) spectra indicated the concentration-dependent truncated for thermostability and typically lack significant regions of the oligomerization by the active form of this protein. We will determine the extracellular loops and the amino- and carboxy-termini. This study directly dynamics and membrane depth parameter of individual residues and oligomer- interrogates the topology of the extracellular loops of full-length SERT in ization state of PHS21. Rigid spin label like TOAC will be used to confirm the its resting state utilizing site-directed photoaffinity labeling and mass spec- orientation and alignment of PHS21. trometry (MS) to provide distance constraints. In every extracellular loop at least one single cysteine mutation was introduced systematically, using 261-Pos a Cys-null rat SERT construct and overexpression in Sf-9 insect cells. Assessing Depth-Dependence of the Azido Vibrational Probe Group in For each mutation, purified rSERT was reconstituted into lipid vesicles Bilayers using Transmembrane Peptides and tagged with MTS-benzophenone. Upon photoactivation isolated rSERT Yanyu Zhao. was proteolyzed and analyzed by tandem MS studies to identify sites of Chemistry, Haverford College, Haverford, PA, USA. non-specific covalent attachment of the photocrosslinker. The network of Characterizing membrane proteins is crucial to understanding the mechanisms identified crosslinks provides direct distance constraints and direct topologi- of a variety of cell functions and diseases. Recently, vibrational spectroscopy cal information regarding loop structure, offering a unique complement to has gained popularity for characterizing the dynamics of membrane proteins high resolution structural determinations. because of its instrinsically fast time scale. Vibrational probe groups (func- . tional groups with unique vibrational frequencies) are useful tools to charac- terize membrane proteins because they can be inserted site-specifically into 264-Pos peptides or proteins and report directly on membrane vs solvent exposure of Crystallization of the E. Coli Ribose ABC Importer Complex the probe groups. This project focuses on the azide probe group and uses a Leang-Chung Choh1, Satchal K. Erramilli2, Nicholas Noinaj1, 23-amino acid polyleucine transmembrane helical peptide, whose helical Cynthia V. Stauffacher1,3. length matches the hydrophobic thickness of POPC bilayers, as model system 1Dept Biological Sciences, Purdue Univ, West Lafayette, IN, USA, 2Dept to locate the probe group at varying burial depths. Peptides with azide groups Biochem & Molecul Bio, Univ Chicago, Chicago, IL, USA, 3Purdue Center were integrated into POPC membranes in both rehydrated bilayers and vesicles. for Cancer Research, Purdue Univ, West Lafayette, IN, USA. Far-UV circular dichroism (CD) indicated that the peptides were properly in- The ribose transporter complex (RbsABC) is one of many ATP-binding serted and helically folded into the POPC membranes. Infrared spectroscopy cassette (ABC) importers which are solely present in prokaryotes. This trans- (IR) was used to study the azide probes’ behaviors at different burial depths: membrane transporter is required to move ribose across the inner membrane all spectra exhibited a narrow, high-frequency peak (likely due to dehydrated of E. coli by utilizing ATP as the energy source. RbsABC is made up of a peri- probe groups) and a lower-frequency, broader spectral subpopulations plasmic ribose binding protein (RbsB), a transmembrane homodimer (RbsC) (possibly due to hydrated azide groups) whose intensity varied with the probe and a nucleotide-binding domain (RbsA) which contains two ATP-binding position. While the azide group exhibits a clear depth dependence and could be sites. While nucleotide-binding domains typically consist of two sites for used as a reporter of burial depth, this model system could also be used as a ATP hydrolysis, one of the RbsA ATP-binding sites possesses ATP hydrolysis spectroscopic ruler to characterize the behavior of other probe groups for activity while the other is inactive. This asymmetric ATP hydrolysis property is possible use in membrane proteins. rarely seen in other bacterial ABC importers. To understand the functional mechanism of theribose transporter complex, we are determined to solve the 262-Pos crystal structure and to use the structure to design further biophysical experi- Mapping the Topological Change of Sarcolipin Upon Sarcoplasmic 2D 2D ments on transporter dynamics. Crystallization screening of theribose trans- Reticulum Ca -ATPase Binding Along the Ca -Transport Cycle by porter complex using lipidic cubic phase, bicelles and different detergents Solid-State NMR was performed. Several conditions have been identified that areable to induce Songlin Wang, Gopinath Tata, Erik Larsen, Daniel Weber, Gianluigi Veglia. crystallization of this complex. From these crystallization hits, several crystals Department of Biochemistry Molecular Biology and Biophysics, University that diffracted at a synchrotron X-ray source to 3.2 A˚ were obtained and initial of Minnesota, Minneapolis, MN, USA. molecular replacement was attempted to solve the structure. During the Sarcoplasmic Reticulum Calcium-ATPase (SERCA) plays an important role in 2þ molecular replacement attempts, the periplasmic ribose-binding protein and muscle relaxation by maintaining Ca homeostatic balance in the sarco- cytoplasmic nucleotide-binding domains were well resolved but not the trans- plasmic reticulum (SR) lumen. Sarcolipin (SLN), which is expressed in cardiac membrane domain. Currently, crystallization of the selenomethionine- and skeletal muscles, modulates SERCA’s function by uncoupling ATP- derivative of RbsABC is being carried out to solve the RbsABC structure using 2þ b hydrolysis from Ca -transport and its phosphorylation in response to -adren- anomalous scattering. In addition, optimization of crystallization conditions is ergic stimulation results in relieved inhibition, making it an invaluable tool in still ongoing in order to obtain crystals with higher diffraction quality which studying SERCA structure-function relationship. By using Oriented-Sample will enable us to solve the crystal structure of the ribose transporter complex (OS) solid-state NMR (ssNMR), we captured the residues of SLN interacting in molecular detail. with SERCA at different states and mapped the topological change of SLN upon SERCA binding along the Ca2þ-transport cycle. The detergent- 265-Pos mediated reconstitution protocol is used to reconstitute SERCA/SLN complex Dimerization of Human Adenosine A2AReceptor - Impact of the C-Terminus into the bicelles. Furthermore, the paramagnetic ion chelated lipid is employed Khanh D. Nguyen1, Susanna Seppala2, Michael Vigers2, in the bicelle preparation to improve spectral sensitivity by shortening the bulk Nicole S. Schonenbach2, Songi Han1,2, Michelle A. O’Malley2. 1 1 HT1 relaxation. The highly resolved OS-ssNMR spectra enable the site- Chemistry and Biochemistry, UC Santa Barbara, Santa Barbara, CA, USA, specific analysis of the SLN/SERCA interaction. Our preliminary results sug- 2Chemical Engineering, UC Santa Barbara, Santa Barbara, CA, USA. gest that the topology of SLN is very sensitive to the different states of SERCA. It has long been recognized that the functions and activities of G protein-coupled Mapping the residues which interact with SERCA and studying which regions receptors (GPCRs) can be regulated by oligomerization. Among this largest fam- of the small peptide SLN are responsible for this inhibition will lead to a deeper ily of receptors, the human adenosine A2A receptor (A2AR) is capable of forming understanding of the increasingly complex system involved in modulating homo-oligomers and hetero-oligomers with other GPCRs, playing an impactful 2þ Ca cycling in the cardiomyocyte. role in several CNS disorders. Crystal structures and previous studies of A2AR

BPJ 9303_9305 Sunday, March 3, 2019 53a usually involve the truncation of the 95-residue long C-terminal tail to enhance Rhodopsin is the visual receptor that is responsible for scotopic vision of verte- conformational and thermostability of the receptor. However, there remain ma- brates [1]. Conventional X-ray crystallography has played a prominent role in jor unknowns in the role of this disordered C-terminus in the dimerization of our understanding of rhodopsin structure, yet there are major gaps in the dynam- A2AR. The ultimate goal of the project is to understand at the molecular level ical basis for its activation mechanism. We hypothesized that light absorption by (1) the impact of the C-terminus to A2AR dimerization, (2) the dimeric interface, rhodopsin leads to an influx of bulk water into the protein core that regulates the and (3) potential functional consequences. Our approach involves the use of size- catalytic binding and unbinding of the cognate G-protein, transducin. By employ- exclusion chromatography (SEC), electron paramagnetic resonance (EPR), and ing a combination of small-angle neutron scattering (SANS) and quasielastic- cryogenic electron microscopy (cryo-EM). neutron scattering (QENS) we investigated how the bulk solvent and the hydra- tion shell governs structural fluctuations during rhodopsin activation [2]. In 266-Pos SANS experiments rhodopsin was solubilized in CHAPS or DDM, and data Structural Insight into CwsA and CrgA Interaction in a Lipid Bilayer with were collected under detergent-contrast matched conditions to isolate the protein ssNMR structural changes upon photoactivation. We discovered how the rhodopsin fluc- Rongfu Zhang1, Huajun Qin1, Riqiang Fu1, Tim Cross1,2. 1 2 tuations are slaved to bulk water and the hydration shell. In the fully hydrated Natl High Magnetic Field Lab, Tallahassee, FL, USA, Chemistry and state, the a-fluctuations are coupled to the bulk solvent, opening up the protein Biochemistry, Institute of Molecular Biophysics, Florida State University, structure and facilitating activation of the G-protein transducin. The QENS exper- Tallahassee, FL, USA. iments compared the b-fluctuations that are coupled to the hydration shell of the Tuberculosis (TB) is a leading infectious disease killer worldwide caused by ligand-free opsin versus the dark-state rhodopsin in partially hydrated (h=0.27) Mycobacterium tuberculosis Mtb ( ). CwsA and CrgA are both small integral powdered protein samples. We discovered that local hydrogen-atom dynamics Mtb membrane proteins that belong to cell divisome. Previous studies by in the light-activated state are on-average slower, indicating greater steric crowd- et al. Plocinski showed that the interaction between CwsA and CrgA might ing versus the dark-state rhodopsin. Our experiments suggest that the light- result in the elongation at the poles and division at midcell. However, the struc- activated state of rhodopsin is swollen, yet locally more compact as a result of tural details on the atomic level of this interaction in a lipid bilayer environment increased interactions between the hydrophobic groups to accommodate the are not clear. Here, we strive to understand this interaction through both penetration of water into the protein core. Thus integrating SANS for the fully oriented (OS) and magic angle spinning (MAS) solid-state NMR (ssNMR) ap- hydrated protein with QENS for the partially hydrated protein reveals unprece- proaches. We first determined the tilt of a careful designed CwsA fragment in a dented insights into the energy landscape of GPCR activation process. POPC/POPG lipid bilayer to be 30 with glass slides supported OS ssNMR. References: [1] S.M.D.C.Perera et al.(2016) J.Phys.Chem.Lett.7,4239. [2] The INEPT-TOBSY pulse sequence was applied to probe the dynamic residues U.Shrestha et al.(2016) J.Phys.Chem.Lett.7,4130. of CwsA and CrgA, the spectra difference between uniformly-13C labeled 13 CwsA with and without CrgA (or uniformly- C labeled CrgA with and without 269-Pos CwsA) were compared to obtain possible interaction regions within CwsA and VDAC1 Conformational Changes Investigated by High Pressure DEER CrgA. The DARR pulse sequence was utilized to probe the transmembrane he- Lucie Bergdoll1, Matthias Elgeti2, Wayne Hubbell2, Jeff Abramson1. lical interactions with different isotope specific labeled CwsA and CrgA. 1Dept Physiology, Univ Calif Los Angeles, Los Angeles, CA, USA, 2Univ Together, MAS ssNMR approach was performed to gain insight into the inter- Calif Los Angeles, Los Angeles, CA, USA. action site at atomic level. The structural characterization of CwsA and CrgA Each day, humans turn over their body-weight equivalent in ATP, most of Mtb interaction will help to understand the mechanism of cell division which is produced in the mitochondria. Thus, ATP and ADP transport in and machinery. out of this organelle is critical for a good homeostasis. This transport is medi- 267-Pos ated by the Voltage-Dependent Anion Channel (VDAC), the most abundant b Cryo-EM Structure of a Mitochondrial Calcium Uniporter protein of the outer mitochondrial membrane. VDAC is a 19 strand -barrel, Jiho Yoo1, Mengyu Wu2, Ying Yin1, Mark A. Herzik Jr2, with an open conformation of high anion selective conductance (4 nS) allowing Gabriel C. Lander2, Seok-Yong Lee1. metabolite transport. Various low-conductance states exist (1.6-1.8 nS), which 1Biochemistry, Duke Univ, Durham, NC, USA, 2Integrative Structural and are cation selective and impermeable to metabolites. While several structures Computational Biology, The Scripps Research Institute, La Jolla, CA, USA. of VDAC in the open conformation have been solved, any other conformations, Calcium (Ca2þ) is a major cellular signaling molecule that participates in such as the closed states, remain to be identified. Here, we used high hydrostatic several many important physiological events, such as muscle contraction, pressure, a powerful method to investigate conformational changes in proteins, cell migration, and cell growth. Calcium plays a crucial role in the mitochon- to stabilize conformations that are otherwise negligibly populated and thus dria, where it is involved in cell apoptosis and ATP production. There have inaccessible to traditional biophysical analysis. Using double electron- been many efforts to understand the molecular mechanism by which mitochon- electron resonance (DEER) spectroscopy we probed structural changes induced drial calcium is regulated in order to achieve mitochondrial calcium homeosta- by high pressure on double-labeled mVDAC1, providing a better understanding sis. Recently, the mitochondrial calcium uniporter (MCU) was identified as a of the barrel flexibility. calcium-selective ion channel that serves to uptake transport calcium from the cytoplasm into the mitochondrial matrix. However, the molecular mecha- 270-Pos nism of calcium uptake by MCU has remained unclear. To understand calcium Elucidating Olfactory Receptor - Odorant Interactions: Leveraging Drug- transport by MCU on an atomic level, we determined the cryo-EM structure of Design Ideations 1 1 1 MCU from Neurospora crassa. The Our MCU structure reveals that MCU is a Chiquito J. Crasto , Anuththara Lokubandara , Vasanth Mandla , 2 tetramer and shows how that MCU adopts thea tetrameric architecture and how Dan Buzatu . 1Center for Biotechnology and Genomics, Texas Tech University, Lubbock, the conserved WDxxEPVTY sequence forms a . However, the soluble N-termi- 2 nal domain has different symmetry with respect to the transmembrane domain. TX, USA, Federal Drug Administration, National Center for Toxicology We found that the WDxxEPVTY sequence is conserved in the MCU pore and Research, Jefferson, AR, USA. forms a a selectivitye filter that exlusivelyexclusively conducts Ca2þ ions. for Drug-design is predicated on the creation of a compound that possesses features calcium uptake. This structure, iIn combination with cell-based mutagenesis (electronic and structural) that enhanced efficacy and diminishing toxicity. 1d- studies, this structure provides insight into the selective calcium uptake mech- and 3-d QSAR, and 1-d and 3-d SDAR methodologies have been used to design anism mediated by MCU in mitochondria. drugs. Design of the drug depends, additionally, on its complementarity with the target receptor protein, which precedes receptor-protein activation or inhi- 268-Pos bition. Ease of synthesis is also critical consideration. Structural Fluctuations in Rhodopsin Activation Revealed by Neutron Olfactory receptors (ORs) constitute the largest gene family in the human and Scattering most higher-order mammalian genomes. ORs, that line the central olfactory Suchithranga M.D.C. Perera1, Udeep Chawla1, Utsab Shrestha2, processing system, provide the link between the atmosphere and the olfaction Debsindhu Bhowmik2, Andrey V. Struts1,3, Shuo Qian4, Xiang-Qiang Chu5, processing centers of the brain. Michael F. Brown1,6. ORs discriminate between thousands of individual odorants or complex odors. 1Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ, Interactions between ORs and odorants are promiscuous—one of the chal- USA, 2Department of Physics and Astronomy, Wayne State University, Detroit, lenges in devising a rational panel of putative odorants to test the functionality MI, USA, 3St. Petersburg State University, St. Petersburg, Russian Federation, of an olfactory receptor. Other challenges include expression of these 4Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, TN, membrane-bound proteins, and the lack of an experimentally determined pro- USA, 5Graduate School of China Academy of Engineering Physics, Beijing, tein structure of the receptor. There are strong parallels between drug-receptor China, 6Department of Physics, University of Arizona, Tucson, AZ, USA. interactions and odorant-receptor interactions.

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We undertook a comprehensive assessment of odorant molecules, using an 273-Pos odorant-centric approach to understand the structural and electronic nature of Exploring (Proteo-) Liposomes for Mass Spectrometry these compounds. We tested odorants that were implicated in activating Melissa Frick1, Caroline Haupt2, Carla Schmidt1. (strongly, moderately or weakly) functionally characterized olfactory recep- 1Interdisciplinary Research Center HALOmem, Charles Tanford Protein tors. The conformational space of these odorants were explored in vacuo; Center, Martin Luther University Halle-Wittenberg, Halle/Saale, Germany, this was followed by unrestricted and targeted docking of these odorants in 2Institute of Biophysical Chemistry, Martin Luther University computational models of variants of these olfactory receptors (wild type and Halle-Wittenberg, Halle/Saale, Germany. mutated). We also carried out extensive QSAR and 1d- and 3d- SDAR assess- Liposomes are phospholipid bilayer vesicles which resemble cellular organ- ments of odorants known to activate ORs to identify nuanced electronic- elles and membranes. Due to their variability in size, composition and amphi- structural features that are potentially responsible for odorant interactions. philic character they are promising mimics of natural membranes. However, The use of drug-design ideations in chemoreception is seminal. due to their heterogeneity and ability to form large clusters in the gas-phase, liposomes were not employed for mass spectrometric analysis to-date. We 271-Pos envision that, in the gas-phase of the mass spectrometer, these clusters can Regulation of Proton Transport in Tetrameric UCP2 by an Intramolecular be dissociated, releasing intact proteins and protein complexes from the lipid Salt-Bridge Network bilayer, allowing their structural analysis. Our goal therefore was to use Afshan Ardalan1, Stephanie Uwumarenogie1, Michael Fish2, liposomes for the analysis of lipids and proteins directly from phospholipid Shahin Sowlati-Hashjin3, Mikko Karttunen4, Matthew D. Smith2, bilayers. To reach this goal, we first analyzed ‘‘empty’’ liposomes under dena- Masoud Jelokhani-Niaraki1. turing and non-denaturing conditions. We prepared liposomes of different sizes 1Chemistry & Biochemistry, Wilfrid Laurier University, Waterloo, ON, using different lipid species in varying concentrations. We found that Canada, 2Biology, Wilfrid Laurier University, Waterloo, ON, Canada, liposomes can be dissociated in the gas phase of the mass spectrometer inde- 3Chemistry, University of Western Ontario, London, ON, Canada, pendently on their size, composition and concentration. Using shotgun lipido- 4Chemistry/Applied Mathematics, University of Western Ontario, London, mics we were able to identify and quantify different phospholipid species from ON, Canada. various lipid classes directly from the lipid bilayer. Employing a mass spec- Uncoupling proteins (UCPs) are carriers responsible for fatty acid regulated trometer modified for transmission of high masses revealed the presence of proton leak across the mitochondrial inner membrane, resulting in uncoupling lipid clusters which dissociate at higher collisional energies. Furthermore, we of electron transport process from ATP synthesis. Similar to other members of could show that proteins and liposomes mixed in solution can be analyzed mitochondrial carrier superfamily (MCF), UCPs possess two salt-bridge net- together in the same mass spectrum. We next prepared liposomes which works facing the mitochondrial matrix and intermembrane space. It has been include soluble proteins encapsulated. We were able to dissociate these lipo- suggested that alternating formation (opening) and disruption (closing) of these somes in the gas phase releasing the encapsulated protein. Currently, we two salt-bridge networks can control the ionic/molecular transport through optimize the mass spectrometric analysis of proteoliposomes which harbor pro- MCFs, including UCPs. In this study, we probed the role of matrix network teins embedded in their lipid bilayer. Using both denaturing and non-denaturing in proton transport function of UCP2 via selective mutation of the lysine conditions, we aim to release the proteins from the liposome membrane and residues (K38Q, K141Q, K239Q, K38Q/K239Q), thereby disrupting one/two study their structures in the gas phase of the mass spectrometer. salt-bridge(s) by eliminating the positive charge(s). These mutants were confor- mationally comparable to UCP2, as verified by CD spectroscopy. Concurrently 274-Pos we found that once reconstituted in lipid bilayers, the monomeric UCP2 (orig- Lipid-Modulation of SthK, a Cyclic Nucleotide-Gated Channel inally in mild detergents) is spontaneously transformed to stable tetrameric Philipp A.M. Schmidpeter1, Jan Rheinberger1,DiWu2, Haiping Tang2, forms. Tetramerization of UCP2 in lipidic milieus (and stability of tetrameric Carol V. Robinson2, Crina M. Nimigean1. vs. monomeric/dimeric forms) was also supported by molecular dynamics 1Dept Anesthesiology, Weill Cornell Medicine, New York, NY, USA, 2Dept (MD) simulations. This ‘‘monomer to tetramer’’ molecular transformation in Chemistry, University of Oxford, Oxford, United Kingdom. lipid bilayers did not happen in the structurally relevant ADP/ATP carrier The function of integral membrane proteins, such as ion channels, depends not (AAC1) protein. Our results show that disruption of salt-bridges in the matrix only on the physical properties of the surrounding lipid bilayer but also the network caused a substantial increase in transport rate (as measured by fluores- identity of lipids, which may interact specifically with the protein, altering cence assays), implying the regulatory role of this network in the proton trans- functional characteristics of the protein. Here, we investigate the lipid require- port function of UCP2. Interestingly, the MD simulations showed that the ment of the bacterial cyclic nucleotide-gated (CNG) channel SthK and how UCP2 tetramer is asymmetric and this asymmetric character suggests that all different lipids affect channel function. four of the UCP2 units may not be (simultaneously) functional. Overall, these Density maps from single particle cryoEM structures of SthK reconstituted into findings show the important role of matrix network in regulating the proton/ion lipid nanodiscs revealed lipids located in close proximity to SthK at the protein- transport function of UCPs, and support the hypothesis that the monomeric membrane interface. In addition, lipid density was also identified along the outside UCPs are not the only functional forms of these proteins in lipid environments. of the inner pore helix, a location less accessible for protein-surrounding lipids. The same lipid densities were observed in SthK structures solved in detergent mi- 272-Pos celles, indicating that these lipids were co-purified with the channel from E. coli. Probing the Active and Inactive Forms of the Bacteriophage S21 Pinholin Thin layer chromatography confirmed the presence of lipids bound to SthK in Protein System using Magnetic Resonance Spectroscopy detergent and, together with mass spectrometry on the same sample, we were Daniel L. Drew1, Tanbir Ahammad1, Rachel Serafin1, Brandon Butcher1, able to identify these lipids as POPG and Cardiolipin. We then systematically Katherine Clowes2, Indra D. Sahu1, Robert McMarrick1, Gary A. Lorigan1. 1 2 analyzed the effects of these lipids on channel activity by using a fluorescence- Chemistry and Biochemistry, Miami Univ, Oxford, OH, USA, Chemistry, based stopped-flow flux assay. In the absence of negatively charged lipids SthK University of Kentucky, Lexington, KY, USA. shows little to no activity indicating that the co-purified lipids are not sufficient The mechanism for the lysis pathway of double-stranded DNA bacteriophages in- to activate the channel. Increasing the amount of POPG or Cardiolipin during volves a small hole-forming class of membrane proteins known as the holins. This the reconstitution leads to significantly increased and stable channel activity. study focuses on a poorly characterized class of holins, the pinholins, of which the The time course of activation with cAMP and the cAMP concentration needed 21 f S protein of phage 21 is the prototype. This study will show the first in vitro for half activation were not significantly affected by the type of lipid employed. synthesis of the active wildtype form of the S21 pinholin, S2168, and the nonfunc- 21 Our structural data combined with the lipid identification by mass spectrometry tional form, S IRS, using solid phase peptide synthesis. Site directed spin label- and the subsequent functional analyses form the groundwork for future studies ing of the pinholin with the nitroxide spin label MTSL allows for the use of both on the molecular determinants of protein-lipid interactions in SthK. continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) spec- troscopy to study the pinholin structure and dynamics. CW-EPR results confirm 275-Pos successful incorporation of the pinholin into proteoliposomes, while 3-pulsed Site-Directed Labeling of Type II Cannabinoid Receptor CB2 for Struc- ESEEM EPR spectroscopy in tandem with circular dichroism (CD) both confirm tural EPR Analysis proper helical secondary structure. Calculated CD molar ellipticity ratios of both Levi T. Hooper. active and inactive forms of the pinholin suggest helical interactions occurring Laboratory of Membrane Biochemistry & Biophysics, National Institute on within the bilayer which is consistent with the proposed oligomerization step in Alcohol Abuse and Alcoholism, Rockville, MD, USA. models for the mechanism of hole formation in the inner bacterial membrane. Human endocannabinoid receptor CB2 is a G protein-coupled receptor (GPCR) Finally, 2H and 31P solid-state nuclear magnetic resonance (NMR) spectroscopy of interest due to its role in numerous physiological processes including were utilized to probe the interactions of wildtype active S2168 and nonfunctional immune response and inflammation. Here we explore its structure using S21IRS pinholin with the lipid bilayer. non-invasive continuous wave-electron paramagnetic resonance spectroscopy

BPJ 9303_9305 Sunday, March 3, 2019 55a

(CW-EPR). Small site-directed nitroxide spin labels were introduced by chemi- KCNE3 (E3) is a b-subunit of voltage gated potassium ion channels (Kv) that cally labelling of cysteines followed by analysis using CW-EPR. CB2 constructs modulates the function of Kv by interacting with its a subunits including for structural analysis were produced by removing non-essential, water-exposed KCNQ1 and KCNQ4 (known as Q1 and Q4). Compared to other Kv channels, cysteine residues and creating a cysteine minimal mutant of CB2 that expressed as E3-Q1 interactions have been well studied in intestinal and tracheal epithelial a fully functional receptor as assessed by ligand binding studies and activation of cells where the complex maintains ion homeostasis. E3 interaction with Q1 al- G protein. Using this template, new cysteine residues were introduced by muta- lows Ky to act as a voltage independent and constitutively active channel that fa- genesis in both intracellular and extracellular positions on the receptor. Cysteine cilitates transepithelial Cl- ion secretion. Mutations in KCNE3 are responsible for CB2 mutants were chemically labelled with 3-(2-Iodoacetamido)-PROXYL and diseases related to salt and fluid disorders including cystic fibrosis. Hence, it is solubilized in Facade-TEG/cholesteryl hemisuccinate detergent micelles. Spec- important to elucidate KCNE3 structural and dynamic information with func- tral analysis revealed decent selectivity of site specific labeling as well as substan- tional studies. Previously, the structure of E3 has been studied in a bicelle envi- tial differences in chemical reactivity of water-exposed cysteines. Polarity of ronment. However, in physiological conditions, lipids are organized into label environment and dynamics of label movements are accessed by measure- bilayers instead of single sheets of molecules. This research has been focused ment of relaxation time, hyperfine splitting and line shape analysis. Results are on protein structure, dynamic and topology of KCNE3 membrane protein in interpreted in terms of CB2 structure and dynamics. POPC/POPG vesicle usingSite-Directed Spin Labeling (SDSL) and Electron Paramagnetic Resonance Spectroscopy (EPR)techniques. EPR coupled with 276-Pos SDSL provides structural and dynamic information about proteins. In this study Lysosomal Exocytosis Impacts Dectin-1 Endocytosis After b -Glucan we have successfully introduced S-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol- Stimulation 3-yl) methyl methanesulfonothioate spin label (MTSL) on several site-specific Akram Etemadi Amin, Aaron Neumann. position of E3 by site-directed mutagenesis to obtain EPR spectra. CW-EPR University of New Mexico, Albuquerque, NM, USA. (Continuous Wave Electron Paramagnetic Resonance)line shape analysis has Dectin-1 is a pattern recognition receptor expressed by myeloid cells that detects been performed to investigate the dynamic of the protein in a lipid bilayer, while b-glucans in Candida species fungal pathogen cell walls. Several recent studies EPR power saturation has been used to study the topology of KCNE3 in a vesicle. have suggested that Dectin-1 associates with liquid ordered domains during its Our data are consistent with the model of KCNE3 in a bicelle and revealed more activation. Ceramide Enriched Domains (CED) are one type of liquid-ordered promising structure and dynamic information for the protein in a native mem- membrane domain, but the potential role of CED in Dectin-1 mediated recogni- brane environment. tion of b-glucan remains unknown. CEDs can be generated upon fusion of pe- ripheral lysosomes with the plasma membrane, leading to local release of acid 279-Pos sphingomyelinase (ASMase), which converts outer leaflet sphingomyelin to cer- Purification and Preliminary Characterization of Human KCNQ1 (100- amide. Our goal is to determine the role that acid sphingomyelinase plays in 370) Potassium Ion Channel in Lipid Bilayers using Solid-State NMR Dectin-1 signaling. Specifically, we hypothesize that Dectin-1 signaling inten- Spectroscopy sity is regulated by receptor internalization dynamics in a CED- and ASMase- Colleen K. Jaycox1, Gunjan Dixit1, Indra D. Sahu1, Robert McMarrick1, mediated manner after activation-dependent fusion of peripheral lysosomes at Charles R. Sanders2, Gary A. Lorigan1. the plasma membrane. We conducted immunofluorescence experiments on 1Chemistry and Biochemistry, Miami University, Oxford, OH, USA, 2Ctr glucan stimulated HEK-293 cells expressing Dectin-1 to measure LAMP1 (a Struct Biol, Vanderbilt Univ, Nashville, TN, USA. luminal marker for lysosomes) exposure in plasma membrane. Furthermore, KCNQ1 (Q1 or Kv7.1 or KvLQT1) is a voltage gated potassium channel we determined Dectin-1/glucan internalization by quantifying co-localization expressed in several tissues of the body and modulated by another membrane of the receptor with EEA1þ early endosomes. Our results show a significant in- protein KCNE1 (E1). Q1 is involved in the cardiac repolarization phase of the crease in LAMP1 exposure on the plasma membrane in stimulated samples heart beat following an action potential. Q1/E1 interaction slows down the compared to unstimulated, which indicates lysosomal fusion with the plasma activation kinetics of KCNQ1 required for proper channel and heart function. membrane. Moreover, we detected rapid endocytosis of Dectin-1 in early endo- Hereditary mutations in Q1 can lead to diseases like Long-QT syndrome somes after stimulation. Our working model is that ASMase delivered by periph- (LQTS), atrial fibrillation, sudden infant death syndrome, cardiac arrhyth- eral lysosomes upon Dectin-1 activation is important for formation of CED, mias and congenital deafness. Human KCNQ1 is a 676-residue membrane which promote membrane curvature necessary to support Dectin-1 endocytosis. protein with six transmembrane helices (260-residues), the first four form- These results suggest a new pathway for Dectin-1 endocytosis involving plasma ing the voltage sensor domain/Q1-VSD (S1-S4), linked to the pore domain membrane remodeling, which may be an important regulator of signaling from (S5-S6) by the S4-S5 linker and the cytosolic N (100-residue) and C-terminal Dectin-1 during innate immune fungal pathogen recognition. (300-residues) domains. In this study, the purification and preliminary biochemical and biophysical characterization of the full length (100-370) 277-Pos channel comprising all 6 transmembrane segments is reported. The purified Characterization of the Interaction between Two Influenza a Proteins protein in 0.5% DPC micelles was analyzed using LC-MS/MS and used to (M1 and M2) Involved in Viral Assembly study the interaction of the protein with POPC/POPG proteoliposomes using Abigail Wong-Rolle, Reham Mahgoub, Elizabeth Erler, 31P solid-state NMR spectroscopy by varying the molar ratios of protein to Kathleen P. Howard. lipid. Department of Chemistry and Biochemistry, Swarthmore College, Swarthmore, PA, USA. 280-Pos Two of the proteins required for the efficient assembly of influenza A virus par- Coarse-Grained Simulations of Transmembrane Domain Interactions in ticles are matrix protein 1 (M1) and membrane-bound matrix 2 protein (M2). Semaphroin-Plexin-Neuropilin Signal System Our work involves developing a detailed biophysical understanding of the Zhiyuan Meng1, Zhenlu Li2, Matthias Buck2. interaction of M1 protein and M2 protein. M2 is a 97-amino acid membrane- 1Biochemistry Department, Case Western Reserve University, Cleveland, bound protein that we have previously characterized using site-directed spin-la- OH, USA, 2Physiology and Biophysics Department, Case Western Reserve bel electron paramagnetic resonance (SDSL-EPR). M1 is a 252-amino acid University, Cleveland, OH, USA. protein that associates with M2 protein during viral assembly and budding. Most cell activities are mediated by membrane proteins. Membrane proteins are Using a multi-pronged biophysical approach, we have probed the interaction activated by the ligand to trigger and regulate intracellular signaling pathways, of M1 protein with membranes both with and without reconstituted M2 protein. e.g. semaphorins and plexins. Both M1 and M2 proteins have been previously shown to have multiple confor- In many tissues and organs, the interaction of semaphorin ligands and plexin mational states. We hypothesize that the interaction of the two proteins prefer- receptors is related to the homeostasis and morphogenesis and plays an essen- entially stabilizes a subset of accessible conformations. tial role in neural connectivity, cancer, cell migration and immune response. For their role as single-pass transmembrane receptors, the signal transmits 278-Pos mostly by transmembrane(TM) helix-helix interactions forming dimer interac- Probing Structure, Topology and Dynamics of KCNE3 in Proteoliposomes tions. Former researchers have found that there are trans-interactions between using Site-Direct Spin Labeling and EPR Spectroscopy SEMA4D and Sema6A, Sema6A and PlxnA2, Sema3 and PlexinA1. However, Fathima Dhilhani Mohammed Faleel1, Indra D. Sahu1, there are few experiments on the mechanism on cis-interactions of semaphorin, Robert M. McCarrick1, Charles R. Sanders2, Gary A. Lorigan1. plexin, and the co-receptor neuropilin. The TM region is recognized as an 1Department of Chemistry and Biochemistry, Miami University, Oxford, OH, essential role to trigger the function of the complex. USA, 2Biochemistry and Center for Structural Biology, Vanderbilt Our research focuses on understanding the membrane signaling platform as- University, Nashville, TN, USA. sembly through TM domains by coarse-grained molecular dynamic

BPJ 9303_9305 56a Sunday, March 3, 2019 simulations. Our results will provide more information for the design of drug superfamily. P-gp is an ATP-dependent efflux pump with broad substrate and novel therapeutic methods. specificity responsible for effluxing xenobiotic substances such as toxins or drugs from the cell. Absorption by the body of orally administered drugs 281-Pos is reduced by the presence of P-gp, which facilitates their elimination from Structural Studies of Membrane Proteins using Pulsed EPR Spectroscopy the body. P-gp consists of two transmembrane domains and two nucleotide- 1 1 1 2 Gary A. Lorigan , Indra D. Sahu , Daniel L. Drew , Gunjan Dixit , binding domains. Our laboratory has extensively characterized this protein Tanbir Ahammad1. 1 2 biochemically using a variety of systems, including native membranes, pu- Dept Chem/Biochem, Miami Univ, Oxford, OH, USA, Cell Molecular and rified in detergent micelles or in proteoliposomes. Here we demonstrate the Structural Biology, Miami University Ohio, Oxford, OH, USA. advantages of using nanodiscs for studying and characterizing P-gp. Nano- Solid-state NMR and EPR spectroscopy are important biophysical techniques discs are synthetic membrane model systems that have been widely used that are being using to study membrane proteins. CW and pulsed EPR spec- to study membrane proteins. They are structurally similar to high-density troscopic techniques coupled with site-directed spin-labeling (SDSL) can lipoproteins and are composed of a belt or membrane scaffolding protein provide important structural information on complicated biological systems derived from apolipoprotein-A1 and phospholipids. Nanodiscs are consid- such as membrane proteins. Strategically placed spin-labels alter relaxation ered optimal membrane mimetic systems because size, composition, times of NMR active nuclei and yield pertinent structural information. EPR and specific functional modifications can be controlled on the nanometer techniques such as Double Electron-Electron Resonance (DEER) and Elec- scale. The incorporation of P-gp into nanodiscs was optimized to achieve tron Spin Echo Envelope Modulation (ESEEM) are powerful structural highly homogenous preparations. We demonstrate that the nanodiscs biology tools. The DEER technique can be used to measure distances between better resemble the behavior of P-gp in membranes when compared to ˚ 2spinlabelsfrom20to70A. However, the application of DEER spectros- protein purified in detergent micelles. Finally, P-gp nanodiscs were used copy to study membrane proteins can be difficult due to short phase memory to study conformational changes associated with the ATP hydrolysis cycle. times (Tm) and weak DEER modulation in more biologically relevant proteo- The UIC2 antibody, which binds to the extracellular region of liposomes when compared to water soluble proteins or membrane proteins in P-gp, was found to dissociate during ATP hydrolysis, indicating that the detergent micelles. The combination of these factors often leads to broad dis- extracellular loops change in conformation as the molecule hydrolyzes tance distributions, poor signal to noise, and limitations in the determination ATP on the opposite side of the protein. All in all, we demonstrate that of longer distances. The short phase memory times are typically due to un- upon optimization, nanodiscs are a valuable platform for studying P- even distributions of spin-labeled protein within the lipid bilayer, which cre- glycoprotein. ates local inhomogeneous pockets of high spin concentrations. Approaches to overcome these limitations and improve the quality of DEER measurements 284-Pos for membrane proteins will be discussed: lipodisq nanoparticles, bi- Structural Study for Membrane Protein using Solution X-Ray Scattering functional spin labels (BSL), and Q-band pulsed EPR spectroscopy. ESEEM Contrast Variation data will be used to probe the secondary structure of membrane proteins. Xiaobing Zuo. ESEEM can be thought of as EPR detected NMR. CW-EPR spectra of X-ray Science Divison, Argonne Natl Lab, Lemont, IL, USA. spin-labeled membrane proteins will be used to investigate dynamics and Membrane protein often needs lipid bilayer membrane to stabilize its struc- the immersion depth in a lipid bilayer. A variety of different membrane pro- ture and to fulfill its functions. However, the presence of lipid bilayer mem- teins will be probed with these state-of-the art magnetic resonance brane makes the structural study on the protein very difficult. Here techniques. we proposed to use X-ray scattering contrast variation method in which 282-Pos membrane protein (with lipid membrane) samples will be measured in a se- Structural and Functional Role of the Surface-Exposed Loops of Ail Dur- ries of solutions with various sucrose concentration. X-ray scattering ing Complement-Mediated Evasion by Y. pestis intensity measures the scattering length/capability difference between sam- Luz Marina Meneghini, Chandan Singh, Kyungsoo Shin, L. Miya Fujimoto, ple/solute and buffer solution/solvent. Since X-ray scattering lengths of pro- Ye Tian, Francesca M. Marassi. tein and lipid are different and scattering length of sucrose buffer changes Sanford Burnham Prebys Medical Discovery Inst, La Jolla, CA, USA. along with its concentration, the ratio of X-ray scattering contribution Yersinia pestis, the causative agent of plague, promotes its proliferation in the from protein part and lipid part varies along with the sucrose concentration. host by expressing various bacterial proteins that inhibit the host innate im- In such way, we can separate the scattering contribution of protein part from mune system. One of these is attachment invasion locus (Ail), which is an that of lipid. We also simultaneously fit the data sets collected from the eight-stranded b-barrel outer membrane protein with four surface exposed sucrose concentration series measurements and obtain the 3-D low re- extracellular loops. Ail is essential for Y. pestis pathogenesis and proliferation solution structure for the protein and the lipid at the same time. Comparing as it recruits host serum proteins to inhibit the complement-dependent bacte- to neutron scattering contrast variation method, this X-ray contrast method riolysis and inflammatory response, and regulates host cell attachment and is less expensive and can provide wider q range due to the availability delivery of Yersinia outer proteins to host tissue. Recently, we demonstrated of high flux X-ray sources. A few membrane protein examples will be that Y. pestis can also recruit vitronectin (Vn) from the human serum in Ail- presented. Figure. SAXS data for membrane protein in various sucrose dependent manner. Vn is a 459-residue multi-domain protein that interacts solution. with various molecules to promote cell adhesion and migration, and inhibits In this presentation, I will also discuss the capabilities of beamline 12-ID-B of formation of the membrane attack complex, indicating that the interaction be- the Advanced Photon Source at Argonne National Laboratory. tween Ail and Vn may be responsible for promoting Y. pestis survival and Acknowledgement: Use of the Advanced Photon Source, an Office of Science pathogenesis. Notably, previous works have indicated that serum proteins User Facility operated for the U.S. Department of Energy (DOE) Office of such as Vn are recruited by Ail via its extracellular loops. Thus, we hypoth- Science by Argonne National Laboratory, was supportedby the U.S. DOE un- esized that Ail’s extracellular loops may also be responsible for binding to der Contract No. DE-AC02-06CH11357. Vn. To characterize this interaction, amino acids in each extracellular loop of Ail were substituted for alanine to individually characterize the loops 285-Pos for Vn binding. The structure and function of each Ail loop mutant was char- Structure and Mechanisms of an Anion Transporter Family acterized in a native like environment inside membrane (detergent micelle Robert M. Stroud, Jonathan Leano, Samir Batarni, Robert Edwards. and nanodiscs) by solution state NMR and ELISA binding assays. These Dept Biochem/Biophys, Univ Calif San Francisco, San Francisco, CA, USA. studies identify specific extracellular loops of Ail that are important for bind- Members of the solute carrier 17 family use divergent mechanisms to ing to Vn, which will guide future developments of therapeutics aimed at concentrate organic anions. Membrane potential drives uptake of the prin- combating Y. pestis infections. cipal excitatory neurotransmitter glutamate into synaptic vesicles, whereas closely related proteins use electroneutral cotransport to drive efflux from 283-Pos the lysosome. To identify the common features of ionic coupling by the þ Nanodiscs as a Platform for the Study of Human P-Glycoprotein in a SLC17 family, we determined the structure of E. coli D-galactonate/H Membrane Environment symporter DgoT in two states: one open to the cytoplasmic side, and the Sabrina Lusvarghi, Suresh Ambudkar. other open to the periplasmic side with substrate bound. The structures LCB/CCR/NCI/NIH, Bethesda, MD, USA. identify a proton translocation pathway conserved from bacteria to mam- þ Multidrug resistance of cancer cells and pathogens is a significant clinical mals. Functional analysis suggests that a transition in the role of H problem. A major contributor to drug resistance in cancer cells is overex- from flux coupling to allostery may underlie the divergence in energy pression of P-glycoprotein (P-gp), a member of the ATP-binding cassette source.

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286-Pos This suggests that A2AD316R does not maintain all active receptor functions Membrane Binding of HIV-1 Accessory Protein Nef on Sparsely-Tethered of the full-length receptor. Here, we characterize ligand binding properties of Bilayer Lipid Membranes: An Spr Study full-length A2AR, A2AD316R, and Rag23, a thermostable A2AD316R variant Christopher Kervick1, Manish Aryal2, Frank Heinrich1,3, with five point mutations designed to favor agonist binding, by purifying the Thomas E. Smithgall2, Mathias Lo¨sche1,3. receptors into detergent micelles. Additionally, ongoing experiments are being 1 2 Physics, Carnegie Mellon University, Pittsburgh, PA, USA, Microbiology performed to characterize ligand binding properties of similarly purified A1R. and Molecular Genetics, University of Pittsburgh, Pittsburgh, PA, USA, Using fluorescence anisotropy and surface plasmon resonance (SPR), we deter- 3NIST Center for Neutron Research, Gaithersburg, MD, USA. mine equilibrium binding, competitive ligand binding, kinetic rates, and recep- Human Immunodeficiency Virus-1 Nef is an accessory protein essential for the tor stability at increasing temperatures. progression of HIV-1 infection by aiding viral production by down-regulating defense mechanisms of the infected cell to help evade immune response. Nef 289-Pos has been shown to interact with Src- and Tec-family kinases at the plasma mem- Characterization of the Extra-Membrane Domains of CrgA in Lipid Bila- brane of infected cells, leading to constitutive kinase activation. For example, yers using Solid State NMR Yiseul Shin1,2, Riqiang Fu2, Huajune Qin2, Timothy A. Cross1,2. blocking Interleukin-2-inducable T-cell Kinase (Itk) activity with pharmacolog- 1 2 ical inhibitors has been shown to result in decreased viral spread. Evidence also Dept Chem & Biochem, Florida State Univ, Tallahassee, FL, USA, The shows that Nef dimerizes on the membrane as it recruits kinases. However, the National High Magnetic Field Laboratory, Tallahassee, FL, USA. exact mechanism of this interaction between Nef and the kinases, or indeed be- Transmembrane (TM) proteins exist in heterogeneous membrane environments tween those two classes of proteins and the membrane, is not much understood. that affect protein structure in various ways due to diverse biophysical properties We used Surface Plasmon Resonance to study the binding of Nef on sparsely- of membrane environment. The extra-membrane domains have varying dynamics tethered bilayer lipid membranes (stBLMs) in preparation for future studies in compared tothe TM domain. Solid state NMR is a powerful technique for studying complex with kinases. Nef exhibits two distinct binding modes, one fast and TM proteins in liquid-crystalline lipid bilayers that better model the biophysical one slow, which we attribute to two different membrane binding sites on Nef. properties of the native membrane. CrgA is a membrane protein known to interact Mycobacterium tuberculosis The fast binding mode is likely that of the expected binding mechanism for with five other proteins in . To become a versatile Nef; insertion into the membrane of a myristoyl tail, whereby the core region binding partner, CrgA is likely to have multiple binding sites in the cytoplasmic of the protein is distant from the membrane surface. The second binding N-terminus and the periplasmic loop. The structure of extra-membrane domains is characterized with different NMR experiments and amino acid specific 13C mode has Nef in much closer proximity to the membrane, with a much higher 15 surface coverage. This in turn blocks the fast binding mode in subsequent con- and N labeling strategies in order to selectively observe the domains. Full length centration additions. For a given concentration, the binding rate is far higher CrgA is reconstituted in POPC/POPG bilayers for NMR experiments. Selective when that concentration is added directly to a neat bilayer, rather than reached NMR experiments resolve signals from 20 residues in an intrinsically disordered region (IDR). To probe the membrane interactions of the IDR, varying amounts of through a series of concentration additions. Previous Neutron Reflectometry 3þ (NR) characterization of membrane-bound Nef showed a concentration- 16:0 PE-DTPA (Gd ) dopant are introduced so that residues interactions with the dependent conformation change, consistent with this interpretation. lipid head group can be detected through Paramagnetic Relaxation Enhancement. These experiments suggest that charge-charge interactions between the positively 287-Pos charged residues and the lipid head group play an important role in the membrane Flexible Linker Region in Endophilin Structure Affects its Thermosta- interactions of the IDR. Also, the loop is not fully exposed to the bulk aqueous bility environment, but exists in the lipid interfacial region. Broad cross peaks from Rui Jin, Tobias Baumgart. the loop indicate that it is not completely disordered despite of significant motion Chemistry Department, University of Pennsylvania, Philadelphia, PA, USA. occurring in the lipid interface. NMR data suggest that a portion of the N-terminus Proteins involved in endocytosis often adopt specific structures to couple with and the loop closely interact with the lipid interface, thus, highlighting the impor- curved lipid membranes. The BAR protein family is a group of important pro- tance of having a liquid-crystalline lipid environment for dynamics and the struc- teins with crescent BAR domains that often bind to the neck of budding vesicles tural studies of CrgA. to narrow the neck before the final scission step. They also contain SH3 do- mains that further recruit downstream proteins, like dynamin. However, the role played by the the unstructured flexible linker between the BAR and SH3 Posters: Protein Structure, Prediction, and domains has received little attention thus far. Recently linker regions in Design some endocytosis related proteins have been reported to also participate in downstream protein recruiting. Here, we focus our research on the important 290-Pos BAR protein, endophilin. We are interested in endophilin’s behavior upon Modeling Epistasis using Protein Biophysics and fX174 membrane binding as well as the potential role of its linker consisting of around Casey M. Beard. fifty residues. Via truncations of the endophilin sequence, we studied the role of Physics, University of Idaho, Moscow, ID, USA. the linker. Interestingly, the unstructured linker seems to influence endophilin Epistatic effects play a key role in the evolutionary dynamics of viruses and bac- thermostability. We are able to localize this characteristic to a short region teria. These effects have previously been observed within single genes, but less is close to the BAR domain, which contains less than twenty residues. known about the effects between interacting genes. Since selection impacts pro- tein complexes, we expect epistasis to impact interacting genes in ways that are 288-Pos important to understanding the fitness landscapes of bacteria and viruses. We Contributions of the C-Terminus and Mutations to Adenosine Receptor have chosen the bacteriophage fX174 as a model to examine these effects. Spe- Activity and Stability cifically, we focus on mutations in two interacting proteins, the viral capsid (F) Kirsten N. Swonger, Anne S. Robinson. and spike (G) proteins. We predicted the biophysical effects of all possible single Chemical and Biomolecular Engineering, Tulane University, New Orleans, and double mutations within and between the F and G proteins using FoldX. LA, USA. FoldX has a semi-empirical scoring function that requires a minimum of compu- G-protein coupled receptors (GPCRs) are seven-transmembrane domain mem- tational resources and can run in parallel on a computer, allowing us to calculate brane proteins that bind with extracellular ligands, activating intracellular all 250,000 folding and binding stability changes. We also included snapshots downstream signaling cascades. This interaction makes GPCRs ideal drug of the F-G protein complex generated via molecular dynamics simulations as targets for a number of conditions including pain, hypertension, and schizo- inputs to FoldX to provide conformational flexibility. Comparing the predicted phrenia. Adenosine receptors are a family of class A GPCRs that are widely effects of double mutants to an additive model of single mutants allows us to expressed throughout the body. There are four types of adenosine receptors, identify pairs of sites experiencing epistasis. We then examine the fitness of these A R, A R, A R, and A R. All four receptors bind a common agonist, aden- 1 2A 2B 3 mutations in empirically derived results of mutated fX174. osine, with varying affinities. A2AR is the most well-characterized of the four receptors and has been crystallized in a number of active conformations to bet- 291-Pos ter understand the conformational changes that occur during ligand binding to Fourier-Transform Infrared (FTIR) Spectroscopy Transmission Tech- the receptor. Presently, all of the crystal structures of A2AR are of a truncated nique for Analysis of Characteristic Infrared Bands of Peptide Linkage D variant of the receptor, A2A 316R, that lacks 96 amino acids from the C-termi- using Dried Proteins nus to facilitate crystallization. Removal of the flexible C-terminus is useful in Jasmeet Kaur, Shalmoli Bhattacharyya. facilitating crystallization; however, our lab has previously shown that the full- Dept Biophysics, Postgraduate Inst Med Education and Research (PGIMER), length C-terminus is critical in activation of downstream signaling cascades. Chandigarh, India.

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Protein activity is well dependent on its conformational structure and thus sec- 294-Pos ondary structure prediction and determination of proteins are vital require- Molecular Models of Human Elastin and Elastin Biomaterials ments. FTIR is rapidly coming up for protein structural studies, as an Anna Tarakanova. alternative to circular dichroism. However, FTIR spectra are primarily recorded Mechanical Engineering, University of Connecticut, Storrs, CT, USA. using protein samples prepared in water, organic solvents, deuterium oxide, and The elastin polymer, assembled from its molecular precursor tropoelastin, is the membranes. FTIR spectra are thus dominated by the solvent spectrum and sub- dominant component of elastic fibers, which confer elasticity and structural traction of the solvent spectrum introduces severe spectral distortions. In integrity to skin, lung, connective and vascular tissue. Historically, elastin’s aqueous protein solutions, a strong water signal overlaps amide I bands dynamic nature has precluded traditional approaches such as X-ray crystallog- (C=O stretching; 1700-1600 cm1), thus masking most of the protein second- raphy to understand its detailed features. Here, I describe recent work using ary structure information. Further, most of the purified proteins including atomistic and coarse-grained models for predicting elastin’s molecular struc- protein drug formulations are more stable in the solid state than in the liquid ture, mechanical properties and mechanisms, as well as dynamics. We use state. Hence there is a need to explore the use of FTIR for various physical the models to probe the function of key molecular regions, investigate disease states of proteins. In the present study, we showed that protein secondary etiology and explore implications for hierarchical assembly. From the materials structure can be analyzed in a dried state; using potassium bromide (KBr) pellet perspective, elastin-based materials display tunable thermal sensitivity, pre- method for dried known proteins (bovine serum albumin, proteinase K, senting opportunities to mimic and control these responsive features for fibrinogen etc). KBr pellet method gave excellent spectra for all the used dried biomedical applications. We characterize the temperature response spectrum proteins; shown by the appearance of characteristic infrared bands of peptide of elastin-like peptides to design synthetic polymers with tunable switching, linkage (Amide A, B, I-VI) without much interference from water, buffers, sta- resolving effects of peptide chemistry, chain length, and solvent environment. bilizing factors, co-factors etc. We also found that KBr pellet method can We also study a chimera silk-elastin-like protein polymer that combines silk’s analyze a range of protein types; globular to fibrous. Thus KBr method is a strength with elastin’s extensibility and responsive features to identify great tool for prediction/determination of the secondary structure of unknown temperature transition effects on molecular-scale mechanics. We analyze the proteins and quality control of solid-state protein drug formulations. associated free-energy landscape with the Bell-Evans model to interpret temperature-induced phase transitions. Such a feedback loop between simula- 292-Pos tion and experiment for predictive biomaterial design may enable new applica- Toward Accurate Prediction and Design of Kinked Alpha Helices in tions in drug delivery and tissue engineering. Membrane Proteins Brittany Lasher, Rebecca F. Alford, Jeffrey J. Gray. 295-Pos Chemical and Biomolecular Engineering, Johns Hopkins University, HPV VLPs as Scaffolds for Vaccine Design Baltimore, MD, USA. Thomas Jordan1, Carolyn Barcellona2, Danielle Basore3, Charlie Clark1, Protein structure prediction and design can provide insight into the structural Zeyuan Guo1, Sharon Isern2, Kripa Nand1, Gabi Rabasa1, basis of protein function. For membrane proteins, this task is especially difficult Terrence Shoemaker1, Giffin Werner1, Kai Xia4, Xinmeng Yuan1, because they reside in a heterogeneous lipid bilayer. Over the past decade, there Robert J. Linhardt4, Scott Michael5, Christopher Bystroff1. have been many advances in developing computational tools toward sub- 1Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY, USA, Angstrom prediction accuracy. One remaining challenge is capturing a-helical 2Biological Sciences, Florida Gulf Coast University, Fort Myers, FL, USA, kinks: distortions that occur in 60% of transmembrane helices and are key to 3Mercy College, Dobbs Ferry, NY, USA, 4Chemistry and Chemical Biology, flexibility and facilitating conformational change. Predicting kinked helices Rensselaer Polytechnic Institute, Troy, NY, USA, 5Florida Gulf Coast is difficult because their location and qualities are determined by a fine balance University, Fort Myers, FL, USA. of intermolecular interactions with neighboring helices and the surrounding Vaccines train the immune system to recognize antigens, preventing disease by lipid bilayer. In this work, we improve sampling and scoring of kinked helices enabling a timely adaptive immune response. Vaccine efficacy and safety may using a fragment-based approach. We implement a protocol that inserts kinked depend on directing the antibodies to specific epitopes, especially if the antigenic protein fragments from the Protein Databank and then scores the conformation target protein is immunologically ‘‘self’’, such as a cancer cell or a sperm cell. using a knowledge-based term that computes the probability of kinking in a Short peptides can focus antibodies to the desired epitopes, but such small anti- particular helix. We then evaluate this protocol on crystal structures of the gens are unstable unless incorporated into a larger protein. We selected human kinked and non-kinked conformation of two membrane-embedded channels papillomavirus (HPV) L1 protein as a carrier for a novel peptide subunit vaccine, and a membrane-embedded enzyme. We anticipate this method will improve constructed by inserting the coding sequences of the desired epitopes into sites of the accuracy of membrane protein structure prediction and enable de novo natural variation in the L1 proteins of the many HPV strains. The native confor- design of membrane proteins that contain flexible hinges and thereby perform mation of these epitopes, based on molecular models, is preserved by disulfide a broader range of functions. linking its endpoints, an act which may also help to preserve the folding pathway of L1. Successful raising of antibodies that bind the target is seen as an indicator of 293-Pos successful native-state antigenic loop modeling. Successful self-assembly into 55 A Thermodynamically-Rigorous, Biologically-Driven Energy Function for nm virus like particles (VLPs) is an indicator that the inserted loops do not inter- Membrane Protein Modeling and Design fere with folding and assembly of the L1 protein. Previous work has shown that 1 2 2 1 Rebecca F. Alford , Patrick Fleming , Karen G. Fleming , Jeffrey J. Gray . VLPs are especially immunogenic scaffolds, and that the polyvalency possible in 1Chemical & Biomolecular Engineering, Johns Hopkins University, 2 VLP display of peptide antigens sometimes improves the immune response. This Baltimore, MD, USA, Dept Biophys, Johns Hopkins Univ, Baltimore, MD, work focuses on the design and testing of VLPs displaying peptides from a sperm USA. specific calcium channel, CatSper, to produce an anti-sperm vaccine for contra- Protein design is a powerful tool for elucidating the structural mechanisms ceptive use. We will discuss the folding and assembly of the designed chimeric underlying protein function. For membrane proteins, this is particularly chal- capsid proteins. lenging because they are embedded within the lipid bilayer and membrane energy functions are based on organic solvent models rather than native 296-Pos bilayer properties. These energy functions capture neither the anisotropic Redefining the Protein Kinase Conformational Space with Machine structure nor the nanoscale dimensions of the membrane. Specifically, the po- Learning larity gradient of a bilayer is not well modeled by organic solvents. In this Peter Man-Un Ung, Rayees Rahman, Avner Schlessinger. work, we address this challenge by developing computational tools that Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New incorporate these two concepts. The first development is to incorporate solva- York, NY, USA. tion energies measured using a native protein in a native lipid bilayer. The Protein kinases are dynamic, adopting different conformational states that are second development is a geometric model that captures the dimensional critical for their catalytic activity. We assess a range of structural features derived quantities of a bilayer. The preliminary results demonstrate a two- to three- from the conserved aC helix and DFG motif to define the conformational space of fold increase in accuracy as measured by standard computational benchmarks the catalytic domain of protein kinases. We then construct Kinformation,a against experimental targets including protein-protein interface prediction, random forest classifier, to annotate the conformation of 3,708 kinase structures ddG calculations, native structure discrimination against decoys, and de in the PDB. Our classification scheme captures known active and inactive kinase novo protein design. In the future, we will implement this model into multiple conformations and defines an additional conformational state, thereby refining computational algorithms to enable tackling of more complex biological the current understanding of the kinase conformational space. Furthermore, questions such as investigating protein function and protein-drug interac- network analysis of the small molecules recognized by each conformation cap- tions, and de novo design of membrane proteins for therapeutic and nanotech- tures chemical substructures that are associated with each conformation type. nology applications. Our description of the kinase conformational space is expected to improve

BPJ 9303_9305 Sunday, March 3, 2019 59a modeling of protein kinase structures, as well as guide the development of tions to estimate their relative stability and compare them with the experimental conformation-specific kinase inhibitors with optimal pharmacological profiles. data. Recently, we executed MD to validate the stability of docking configura- tions [3] and substrate binding structures [4]. Here, high temperature MD 297-Pos simulations at 400 K and 500 K were executed with simulations at 300 K as Elongated Denatured Conformation of Apoazurin: Crowding and Urea control. The fraction of native atomic contacts, Q, measured for the 400 K sim- Interplay ulations showed a fairly good correlation with the Tm-values. Interestingly, 1,2 2 3 Dirar M. Homouz , Fabio Zegarra , Michael Kovermann , when the residues were classified by their hydrophobicity and size, the 2 2 4 Andrei G. Gasic , Lucas Babel , Pernilla E. Wittung-Stafshede , Q-values of hydrophilic residues exhibited an even better correlation. 2,5 Margaret S. Cheung . Measuring the Q-value on a per-residue level enabled us to identify residues 1Dept Applied Math Sci, Khalifa Univ, Abu Dhabi, United Arab Emirates, 2 3 that contribute significantly to the instability and demonstrate how our analysis Physics, University of Houston, Houston, TX, USA, Chemistry, University can be used in a mutant case-study. [1] H. Nishigami et al. Protein Eng. Des. of Konstanz, Konstanz, Germany, 4Dept Biol & Biol Eng, Chalmers Univ, 5 Sel. 29, 477-484 (2016). [2] N. Shimba et al. J. Chem. Inf. Model. 56, 2005- Gothenburg, Sweden, Center for Theoretical Biological Physics, Rice 2012 (2016). [3] G.-J. Bekker et al. J. Chem. Theory Comput. 13, 2389-2399 University, Houston, TX, USA. (2017). [4] N. Numoto et al. Biochemistry 57, 5289-5300 (2018). We have shown by a nuclear magnetic resonance measurement that the urea- denatured protein apoazurin becomes elongated when the synthetic crowder, 300-Pos dextran 20, is present, which opposes a theoretical prediction based only on A Multi-Objective Stochastic Optimization Approach for Decoy Genera- the consideration of volume exclusion. To explore complex interactions tion in Template-Free Protein Structure Prediction beyond volume exclusion, we have employed coarse-grained molecular dy- Ahmed Bin Zaman, Amarda Shehu. namics simulations to explain the elongated conformation of protein apoazurin Computer Science, George Mason University, Fairfax, VA, USA. resulting from the interplay of an effective attraction between a protein and Though protein structure determines to a great extent its biological function in crowders and the shape of a crowder. With a volume-conserving crowder, the cell, the disparity between the number of protein-encoding gene sequences we show that the shape of a crowder provides an anisotropic direction of the available in genomic databases and the number of biologically-active protein depletion force, in which a bundle of surrounding rod-like crowders stabilizes structures deposited by wet laboratories is more than an order of magnitude. the elongated conformation of apoazurin in the presence of effective attraction. For this reason, computational research in in-silico protein structure determina- tion is very active. In particular, great progress is being made in template-free 298-Pos protein structure prediction, where the goal is to determine the native tertiary Extension Protein Engineering Improves Protein Stability and Binding structure from a given amino-acid sequence that lacks sequence homologs Matthew J. Dominguez1, Zoey L. Sharp2, Valeria Jaramillo Martinez1, with known native structures. One of the key challenges in template-free Benjamin J. Lantz3, Elliott J. Stollar4. protein structure prediction is to generate a good-quality decoy dataset that cap- 1 2 Texas Tech University HSC, Lubbock, TX, USA, Eastern New Mexico tures the underlying energy landscape available to a given protein sequence 3 University, Portales, NM, USA, University of New Mexico HSC, and, more importantly, contains enough native and near-native decoys. This 4 Albuquerque, NM, USA, University of Liverpool, Liverpool, United Kingdom. task is typically pursued under the umbrella of stochastic optimization, as pro- It is desirable to modify proteins to have higher protein stability and stronger tein energy landscapes are high-dimensional and multimodal. In this work, we interactions with their binding partners and protein termini still remain an present a stochastic optimization algorithm that pursues several optimization unexplored region for optimization. We hypothesized that certain termini ex- objectives in order to effectively apportion computational resources between tensions will form desirable intra-molecular interactions, while maintaining exploration, seeing more of the landscape, and exploitation, drilling deeper the protein’s original structure/function. As such, we have developed a method down in the landscape. We carry out a principled evaluation on a benchmark called extension protein engineering (EPE) which screens libraries of variants dataset of twenty target sequences, pitching this multi-objective optimization containing artificial extensions to their natural sequence for improved stability algorithm against the Rosetta decoy generation algorithm and several other and binding. EPE was applied to a hybrid protein that connects the yeast SH3 state-of-the-art algorithms. The comparison shows that the proposed algorithm domain found in Abp1 (AbpSH3) to its target peptide from Ark1 (ArkA15) has high exploration capability and generates better decoy datasets, warranting using a flexible linker. In the hybrid protein, we first mutated the last peptide further research on multi-objective optimization for decoy generation in residue, K(-8), at the C-terminus of the hybrid, to 15 other amino acids (and template-free protein structure prediction. a stop codon) and found little improvement in stability. The ArkA15 peptide was then extended beyond this position for an additional 4 residues using a 301-Pos limited library. Each 4-residue extended variant in the library contains one of Experimental Characterization of ‘‘Metamorphic’’ Proteins Predicted 4 residues (Y, S, P, H) at each position generating a library size of 256. A num- from an Ensemble-Based Thermodynamic Description ber of extended peptides (enriched in tyrosine) were identified from the library James O. Wrabl1, Miranda Russo2, Jordan Hoffmann3, Keila Sheetz1, that increased hybrid stability through slower unfolding rates compared to wild- Andrew Munoz4, Vincent J. Hilser1,2. 1 2 type. EPE with a similar 256 variant library was also used at the N-terminus of Dept Biology, Johns Hopkins Univ, Baltimore, MD, USA, Dept 3 AbpSH3 alone to yield 4-residue extended variants also enriched in tyrosine Biophysics, Johns Hopkins Univ, Baltimore, MD, USA, Graduate School of 4 with increased domain stability; however, these were predominantly stabilized Arts & Sciences, Harvard Univ, Boston, MA, USA, Dept Biology, George through faster folding rates compared to wild type. Taken together, simple arti- Fox Univ, Newberg, OR, USA. ficial extensions can indeed be optimized to increase protein stability and pep- The emerging biological phenomenon of ‘‘metamorphic’’ proteins, single tide binding without affecting core structures/functions. EPE promises to be a amino acid sequences that adopt two physiologically distinct structures and simple method to improve the properties of many other proteins or peptides. functions, challenges current prediction methods largely reliant on sequence similarity. To address this problem, we develop an innovative metric for 299-Pos sequence-structure compatibility, using energetic information derived from Thermal Stability of Single-Domain-Antibodies Estimated by MD an experimentally validated ensemble description of protein thermodynamics. Simulations The simulated ensemble’s unique information, i.e. the locations of high and low Narutoshi Kamiya1, Benson Ma2, Gert-Jan Bekker3. stability, enthalpy, and entropy regions within a protein, is reduced to an eight- 1Grad Sch Simulation Studies, Univ Hyogo, Kobe, Japan, 2Sch Comput Sci symbol code that permits efficient scoring of any structure against any amino and Eng, Georgia Inst of Tech, Atlanta, GA, USA, 3Institute for Protein acid sequence. Ensemble-based information from both native and denatured Research, Osaka Univ, Suita, Japan. states is incorporated, with separate calibration of Gaussian probability distri- Antibodies bind to antigens with high specificity and affinity and have butions for background scores in each state. High-identity sequences, complementarity-determining region (CDR) loops, which play an important previously shown in vitro to adopt either Streptococcus protein GA or GB folds, role in antigen binding. We previously predicted the structure of the CDR- were correctly recapitulated, demonstrating that this ensemble-based com- H3 loop by using MD simulations [1] and used MD for the refinement and patibility metric indeed reflected the energetic determinants of fold. To further the ranking of decoys generated by rigid-body antibody-antigen docking [2]. test this model, arbitrarily chosen uncharacterized members of the high-identity Single-domain-antibodies, sdAbs, function like regular antibodies, however sequence space were expressed and purified; most were found to be consistent consist of only one domain. Because of their low molecular weight, sdAbs with their predicted folds as assessed by circular dichroism spectroscopy. have advantages with respect to production and delivery to their targets. For ap- Several additional designed proteins, each containing a single Glycine plications such as antibody drugs and biosensors, an sdAb with a high thermal mutation, appear to enable a fold switch between the GA and GB conforma- stability is required. In this work, we chose seven sdAbs, which have a wide tional ensembles. Stability measurements, nuclear magnetic resonance charac- range of Tm-values and have known structures. We then applied MD simula- terizations, and single-molecule studies are underway to confirm these

BPJ 9303_9305 60a Sunday, March 3, 2019 conclusions. Since this ensemble-based scoring framework is applicable to any access sequences which allow for incorporation of amino acids that are very desired fold, it may be practically useful for the future targeted design, or large- different from the natural ligand. To evolve aminoacyl-tRNA synthetases scale proteomic detection, of novel metamorphic proteins. more efficiently for new amino acids, we are designing a Monte Carlo-based protocol in the PyRosetta protein modeling software that samples large 302-Pos numbers of aminoacyl-tRNA sequences. The simulation involves assessing Biomolecular Simulations for Structural Biology: Integrating Co- binding interactions of the enzyme with the desired ncAA and the twenty ca- Evolution, Saxs and FRET nonical amino acids to select for enzymes that incorporate the desired amino Alexander Schug. acid efficiently and selectively. Additionally, enzyme kinetics are modeled € € € Julich Supercomputing Centre, Forschungszentrum Julich, Julich, Germany. and are being used to discriminate between effective and ineffective synthe- Exploring the interrelationship of structure and function is crucial for the under- tases. Currently, the Methanocaldococcus jannaschii tyrosyl tRNA synthetase standing of molecular life. Yet despite significant progress of experimental is being evolved for a new acridone-based amino acid. methods, full characterization of functional cycles for proteins and RNA re- mains an ongoing challenge. Biomolecular simulations offer a complementary option to experiments. One can use such simulations akin to an atomically 305-Pos resolved microscope to gain insight into the dynamical motion of biomolecules. Investigating the Structure of Laccasses for Biofuels It is also possible to include additional information as constraints into such Shahla H. Partowmah1, Robert Collins2, Alexei Soares3. 1 2 simulations. The increasingly ubiquitous availability of sequential information Premed, SUNY College at Old Westbury, Westbury, NY, USA, Physical 3 and novel statistical analysis has allowed to trace the co-evolution of residues Sciences, Quinnipiac University, Hamden, CT, USA, Photon Sciences, [1], which can be exploited in structure prediction tools and is, e.g., sufficient Brookhaven National Laboratory, Upton, NY, USA. for the blind prediction of proteins and RNA[2]. Similarly, one can use The purpose of this paper is to discover the structure of various bacterial and low-resolution experimental information such as SAXS data to model suitable archaea laccases for alteration and use in biofuel technology. This project is biomolecular conformations [submitted] or compare data from simulations currently pending results which are being reached through protein crystallog- directly against experimental measurements such as smFRET data [3]. raphy and x-ray diffraction analysis. The whole of this experiment aims to solve References the structure of a specific bacterial laccase using protein crystallography and [1] Weigt M et al., Proc Nat Acad Sci USA (2009) 106, 67-72; Schug A et al., x-ray diffraction to understand laccase function and characteristics. The Proc Nat Acad Sci USA (2009); F. Morcos et al., Proc Nat Acad Sci (2011) 108, EchoÔ (labcyte) is used to eject nano-liter sized samples from crystallization E1293-E1301 kits and protein samples using sound waves travelling through water on to a [2] Dago A et al., Proc Nat Acad Sci USA (2012), 109: E1733-42; E. De plate which is routinely analyzed for crystals. Currently, the best crystals Leonardis et al., Nucleic acids research (2015); M. Figliuzzi et al., MBE have been diffracted at approximately 1.8 A˚ ; and the data is being analyzed (2016); G. Uguzzoni et al., Proc Nat Acad Sci USA (2017) by expert crystallographers and beamline scientist to evaluate the structure of [3] I . Reinartz et al., J Chem Phys (2018) the molecule. Laccase proteins, copper-containing enzymes which catalyze oxidation reactions, greatly benefit bioremediation as their only byproduct is 303-Pos water. After results, research will advance into the manipulation of the laccase Computational Investigation of the Dissociation Pathways of Peptides to function in different environments. Mary C. Sherman, Luke Metzler, Michael J. Van Stipdonk. Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA, USA. 306-Pos Our ability to determine the sequence of amino acids is essential to our under- Expression, Purification, and Crystallization of the Human Oxidoreduc- standing of the underlying chemical processes dictating the nature of proteins. tase, Pyrox-D1: A New Described Cause of Early-Onset Myopathy in De-novo peptide sequencing carried out with collision-induced dissociation Humans (CID) and tandem mass spectrometry (MS/MS), with subsequent database Isaac L. Scott, Roger Sutton. matching is able to accurately identify proteome and peptidome sequences. Molecular Biophysics, Texas Tech University HSC, Lubbock, TX, USA. One aspect of our principal objective is to use molecular dynamics simulations Mutations in the oxidoreductase, Pyrox-D1, have been linked to a newly to gain a better understanding of the peptide fragmentation mechanisms occur- described, early-onset recessive myopathy from five families with four ring in the dissociation process, in particular, the behavior of the mobility and different recessive variants. This new myopathy presents with a histopathology migration patterns of the transferring proton. that is distinctive in that it combines multiple pathological hallmarks character- istic of different myopathies; central and minicore disease, centronuclear, 304-Pos myofibrillar and nemaline myopathy. Patients present in infancy with slowly In Silico Evolution of Aminoacyl-tRNA Synthetases for Incorporation of progressive proximal and distal weakness, facial weakness, nasal speech, swal- Noncanonical Amino Acids lowing difficulties, and mild to moderately elevated serum Creatine Kinase Tiberiu S. Mihaila, E. James Petersson. (CK) levels. The link between the mutations in the Pyrox-D1 gene and the Department of Chemistry, University of Pennsylvania, Philadelphia, PA, disease phenotype are not yet understood. This project involves expression, USA. purification, and crystallization of Pyrox-D1 for eventual characterization by Noncanonical amino acids (ncAAs) have unique side chains that can function X-ray diffraction. Following crystallization, preliminary characterization will as useful chemical probes of protein structure, dynamics, and function. These involve spectroscopic and enzymatic analysis to determine the co-factors molecules may be site-specifically incorporated into endogenously expressed used by this enzyme, and eventual structure determination by X-ray crystallog- proteins in live cells through amber stop codon suppression. The technique raphy. Understanding the co-factors and biophysical characteristic of this relies on the cellular introduction of a suppressor-tRNA with an anti-amber protein could help in developing new treatments in the future for this form anticodon, and an associated aminoacyl-tRNA synthetase, which will couple of myopathy. To date, we have purified and crystallized Pyrox-d1. Going the unique suppressor-tRNA with the ncAA. Critically, the introduced machin- forward, the goal is to determine the molecular structure of Pyrox-D1, which ery must be orthogonal to the natural translation machinery of the cell, meaning will help elucidate the protein’s function. The structure and function can be that the new synthetase and tRNA must not interact with naturally-expressed utilized to develop pharmaceutical treatments for patients presenting with synthetases, tRNAs, and amino acids. Several species of methanogenic archaea this idiopathic dystrophy. We have shown that Pyrox-D1 functions at least express tRNA/synthetase pairs that are orthogonal to bacterial and/or mamma- in part by facilitating the transition between oxidation states of NADP and lian translation machinery. One of the main bottlenecks on the incorporation of does so with the co factor FAD. A potential homologous structure has been new ncAAs in cells has been engineering effective and specific orthogonal created for Pyrox-D1. Two mutations have been identified in idiopathic aminoacyl-tRNA synthetases. Traditionally, this has been accomplished muscular dystrophy patients, which we believe will be found in Pyrox-D1 through directed evolution of orthogonal methanogen synthetases. Many of once we have determined the molecular structure. The protein has been the ncAAs that have been incorporated successfully resemble the natural sub- successfully crystalized and is currently being refined to undergo X-Ray strate amino acids of the enzymes, suggesting that there may be large barriers to diffraction.

BPJ 9303_9305 Sunday, March 3, 2019 61a

Posters: Protein Assemblies I along with flow cytometry to determine the concentration of the complex in cells. 307-Pos A Regulatory Metabolic Complex for Glucose Metabolism in Living Cells: 310-Pos the Glucosome Kinetic Trapping and Robustness in Proteasome Assembly Songon An. Anupama Kante, Eric J. Deeds. Chemistry and Biochemistry, University of Maryland, Baltimore County Molecular Biosciences, University of Kansas, Lawrence, KS, USA. (UMBC), Baltimore, MD, USA. Many macromolecular machines are involved in cellular functions like protein Sequential metabolic enzymes in glucose metabolism have long been hypoth- homeostasis, cellular transport, cell fate determination, etc. These machines are esized to form multienzyme complexes that regulate glucose flux in living synthesized as a set of subunits that must be assembled in order for the complex cells. However, it has been challenging to directly observe these complexes to be functional. Kinetic trapping, where large intermediates are formed that and their functional roles in living systems. In this work, we have used cannot proceed to the fully-assembled structure, can drastically reduce assem- various fluorescence microscopic techniques to investigate the spatial organi- bly yields. It is thus likely that macromolecular machines have evolved assem- zation of metabolic enzymes that participates in glucose metabolism in hu- bly pathways that avoid kinetic trapping. In this study we have chosen the 20S man cells. We provide compelling evidence that human liver-type proteasome Core Particle (CP) from the actinomycete Rhodococcus erythrop- phosphofructokinase 1 (PFKL) forms various sizes of cytoplasmic clusters olis as our model system to characterize kinetic trapping, since it’s subunits can in human cancer cells, independent of protein expression levels and of the be purified and they readily self-assemble into functional CPs in vitro. The pro- choice of fluorescent tags, which is now also supported by others. We further teasome CP is made up of four stacked rings of either seven a or seven b sub- a b b a report that these PFKL clusters colocalize with other rate-limiting enzymes in units; these rings are arranged in an 7 7 7 7 order. Using mathematical both glycolysis and gluconeogenesis. Subsequent biophysical characteriza- models and experimental data, we have shown that CP assembly in Rhodococ- tions strongly support the formation of a multienzyme metabolic complex, cus likely involves a trade-off between assembly speed and robustness to ki- the glucosome, in living cells. Importantly, quantitative high-content imaging netic trapping. These findings not only provide insight into the evolutionary assays indicate that the direction of glucose flux between glycolysis, the pressures on macromolecular assemblies but also can be used to develop novel pentose phosphate pathway and serine biosynthesis is governed by the forma- drugs that inhibit CP assembly (particularly for the treatment of Mycobacte- tion of the multienzyme complexes in a cluster size-dependent manner. Their rium tuberculosis infections). Our results are also important for the engineering size-dependent functional contribution to cells is also validated by mathemat- of protein nano machines that self-assemble quickly and efficiently. ical modeling approaches. Collectively, our results reveal a functionally rele- vant, multienzyme metabolic complex for glucose metabolism in living 311-Pos human cells. Improving the Reconstruction of Low-Resolution CryoEM Map using Enhanced Molecular Dynamics Simulations 308-Pos Cesar A. Lopez1, Mark Swift2, Xiao-Ping Xu3, Dorit Hanein4, Niels Volkmann4, S. Gnanakaran1. Self-Assembly of GAG in Ethanol/Water Mixtures Examined by Molecu- 1 2 lar Dynamics Dept Bio/Biophys, Los Alamos Natl Lab, Los Alamos, NM, USA, Dept Shuting Zhang1, Cuong Trinh1, Reinhard Schweitzer-Stenner2, Bio/Biophys, Sanford Burnham Prebys Medical Discovery Inst, La Jolla, CA, 3 Brigita Urbanc1. USA, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, 4 1Dept Phys, Drexel Univ, Philadelphia, PA, USA, 2Dept Chemistry, Drexel USA, Bioinformatics and Structural Biology Program, Sanford Burnham Univ, Philadelphia, PA, USA. Prebys Medical Discovery, La Jolla, CA, USA. Short peptides have emerged as a new class of fibril- and hydrogel-forming B-cell lymphoma-2 associated X protein (BAX) plays a pivotal role in trig- materials. Although lacking aromatic amino acids, glycylalanylglycine gering cell apoptosis by permeabilizing the mitochondrial outer membrane. (GAG) was recently discovered to form a hydrogel in ethanol/water mixtures Contrary to previous findings, recent electron-microscopy (EM) experiments with at least 55mol% ethanol in acidic pH at concentrations surpassing showed that BAX monomers are able to perturb phospholipid nanodiscs 200 mM. Here, we examine GAG self-assembly using a multiscale molecular (NDs) by forming lipidic pores. In this study, we considered the dynamics dynamics (MD) simulations, which combine: (a) a four-bead (4B) peptide and thermodynamics of DPPC NDs with and without an embedded full- model in implicit solvent (HYDRA) studied by efficient discrete MD (the length BAX monomer using long timescale coarse-grained (CG) and all- DMD4B-HYDRA approach) and (b) explicit-solvent all-atom MD using atom (AA) molecular dynamics (MD) simulations, and directly compared the the OPLS-AA force field with OPLS ethanol and TIP4P/2005 water models. computational results with cryo-EM reconstructions of the same construct. By calibrating the implicit solvent ‘‘hydropathy’’ parameter in the DMD4B- Our results suggest that entropic and other thermodynamic effects due to the HYDRA approach to ethanol/water solvent conditions, monomers are confinement of lipids by the protein belt play a significant role in the physical observed to self-assemble into a long ordered GAG fibril with a three-fold and structural properties of bare and BAX-embedded NDs. symmetry and a twist. This ordered GAG fibril is used as an initial confor- mation for all-atom MD simulations in several ethanol/water mixtures, 312-Pos including pure water and pure ethanol. Preliminary results show that the or- The Mechanism of Action for Drugs that Undermine HIV-1 Viral Capsid dered GAG fibril remains stable in pure ethanol at peptide concentrations sur- Formation and Activity: Insights from Large-Scale Coarse-Grained passing 500 mM. For comparison, we also examine GAG self-assembly from Simulations monomers using all-atom MD in explicit solvent to show that GAG self- Alexander J. Pak, John M.A. Grime, Gregory A. Voth. assembles only for ethanol fractions surpassing 80% whereby the resulting Chemistry, University of Chicago, Chicago, IL, USA. GAG assembly is not ordered. Our preliminary findings are qualitatively Complex enveloped viruses have an intriguingly robust ability to assemble, consistent with experimental observations and provide a quantitative assess- package, and release viral materials from host cells as virions to spread infec- ment of the MD force field for studies of short unfolded peptides in ethanol/ tion. Human immunodeficiency virus type-1 (HIV-1) is one paradigmatic water mixtures. example, and a plethora of small molecule drugs have been developed to disrupt the many stages of the viral lifecycle. The current paradigm involves 309-Pos competitive inhibition of viral enzymes and receptors. However, drug- Quantifying a Protein-Protein Interaction in Living Cells resistance due to genetic polymorphism remains a key challenge. Instead, an Shannon L. Speer, Alex J. Guseman, Gary J. Pielak. alternative strategy is to target the main structural polyprotein known as Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, Gag, which coordinates several steps of viral morphogenesis and conserves NC, USA. over 70% of its residues amongst subtypes. A class of drugs known as capsid Two-thirds of disease associated mutations disrupt protein complexes, yet inhibitors, e.g., PF-74 from Pfizer and GS-CA1 from Gilead Sciences, have there is a lack of quantitative thermodynamic information about their stability recently emerged with this aim in mind. Nonetheless, their molecular mecha- in cells, where the concentration of macromolecules can exceed 300 g/L. We nisms of action have remained elusive. For example, different studies involving are quantifying the dissociation of a protein-protein interaction in Escherichia PF-74 suggest many potential mechanisms, which include the drug destabiliz- coli cells using a variant of the B1 domain of protein G that forms a domain- ing viral cores during infection, abrogating nuclear transport, over-stabilizing swapped homodimer labeled at its sole tryptophan with fluorine. We are us- cores to prevent reverse transcription, and preventing core assembly. In this ing 19F NMR to quantify the amounts of monomer and dimer, Tuner(DE3)tm talk, we will describe the use of large-scale coarse-grained (CG) molecular dy- cells and inducer concentration to modulate protein expression, and LC-MS namics simulations to investigate the putative mechanisms of capsid inhibitors,

BPJ 9306_9309 62a Sunday, March 3, 2019 in which we explore the complete assembly of capsid proteins during viral Although published structural models of virus capsids generally exhibit perfect maturation and their disassembly during uncoating. Our simulations broadly symmetry, defects might be expected for several reasons including the finite- suggest that capsid inhibitors accelerate capsid assembly, thereby undermining temperature, fluctuating environment in which capsids assemble and, in some this necessarily-controlled process. As a result, several mechanisms of action cases, the requirement for disassembly during the infection cycle. Different are expressed, including malformed core formation and spontaneous core lines of evidence also suggest the presence of defects: the observation of defec- uncoating. Taken together, our results provide fundamental biophysical under- tive structures in computer simulations, and the presence of imperfect capsids standing into supramolecular protein assembly and elucidate a broad-spectrum in single-particle cryo-EM studies. To begin to quantify the conditions under biomedical strategy to combat viral infection. which defects might be expected, we develop a simple equilibrium theory for capsids based on a lattice model allowing for both ideal and defective sites. 313-Pos Both analytical and numerical calculations with the model show that a signif- E. coli High Throughput Assay Identifies Regulators of Endothelial icant population of defects appears below a fairly sharp threshold value of a Barriers key parameter: the difference in effective pair-binding affinities between Dario Mizrachi. ideally oriented and mixed ideal/defective pairs of interacting sites. The gen- Physiology Developmental Biol, Brigham Young Univ, Provo, UT, USA. erality of the model enables us to estimate threshold values for a wide range Regulation of endothelial barrier function is critical for vascular homeostasis, of virus capsids, providing potential guidance for future experimental studies. as dynamic and local control of vascular permeability permits macromolecular Beyond furthering our understanding of potential structural heterogeneity in transport, immune surveillance, and deposition of a fibrin barrier to contain virus capsids, the findings suggest a novel strategy for assembly inhibition infection at sites of inflammation. Many of the signaling pathways promoting by enhancing ‘off-lattice’ interactions among capsid proteins via adaptor useful vascular permeability, however, are also triggered during disease, result- molecules. ing in prolonged or uncontrolled vascular leak. Hyper-permeability triggered by inflammation or ischemia in the heart, brain, or lung promotes edema, exac- 316-Pos erbates disease progression, and impairs recovery. Currently there is no clinical Structures and Functions of the HIV-1 Pre-Integration Complexes solution for the regulation of the endothelial barriers. A main reason for the lack Julien Batisse1, Eduardo Bruch1, Nicolas Levy1, Patrice Gouet2, of translational solutions is the technical difficulties (e.g. low throughput, and Stephane Emiliani3, Vincent Parissi4, Marc Ruff1. lengthy assays) to explore libraries of small compounds to begin drug develop- 1Dept Struct Biol/Genomics, IGBMC CERBM, Illkirch, France, 2MMSB- ment. Regulating endothelial barriers is crucial in the prevention of disease, IBCP, UMR 5086 CNRS Universite Lyon 1, Lyon, France, 3Institut Cochin, disease progression, and the discovery and implementation of new therapies. Paris, France, 4UMR5234 CNRS-Bordeaux University, Bordeaux, France. Claudins, and occludin, are Tight Junction membrane proteins (TJ-MP) that After retroviral infection of a target cell, during the early phase of replication, seal the paracellular space creating the endothelial barriers. the HIV-1 genomic RNA is reverse transcribed by the viral RT to generate the Our laboratory has devised a strategy to express TJ-MPs in the outer membrane double-stranded DNA that interact with viral and cellular proteins to form the of E. coli. TJ-MP expression drives cell-cell interactions above the unicellular pre-integration complex (PIC). Viral integrase (IN) is the key component of behavior of E. coli. TJ-MPs force this aggregation in a manner that correlates the PIC and is involved in several steps of replication notably in reverse tran- with their strength in the native endothelial barriers. The aggregation of E. coli scription, nuclear import, chromatin targeting and integration. Viral compo- can be quantified using flow cytometry. Our preliminary results suggest that our nents such as IN cannot perform these functions on their own and need to assay has sensitivity to detect hyper-permeability (barrier opening) as well as recruit host cell proteins to efficiently carry out the different processes. IN is hypo-permeability (barrier closing or strengthening). a flexible protein, property allowing its interaction with multiple partners and enabling its multiple functions. To study the molecular mechanisms of 314-Pos viral integration we use a bottom - up strategy by assembling in vitro and/or Tetrameric Assembly of the Oncogenic C-Terminal Binding Proteins in cellulo multiprotein complexes around the integrase protein (core protein William E. Royer1, Andrew G. Bellesis1, Anne M. Jecrois1, of the PIC) and DNA. This strategy enabled us to solve cryo-EM structures Brendan J. Hilbert1, Martin M. Dcona2, Steven R. Grossman2, of the IN/LEDGF and IN/LEDGF/INI1 complexes at low resolution [1, 2]. Celia A. Schiffer1. With the recent progress of the cryo-EM techniques and our improvement in 1Dept Biochem/Molec Pharm, Univ Massachusetts Med, Worcester, MA, the complex preparations [3] new cryo-EM datasets on the IN/LEDGF/DNA USA, 2Massey Cancer Center, Virginia Commonwealth University, and IN/LEDGF/nucleosome complexes are collected which will enable us to Richmond, VA, USA. increase the structure quality to near atomic resolution for the IN/LEDGF/ C-terminal binding proteins (CtBP1 & 2) are co-transcriptional factors that DNA complex. Moreover, the efficient production of the proteins in mamma- have been implicated in progression of a number of cancers. CtBP possesses lian cells allowed us to identify PTMs on HIV integrase. Their effect on viral a D-isomer specific 2-hydroxyacid dehydrogenase (D2-HDH) domain that replication and 3’ processing are analyzed. The diverse structures and func- binds NAD(H) and provides an attractive target for small molecule interven- tions of HIV-1 integrase induced by protein and DNA interactions as well as tion. Our crystal structures of CtBP1 and CtBP2 with bound substrate led to by PTMs will be presented and discussed. [1] Michel et al. (2009) EMBO the design of an inhibitor (HIPP) with 300nm binding affinity. Experiments J., 28, 980-991. [2] Maillot et al. (2013) PLoS ONE 8(4): e60734. [3] Levy using HIPP in APCmin (colon cancer) mouse models suggest that CtBP inhib- et al. (2016) Nature comm. 7 : 10932. itors could be efficacious agents in a number of cancers. NAD(H) linked olig- omeric assembly of CtBP has been suggested to contribute to co-transcriptional 317-Pos signaling, although the biologically relevant level of CtBP assembly has been Probing and Differentiating the Shell and Enzyme Proteins of the Bacterial unclear. Using multi-angle light scattering (MALS), we find that CtBP assem- Microcompartment by Thermal Shift Assay bles from dimers into tetramers as a function of NAD(H) concentration with Naimat Kalim Bari, Gaurav Kumar, Simerpreet Kaur, Sharmistha Sinha. Biological Science, Institute of Nano Science and Technology, Mohali, India. EC50 values in the range of 50-150 nM, depending on the construct used for CtBP1 and CtBP2. Although crystal structures of CtBP1 and CtBP2 have Bacterial microcompartments are prokaryotic organelles, distributed across been described in terms of dimers, due to extensive intradimeric interactions, bacterial species and are involved in several metabolic pathways. These are examination of the distinct crystal lattices for CtBP1 and CtBP2 reveal a proteinaceous in nature and have a distinct shell-core structure. The core is very similar tetrameric assembly in both paralogs. Site-directed mutations of made up of enzymes which are encapsulated by an outer coat made up of shell residues involved in the observed lattice contacts support the lattice tetramer proteins. The shell proteins self-assemble to form an extended sheet which as the solution tetramer. We propose that this tetramer is the co- folds to form a closed shell. The shell protein units have vivid concave and transcriptionally active form of CtBP and are investigating inhibitors that convex surfaces. These two surfaces differ in the distribution of hydrophobic disrupt this tetrameric assembly as potential lead compounds for a therapeuti- and hydrophilic residues. In this work, we employ a thermal shift assay to cally useful CtBP specific inhibitor. distinguish between the shell proteins and the internal enzyme clusters of bac- terial microcompartment. Thermal shift assay is a well-known technique for 315-Pos studying protein unfolding using a fluorophore. We observe a few interesting Should Virus Capsids Assemble Perfectly? A Simple Equilibrium Model differences between the signature fluorescence spectra of enzymes and shell for Defects proteins as probed by fluorescent dyes. Unlike the encapsulated globular en- Justin M. Spiriti1, James F. Conway2, Daniel M. Zuckerman3. zymes, these shell proteins show high initial fluorescence. The enzyme clusters 1Dept of Biomedical Eng, Oregon Hlth Sci Univ, Portland, OR, USA, being globular in nature give their characteristic transition peaks at their 2Structural Biology, University of Pittsburgh, Pittsburgh, PA, USA, 3Oregon melting temperatures, interestingly not observed in case of shell proteins. Hlth Sci Univ, Portland, OR, USA. This difference is because of the variation in the distribution of hydrophobic

BPJ 9306_9309 Sunday, March 3, 2019 63a and hydrophilic residues on the surfaces of globular and the shell proteins. In The autoprotease mutant, EDDIE, was used to express and subsequently isolate the case of globular proteins, the hydrophobic residues are buried and get a 13-residue sequence. This sequence is referred to as the KD peptide, a natu- exposed only at the transition temperature resulting in high fluorescence. On rally derived hydrogel forming sequence from the human semen protein, seme- the other hand, the shell proteins have their hydrophobic residues exposed on nogelin I. A plasmid was obtained with the KD peptide attached to the either of the two surfaces resulting in high initial fluorescence. The shell pro- N-terminus of EDDIE. This plasmid was transformed into E.coli cells, which teins do not have a buried hydrophobic core like globular protein so no signa- underwent overnight expression to grow and clone the EDDIE-KD complex. ture transition curve. We propose a thermal shift assay as a method to study the The complex was isolated in inclusion bodies and purified via sonication and morphology of different proteins. anion exchange chromatography on a Fast Protein Liquid Chromatography (FPLC) system. A series of reaction conditions were performed to optimize 318-Pos the cleavage between EDDIE and the KD peptide of the purified complex. Modeling Proteasome Assembly Pathways in Bacteria Purification and reaction rates were analyzed by Sodium Dodecyl Sulfate Poly- Pushpa Itagi1, Eric J. Deeds2. acrylamide Gel Electrophoresis (SDS-PAGE) and High-Performance Liquid 1Dept Compu Biol, Univ Kansas, Lawrence, KS, USA, 2Dept Mol Biosci, Chromatography (HPLC). A His-tagged EDDIE-KD plasmid was also trans- Univ Kansas, Lawrence, KS, USA. formed and isolated. Preliminary work on cleaving this complex via a Nickel Cells employ various systems to remove unwanted or damaged proteins. The resin column was begun. proteasome is one such system, which is a large macromolecular machine that functions in degrading proteins and regulating cellular processes. It con- 321-Pos sists of a 20S Core Particle (CP) capped by a Regulatory Particle (RP). The Unveiling the Impact of the Negative Arm of the Circadian Clock on CP is comprises of four stacked rings each made up of seven monomers (a Output in Neurospora crassa and b) stacked in a barrel-shaped architecture. Modulating proteasome func- Alexander E. Mosier, Jennifer M. Hurley. tion for therapeutic purposes has gained significant interest during the past Biological Sciences, Rensselaer Polytechnic Institue, Troy, NY, USA. decade to combat diseases like cancer and drug resistant tuberculosis. Recent Circadian rhythms are cellular oscillations that tune biology to Earth’s 24-hour findings suggest that Mycobacterium tuberculosis(Mtb) is an attractive target light/dark cycle, aiding in the anticipation of environmental changes. These for proteasome inhibitors. The CP of Rhodococcus erythroplisshares about rhythms are maintained by an evolutionarily conserved transcriptional/transla- 64% sequence identity to Mtband its assembly has been well-characterized tional negative feedback loop, referred to as the core circadian clock. In this experimentally. In all organisms, CP assembly follows a hierarchical pathway core clock, positive arm proteins act to transcriptionally activate negative where two Half Proteasomes (HP: a7b7) are formed first and then dimerize to arm proteins that act back on the positive arm to halt the negative arm’s acti- form CP (a7b7b7a7). It is hypothesized that HP formation follows different vation before degradation and re-initiation, thus setting the period of the clock. pathways in archaea, bacteria and eukaryotes, but we currently lack definitive Most research into circadian regulation on output has focused on transcriptional evidence for these hypotheses. In this work, we employed mathematical activation by the positive arm proteins. However, recently published work has models to study the CP assembly pathway in R. erythropolis. Specifically, demonstrated that there are many oscillating proteins for which the correspond- we found that more hierarchical assembly pathways provide the system ing mRNA is not rhythmic. This has led to the theory that the post-translational with greater robustness against kinetic trapping at high concentration. The regulation of circadian output exists, which we hypothesize is imparted by the robustness of these pathways comes at the cost of significantly slower assem- negative arm proteins. bly kinetics. Comparison of our simulations with experimental data suggests In the model organism Neurospora crassa, the negative arm includes that the CP in R. erythropolisemploys an assembly pathway that is much less FREQUENCY (FRQ). FRQ has been computationally and experimentally hierarchical than has been previously proposed. Further experimental and proven to be an intrinsically disordered protein (IDP). Aside from lacking theoretical investigation of CP assembly will be crucial to efforts aimed at a predetermined three-dimensional structure, IDPs play key roles in cellular inhibiting or modulating proteasome function in bacteria and other signaling and regulation due, in part, to the transient but highly specific and organisms. varied interacting partners they have. These attributes make FRQ a prime candidate to impart circadian regulation post-translationally. The aim of 319-Pos this study is to characterize protein complexes, centered around FRQ, that Effects of Chromogranin A and B Association on the Anion Channel change in a time specific manor to determine the impact these interactions Function in the Regulated Secretory Pathway could impart on circadian regulatory pathways. To this end, Neurospora Sutonuka Bhar, Gaya P. Yadav, Mahesh S. Chandak, Qiu-Xing Jiang. samples where crosslinked and purified through co-immunoprecipitation Microbiology and Cell Science, University of Florida, Gainesville, FL, USA. before protein identification through liquid chromatography mass spectrom- Chromogranins A and B (CHGA and CHGB), of the granin superfamily, are etry. Initial results suggest that FRQ plays a role in regulation on meta- pivotal for the biogenesis, maturation and release of secretory granules in the bolism, the cell cycle, and a variety of other functions through novel regulated secretory pathway. They have been used as biomarkers for neuroen- protein interactions. docrine tumors, cardiovascular diseases, and are genetically associated with Type 2 diabetes and psychiatric disorders. Although their roles in regulated 322-Pos secretion are conserved within the kingdom of eukaryotes, the molecular mech- Resolving the Transition States of Human Hemoglobin Assembly through anisms underlying their functions inside the cells are unclear, which makes it a Combination of Spectroscopic Studies and All-Atom Molecular Dy- difficult to target them for specific diseases. Chromogranin B was demonstrated namics Simulations to function as an anion channel in membrane. It is proposed to serve the long- Premila P. Samuel Mohan Dass1, George N. Phillips2, John S. Olson2, sought anion conductance that is needed to shunt positive potential developed David A. Case1. by Hþ-ATPase-drive proton pumping into dense-core secretory granules and 1Proteomics, Rutgers University, Piscataway, NJ, USA, 2Department of maintain a more stable acidic intra-vesicular environment at pH 5.5. Biosciences, Rice Univ, Houston, TX, USA. CHGA, on the other hand, was found to be in both a membrane-bound state The assembly of hetero-tetrameric HbA is a multi-faceted pathway emphasized and a soluble state. The membrane-bound CHGA alone does not conduct by both quaternary structure formations via the interacting alpha and beta sub- ions. Because CHGA and CHGB are usually present together in the secretory units of HbA as well as the oxygen coordinating heme (Fe(II)-protoporphyrin granules, we are studying the interactions between CHGA and CHGB in mem- IX) insertion within the heme cavity of each subunit. Folding transition states at brane, and investigate the roles of the CHGA/CHGB complex in granule matu- different stages of this pathway could act as precursors for misassembly of ration in neuroendocrine cells. Further, we are developing high-through screen HbA, leading to hemoglobinopathies and disruption of oxygen transport within assays to search for specific binders to the CHGA/CHGB complex and resolve the cardiovascular system. Simultaneous circular dichroism and visible absor- the structural basis for its function using cryo-electron microscopy. bance measurements of guanidine hydrochloride induced unfolding of HbA showed that the initial formation of the alpha-beta dimer interface occurs via 320-Pos a molten globule heterodimer state with 30% helical content. Reversible he- Using NProAutoprotease Fusion Technology to Express a Semenogelin I min (Fe(III)-protoporphyrin IX) binding to the melted heme pockets in this Peptide transition state results in a six coordinate, low spin iron state known as a hemi- Fiona Berry1, Birgitta Frohm2, Sara Linse3, Karin Akerfeldt4. chrome, which stabilizes the hetero-dimer interface. Atomic level modeling of 1Haverford College, Haverford, PA, USA, 2Lund University, Lund, Sweden, hemin disassociation from native HbA using the Amber 2018 molecular 3Dept Biophys Chem, Lund Univ, Lund, Sweden, 4Dept Chemistry, dynamics (MD) package showed that these hemichrome transition states Haverford Col, Haverford, PA, USA. can also occur in both the folded alpha and beta subunits at 37 C via

BPJ 9306_9309 64a Sunday, March 3, 2019 hexacoordination of the iron by internal histidines within the heme cavity. Alpha-synuclein (a-syn) is an intrinsically disordered protein, the purported These atomic level simulations enable us to characterize the transitionary na- physiological function of which is to mediate synaptic vesicle trafficking. Ab- ture of bond breaking and formation with metal cofactors that is beyond the res- normalities in a-syn’s interactions with lipid vesicles and membranes lead to olution limits of solution spectroscopy and X-ray crystallography. Modeling the formation of non-functional, misfolded fibrils, which are implicated in Par- studies involving thermodynamics integration are also currently being under- kinson’s disease. A variety of post-translational modifications (PTMs) – cova- taken to measure free energy change for HbA tetramer interface formation, lent modifications made to proteins through enzymatic and non-enzymatic which markedly enhances heme affinity of HbA. processes – regulate protein activity, structure, and cellular localization. Many PTMs have been identified and studied in a-syn, the most common of 323-Pos which is phosphorylation. A variety of other modifications such as nitration, ar- Interactions between Thrombomodulin and the Complement System Stud- ginylation, and N-terminal acetylation also affect the structure and behavior of ied by Surface Plasmon Resonance and Deuterium Exchange Mass Spec- a-syn and its interactions with other biomolecules. To illustrate - the phosphor- trometry ylation of a tyrosine on a-syn has been found to alter a-syn’s helical conforma- Jose Giler, Mary Catherine Rice, Vanessa Wiltsie, Kyler Anderson, tion state when bound to lipid vesicles, while arginylation of glutamate residues Julia R. Koeppe. in a-syn is thought to protect against fibril formation. We combine protein Dept Chemistry, State Univ New York Oswego, Oswego, NY, USA. semi-synthesis and single molecule fluorescence spectroscopy techniques to Complement component 3 (C3) is at the junction of three different complement determine the effect of various PTMs on the ability of a-syn to mediate vesicle activation pathways (classical, lectin, and alternative). Activation to C3b is a fusion. Using peptide synthesis, recombinant production of proteins with unnat- key step in the innate immune response that allows for the formation of impor- ural amino acids, and native chemical ligation, we produce modified, fluores- tant multi-protein complexes that ultimately participate in pathogen clearance. cently labeled a-syn. We then determine changes in its binding affinity for When misregulated, however, complement can lead to inflammatory disease lipid vesicles through Fluorescence Correlation Spectroscopy and validate and autoimmune disorders. Several regulatory proteins for C3b are known, the structural changes induced by the PTMs via single molecule FRET in the but the molecular details of interactions between these proteins have not yet presence of lipid vesicles. We examine the effects of these structural and inter- been elucidated. Thrombomodulin (TM), and specifically its N-terminal actional changes on protein function by assaying the fusion of fluorescently lectin-like domain (TMD1), has been identified as a possible regulator of com- labeled vesicles mediated by modified a-syn. Our study provides insight into plement through interactions with C3 or C3b, and the known regulator CFH the impact of PTMs on protein structure, function, and potential contribution may also be required. We have used surface plasmon resonance (SPR) and to disease. hydrogen/deuterium exchange mass spectrometry (HDXMS) to study the inter- action of TMD1 with C3 and C3b. Using SPR, we see that C3 binds to a surface 326-Pos coated with TMD1, and full kinetic studies are underway. Using HDXMS, we Mechanistic Study of Prenylated Flavin Mononucleotide Formation see that TMD1 interacts with C3b, and there is a lesser interaction with C3. Szymon Zaczek, Agnieszka Dybala-Defratyka. TMD1 tends to make C3b more accessible to deuterium exchange, while C3 Faculty of Chemistry, Institute of Applied Radiation Chemistry, Lodz tends to be less accessible to deuterium exchange in the presence of TMD1. University of Technology, Lodz, Poland. This difference suggests a possible role for TMD1 in regulating a key step Prenylated flavin mononucleotide (prFMN) is a recently discovered cofactor, along the complement pathway. We will next investigate the role of CFH in which catalyzes the first known enzymatic 1,3-dipolar cycloaddition reaction. these interactions. The formation of prFMN is facilitated by UbiX, which acts as a prenyltransfer- 324-Pos ase, catalyzing an addition of a fourth six-membered ring to flavin mononucle- SGEF GEF Activity and its Regulation by Scribble and Dlg1 otide (FMN). Even though the mechanism of prFMN biosynthesis by UbiX Ashley Simpson. was proposed (based solely on the obtained crystallographic structures), to Bay Path University, Longmeadow, MA, USA. the best of our knowledge, no further insight into the process was reported We discovered a cell polarity complex comprised of SGEF, a guanine nucleo- till today. tide exchange factor (GEF) for the RhoG GTPase, and the scaffolding proteins Herein, we report our findings regarding the initial steps of prFMN biosyn- Scribble and Dlg1. SGEF binds the Scribble PDZ1 domain and the guanylate thesis by UbiX. In particular, we investigate the following steps of the pro- kinase (GUK) domain of Dlg1. SGEF-catalyzed nucleotide exchange of cess, which were suggested to occur in a concerted fashion: proton transfer RhoG is critical for cell junction polarity. Complications with cell polarity from S15 to E49, proton transfer from FMN to S15, bond cleavage between are associated with metastasis in cancer. However, the mechanism(s) for O from the phosphate group and C from the dimethylallyl moiety in dime- SGEF regulation and the role of Scribble and Dlg1 in this regulation remain un- thylallyl monophosphate (DMAP), bond formation between N from FMN known. Previous work by Ellerbroek et. al. (2004) suggests that the N-terminus and C from the dimethylallyl moiety in DMAP, protonation of the phosphate of SGEF is auto-inhibitory to its GEF activity. We hypothesize that the N-ter- group in DMAP. For these purposes, we employ computational chemistry minus of SGEF blocks the GEF catalytic site from RhoG and that SGEF bind- tools, mainly free energy calculations performed with Umbrella Sampling ing to Scribble and/or Dlg1 relieves this auto-inhibition. Here, we begin to test and Adaptive String Method using the QM/MM scheme. We determine the this model using in-vitro GEF exchange assays with SGEF constructs in the nature of the considered chemical steps (if they are all concerted, or happen presence and absence of the Scribble PDZ1 domain and Dlg1 GUK. The partially in a step-wise fashion) and the associated energy barriers. Further- SGEF catalytic domain (DH-PH domain) was expressed, the FL-SGEFD5, more, it was suggested that DMAP should abstract hydrogen from E140, the Scribble PDZ1 and Dlg1 GUK domains. Next, we conducted GEF- which is precluded on the basis of pKa calculations performed with propKa exchange assays that measure the change in fluorescence upon the incorpora- software, which predicted this residue to be in its anionic form. According to tion of MANT-GDP into RhoG. These assays showed that purified RhoG our initial MD study and propKa’s predictions, the positioning and proton- was active and that the SGEF DH-PH region increased the rate of nucleotide ation states of K129 and Y169 make them feasible candidates for being exchange. As a control, we used SGEF DH-PH in the presence of Scribble hydrogen donors towards the prFMN formation. Therefore, we also investi- PDZ1 and Dlg1 GUK. Unexpectedly, the rate of RhoG nucleotide exchange gate which residue is required for the DMAP protonation prior to the prFMN increased further. A preliminary assay of the FL-SGEFD5 showed a marked formation. decrease in the rate of nucleotide exchange compared to the DH-PH construct, consistent with auto-inhibition. Future experiments will focus on examining the 327-Pos nucleotide exchange rate of FL-SGEFD5 in the presence of Scribble PDZ1 and/ Study of the Lysine Deprotonation Mechanism in Ubiquitin Conjugating or Dlg1 GUK. These studies will help understand the role of SGEF/Scribble/ Enzyme Ubc13 Dlg1-polarity-complex in regulating SGEF GEF activity. Katherine Elliott1, N. Cole Seward1, Heath Hampton2, Isaiah Sumner1. 1Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, VA, USA, 2Department of Biology, Galludet University, Posters: Enzyme Function, Cofactors, and Post- Washington D.C., VA, USA. translational Modifications Ubiquitin (Ub) is a regulatory protein with the ability to flag proteins to be degraded by the proteosome. Ub is covalently attached to a lysine on the target 325-Pos protein by a series of reactions catalyzed by three types of enzymes: ubiquitin Investigating the Effect of Alpha-Synuclein Post-Translational Modifica- activating enzymes, E1; ubiquitin conjugating enzymes, E2; and ubiquitin li- tions on Synaptic Vesicle Trafficking gases, E3. Before Ub is transferred to its target, it is bonded to the E2 via a thio- Buyan Pan, E. James Petersson, Elizabeth Rhoades. ester linkage. In this study, we examine the E2 enzyme, Ubc13, which catalyzes University of Pennsylvania, Philadelphia, PA, USA. the formation of K63-linked polyubiquitin chains. The chains are formed when

BPJ 9306_9309 Sunday, March 3, 2019 65a a lysine on the target Ub (K63) attacks the thioester bond between Ubc13 and other serine/threonine phosphorylation sites, phosphorylation sites with the substrate Ub. To initiate this reaction, K63 on the target Ub must be depro- (S/T)-P motif could be distinguished by distinct sequence profiles and biophys- tonated, turning it into an active nucleophile. There are two possible deproto- ical properties. One of the notable features of (S/T)-P motif is its strong nation sites: a conserved aspartate in Ubc13 (D119) and a conserved association with intrinsically disordered regions (IDR): not only most phos- glutamate in the target ubiquitin (E64). In order to determine any preference phorylation sites with (S/T)-P motif are found in long unfolded/non-globular between D119 and E64, we used classical molecular dynamics, Born- regions, the frequency of (S/T)-P motif is positively correlated with fraction Oppenheimer molecular dynamics and single point QM/MM to model K64 de- of IDR within proteome. On the other hand, relatively small number of corre- protonation in several E2Ub conjugates. sponding kinases and higher sequence diversity imply that phosphorylation of (S/T)-P motif is rather promiscuous. Phenotype studies also show that missense 328-Pos mutation of (S/T)-P motif has lower frequency of being pathogenic than muta- Molecular Dynamics Simulations Reveal the Origins of the Distinct Activ- tion of other phosphorylation sites.Based on all these findings, we hypothesized ities and Substrate Selectivities of E. coli Dehydratases FabA and FabZ that phosphorylation of (S/T)-P motif plays an undiscovered fundamental role Greg J. Dodge1, Kara L. Jaremko2, Ashay Patel2, J. Andrew McCammon2,3, in the cellular environment via a simple biophysical mechanism, specifically Janet L. Smith1, Michael D. Burkart2. IDR aggregation avoidance. Phosphorylation of (S/T)-P motif promotes the 1Department of Biological Chemistry and Life Sciences Institute, University formation of alpha-helix and polyproline II (PII) conformations locally, thereby of Michigan, Ann Arbor, MI, USA, 2Department of Chemistry and preventing accumulation of beta-strands and lower the possibility of adopting Biochemistry, University of California, San Diego, La Jolla, CA, USA, amyloid-like structures. This hypothesis may provide explanation to two ques- 3Department of Pharmacology, University of California, San Diego, La Jolla, tions: how this motif is associated with IDR, and why this motif is enriched in CA, USA. the eukaryotic proteome. Unsaturated fatty acids are vital constituents of cell membranes. Their rela- tive abundance modulates membrane fluidity, providing organisms with 331-Pos mechanisms by which to maintain cell membrane homeostasis in response Investigating the Catalytic Base Residues in the Phosphoglucose Isomerase to changes in the external environment. In E. coli, unsaturation is introduced From Thermotoga maritima into fatty acids during - not after - de novo fatty acid biosynthesis by FabA, a Katherine Lake, Nicole Swope, Linda Columbus. dehydratase that not only catalyzes the dehydration of a key substrate in the University of Virginia, Charlottesville, VA, USA. pathway but also isomerizes the resulting product to yield a cis-unsaturated Phosphoglucose isomerases (PGIs) are a class of enzymes that catalyze the intermediate. A related dehydratase, FabZ, unlike FabA, has no isomerase ac- reversible isomerization of fructose-6-phosphate (F6P) and glucose-6- tivity, but complements FabA in fatty acid biosynthesis by processing a phosphate (G6P). PGIs are crucial in glycolysis and gluconeogenesis path- unique set of substrates. Though this presentation will emphasize the use ways and have been proposed as ‘‘moonlighting’’ proteins in different organ- of molecular dynamics (MD) simulations to delineate the origins of the isms. In humans, an inactive or inefficient PGI leads to nonspherocytic distinct activities and substrate selectivities of these dehydratases, we hope hemolytic anemia, and PGI elevation is a known marker for tumor metastasis, it will also illustrate the value of using chemical, structural, and computa- which highlights the importance of understanding the function of PGIs. The tional biology in concert to resolve the mechanisms by which biomolecules proposed function of Thermotoga maritimaPGI (TmPGI), based on structural function. similarity, was confirmed by kinetic and colorimetric assays in addition to 1H nuclear magnetic resonance (NMR) spectroscopy. Evidence of solvent ex- 329-Pos change in the 1H NMR spectra supports that TmPGI isomerization proceeds Can Photolysis of the Co-C Bond in Coenzyme B12-Dependent Enzymes be through the ene-diol intermediate mechanism, rather than the alternative direct Used to Mimic the Native Reaction ? hydride shift. The ene-diol mechanism consists of three basic steps: ring open- Abdullah Al Mamun, Pawel M. Kozlowski. ing, isomerization, and ring closure. A glutamate (E281) and a histidine Chemistry, University of Louisville, Louisville, KY, USA. (H310) residue in the active site each have been proposed as the catalytic Coenzyme B12 (Adenosylcobalamin = AdoCbl)-dependent enzymes catalyze base that forms the ene-diol intermediate based on previous crystal structures complex molecular transformations where cleavage of the Co-C bond initiates of PGIs. To determine which of these residues is the PGI residue critical for the catalytic cycle. Alternatively, the Co-C bond can also be cleaved with base catalysis, alanine mutations, E281A and H310A, corresponding to the light. In both cases, rupture of the Co-C bond results in the formation of TmPGI residues in the active site. The E281A mutant activity and solvent ex- Co(II)/Ado radical pair (RP). There has been a suspicion that the photolytic change behavior were consistent with wild-type TmPGI at high enzyme con- cleavage of the Co-C bond can be used to mimic the RP formation that occurs centrations (mM), suggesting that the proposed catalytic glutamate is not in enzymes. Within the field of B12 chemistry, there has been a lack of critical for PGI function. In addition to the catalytic base activity, TmPGI consensus as to whether or not photolytic cleavage can be used as a probe H310 has been proposed as the residue performing the first ring opening or a direct comparison to the native reaction. Herein, we seek to resolve step; thus, the proposed active site histidine residue may be performing what the connection between light-induced RP formation and the native cat- both ring-opening and isomerization steps of PGIs that utilize the ene-diol alytic cycle is. We used a combined QM/MM approach to construct PESs for pathway. Preliminary results with H310A will be presented to investigate AdoCbl-dependent ethanolamine ammonia-lyase (EAL) as a function of axial this hypothesis. bonds to describe the reaction mechanism. We have found that there is no direct comparison that can be made between photolysis and enzymatic cleav- 332-Pos age. However, we offer an alternative explanation which involves considering A Kinetic Study of Ligand Binding and Conformational Changes in Induc- the reduced cofactor. The topology of the PES (D1) for the one electron ible Nitric Oxide Synthase reduced cofactor is very similar to the PES associated with photo-induced Karin Nienhaus, Michael Horn, G. Ulrich Nienhaus. cleavage (S1). Both surfaces contain two energy minima that are, similarly, Dept Appl Phys, Karlsruhe Inst Tech, Karlsruhe, Germany. the result of two distinct electronic states. Thus, it can be concluded that The mammalian inducible nitric oxide synthase (iNOS) catalyzes the produc- the reaction mechanism associated with the D1 surface and the S1 surface tion of nitric oxide (NO) in a two-step monooxygenation of L-arginine (L- are very similar, providing the connection between photolysis and the native Arg) to NO and L-citrulline via the intermediate Nu-hydroxy-L-arginine reaction. (NOHA). The reaction starts with the formation of a ternary complex consisting of the enzyme, an O2 ligand bound to the heme iron and the L-Arg substrate 330-Pos residing close to the heme-bound ligand. We have blocked the enzymatic turn- Phosphorylation Sites with S/T-P Motif: Possible Basal Anti-Aggregation over by replacing O2 by carbon monoxide (CO), because it resembles O2 in size Mechanism and heme binding properties, and then studied CO binding to the iNOS oxygen- Min Hyung Cho, James Wrabl, Vincent Hilser, James Taylor. ase domain, iNOSoxy, by using flash photolysis. The P420 and P450 forms of Department of Biology, Johns Hopkins University, Baltimore, MD, USA. the enzyme, assigned to a protonated and unprotonated proximal cysteine, Protein phosphorylation is the most common post-translational modification through which the heme is anchored to the protein, show markedly different (PTM) found in eukaryotic proteome. One of the common sequence motifs CO rebinding properties. Only very few CO ligands can escape from P420. found in phosphorylation sites is serine/threonine phosphorylation sites fol- They rebind very slowly, suggesting that P420 has a widely open distal pocket lowed by proline residue ((S/T)-P). While this motif is found in up to 1/3 of that admits water. CO rebinding to the P450 form strongly depends on the pres- u whole human phosphorylation sites, individual sites with this motif are less ence of the substrate L-Arg, the intermediate N -hydroxy-L-arginine and also well characterized, leaving most of its functions unknown.When compared to the cofactor tetrahydrobiopterin. After adding these small molecules to the

BPJ 9306_9309 66a Sunday, March 3, 2019 enzyme solution, the CO kinetics change slowly over hours. This process can NADPþ/H decreased. In the reduced form, the absorption band centered at be described as a relaxation from a fast rebinding, metastable P450 species to a around 450 nm appeared in all RpFNRs, and the wavelength of the absorption slowly rebinding, thermodynamically stable P450 species, which we associate maxima shifted toward shorter wavelength in the mutants. Comparison with with the enzymatically active form. Our results allow us to determine kinetic other homo-dimer type FNRs suggested that the presence of this band relate parameters of L-Arg binding to ferrous deoxy iNOS protein, the L-Arg disso- to the rapid CT complex formation. We will discuss about the relation be- ciation rate coefficient,= 15.4 5 0.4 s1, the bimolecular L-Arg association tween the reversibility and the conformation change of the isoalloxazine rate coefficient, = 1153 5 71 mM1s1, and by taking the ratio of the two ring portion of FAD prosthetic group with the data of other homodimer- values, the equilibrium dissociation coefficient of L-Arg and ferrous iNOSoxy, type FNRs. KD = 13.4 5 0.9 mM. 335-Pos 333-Pos Substrate Specificity of an Isopentenyl Phosphate Kinase Essential Determinants of the Substrate Specificity of PTEN Shanteri Singh, Erin M. Scull. Kirstin Hobiger1, Michael G. Leitner2, Dominik Oliver1, University of Oklahoma, Norman, OK, USA. Christian R. Halaszovich1. Isopentenyl diphosphate and dimethylallyl diphosphate are the fundamental 1Dept Physiology, Philipps Univ Marburg, Marburg, Germany, 2Dept. of biosynthetic precursors for >80,000 diverse isoprenoids including NPs, Physiology and Medical Physics, Medical University of Innsbruck, cholesterol, coenzyme Q, chlorophyll, and vitamin A, of all domain of Innsbruck, Austria. life. These precursors are biosynthesized via methylerythritol phosphate PTEN antagonizes the PI3-kinase/Akt pathway through its D3-site phosphatase (MEP) pathway in bacteria and mevalonate (MVA) pathway in eukaryotes, activity toward PI(3,4)P2 and PI(3,4,5)P3, thereby acting as a tumor suppressor. archaea, some eubacteria. Recently, an alternate MVA pathway has been The structural determinants of its specificity remain elusive. Interestingly, identified in archaea and plants, that involves phosphorylation of isopentenyl PTEN shows remarkable homology with voltage-sensitive phosphatases monophosphate by isopentenyl phosphate kinase (IPK) to yield isopentenyl (VSPs) that dephosphorylate D5- and/or D3-sites of PI(4,5)P2, PI(3,4)P2, and diphosphate. In this poster we will demonstrate IPK to have broad substrate ˚ PI(3,4,5)P3. Since the catalytic centers of PTEN and VSPs differ markedly specificity and present a 2.1A crystal structure of IPK from Candidatus only in TI/gating loop and active site motifs, we wondered whether these dif- methomethylophilus alvus, that identifies molecular factors responsible for ferences could explain the differing substrate specificities. broad substrate specificity among IPK. In addition, we will demonstrate To address this question, we introduced mutations into PTEN, mimicking the variant IPK generated based on structure-guided engineering to have corresponding VSP sequences, and studied the resulting phosphatase activity new activity. in living cells utilizing engineered, voltage-switchable PTENCiV, a Ci-VSP/ PTEN-chimera. Substrate specificities of these mutants were analyzed using 336-Pos total internal reflection fluorescence microscopy (TIRF-M) in conjunction A Microfluidics-Based Assay for Mapping Connectivity in Highly Profi- with genetically encoded fluorescent phosphoinositide sensors. In PTENCiV, cient Enzymes Reveals Functional Modularity mutating TI167/168 in the TI loop into the corresponding ET-pair of VSPs Craig J. Markin, Daniel A. Mokhtari, Fanny Sunden, Daniel Herschlag, induced VSP-like D5-phosphatase activity toward PI(3,4,5)P3, but not toward Polly M. Fordyce. PI(4,5)P2. Combining TI/ET-mutations with an A126G-exchange in the Biochemistry, Stanford University, Palo Alto, CA, USA. active site removed major sequence variations between PTEN and VSPs Enzymes are some of the most proficient and most specific catalysts and resulted in D5-activity toward PI(4,5)P2 and PI(3,4,5)P3 of PTENCiV, known; however, a complete understanding of how these enormous rate en- fully reproducing the substrate specificity of native VSPs. Experiments per- hancements are achieved is lacking. Enzymes are large and highly cooper- formed with non-chimeric PTEN mutants in living cells as well as quantifi- ative, containing functional connections throughout their entire structures. cation of phosphoinositide levels by ELISA confirmed the PI(3,4,5)P3 D5 However, due to the cost and labor required to prepare and assay the num- activity of PTEN(A1267G, TI167/168ET). In contrast to PTEN, reciprocal ber of mutants required to characterize this connectivity, it has been diffi- mutations in VSPs did not alter their substrate specificity, but reduced phos- cult to obtain the large sets of data necessary for improving rational phatase activity. enzyme design; as a result, the proficiencies of designed enzymes fall In summary, A126 in the active site and TI167/168 in the TI loop are essential well short of their natural counterparts. To address these experimental lim- determinants of PTEN’s substrate specificity, whereas additional features itations, we have developed a microfluidic assay, HT-MEK (High- might contribute to the enzymatic specificity of VSPs. Throughput Microfluidic Enzyme Kinetics), in which we express, purify, This work was supported by the Grants of the University Medical Center and quantitatively characterize >1000 enzyme mutants in a single experi- Giessen and Marburg to M.G.L. (UKGM 13/2016MR) and to C.R.H. ment across multiple kinetic parameters. This platform greatly reduces (UKGM 32/2011MR) and of Deutsche Forschungsgemeinschaft to D.O. both time and cost compared to traditional assays, while providing data (GRK 2213). of comparable precision. Using this technology, we performed systematic mutagenesis over the entire scaffold of the highly proficient phosphatase 334-Pos PafA. We measured the activity of each mutant against multiple substrates Role of the Re-Face Tyr Residue Stacked on the FAD Prosthetic Group in and inhibitors, obtaining the same quantitative biochemical parameters as D Ferredoxin-Nadp Oxidoreductase from Rhodopseudomonas palustris obtained from traditional assays: kcat, KM, kcat/KM,andKI. Together, these during Catalysis with NADPD/H measurements define a quantitative functional ‘fingerprint’ for each muta- Daisuke Seo, Issei Hayashi. tion. Mutants having similar fingerprints distinctly cluster around the spe- Div Material Sci, Kanazawa Univ, Kanazawa, Japan. cific active site residues that display similar mutational fingerprints when Ferredoxin-NAD(P)þ oxidoreductase (FNR) is a soluble flavoprotein cata- themselves mutated. These comparisons reveal functional connections be- lyzing the redox reaction between NAD(P)þ/H and small iron sulfur protein tween individual active site residues and those throughout the structure, ferredoxin. Among homo-dimer type FNRs which has a significant structural and suggest a high degree of modularity within the enzyme. The ability homology with the bacterial NADPH-thioredoxin reductases, FNR from Rho- to rapidly characterize thousands of mutants with the quantitative precision dopseudomonas palustris (RpFNR) is distinctive for the presence of the Tyr of traditional assays will allow us to efficiently test models from computa- residue on the re-face of the isoalloxazine ring portion of FAD prosthetic tion and phylogeny, while providing the necessary data to improve future group. We report the reactivity of the wild type and Tyr mutants with design efforts. NADPþ/H evaluated by a stopped-flow spectrophotometry. Mixing oxidized wild type and mutated RpFNRs with NADPH provided a 337-Pos rapid charge transfer (CT) complex formation followed by the reduction of Structural and Dynamical Effects of Ubiquitination of Key Lysine Resi- FAD. Reduction of RpFNR occurred in part even in the presence of 50-fold dues in the Human Myeloperoxidase. Understanding the Signaling excess NADPH at equilibrium. Mixing reduced wild type and mutated Mechanisms Involved in Autoimmune Responses in Systemic Lupus þ RpFNRs with NADP also resulted in a rapid CT complexes formation fol- Erythematosus lowed by the oxidation of FAD. Kinetic analysis indicated that the rate- Daniel Carrillo-Va´zquez1, Diana Go´mez-Martı´n1, limiting step was the hydride-transfer process in both directions and the rates Eduardo Jardo´n-Valadez2. of both directions were comparable. Replacement of the Tyr residue did not 1Immunology and Rheumatology, Instituto Nacional de Ciencias Medicas y affect the rate constants of the hydride transfer step significantly, but the Nutricio´n Salvador Zubira´n, Ciudad de Mexico, Mexico, 2Earth Resources, observed rates responding to the following release and re-association of Universidad Auto´noma Metropolitana, Lerma, Mexico.

BPJ 9306_9309 Sunday, March 3, 2019 67a

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease research project was conducted to identify the function of the protein 3DS8 affecting organs and tissues, such as kidney, joints and skin. The etiology of in the Protein Data Bank and to further the understanding of the relationship lupus is unknown, and diverse abnormalities in cellular responses, such as between protein sequence, structure, and function. A putative enzyme func- oxidative stress and enhanced proinflammatory responses to self-antigens, tion has been identified through computational techniques using PyMol and have been associated with the development of symptoms. Abnormal innate im- ProMol (which searches a library of enzymatic motifs for local structural mune responses, such as enhanced NETosis play a key role in the pathogenesis homologs), BLAST and Pfam (servers that identify global sequence homo- of SLE. Neutrophil extracellular traps (NETs) are molecular complexes that are logs), and Dali (a server that identifies structural homologs). From these composed by extracellular DNA fibers combined with nuclear proteins that are techniques, this protein, 3DS8, was found to be from the gene Lin2722 of externalized by neutrophils upon diverse stimuli, such as microbes and cyto- the Listeria innocua organism and to have a potential alpha/beta hydrolase kines. One of the most abundant protein in these NETs is myeloperoxidase function. The protein is being expressed in vitro, and its enzymatic function (MPO), an enzyme that synthesizes hypochlorite ions as antibacterial agent. is being detected using p-nitrophenyl colorimetric assays. Cleavage of p-ni- Our group has recently demonstrated the presence of ubiquitin in the NET com- trophenyl substituted substrate analogues produces p-nitrophenol, which plex, more specifically bonded to the MPO enzyme. In order to elucidate the canbemeasuredusingUV-Visspectroscopy, thus confirming enzymatic role of ubiquitination of MPO, we explored the structure and conformational activity. dynamics of the MPO-UBQ dimers using molecular dynamics methods. We set up three MPO systems in solvent water molecules and 0.15 M NaCl, 340-Pos namely, I:MPO, II:MPOLys129-UBQ, III: MPOLys488-UBQ. Configurational Insight into Weak Interactions between Carrier Proteins and their Sub- space was sampled during 100 ns for each system, at 37oC and 1 bar strates by Chemo-Enzymatic and Improved NMR Methods 1 2 2 1 (isothermal-isobaric ensemble). The MPO structure and dynamics were sensi- Indrani Pal , Yousang Hwang , David Meyers , Dominique P. Frueh . 1Dept Biophy/Biophys Chem, Johns Hopkins Sch Med, Baltimore, MD, tive to the presence of UBQ in terms of its structural fluctuations and its prin- 2 cipal components (PCA), dynamic cross-correlations and network community USA, Pharmacology and Molecular Sciences, Johns Hopkins Sch Med, analysis, and the solvent accessible area of key residues and the active site. Baltimore, MD, USA. Computational results were compared to biological assays of samples taken Non-ribosomal peptides are a class of microbial secondary metabolites syn- from SLE patients, allowed us to evaluate the impact of pos-traslational mod- thesized by non-ribosomal peptide synthetases (NRPSs) often with pharma- ifications in the MPO enzyme. ceutical and medicinal properties. There is an emerging demand to engineer NRPSs to develop novel pharmaceuticals but gaps in understanding 338-Pos molecular mechanisms hamper progress. During synthesis, substrates are Investigating the Key Structural Elements that Confer Specificity to the attached to 20 A˚ phosphopantetheinyl moieties of carrier protein (CP) do- Acetyltransferases Enzyme Family mains via thioester bonds. CPs then visit partner domains in a series of Sara K. Lowe1, Kole J. Runyan2, Patricia Soto3, Yadilette Rivera-Colo´n4. sequential transient interactions. Substrates were seen to dock with CP pro- 1Science, Bay Path University, Longmeadow, MA, USA, 2Chemistry, tein cores in a manner that may affect domain interactions. Further, such Creighton University, Omaha, NE, USA, 3Physics, Creighton University, docking events are yet to be observed for the canonical NRPS substrates, Omaha, NE, USA, 4Bay Path University, Longmeadow, MA, USA. amino acids. However, substrates often rapidly fall off CPs due to hydrolysis Enzymatic-acetylation is a process that is mediated by acetyltransferases of the thioester bond, thus challenging structural studies. Here, we employ a where an acetyl group is transferred from acetyl-CoA to a target protein. chemo-enzymatic route to load a cysteine onto a carrier protein through an Protein acetylation is affects many biological processes such as homeostasis, inert amide bond. This approach overcomes disulfide bond formation, trans- metabolism, transcription factor-function, protein-protein interactions, pro- thioesterification and disulfide-thiol exchange inherent to the thiol functional tein stability, and enzyme activity. Defects in acetylation could result in dis- group of cysteines. Using NMR spectroscopy, we identified weak but well- eases that could be fatal and chronic like cancer and neurodegenerative defined chemical shift perturbations indicating a transient interaction between diseases. To understand defects in acetylation, it is important to understand the substrate and the protein core. To describe this interaction at the molec- the molecular mechanisms of acetylation by acetyltransferases. We have yet ular level, we developed a time-shared filtered NOESY NMR experiment to to determine the parts of the acetyltransferases structures that provide spec- measure distances between substrates and CP cores. Our results will help un- ificity. Understanding what confers specificity to this enzyme family will derstand whether and how different substrates interact with CPs as they are allow us to investigate their binding patterns and overall activity. To iden- presented to partner domains, thus guiding NRPS engineering to produce tify and test structural features in acetyltransferases that could be in charge novel metabolites. of specificity, three single point-mutations were done using Naa50p to change the active site to resemble the active site of a-TAT bysecondary- 341-Pos structure alignment. Since Naa50p is an N-terminal acetyltransferase, we Light-Induced Activation of Organo-Metallic Co-C Bond in MeCbl- want to test if we can change the specificity to that of a lysine acetyltrans- Dependent Methionine Synthase- QM/MM Study ferase (a-TAT). Naa50p mutants should show more activity towards a-TAT Arghya P. Ghosh, Abdullah Al Mamun, Pawel M. Kozlowski. substrates and less activity towards Naa50p substrates. The Naa50p mutants Chemistry, University of Louisville, Louisville, KY, USA. were generated by mutagenesis PCR on human Naa50pDNA and will be Methylcobalamin (MeCbl)-dependent enzyme methionine synthase (MetH), used to express the proteins in bacteria. The mutants will be purified by af- plays a critical role in the catalysis of methyl group transfer from methyltetra- finity chromatography using a GST-tag. The Naa50p mutants will be tested hydrofolate to homocysteine. It often performs a side reaction to generate cob with fluorescence-assays for change in activity. Using structural bioinfor- (II)alamin through photolysis of organometallic Co-C bond. A hybrid QM/MM matics, we will identify the effect of point mutations on the structural method (DFT/MM and TD-DFT/MM) has been performed to explore the dynamics and local network of residue interactions of the enzymes. The photochemistry of MeCbl-bound MetH. The manifold of low-lying excited goal study the effect of the mutations on Naa50p activity to further under- states and corresponding potential energy surface (PES) of electronically stand the biochemical and structural characteristics of acetyltransferases to excited S1 state has been constructed as a function of axial bond lengths to lead to the development of new therapies for acetyltransferase-related elucidate the mechanism of photoinduced activation of Co-C bond inside diseases. enzyme to investigate the photolytic properties of MeCbl-bound MetH. The analysis of S1 PES has revealed that the two different electronic states, namely 339-Pos metal-to-ligand charge transfer (MLCT) and the ligand field (LF), of S1 PES are Function Identification of the Protein Product of Gene Lin2722 from relevant to the photodissociation of Co-C bond. There are two possible path- Listeria innocua using Computational and In-Vitro Techniques ways identified, Path A and Path B, that connect the MLCT to LF state that Mary Sharkawy1, Andrea A. Carter2, Paul Craig3. represent possible photodissociation mechanisms. In the case of MetH, one 1Meredith College, Raleigh, NC, USA, 2Chemistry Physics and Geo, possible photodissociation pathway (Path B) was identified based on the ener- Meredith College, Raleigh, NC, USA, 3Chemistry, Rochester Institute of getics of MLCT and LF states.The energetically accessible Path B involves Technology, Rochester, NY, USA. initial the detachment of Co-NIm followed by a displacement of the Co-C Identifying the functions of proteins is an essential component of biochem- bond prior the formation of cob(II)alamin / CH3 radical pair (RP). The photo- istry, structural biology and bioinformatics, since proteins play an important chemical data of base-on MeCbl was compared with the computed result of role inside the body. Proteins are used to build and repair tissues, act as en- MeCbl-bound MetH to understand the effect of enzymatic environment on zymes and hormones, as well as performing other functions. In collaboration the photolytic properties of MeCbl. In compare to MeCbl in solution, the LF with Dr. Paul A. Craig’s lab at Rochester Institute of Technology (RIT), this state is energetically higher in energy inside the enzyme which makes it easier

BPJ 9306_9309 68a Sunday, March 3, 2019 to form RP for isolated cofactor in solution. Therefore the enzymatic environ- Designed proteins often differ from natural proteins in their extraordinary sta- ment has played a role in the destabilization of the LF state which impede the bility and their low level of dynamic behavior. Such dynamic motion is an un- photolysis of MeCbl-bound MetH. derappreciated but critical aspect of functional computational protein design. Here we report the rational redesign of DF2, an extremely rigid, computation- 342-Pos ally designed homodimeric diiron protein, into the functional enzyme Identifying the Function of Protein 2O14 using Bioinformatic and CDM13, a single-chain dimetal-containing four-helix bundle, by increasing Biochemical Techniques the dynamic motion of the protein via the reversal of the amino acid sequence Antonina Flair1, Andrea A. Carter2, Paul A. Craig3. of two of the helices, greatly reducing the packing complementarity in the pro- 1Chemistry, Meredith College, Raleigh, NC, USA, 2Chemistry Physics and tein core. The diiron form of CDM13 activates oxygen, forming m-oxo- Geo, Meredith College, Raleigh, NC, USA, 3Chemistry, Rochester Institute bridged diferric clusters at a rate 33 times greater than DF2, at a kcat of of Technology, Rochester, NY, USA. .117 per minute versus a kcat of .0035 per minute. This form also reacts Understanding the relationship between protein sequence, structure, and func- with 4-aminophenol, producing benzoquinone monoimine with a kcat of .02 tion has helped advance many scientific fields and proven integral to related per minute. The reduced form of dimanganese-containing CDM13 oxidizes health disciplines. Nonetheless, there is still much about protein folding and hydrogen peroxide by two electrons at a rate greater than 1000 s(-1), more its relationship to function that remains unknown. With the development of than 10000 times faster than the oxidized form reduces it to water. Thus, genome sequencing, large numbers of previously unidentified open reading CDM13 promises to serve as a unique alternate version of an oxygen- frames have been found. Many were then studied and characterized by the Pro- evolving cluster in the design of new photoautotrophic organisms which tein Structure Initiative; however, thousands of protein structures in the Protein perform carbon fixation utilizing light and low energy electrons originating Data Bank (PDB) still have an unknown function. The aim of this research from what is typically considered to be a toxic intercellular waste product. study is to identify the function of protein 2O14 in the PDB. A computational The ability of these enzymes to catalyze the oxidation of methane to methanol analysis of the protein is being performed using bioinformatics tools (BLAST, will also be discussed. The creation of such a broad range of catalytic activity Pfam, Dali, ProMOL, and Autodock) to assign a putative function. Expressed in a multifunctional active site like the dimetal site of DF2 merely by and purified protein is being analyzed with a hydrolase assay to further study increasing dynamic motion underscores the need to move beyond the para- and confirm the function. In this assay, p-nitrophenyl substituted substrate an- digm of structure in protein design to encompass a more realistic view that alogues are used to detect hydrolase activity; liberation of p-nitrophenol is encompasses protein and enzyme dynamics. detectable with UV-Vis spectrophotometry. Current results will be presented and discussed. Posters: Chromatin and the Nucleoid

343-Pos 345-Pos Structure-Based Prediction of Polypeptide Substrate Specificities of Glyco- The Removal of Histones from Ultrafine Anaphase Bridges Studied by syltransferases Optical Tweezers 1 2 2 Yashes Srinivasan , Sai Pooja Mahajan , Jason Labonte , Dian Spakman1, Andreas S. Biebricher1, Graeme A. King1, Kata Sarlo´s2, 3 2 Matthew P. DeLisa , Jeffrey J. Gray . 2 1 1 1 Ian D. Hickson , Erwin J. Peterman , Gijs J. Wuite . Chemical and Biomolecular Engineering, University of California Los 1 2 Department of Physics and Astronomy, Vrije Universiteit Amsterdam, Angeles, Los Angeles, CA, USA, Chemical and Biomolecular Engineering, 2 3 Amsterdam, Netherlands, Department of Cellular and Molecular Medicine, Johns Hopkins University, Baltimore, MD, USA, Chemical and University of Copenhagen, Copenhagen, Denmark. Biomolecular Engineering, Cornell University, Ithaca, NY, USA. During mitosis, duplicated DNA has to be faithfully segregated into two Glycans are some of the most abundant and diverse biopolymers, important in daughter cells. In anaphase, regions of entangled double-stranded DNA be- a number of biological processes including cell adhesion, receptor activation, tween segregating sister chromatids can give rise to DNA threads known as ul- molecular trafficking, endocytosis, and signal transduction. Protein glycosyla- trafine anaphase bridges (UFBs). Failure to resolve UFBs can result in genome tion is primarily mediated by enzymes known as oligosaccharyltransferases instabilities. It has been proposed that a key step in UFB resolution is the (OSTs) and glycosyltransferases (GTs). Polypeptide N-acetylgalactosaminyl- removal of histones, and that this is facilitated by the SNF2-family protein transferases (GalNAc-Ts) are higher eukaryotic retaining GTs that transfer a PICH. The mechanism of nucleosome remodeling by PICH has, however, re- GalNAc moiety from uridine diphosphate N-acetylgalactosamine (UDP-Gal- mained elusive. Here, we present a single-molecule assay that recapitulates NAc) onto Ser/Thr residues. This enzyme family initiates the most common chromatin re-organization during UFB resolution. In vitro chromatin constructs type of O-glycosylation and thus affects the structure, stability, and function consisting of 4 to 12 high affinity histone binding sites (601 sequences) within of many proteins. Structure-based prediction tools have the potential to an 8.5 kbp DNA molecule were prepared using a combination of restriction- advance our understanding of glycan-related phenomena as well as enable enzyme based cloning and Gibson assembly. After reconstitution with human the design of novel glycoconjugates. Here, we present new computational pro- histones, these constructs were manipulated within a multi-channel microflui- tocols that allow rapid computational screening and scoring of polypeptide dic flow cell using dual-trap optical tweezers. This approach allows us to mea- substrates of GTs such as GalNAcT2 and human-OGT. Our Rosetta-based al- sure tension-induced nucleosome unwrapping in the presence or absence of gorithm docks a fully flexible glycosylated polypeptide substrate to the cata- PICH. Our results suggest that, under moderate tension (10 pN), PICH facili- lytic site of the GT, exhaustively sampling polypeptide and glycan tates the unwrapping of nucleosomes. In further experiments, we aim to corre- conformational space, generating thousands of decoys for each docked poly- late nucleosome stability with the dynamics of single fluorescently-labelled peptide followed by ranking of decoys using the Rosetta score function. Using PICH proteins on DNA. Taken together, our single-molecule approach pro- this protocol, we are able to predict relative specificities for polypeptide glyco- vides new insights into the mechanisms by which PICH is involved in anaphase sylation for all possible amino acid permutations at substrate positions X-1 and UFB resolution. Xþ1 for GalNAc-T2 and human-OGT. We compared predictions to experi- mental data (Kightlinger, W. et al., Nat. Chem. Biol.14, 627-635 (2018)) for 346-Pos all 400 polypeptide substrates per enzyme. Using a receiver operating curve Sequence-Dependent Asymmetric Unwrapping of Nucleosomes of Yeast analysis, the predictions were found to have an area-under-curve above 0.80, Hidetoshi Kono1, Di Luo1, Daiki Kato2, Jumpei Nogami3, indicating that the method can often distinguish true and false positives. To Yasuyuki Ohkawa3, Hitoshi Kurumizaka4. the best of our knowledge, our protocol is the first example of a structure- 1Molecular Modeling & Simulation, QST, Kyoto, Japan, 2Waseda Univ, based computational protocol for predicting polypeptide substrate specificities Tokyo, Japan, 3Kyushu Univ., Fukuoka, Japan, 4Univ of Tokyo, Tokyo, of GTs. Japan. The first nucleosomes in the downstream of transcription starting sites are 344-Pos called þ1 nucleosomes, which are expected to be readily unwrapped for Engendering Catalytic Activity by Increasing Dynamics in a Designed DNA transcription. To characterize DNA accessibility in þ1 nucleosomes, Enzyme MNase-seq experiments were carried out with 20 reconstituted þ1 nucleo- Jonathan Preston1, Bernard Everson1, Fabien Giroud2, David Vinyard3, somes of budding yeast. Although MNase has been known for its sequence Kelly Greenland1, Emma Bjerkefeldt1, Shelley Minteer2, Gary Brudvig3, preference in DNA digestions, we confirmed that this sequence preference is Ronald Koder1. overwhelmed by DNA accessibility by identifying the sequence-driven and 1Physics, The City College of New York, New York, NY, USA, 2The accessibility-driven cleavages. Specifically, we find that sequences favored University of Utah, Salt Lake City, UT, USA, 3Yale University, New Haven, by MNase at the end regions such as TA dinucleotide are prohibited from cleav- CT, USA. age at the internal sites in the early stage (less than 3 min.) of digestion.

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Nevertheless, sequences less favored by MNase at the end regions such as AA/ properties of the living cell nucleus. Our natural probes enable to analyze TT dinucleotide are predominantly cleaved at the internal sites in the early the local diffusion behavior in the living cell nucleus without causing damage stage of digestion. Since our MD results showed that AA/TT is one of a rigid to the cell. dinucleotide step resistant to DNA bending, these internal cleavages reflect the local site exposures induced by DNA mechanics. As the DNA entry site of þ1 349-Pos nucleosomes in highly expressed genes of yeast is found AA/TT-rich, this Microrheology of Interphase Nuclei: A Computer Simulation Study sequence element may play a role in gene activation by reducing DNA- Andrea Papale, Angelo Rosa. histone affinities along the direction of DNA transcription. SISSA, Trieste, Italy. The nucleus stands as one of the most investigated organelle of the cell. To a 347-Pos large extent though, it remains poorly understood. In general, the nucleus is Uncovering a Novel Folding Landscape of the Drosophila Genome crowded with chromatin, proteins and other complexes: while the former is Through Hi-C Normalization via Fractal Monte Carlo Deep Sampling typically confined to specific regions of the nucleus, the latter are mostly mo- Qiu Sun1, Alan Perez-Rathke2, Daniel Czajkowsky3, Zhifeng Shao3, bile so to target selected chromatin sequences. How proteins or complexes Jie Liang2. diffuse inside the nucleus is enormously important. To answer this question, re- 1Shanghai Center for System Biomedicine, Shanghai Jiao Tong University, searchers have been applying microrheology: artificial nanoprobes are intro- Shanghai, China, 2Bioengineering, Univeristy of Illinois at Chicago, duced inside the nucleus and their diffusive motion tracked by fluorescence. Chicago, IL, USA, 3School of Biochemical Engineering, Shanghai Jiao Tong Here, we employ molecular dynamics computer simulations to describe nanop- University, Shanghai, China. robe motion inside the nucleus and offer an interpretation to experimental data. Hi-C measurements provide rich information on genomic 3D structures. Mimicking experimental diffusive dynamics, we consider a polymeric model However, genomic-distance dependent biases pose significant challenges. accounting for chromatin crowding of interphase nuclei where hundreds of To reduce such biases, Hi-C frequencies are often normalized through Brownian free particles are immersed in. We consider several nanoprobe sizes, enrichment calculations, where observed contact probabilities are compared from the diameter of the chromatin fiber up to the mesh size of the chromatin to expected contact probabilities. However, only a fraction of the distance- solution. dependent biases can be removed in this way, which is particularly prob- Tracking the motion of these fictitious nanoprobes, we characterize dynamical lematic for long-range contacts. Here we describe a novel normalization properties of the nucleus covering several orders of magnitudes in length- and method based on deep sampling of physical chromatin chains via fractal time-scales. In particular, we investigate a possible variation of the simple Monte Carlo calculations. Instead of taking the average of all contacts at polymeric model which includes a set of springs modifying dramatically the a specific genomic distance as the expected contact probability, we viscoelastic properties even for small values of the elastic constants. Interest- ^ construct physical models of large ensembles (210 5) of randomly folded ingly, we observed the cage-and-escape dynamics peculiar for short times dy- chromatin chains with appropriate physical properties (confinement, namics of nanoprobes seen in experiments. excluded volume, and persistence). By taking the random contact probabil- ities derived from calculations of Euclidean distances on chromatin chains 350-Pos in the ensembles as the expected probabilities, we achieve a significant Ledgf/P75 Decreases Nucleosome Accessibility reduction in distance-dependent biases. Using a recently obtained high- Khan Cox1, Matthew D. Gibson2, Mamuka Kvaratskhelia3, resolution Hi-C map of the Drosophila genome, we show this approach Michael G. Poirier1. is much more sensitive at detecting diverse functional relationships, 1Physics, The Ohio State University, Columbus, OH, USA, 2Ohio State Univ, including regions rich in active promoters, heterochromatin, as well as Columbus, OH, USA, 3Structural Biol/Molecular Biol, University of silent domains. We discuss details of a new pattern of enriched con- Colorado, Denver, CO, USA. tacts among regions of biological significance, including a distance- LEDGF/p75 is a chromatin-binding protein that has been tied to integration of independent enrichment of promoter-promoter contacts. This is in contrast HIV-1 cDNA into human chromosomes and reduction of cellular stress- with a dramatic depletion with increasing genomic distance found when us- induced apoptosis. While the structural basis for the binding of the N-terminal ing a currently widely used normalization method. We also show that our PWWP domain of LEDGF to chromatin has been detailed, the binding mech- method has the potential of predicting novel long-range specific contacts anism and effects of LEDGF binding to chromatin is still not well understood. associated with functional chromatin states. Overall, normalization through Here we detail the effects of LEDGF binding to nucleosomes through fractal Monte Carlo deep sampling reveals a novel folding landscape of the ensemble and single-molecule FRET assays and report that LEDGF binding Drosophila genome. to nucleosomes decreases accessibility of nucleosomal DNA to transcription factors. 348-Pos Diffuion Behavior of Supuramolecular Protein Assemblies in the Living 351-Pos Cell Nucleus The Effects of Linker Histone Isoforms on the Structure and Dynamics of Shuji Fujii1, Irena Bronshtein2, Yuval Garini3, Michael Elbaum4. the Chromatosome 1Hokkaido Univ, Sapporo, Japan, 2Bar Ilan Univ, Ramat Gan, Israel, 3Physics Dustin C. Woods1, Jeffery M. Wereszczynski2. Department, Bar Ilan University, Ramat Gan, Israel, 4Dept Matl/Interfaces, 1Chemistry, Illinois Institute of Technology, Chicago, IL, USA, 2Dept Weizmann Inst Sci, Rehovot, Israel. Physics, Illinois Inst Tech, Chicago, IL, USA. Cell nucleus is filled with chromatin which has uncondensed polymeric confor- Experiments have shown that linker histones (LH) can bind to the nucleosome mation without intermolecular entanglement. In spite of the lack of the entan- core particle (NCP) in either an on- or off-dyad mode, and its presence can have glement, chromatin is compartmentalized in highly organized manner. cascading effects that may influence greater chromatin structure and dynamics. Previous studies revealed that the chromatin moves coherently with long- However, the molecular mechanisms that drive the binding mode, how these range correlations on the scale of microns. The spatiotemporal organization re- modifications are influenced by specific linker histone residues, and the major mains an open question in cell biology. To elucidate such long scale motion biological implications of these variations in chemical composition and confor- under highly organized structural regularity, we need to know its viscoelas- mation, remain poorly understood. Our goal is to elucidate the specific struc- ticity in micro and meso scales. However, we have only limited measurement tural dynamics and energetic contributions of each LH in complex with the methods for the viscoelasticity of confined in such narrow space. One useful NCP in both binding modes. Here, we report on the chemical-physical proper- method for measuring the viscoelasticity is the particle tracking, which extract ties of the chromatosome from a series of molecular dynamics simulations of shear moduli by analyzing diffusive motion of particles embedded in the me- two different linker histone isoforms bound in on- and off-dyad binding modes. dium. However, injecting particles inside the nucleus induces serious stress to Results show that the flexibility of the linker DNA is reduced upon linker his- the cell. To perform the particle tracking in the living cell nucleus without any tone binding, leading to increased chromatosome compaction. Additionally, an stress, we need a natural probes. In this study, we use genetically encoded su- analysis of relative binding energies suggests that Globular Histone H5 (GH5) pramolecular protein assemblies as natural probes [Bellapdorona et al., Angew. exhibits a stronger binding affinity to the NCP than the other linker histone, Chem. Int. Ed. 2014, 53, 1534]. Expressed supramolecular protein assemblies Globular Histone H1 (GH1), and favors the on-dyad binding mode. Moreover, form spherical aggregates with a diameter of 0.5 - 1.0 micron in the nucleus. evaluation of the linker histone tertiary structure shows that GH1 samples a Particle tracking method reveals that the protein assemblies show an anoma- distinct conformational space apart from histone GH5, independent of binding lous diffusion at short time range but shifts to the normal Brownian motion mode. Overall, these atomistic simulations provide insight into the physical and at long time range. Our study presents a noninvasive approach of using a su- chemical effects of linker histone isoforms on the structure and dynamics of the pramolecular protein assemblies and their dynamics to investigate material chromatosome.

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352-Pos 354-Pos Chromatin Structure Regulation by an Epigenetic Switch Tuning the Flex- Phase-Separation Driven Heterochromatin Formation: Experiment and ibility of the H1 C-Terminal Domain Theory Akshay Sridhar1, Stephen Farr1, Guillem Portella2, Tamar Schlick3,4, Dan Deviri1, Amy R. Strom2, Serafin Colmenares3, Shelby Wilson3, Modesto Orozco5,6, Rosana Collepardo-Guevara1. Collin Hickmann3, Gary Karpen4, Samuel Safran1. 1University of Cambridge, Cambridge, United Kingdom, 2Department of 1Chemical and Biological Physics, Weizmann Inst Sci, Rehovot, Israel, Chemistry, University of Cambridge, Cambridge, United Kingdom, 3Courant 2Princeton University, Princeton, NJ, USA, 3UC Berkeley, Berkeley, CA, Institute of Mathematical Sciences, New York University, New York, NY, USA, 4BioScis, Lawrence Berkeley National Lab, Berkeley, CA, USA. USA, 4Department of Chemistry, New York University, New York, NY, In the flow of information in biological systems, DNA molecules are tran- USA, 5Joint IRB-BSC Program on Computational Biology, Institute for scribed to RNA molecules, which are then translated to functional proteins. Research in Biomedicine, Barcelona Institute of Science and Technology, Although transcription is well understood at the in-vitro level, the mechanisms Barcelona, Spain, 6Department of Biochemistry and Biomedicine, University that control transcription within the nucleus depend on DNA spatio-temporal of Barcelona, Barcelona, Spain. organization, which is poorly understood. The organization of DNA (in the Inside Eukaryotic Cells, DNA is complexed with histone proteins forming form of chromatin) in the nucleus presents many non-trivial physical phenom- chromatin - an array of nucleosomes separated by protein-free linker DNA. ena, e.g. fractal organization, glassy dynamics, and out-of-equilibrium steady H1 Linker Histones (LH) are an essential component of chromatin where states. At the largest length scale of the nucleus itself, the genome is broadly they bind to the surface of nucleosomes, transforming the conformation of nu- organized into two distinct chromatin states: Heterochromatin, which is dense cleosomes with respect to one another. H1 proteins are formed by a structured and transcriptionally repressed, and euchromatin, which is less compact and globular domain (GD) and unstructured N- and C-terminal domains (CTD). transcriptionally active. In this work, we review experimental evidences While high-resolution structural studies have characterized the binding of the showing that small protein (HP1) phase-separation within the nucleus is crit- H1 GD to nucleosomes, truncation mutant experiments demonstrate a depen- ical for heterochromatin domain formation. We will present a physics-based, dence of chromatin compaction on the unstructured terminal domains. These minimal, theoretical model of HP1 phase-separation that drives formation of intrinsically disordered terminal domains are also the sites of multiple post- HP1-dense heterochromatin. Motivated by experimental findings, we consider translational modifications that can further regulate chromatin structure and that HP1 protein can exist in two distinct configurations (open or closed), each thereby gene expression. having a different valency that determines its ability to interact with itself or Here we determine the influence of the terminal domains on chromatin struc- with chromatin. We show that this minimal model is sufficient to recapitulate ture through a novel multiscale strategy that combines nucleosome-scale many aspects of heterochromatin formation such as the mobility of HP1, chro- multi-microsecond atomistic enhanced-sampling Molecular Dynamics simula- matin compaction, and the observed, inverse relation between concentration tions with coarse-grained Monte-Carlo simulations of 100 nucleosome arrays. of DNA within globules and HP1 phosphorylation. We use a novel method In the atomistic scale, we use a Biased-exchange Metadynamics setup biasing to measure the in vivo response of heterochromatin to temperature changes, the CTD’s secondary structure and its interactions with DNA. These results are which provides an important test of the theory. In addition, by considering used to develop LH parameters for integration into the successful NYU chro- chromatin mechanics, we predict that external compressive forces applied matin coarse-grained model. Our results suggest that the CTD bridges DNA to the nucleus can promote heterochromatin formation. This implies that through flexible loops and abets the formation of an asymmetric chromatosome the process that forms heterochromatin is inherently mechanosensitive, and with curved linker DNA arms - a result challenging the canonical view that LHs may play a key role in the ability of a cell to respond to and resist mechanical induce a rigid and symmetric DNA stem but more consistent with the emerging stimuli. view of chromatin polymorphism. The chromatosome asymmetry allows the formation of polymorphic structures remarkably similar to those from recent 355-Pos Chrom-EMT images. Extending the multiscale strategy to investigate H1 In Vivo Chromatin Compaction Changes as Detected by Water Dipolar CTD phosphorylation, we demonstrate that this epigenetic modification’s Relaxations: the Molecular Crowding Role Revealed by the Acdan cyclical effect on chromatin compaction is tied to its influence on the disor- Fluorescence dered CTD’s flexibility. Leonel S. Malacrida1,2, Lorenzo Scipioni1, Enrico Gratton1. 1Department of Biomedical Engineering, Univeristy of California, Irvine, 353-Pos CA, USA, 2Departamento de Fisiopatologı´a, Univesidad de la Repu´blica, Binding Dynamics of Disordered Linker Histone H1 with a Nucleosomal Montetevideo, Uruguay. Particle Chromatin occupies territories in the nucleus in regards to its different status, Hao Wu1, Yamini Dalal2, Garegin A. Papoian3. hetero and euchromatin. Chromatin dynamics responds to chemical and phys- 1Biophysics Program, University of Maryland, College Park, MD, USA, ical inputs, producing changes in the molecular crowding and thermodynamic 2National Cancer Institute, National Institutes of Health, Bethesda, MD, parameters. Using ACDAN photophysics we demonstrate the correlation be- USA, 3Department of Chemistry and Biochemistry, University of Maryland, tween the water dipolar relaxation and the chromatin status(Malacrida-2017). College Park, MD, USA. ACDAN was synthesized to sense polarity and dipolar relaxation in Eukaryotic genomes are packaged via formation and condensation of chro- proteins(Weber-1979). Inside the cell one of the main dipolar relaxation matin fibers, which in turn are assembled from nucleoprotein complexes, active molecules is water. Using spectral phasors we analyzed the ACDAN called nucleosomes that are comprised of histone proteins and DNA. Nucle- spectral shift due to modification of chromatin compaction using chemical osomes and chromatin are further regulated by the H1 linker histones, which and physical stimuli. First, we produce double-strand DNA break (DSB) by are essential for functioning of eukaryotic chromatin. Consisting of a rigid irradiating a small ROI (780nm 25mW, 10x10pixels, 150nm/pixel) inside globular domain and two disordered terminal domains, H1 linker histones the nucleus. Using Trichostatin-A (TSA, a histone deacetylase inhibitor) we regulate the higher-order chromatin structure via binding to nucleosome seg- produced chromatin decompaction by the unfolding of the heterochromatin ments and linker DNA. Multiple H1 binding modes have been discovered in driven by the inhibition of removal of acetyl group from histones. To induce different studies, where detailed binding mechanisms are still under debate. In chromatin compaction we used Okadaic acid (OA, a protein phosphatase in- particular, disordered H1 C-terminal domain (CTD) is believed to stabilize hibitor). OA can inhibit serine/threonine protein phosphatases and thus modi- the binding sites. In this work, we used AWSEM-IDP, a new branch of the fication of Ser10 phosphorylation on Histone-3. The DSB treatment in NIH- coarse-grained (CG) protein force field AWSEM, in combination with another 3T3 cells produces a decrease in the water dipolar relaxation as detected by CG DNA force field 3SPN.2C, to simulate the H1-nucleosome complex. ACDAN on the irradiated ROI. When cells were incubated with 25mg/mL More specifically, we performed the following molecular dynamics studies of TSA for 1 hour the ACDAN fluorescence indicates a decrease in dipolar of a canonical nucleosome: (1) without the H1; (2) with the H1 globular relaxation. However, cells incubation for 1 hour with 50nM of OA show an domain; (3) with the full-length H1 to investigate the binding mechanisms increase in the dipolar relaxation indicated by ACDAN spectral shift. These of different H1 domains. Our results indicate that full-length H1 forms a results indicate that chromatin compaction/decompaction correlates with more rigid complex with the nucleosome than the H1 globular domain. We changes in molecular crowding, and thereby with water activity. The water also found that H1 CTD binds with linker DNA arms via strong electrostatic activity and its modulation can be related with biochemical processes inside interactions, which stabilize the entire complex. Overall, our report sheds the nucleus. (Supported by NIH P41-GM103540 ) Malacrida et al. Biophysi- light on the dynamics of H1 CTD in the nucleosomal context at near- cal Journal.(2017) 112 (3),p218a. Weber, G., Farris, FJ. Biochemistry.(1979), atomic resolution. 18 (14),pp3075-3078.

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356-Pos tion was implemented. A two nucleosome system with varying DNA-linker Modulation of the DNA Accessibility in the Nucleosome – Insights from length to elucidate the effects of HP1 on adjacent nucleosomes. A Martini Physics Models Coarse Grain MD simulation was implemented to extend that sampling ability Alexey V. Onufriev. of the all-atom simulation; as well as, allow for an increase in length of the Dept Comp Sci/Phys, Virginia Tech, Blacksburg, VA, USA. chromatin fiber. The nucleosome, a complex of 147 base-pairs of DNA with eight histone pro- An ultra-CG simulation was utilized to study the heterochromatin fiber on teins, must protect its DNA, but, at the same time, allow on-demand access to it a kilobase scale. The dynamics and structure was observed similar to when needed by the cell. The exact mechanism of the control remain unclear. the previous simulation scales; however, the ultra-CG allowed to simulate A simplified coarse-grained electrostatic model of the nucleosome reveals that chemical transformation of regulatory functions of chromatin under varying at physiological conditions the complex is close to the phase boundary sepa- physiological conditions. To understand the dynamics of the folding rating it from the ‘‘unwrapped’’ states where the DNA is more accessible. A polymer, we first simulated a heterochromatin fiber without the presence small drop in the charge of the globular histone core (e.g. through acetylation of histone methyltransferase (HMT) enzymes. Then, we simulated the system of a lysine) can significantly lower the nucleosome stability, and thus increase with HMTs to understand the effect of the H3K9 trimethylization alters the DNA accessibility. the dynamics of the heterochromatin fiber. We then altered the presence A follow-up multi-state atomistic model explores virtually all possible charge- of HMTs to understand the effects that the various HMTs have on altering post-translational modifications (PTMs) in the globular histone core. heterochromitization. The model reveals a rich and nuanced picture: the effect of PTMs varies greatly depending on location, including counter-intuitive trends such as 359-Pos decrease of DNAaccessibility for some lysine acetylations in the core. The Surface Fluctuations and Coalescence of Nucleolar Droplets in the Human findings suggest that charge-altering post-translational modifications in the Cell Nucleus histone core might be utilized by the cell to modulate accessibility to its Christina M. Caragine, Shannon Haley, Alexandra Zidovska. DNA at the nucleosome level. A connection to transcription regulation in- Center for Soft Matter Reseach, Department of Physics, New York vivo is made. University, New York, NY, USA. The nucleolus is a membraneless organelle embedded in chromatin solution in- 357-Pos side the cell nucleus. It is a site of ribosomal biogenesis, plays a key role in cell Structure and Dynamics of the Telomeric Nucleosome and Chromatin cycle progression and stress response, and acts as an indicator in many human Lars Nordenskio¨ld1, Aghil Soman1, Nikolay Korolev1, Surya Wahyu1, diseases and aging. However, the physical nature of the human nucleolus re- Sook Yi Wong1, Chong Wai Liew1, Simon Lattmann1, Hsian Ling Teo1, mains an open question. Elucidating nucleolar dynamics, such as their shape John Van Noort2, Daniela Rhodes1. fluctuations and fusion, could not only help answer this question, but also pro- 1Sch Biol Sci, Nanyang Tech Univ, Singapore, Singapore, 2Phys Life vide insight into the material properties of the surrounding nucleoplasm. In this Processes, Leiden Univ, Leiden, Netherlands. work, we analyze the surface dynamics and fusion kinetics of nucleoli in live Eukaryotic genomic DNA is condensed into chromatin: 146bp DNA wraps human cells[1]. Nucleolar surface fluctuations inform on the nucleolus- around the histone octamer (HO) comprising two copies each of the core his- nucleoplasm interface, while nucleolar fusion might resolve whether nucleoli tones H2A, H2B, H3 and H4 forming the nucleosome core particle (NCP). A aggregate like solid particles or coalesce like liquid droplets. We find that string of nucleosomes folds into the so-called called ‘‘30nm’’ chromatin fibre, the surface exhibits subtle, but measurable, fluctuations and that the neck con- which compacts into metaphase chromosomes. necting two fusing nucleoli grows as r(t)t1/2. This is consistent with liquid Human telomeres constitute the protective structure at the end of the chromo- droplets with low surface tension 106 Nm1 coalescing in an outer fluid somes with repetitive TTAGGG sequences that are about 10kbp. Little is of higher viscosity 103 Pa s, i.e. chromatin solution. We hypothesize that known about the consequences of this unique sequence for chromatin structural the high viscosity of chromatin solution slows down the nucleolar coalescence and dynamic properties. Although it is established that telomeric DNA is pack- kinetics to facilitate transcription inside nucleoli. Interestingly, we find the neck aged in chromatin with an unusually short (157 bp) nucleosome repeat length velocity, dr/dt, to be comparable to the velocity of chromatin solution[2]. Sur- (NRL) and can form nucleosomes and chromatin, almost nothing is known prisingly, nucleolar coalescence occurs in an active fluid, yet can be described about the telomeric nucleosome core and telomeric chromatin structure at the by coalescence theory for passive liquid droplets, suggesting that the measured detailed molecular level. viscosity and surface tension might be effective quantities. Our study presents a We have characterised the telomeric human HO 145 bp DNA NCP (Telo- noninvasive approach of using natural probes, such as nucleoli, and their dy- NCP), using biophysical methods and determined its structure at 2.1 A˚ resolu- namics to investigate material properties of the cell and its constituents[1]. tion with X-ray crystallography. Although the structure is largely similar to the 1. Caragine CM et al., Phys. Rev. Lett., In Press, 2018. well-known published atomic resolution NCP structures, several differences 2. Zidovska A et al., Proc. Natl. Acad. Sci., 110(39), 15555-60, 2013. can be identified, e.g. DNA stretching on the Telo-NCP is different from that of the ‘‘601’’ (high-affinity nucleosome positioning sequence) and alpha satel- 360-Pos lite NCPs of the same length. A Platform for Investigating Nuclear Organization and its Changes During We have designed and prepared 20157 bp telomeric DNA template repeats, Human iPSC Differentiation reconstituted these with human HO and performed characterisation of the struc- Susanne M. Rafelski. tural and dynamic properties of the telomeric chromatin fibres with EM and sin- Allen Institute for Cell Science, Seattle, WA, USA. gle molecule magnetic tweezer experiments. Telomeric chromatin fibres are The Allen Institute for Cell Science is developing a state space of stem cell considerably more homogeneous than corresponding 157 bp repeat fibres re- structural signatures to understand the principles by which cells reorganize dur- constituted from 601-DNA array templates. However, the structural and dy- ing the cell cycle and differentiation. We have developed a pipeline to generate namic properties of individual telomeric arrays saturated with 20 high-replicate, dynamic image data on cell organization and activities using nucleosomes, appear similar to equivalent 601 chromatin fibres under the influ- endogenous fluorescently tagged human induced pluripotent stem cells ence of force in single molecule pulling experiment. (hiPSCs) (the Allen Cell Collection at www.allencell.org). Each of the 25 lines express a monoallelic EGFP-tagged protein representing a cellular struc- 358-Pos ture. For each structure, we take advantage of thousands of replicate high- Multi-Scale Simulation of the Chromatin Fiber to Elucidate Oct4 Gene resolution 3D images to develop quantitative image-based assays, segmenta- Repression tions, analyses, and computational models. We are initiating an analogous Michael R. Williams, Dmitri Kireev. study of the nucleus, with a goal of creating a ‘‘state space of nuclear signa- Center for Integrative Chemical Biology and Drug Discovery, Eshelman tures.’’ We are endogenously tagging an initial set of eight key nuclear land- School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA. mark proteins with EGFP and analyzing their sub-nuclear localization as the The structural and chemical transformations of the chromatin fiber are crucial cells progress through the cell cycle and differentiate into cardiomyocytes. to the understanding of cellular functions. We investigated the repression of the These landmarks include the nucleolus, nuclear lamina, nuclear pores, cohesin, gene Oct4, which is key in regulating embryonic stem cell pluripotency. The CTCF, histones, and heterochromatin. We are also developing methods to repression occurs due to the introduction of posttranslational modifications re- quantify the change in localization patterns for these nuclear landmark proteins sulting in the recruitment of heterochromatin protein 1 (HP1). in the undifferentiated hiPSCs vs. cardiomyocytes derived from them. Prelim- We applied a multi-scale simulation methodology to elucidate chromatin func- inary image analyses, for example, reveal that H2B in cardiomyocytes displays tions at varying levels of detail. An all-atom molecular dynamic (MD) simula- less variation in both signal intensity and local spatial structure, generating a

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‘‘smoother’’ protein distribution pattern within the nucleus and suggesting that [1] Branco, MR et al, PLos Biol., 2006.[2] Sinha, DK et al, Biophys J., 2008.[3] our analysis approaches can detect changes in localization patterns of nuclear Germier, T et al, Biophys J., 2017.[4] Schubeler,€ D et al, Genes Dev., 2004.[5] landmark proteins. We are also using single-cell RNAseq, FISH, and single- Narayan, G et al, Gene Chromosome Canc., 2007. cell activity assays to conjoin gene expression with organization and pheno- type. These cell lines and our analyses will further be integrated with 363-Pos genome-wide assays of chromatin architecture together with the 4D Nucleome Dynamical Signatures of Local DNA Damage in Live Cells Project and once fully characterized will be distributed publicly. Jonah Eaton. Dept Physics, New York Univ, New York, NY, USA. 361-Pos The dynamic organization of chromatin inside the eukaryotic cell nucleus is Intensity Sorted Fluorescence Correlation Spectroscopy: A Novel Method essential to the regulation of the genome such as maintaining its integrity, to Probe Nuclear Dynamics and Chromatin Organization in Living Cells gene expression and replication. However, the physical picture of chromatin Melody Di Bona1,2, Simone Pelicci1,2, Isotta Cainero1,2, structure and dynamics remains unclear and requires identifying the dynam- Giuseppe Vicidomini3, Davide Mazza4, Michael A. Mancini5, ical and structural signatures of specific biological processes. In this work, Alberto Diaspro1,2, Luca Lanzano’1. we study the chromatin dynamics in interphase, time between two cell divi- 1Nanoscopy, Istituto Italiano Tecnologia, Genova, Italy, 2Department of sions, in response to DNA damage. As shown previously, chromatin dynamics Physics, University of Genoa, Genova, Italy, 3Molecular Spectroscopy and across the whole nucleus change dramatically after DNA damage; chromatin’s Microscopy, Istituto Italiano Tecnologia, Genova, Italy, 4The European coherent motion is eliminated, while its local mobility increases and its Center for Nanomedicine, Ospedale San Raffaele, Milano, Italy, 5Mol and compaction decreases [1]. Here, we explore the local chromatin dynamics Cell Biology, Baylor College of Medicine, Houston, TX, USA. and compaction in response to local DNA damage in human cells. To do The architectural organization of chromatin can play an important role in gene so, we induce double stranded DNA breaks (DSBs), which we visualize using expression by regulating the diffusion of molecules via binding interactions and 53BP1-mCherry, and observe the spatiotemporal evolution of their dynamics molecular crowding [1]. In this respect, understanding how variations of chro- using single particle tracking. Simultaneously, we use histone H2B-GFP as a matin architecture affect nuclear dynamics is of fundamental importance. Fluo- reporter on the chromatin compaction, to compare DSB dynamics inside the rescence Correlation Spectroscopy (FCS) is able to probe chromatin heterochromatin and euchromatin. To elucidate the dynamic signature of accessibility and molecular crowding in live cells by measuring fast diffusion the local DNA damage response, we vary the number of DSBs as well as of molecules in the range between microseconds and milliseconds [2]. In explore the ATP-dependence of these processes. Our preliminary results particular, single point FCS (spFCS) has a high temporal resolution but lacks show the local subdiffusive dynamics of the DSBs changes with an increasing spatial information. Conversely, spatially-resolved methods, like scanning DSB count inside the cell nucleus. We also explore the relationship between FCS, have in general limited temporal resolution. Here, we introduce a novel the protein aggregation, diffusion coefficient and chromatin compaction at a technique that samples several nuclear positions by slowly scanning the FCS DSB. observation spot in a continuous line: by analyzing the data in short time se- [1] Zidovska A, Weitz DA & Mitchison TJ, Proc. Natl. Acad. Sci., 110(39), quences, we preserve the high temporal resolution of spFCS while probing 15555-60, 2013. different nuclear compartments in the same cell. We use the intensity as a refer- 364-Pos ence to sort the short sequences into Auto-Correlation Functions associated to Destabilizing Nucleosomes and the Role of HMGB Proteins different nuclear regions. Interestingly, with respect to different chromatin Micah J. McCauley1, Ran Huo1, Emily Navarrete1, Nicole Becker2,QiHu2, states, we show that the mobility of a small inert probe like monomeric GFP Molly Nelson Holte2, Uma Muthurajan3, Ioulia Rouzina4, Karolin Luger3,5, is reduced in heterochromatin vs euchromatin, especially in the perinucleolar Georges Mer2, L. James Maher III2, Nathan Israeloff1, Mark C. Williams1. heterochromatin; moreover, we show that compaction due to ATP depletion 1Physics, Northeastern University, Boston, MA, USA, 2Biochemistry and or hyperosmolar treatment affects this ratio in a different way. More mechanis- Molecular Biology, Mayo Clinic College of Medicine and Science, tically, we also use this method to characterize hormone-induced changes in the Rochester, MN, USA, 3Chemistry and Biochemistry, University of Colorado, mobility and binding of estrogen receptor-a to an engineered prolactin gene Boulder, CO, USA, 4Chemistry and Biochemistry, Ohio State University, array. Finally, we discuss coupling with STimulated Emission Depletion Columbus, OH, USA, 5Howard Hughes Medical Institute, Chevy Chase, MD, (STED), for performing spot-variation FCS in different nuclear regions at USA. sub-diffraction spatial scales[3]. [1] Misteli T., Science 2001 [2] Elson E., Bio- Nucleosome disruption plays a key role in many nuclear processes including phys J, 2011 [3] Lanzano` et al, Nat Commun 2017. transcription, DNA repair and recombination. We combine atomic force micro- scopy (AFM) and optical tweezers (OT) experiments to show that the histone 362-Pos chaperone protein FACT (facilitates chromatin transcription) disrupts nucleo- Effect of Different Transcriptional States on the Single Gene Dynamics somes, revealing a mechanism for regulation of chromatin accessibility. We Fang-Yi Chu1,2, Alexandra Zidovska1,2. find that FACT (including the subunits Spt16 and SSRP1) binds directly to 1 Center for Soft Matter Research, Department of Physics, New York Univ, the nucleosome, specifically disrupting the H2A-H2B dimer interaction with 2 New York, NY, USA, Department of Physics, New York University, New DNA, leading to the release of that DNA from the nucleosome and the ejection York, NY, USA. of these dimers from the octamer. High Mobility Group B (HMGB) domains Chromatin structure undergoes large architectural changes upon transcription are key components of FACT. We find that both isolated double box Hmo1 activation in eukaryotic cells: DNA molecule becomes increasingly more and the single box Nhp6A display similar binding affinities for nucleosomes accessible to the transcription machinery, e.g., the transcription factors and as full-length FACT, and both HMGB proteins destabilize and unwind DNA RNA polymerase II [1]. More specifically, recent studies in mammalian cells from the H2A-H2B dimers. Unlike Nhp6A, Hmo1 also releases half of the found that while the chromatin structure is more "open’’ at active transcription DNA held by the (H3-H4)2 tetramer. Furthermore, in contrast to full-length sites [2], the mobility of an actively transcribed gene seems confined [3]. FACT, the octamers appear intact and the remaining (H3-H4)2 tetramer inter- Conversely, the presence of the epigenetic code in form of histone modifica- actions with the DNA are also destabilized. Differences in nucleosome desta- tions suggests that every gene carries information about its expression level, bilization point to complementary roles that FACT subunits play in which can vary across different genes, and, thus, lead to different transcrip- chromatin remodeling. While the HMGB proteins are important, other subunits tional states [4]. How such different transcriptional states affect the dynamics are required for full displacement. Furthermore, these differences may explain of single genes remains an open question. In this work, we study dynamics why Nhp6A and Hmo1 function at different genomic sites. Hmo1 is enriched at of two distinct genes in different transcriptional states, MUC4 encoding for a highly transcribed ribosomal genes, known to be depleted of histones. In glycoprotein, which constitutes mucus on the epithelial cell layers, and IL6 en- contrast, Nhp6A is found across euchromatin, pointing to a significant differ- coding for an interleukin, which acts as a pro-inflammatory cytokine in live ence in cellular function. HeLa cells. In contrast to IL6, MUC4 is known to be up-regulated in cervical cancer [5], thus these two genes are in different transcriptional states already 365-Pos before their activation. Using CRISPR/dCas9 we visualize the position of Chromatin Folding Heterogeneity Inferred from High-Resolution Nucleo- both genes while we monitor their respective mobility before and after their some Structures transcriptional activation using spinning disk confocal microscopy followed Stefjord Todolli1, John Yager2, Wilma K. Olson1. by single particle tracking. Our preliminary results show that while both genes 1Center for Quantitative Biology, Rutgers University, Piscataway, NJ, USA, behave subdiffusively, they exhibit different dynamical signatures both before 2Biomedical Engineering, Rutgers University, Piscataway, NJ, USA. and after their transcriptional activation. This suggests that mobility of a gene is DNA in eukaryotic cell nuclei occurs in a highly compact chromatin structure related to its transcriptional level and state. where the basic packaging unit is the nucleosome. This dynamic structure

BPJ 9306_9309 Sunday, March 3, 2019 73a displays a wide range in compaction and provides a regulatory mechanism for the existence of significant many-body chromatin interactions in topologi- many biologically relevant processes, such as gene expression. Since the first cally associating domains (TADs) bounding SE regions. Specifically, we inves- description of nucleosome-decorated DNA as beads on a string condensing tigate: i) the prevalence of significant many-body chromatin interactions into a 30 nm fiber in vitro, there have been many attempts to understand the beyond random polymer folding; ii) the extent of enrichment of many-body in- nature of chromatin compaction and to create models at this scale. However, teractions in SE regions; iii) which epigenetic markers are predictive of many- the proposed models for the 3D organization of the nucleosomes and inter- body interactions. Our analysis is performed on GM12878 and K562 cell lines vening DNA vary quite significantly, and the very existence and relevance of at 5 KB resolution. a 30 nm structure in vivo has been highly debated. An analysis of the available high-resolution nucleosome core particle (NCP) structures shows subtle, yet 368-Pos significant differences in DNA wrapping around the histone octamer core, Quantitative Measurement of Nucleosome Occupancy and DNA Accessi- potentially due to DNA and histone modifications, presence of NCP-binding bility proteins, experimental crystallization/cryoEM conditions, intrinsic flexibility, Razvan V. Chereji1, Terri D. Bryson2, Steven Henikoff2. etc. Monte Carlo simulations of regular nucleosomal arrays using a meso- 1NICHD, NIH, Bethesda, MD, USA, 2Fred Hutchinson Cancer Research scale representation of chromatin suggest that these local differences can result Center, Seattle, WA, USA. in large changes in global structure, comparable to the effect of changes in Micrococcal nuclease (MNase) is typically used to digest free DNA in order to nucleosome spacing by about 2-3 base pairs. Our results suggest that a regular map nucleosome positions genome-wide. Unfortunately, MNase has a strong nucleosome array with a 177-base pair repeat can display either a loose three- DNA sequence specificity, and different levels of digestion release from chro- stack or a more compact two-stack arrangement, depending on the mode of matin different ensembles of nucleosomes [1,2]. Mild digestions release from DNA wrapping in the nucleosome. Insights from our work may help bridge chromatin the more accessible nucleosomes, while extensive digestions destroy the significant divide in the proposed chromatin models based on various exper- the former ones and release the less accessible nucleosomes. Recently, we have imental results. developed a new nucleosome mapping method that does not use MNase, and eliminates the biases introduced by this nuclease [3]. Nevertheless, MNase- 366-Pos seq remains the most used method of mapping nucleosomes, although its results Histone Tail-DNA Interactions: Charge Regulation and Sequence Speci- are not quantitative and are strongly affected by the level of digestion. Using a ficity rigorous mathematical treatment of all the steps involved in DNA digestion by Raju Timsina, Xiangyun Qiu. nucleases, we have developed a quantitative framework of obtaining the nucle- Department of Physics, The George Washington University, Washington, osome occupancy and DNA accessibility genome-wide, at the single- DC, USA. nucleosome level. We show that the traditional way of analyzing MNase-seq Histones not only assemble DNA in the form of nucleosomes, basic structural experiments is flawed, and the coverage obtained by stacking the undigested units of chromatin, but dynamically participate in gene regulation through DNA fragments obtained at any level of digestion is not proportional to the post-translational modifications of histone tails. Histone tails are poly- real nucleosome occupancy. Therefore, the MNase-seq coverage is an inaccu- cationic, unstructured terminal domains protruding from the nucleosome rate measure of the fraction of cells containing a nucleosome at a given posi- core and they are known to interact with nucleosomal DNA, linker DNA, tion. Using a series of digestions, we determine the real fraction of cells as well as the nucleosome core. As such, the role of histone tails in the struc- containing a nucleosome at each position, DNA accessibility, and the rates ture and dynamics of nucleosome and chromatin has been studied exten- of MNase digestion corresponding to each position in the genome of sively; however, there is dearth of quantitative measurements of the Drosophila melanogaster. molecular interactions directed by histone tails of considerable diversity. [1] Chereji RV et al. - Nucleic Acids Res 44, 1036 (2016) Here we probe the interactions between histone tails and DNA through quan- [2] Chereji RV et al. - Mol. Cell 65, 565 (2017) titative measurements of DNA-DNA forces modulated by histone tails. Spe- [3] Chereji RV et al. - Genome Biol. 19, 19 (2018) cifically, ordered DNA arrays are charged neutralized by defined peptide sequences derived from histone tails, osmotic stress is used to vary the 369-Pos DNA-DNA forces, and x-ray diffraction is used to determine the DNA- The Chromosome’s Fight Against Disorder in E. coli DNA spacing. Remarkably, such DNA force-spacing curves are observed Christopher H. Bohrer, Elijah Roberts, Jie Xiao. to sensitively depend on both total peptide charge and the actual sequence Johns Hopkins Univ, Baltimore, MD, USA. of peptides with the same total charge, evidencing non-specific (charge regu- An interesting structure within the nucleoid of E. coli is the presence of lated) and specific (sequence/structure dependent) interactions between his- approximately 400 topologically constrained chromosomal DNA loops. tone tails and DNA. Furthermore, a subset of histone tail sequences are These loops, approximately 10kb in size, are predicted to contribute to the able to spontaneously co-assemble with DNA into compact condensates, correlations in gene expression between neighboring genes, and are thought while reducing the total charge or varying the sequence would abolish such to lead to coordinated ‘‘bursty’’ mRNA production, due to the buildup of action. We speculate on the physics of histone tail-DNA interactions in terms supercoiling. These hypotheses, however, have not been directly tested in of charge patters and structural congruence. The biological implications of vivo in a living cell . In this work we integrated into the E. coli chromosome our observations are discussed with respect to chromatin conformation and a synthetic system in which the formation of a topological domain can epigenetic regulation. be controlled. We then investigated the effect of topological constrain, pro- moter activity and gene order on the expression correlation of three genes 367-Pos within or outside of the topological domain using single-molecule detection Many-Body Chromatin Interactions in Super-Enhancer Tads based gene expression reporting systems. Our work provided the first-of-its- Alan Perez-Rathke1, Qiu Sun2, Valentina Boeva3, Jie Liang1. kind insight into a possible gene regulation mechanism by topological 1Bioengineering, Univ Illinois Chicago, Chicago, IL, USA, 2Shanghai Center organization. for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China, 3Institut Curie, Paris, France. Chromatin interactions are thought to be important for gene regulation Posters: DNA Replication, Recombination, and via enhancer-promoter looping as well as for critical functions such as cellular Repair specialization. There is now emerging evidence that many-body (>2) chromatin interactions may be an important feature of super-enhancer (SE) re- 370-Pos gions - for example, condensing the SE region into a cohesive transcriptional DNA Mismatch Repair Relies Entirely on Stochastic Transactions apparatus. Chromosome conformation capture techniques such as Hi-C have Jiaquan Liu1, Ryang-Geun Lee2, Brooke Britton1, James London1, greatly contributed to our understanding of the chromatin folding landscape. Jeungphill Hanne1, Jong-Bong Lee2, Richard Fishel1. However, Hi-C has limitations as it only captures pairwise chromatin interac- 1The Ohio State University, Columbus, OH, USA, 2Pohang University of tions and the interaction frequencies mostly represent population averages. Science and Technology, Pohang, Republic of Korea. Therefore, it is generally not possible to directly infer the existence of signifi- DNA mismatch repair (MMR) is a highly conserved excision-resynthesis sys- cant many-body chromatin interactions. With the goal of solving these prob- tem that corrects polymerase misincorporation errors. Defects in human MMR lems, we have developed a computational model which utilizes physical genes cause Lynch Syndrome or hereditary non-polyposis colorectal cancer. In properties of chromatin folding (e.g. nuclear confinement and self-avoidance) prototypical Escherichia coli (E. coli), cascading MutS and MutL sliding as well the experimental Hi-C data to reconstruct the corresponding ensemble clamps initiate MMR and recruit MutH to locate and incise a hemi- of 3-D polymers. We deeply sample from a Bayesian generative model to infer methylated GATC site, which then serves as an entry point for MMR strand

BPJ 9306_9309 74a Sunday, March 3, 2019 excision. The UvrD helicase unwinds the double-stranded DNA (dsDNA) in meric dumbbell structure formed by a central N-terminal domain (NTD) con- the direction towards the mismatch while the single-stranded DNA (ssDNA) nected to peripheral C-terminal domains via flexible coils. Electron containing the misincorporation is further degraded by an exonuclease. Single Microscopy data support the dumbbell-shape arrangement observed in AFM molecule imaging of the complete E. coli MMR excision process revealed that micrographs. In addition, AFM images of CtIP-DNA binding reactions show the ATP-bound MutL sliding clamp exclusively captured the UvrD helicase at the formation of long molecules as a consequence of association of shorter the site of ssDNA-dsDNA junction, followed by tethering the UvrD to DNA DNAs by CtIP acting as a bridge. Bridging events are dependent on [hCtIP] junction as an unwinding processivity factor. The direction of the unwinding and on the oligomeric state of hCtIP, as corroborated with mutants. Interest- appeared to be random, which is determined by the side of the strand scission ingly, in vitro fluorescence anisotropy studies indicate a higher CtIP avidity the MutL clamp was sliding prior to its interaction with UvrD. Moreover, the for complex DNA ends. All together, we propose a model where CtIP binds single-stranded DNA-binding protein (SSB) regulated the translocation of DNA at internal positions and slides along the duplex before being captured UvrD helicase, resulting in highly dynamic and reversible strand unwinding/re- at DNA non-canonical ends. CtIP interacts directly with other proteins involved zipping tracts. The exonuclease (Exo I or Exo VII) activity was poor during in DNA damage repair and signalling, and may therefore act as a platform to strand unwinding and ultimately may be superfluous with multiple GATC in- co-ordinate the repair of two-ended DNA breaks via HR. cisions. These observations suggest that the traditional MMR model have missed several essential mechanical operations and may not accurately reflect 373-Pos MMR in vivo. In addition, our results demonstrate that MMR is governed by the Novel Assay Resolves D-Loop Processing Pathways by E. Coli RecQ and stochastic nature of DNA interactions and the plasticity of protein complexes Human BLM Helicases that employ thermal diffusion. Gabor M. Harami1, Janos Palinkas1, Yeonee Seol2, Mate Gyimesi1, Zoltan J. Kovacs1, Keir C. Neuman2, Mihaly Kovacs1. 371-Pos 1Department of Biochemistry, Eo¨tvo¨s Lora´nd University, Budapest, Deciphering the Essential Interaction between Primase and Helicase in Hungary, 2Laboratory of Single Molecule Biophysics, National Heart, Lung Mycobacterium tuberculosis and Blood Institute, National Institutes of Health, Bethesda, MD, USA. Dhakaram P. Sharma, Ramachandran Vijayan, Syed Arif Abdul Rehman, Via the process of homologous recombination (HR) cells can repair various Samudrala Gourinath. types of DNA damage. Moreover, HR is essential for the generation of ge- School of Life Sciences, Jawaharlal Nehru University, Delhi, India. netic diversity in meiosis. In both cases quality control is required to avoid The helicase-primase interaction is an essential event in DNA replication deleterious consequences of imprecise recombination and to regulate the fre- and is mediated by highly variable C-terminal domain of primase (DnaG) quency of recombination events. Human Bloom’s syndrome (RecQ-family) and N-terminal domain of helicase (DnaB). To understand the functional helicase (BLM) has been implicated in both the quality control and efficient conservation with a low sequence homology of the DnaB binding domain progression of HR events. BLM can disrupt recombination intermediate joint of the DnaG among the eubacteria, we determined the crystal structure of DNA molecules (e.g. D-loops), possibly to selectively inhibit imprecise the helicase binding domain of DnaG from M. tuberculosis (MtDnaG- recombination during repair and to limit the number of chromosome cross- CTD) at 1.58 A˚ . The overall structure of MtDnaG-CTD consists two subdo- overs during meiosis. Intriguingly, BLM is also implicated in the stabilization mains, the N-terminal globe region (GR) and the C-terminal helix hairpin and extension of D-loops. To resolve the underlying mechanism producing region (HHR) connected with a small loop. On comparing with other these antagonist behaviors, we developed a new kinetic assay that precisely DnaG-CTDs structures, the MtDnaG-CTD structure shows conservation of resolves all possible outcomes of D-loop processing by helicases. By the folds primarily in the GR. Further, to insight into the crucial helicase- combining this assay with single-molecule DNA unwinding experiments, primase interaction in M. tuberculosis, a complex was modeled using the we show that human BLM maintains a balance between disruption and stabi- MtDnaG-CTD and MtDnaB-NTD crystal structures. Two nonconserved hy- lization of D-loops. Interestingly, the activity profile of BLM markedly dif- drophobic residues (Ile605 and Phe615) of MtDnaG were identified as fers from that of bacterial (Escherichia coli) RecQ helicase that is strongly potential key residues interacting with MtDnaB. Biosensor binding studies biased toward disruption of D-loops. Based on these results we hypothesize show ten-fold decrease in binding affinity of MtDnaB-NTD with mutant that the D-loop disruption or stabilization activities of BLM are selectively Ile605Ala MtDnaG-CTD (KD-250 nM) compared to native MtDnaG-CTD utilized by the cell to halt or channel HR into different pathways, respec- (KD-2.5 mM). The loop, connecting both helixes of the HHR, was largely tively. Our assay is also suitable for elucidating the influence of additional responsible for the stability of DnaB-DnaG complex. The MtDnaB-NTD factors regulating BLM’s predisposition towards different D-loop processing shows the micromolar affinity with DnaG-CTDs from E. coli and H. pylori activities. while the MtDnaB-NTD shows unstable binding with DnaG-CTD from V. cholerae. The interaction pattern of DnaG and DnaB seems to be unique 374-Pos for an organism. The conserved catalytic domains and highly variable inter- Mechanism of SSB Displacement by Replicative DNA Polymerases During acting domains of both DnaG and DnaB suggest the species-specific evolu- Lagging Strand Synthesis tion of the system. Fernando Cerron1, Grzegorz L. Ciesielski2, Laurie S. Kaguni3, Francisco J. Cao4, Borja Ibarra1. 372-Pos 1IMDEA Nanociencia, Madrid, Spain, 2Auburn University at Montgomery, AFM Shows That Human Ctip Forms a Tetrameric Dumbbell-Shaped Montgomery, AL, USA, 3Michigan State University, East Lansing, MI, USA, Particle which Binds and Bridges DNA Ends 4Universidad Complutense, Madrid, Spain. Alejandro Martin-Gonzalez1, Oliver Wilkinson2, Hae Joo Kang3, Genome replication induces the generation of large stretches of single- Sarah Northall2, Dale Wigley4, Mark S. Dillingham5, stranded DNA (ssDNA) intermediates that are rapidly protected by single- Fernando Moreno-Herrero6. stranded DNA binding (SSB) proteins, which bind ssDNA with great affinity. 1Macromolecular Strcutures, Ctr Nacional de Biotecnologia, Madrid, Spain, The mechanism by which tightly bound SSBs are removed from ssDNA by 2DNA:Protein Interactions Unit, School of Biochemistry, University of the lagging strand DNA polymerase without compromising the advance of Bristol, UK., Bristol, United Kingdom, 3Imperial College London, London, the replication fork remains unresolved. We aimed to address this question, China, 4Dept Molec Enzymol, London Res Inst, Potters Bar, United by measuring with optical tweezers the real-time replication kinetics of the Kingdom, 5Sch Biochemistry, Univ Bristol, Bristol, United Kingdom, 6Dept human mitochondrial and bacteriophage T7 DNA polymerases on free- Macromolec Struct, Natl Ctr Biotech, Madrid, Spain. ssDNA, in comparison with ssDNA covered with homologous and non- Homologous recombination (HR) is one of the main cellular mechanisms for homologous SSBs under mechanical tension. We find important differences DNA damage-repair. Because it uses a homologous donor DNA as a template between the force dependencies of the instantaneous replication rates of it is a potentially error-free repair mechanism. The process of repair involves each polymerase on different substrates. Modeling of the data supports a the concerted action of multiple proteins; therefore the handling and mainte- mechanism in which specific polymerase-SSB interactions are required for nance of the DNA ends in close proximity is critical. A key early factor in the polymerase to dislodge the tightly bound SSB from the template, without HR is CtIP. Little is known about this protein apart from its role in the initial compromising its instantaneous replication rate. When these interactions are steps of HR and its tetrameric oligomeric state in solution for a small part of established, elimination of the template secondary structure by SSB binding the protein. Here we have studied the oligomeric state of human CtIP (hCtIP) facilitates the maximum replication rate of the lagging strand polymerase. and its interaction with DNA using Atomic Force Microscopy (AFM). AFM In contrast, in the absence of specific polymerase-SSB interactions, the tightly imaging of hCtIP shows five classes of particles classified according to their bound SSB interferes with the advance of the polymerase, slowing down morphology and volume analysis. The largest class is consistent with a tetra- DNA synthesis.

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375-Pos A$T(H) and reverse Hoogsteen A$T(rH), in particular: - Unconventional Single-Molecule Movies of the Interplay between DNA Polymerase and conformational states - A$T(wWC), A$T(wrWC), A$T(wH) and A$T(wrH), in Single Strand DNA Binding (SSB) Protein which the amino group of the A DNA base acts simultaneously as a donor Longfu Xu, Julia Bakx, Seyda Acar, Andreas Biebricher, Erwin J. Peterman, and an acceptor of the H-bonding [1];- Non-dissociative conformational transi- Gijs J. Wuite. tions - A$T(WC) / A$T(rWC)4A$T(H) / A$T(rH), A$T(WC)4A$T(rWC) / Department of Physics and Astronomy and LaserLaB Amsterdam, Vrije A$T(rH) and A$T(H)4A$T(rH) / A$T(rWC), which link A$T DNA base pairs Universiteit Amsterdam, Amsterdam, Netherlands. and their novel conformers in different combinations [2, 3];- Novel pathways of Reliable DNA replication is completed by the orchestrated activation of a the intrapair tautomerisation in the A$T DNA base pairs through the quasi- multi-protein machinery, the replisome, in a highly coordinated and dynamic orthogonal transition state - A$T(WC) / A$T(rWC)4A*$T(rwWC)/ fashion. The bacteriophage T7 gene 2.5 protein (gp2.5) works as a single A*$T(wWC) and A$T(H) / A$T(rH)4A*N7$T(rwH)/A*N7$T(wH) [4] - or strand DNA(ssDNA) binding protein and has been demonstrated to play a through the protonated amino group of the A DNA base - A$T(wWC) t t t key role in the maintenance of ssDNA stability as well as the suppression 4A$T*(w WC), A$T(wrWC)4A$T*O2(w rWC), A$T(wH)4A$T*(w H) t of hairpin formation during each cycle of Okazaki fragment synthesis. After and A$T(wrH)4A$T*O2(w rH) [5]. We have also established scientifically- gp2.5 binding to ssDNA, gp2.5 might act as a road block for DNA polymer- based explanation, why Nature exploits exactly A$T(WC) DNA base pair for ase to proceed. Bulk biochemical assays, however, demonstrate that the pres- the construction of the genetic material [6]. The biophysical significance and ence of gp2.5 increases the processivity and rate of template extension, while experimental confirmation of the results are thoroughly discussed. 1. Brovar- a mutant gp2.5 without c-terminus decreases the activity. Here we present ets’, O.O., Tsiupa, K.S. Hovorun, D.M. Front. Chem., 2018, 6:8; doi: movies of the single-molecule dynamics of T7 DNA polymerase and gp2.5 10.3389/fchem.2018.00008. 2. Brovarets’, O.O., Tsiupa, K.S., Hovorun, using optical trapping combined with confocal fluorescence microscopy. D.M. Sci. Repts., 2018, V. 8, P.10371. 3. Brovarets’, O.O.; Tsiupa, K.S., Hov- First, we investigated at single molecule level the effect of presence of orun, D.M. Int. J. Quantum. Chem., 2018, V. 118, P.e25674. 4. Brovarets’, wide type and mutant gp2.5 on DNA polymerase activity under various con- O.O., Tsiupa, K.S., Hovorun, D.M. PLoS ONE, 2018, V.13, P.e0199044. 5. ditions. Then, we observed in real-time the labelled gp2.5 binding and un- Brovarets’, O.O., Tsiupa, K.S., Dinets, A., Hovorun, D.M. Front. Chem., binding dynamics interacting with DNA polymerase. It demonstrates that 2018, (in press). 6. Brovarets’, O.O., Tsiupa, K.S., Hovorun, D.M. RSC Adv., gp2.5 binds with ssDNA at low force (<30pN) but that its binding to ssDNA 2018, V. 8, P.13433. decreases significantly at higher force (>30pN). Beside, fluorescence data shows labelled DNA polymerase appearing at multiple places on the DNA 378-Pos which indicates the DNA polymerase might bind to DNA at multiple sites. Muts Homolog Sliding Clamps Shield the DNA from Binding Proteins These results combined with further visualization of the two labeled proteins Jeungphill Hanne1, Brooke M. Britton1, Jonghyun Park2, Jiaquan Liu1, will help us better understand the interplay between DNA polymerase and Juana Martin-Lopez1, Nathan Jones1, Matthew Schoffner1, Piotr Klajner1, gp2.5. Ralf Bundschuh1, Jong-Bong Lee2, Richard Fishel1. 1The Ohio State Univ, Columbus, OH, USA, 2Pohang University of Science 376-Pos and Technology (POSTECH), Pohang, Republic of Korea. Elucidation of the Role of the Interaction between RecQ Helicase and SSB Sliding clamps on DNA consist of evolutionarily conserved enzymes that co- Protein ordinate DNA replication, repair, and the cellular DNA damage response. Gabor M. Harami1, K. Maria Mills2, Zoltan J. Kovacs1, Yeonee Seol2, MutS homolog (MSH) proteins initiate mismatch repair (MMR) by recog- Veronika Barath1, Julianna B. Nemeth1, Hajnalka Harami-Papp1, nizing mispaired nucleotides and in the presence of ATP form stable sliding Keir C. Neuman2, Mihaly Kovacs1. clamps that randomly diffuse along the DNA. The MSH sliding clamps subse- 1Department of Biochemistry, Eo¨tvo¨s Lora´nd University, Budapest, quently load MutL homolog (MLH/PMS) proteins that form a second Hungary, 2Laboratory of Single Molecule Biophysics, National Heart, Lung extremely stable sliding clamp, which together coordinate downstream and Blood Institute, National Institutes of Health, Bethesda, MD, USA. MMR components with the excision-initiation site that may be hundreds to Single-stranded DNA binding proteins (SSBs) play essential roles in genome thousands nucleotides distant from the mismatch. Specific or non-specific maintenance by covering single-stranded (ss) DNA to protect it against poten- binding of other proteins to the DNA between the mismatch and the distant tially harmful chemical reactions. SSBs also interact with numerous DNA- excision-initiation site could conceivably obstruct the free diffusion of these processing enzymes including RecQ helicases that are key players in MMR sliding clamps, inhibiting their ability to initiate repair. Here, we em- recombination-based DNA repair. SSB both recruits RecQ helicases to DNA ployed bulk biochemical analysis, single-molecule fluorescence imaging, and and stimulates RecQ helicase activity. However, the molecular mechanism mathematical modeling to determine how sliding clamps might overcome and the physiological function of the SSB-RecQ interaction are poorly under- such hindrances along the DNA. Using both bacterial and human MSH pro- stood. Via single-molecule magnetic tweezers and rapid kinetic experiments, teins, we found that increasing the number of MSH sliding clamps on a we previously showed that the interaction of E. coli RecQ with the SSB C-ter- DNA decreased the association of the E.coli transcriptional repressor LacI to minal segment changes the DNA-binding mode of SSB. This allows RecQ to its cognate promoter LacO. Our results suggest a simple mechanism whereby gain access to SSB covered ssDNA to efficiently initiate unwinding. We thermal diffusion of MSH sliding clamps along the DNA alters the association have recently developed a novel single-molecule total internal reflection fluo- kinetics of other DNA-binding proteins over extended distances. These obser- rescence (TIRF) assay that permits simultaneous real-time visualization of vations appear generally applicable to any stable sliding clamp that forms on labeled SSB-nucleoprotein filaments and individual RecQ molecules. To eluci- DNA. date the cellular roles of the interaction between RecQ and SSB we combined in vitro and in vivo measurements of RecQ mutants that specifically and selec- 379-Pos tively disrupt the RecQ-SSB interaction. E. coli RecQ interacts via its winged- Single-Molecule Studies Reveal New Replication Reactivation Pathways of helix domain (WHD) with the C-terminal peptide of SSB. We have produced Bacteriophage T7 two RecQ WHD point mutants (RecQR425A, RecQR499A) that are predicted to Bo Sun1, Michelle D. Wang2. compromise SSB binding. Both mutants exhibit significantly decreased binding 1School of Life Science and Technology, ShanghaiTech University, affinity to the SSB C-terminal peptide, but retain near wild-type helicase activ- Shanghai, China, 2Dept Physics, Cornell Univ, Ithaca, NY, USA. ities. These RecQ mutations can be used to selectively test the role of the RecQ- Replication fork arrest or collapse occurs when the replisome encounters ob- SSB interaction in vitro and in vivo without significantly altering the individual stacles such as DNA damage, DNA-bound complexes, or stable DNA second- activities of RecQ or SSB. ary structures. To ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate. Several mech- 377-Pos anisms have evolved to avoid replication failure from fork barriers and to Novel Conformationally-Tautomeric Properties of the Biologically Impor- restart the arrested replication forks in ways that are independent of the tant at DNA Base Pairs replication origins. The existence of these multiple pathways highlights the Ol’ha Brovarets’, Kostiantyn Tsiupa. importance of replication fork recovery. Our understanding of these mecha- Molecular Biology and Genetics, Kyiv, Ukraine. nisms that underlie replication reactivation has been aided through unique For the first time we have revealed at the MP2/aug-cc-pVDZ//B3LYP/6- dynamic perspectives offered by single molecule techniques. Using single- 311þþG(d,p) level of theory the novel routes for the conformationally- molecule optical-trapping technique and the bacteriophage T7 replisome, tautomeric transitions for the four biologically important A$T DNA base we report two new replication restart pathways. We demonstrate that T7 heli- pairs - Watson-Crick A$T(WC), reverse Watson-Crick A$T(rWC), Hoogsteen case can assist DNA polymerase (DNAP) in directly replicating through a

BPJ 9306_9309 76a Sunday, March 3, 2019 leading-strand lesion. In addition, a non-replicating T7 DNAP, together with 383-Pos its helicase, is able to actively disrupt a stalled transcription elongation com- Exog Displays 50-Exonuclease Activity on Both ssDNA and ssRNA plex, and then initiates replication using the RNA transcript as a primer. These Anna Karlowicz, Michal R. Szymanski. observations exhibit T7 helicase’s novel roles in assisting replication over- Intercollegiate Faculty of Biotechnology University of Gdansk and Medical coming obstacles. University of Gdansk, Gdansk, Poland. Human mitochondrial nuclease, EXOG (hEXOG), is essential for repair of 380-Pos damaged mtDNA. It shares sequence homology with the apoptotic endonu- Nanopore Detects Compromise between Speed and Processivity of PcrA clease EndoG. However, apart from being endonuclease, hEXOG also Helicase possesses 50-exonuclease activity which is necessary for the 50-end damage Momcilo Gavrilov, Ram Tippana, Dmitriy Bobrovnikov, Taekjip Ha. removal and thus robust DNA repair in human mitochondria. Recently the Biophysics, Johns Hopkins University, Baltimore, MD, USA. X-ray crystal structures of hEXOG in apo and dsDNA-bound forms were Nanopore sequencing is a method that can capture a movement of one DNA solved. It was shown that a Wing domain of hEXOG confers a specific strand through a nanopore, where an enzyme such as a helicase or a polymerase 50-dsDNA exonuclease activity which provides an optimal substrate for sub- is responsible for the DNA movement. This method has been applied to study sequent gap-filling and strand ligation steps, thus efficient base-excision the activity of enzymes and detect single steps of polymerases or translocating repair (BER). Still, the molecular mechanism underlying mitochondrial helicases. Here, we use a nanopore method to study unwinding activity of BER has yet to be clearly defined. Here we report structural and biochemical different mutants of PcrA helicase. Mutations in the single-strand binding re- evidence of 50-exonuclease activity of hEXOG on ssDNA as well as gion change the speed and the processivity of a helicase. We find that the faster ssRNA. Our results demonstrate that hEXOG acts as a multifunctional mutant tends to be less processive and more prone to stalling and making back- enzyme which may contribute not only to the 50-end damage removal in steps. This observation can help us understand better whether helicases have mitochondrial BER but also to elimination of unnecessary single-stranded evolved to maximise the processivity over speed. DNA and RNA molecules that may arise during nucleic acid processing in mitochondria. 381-Pos Replisome Preservation by a Single-Stranded DNA Gate in the CMG 384-Pos Helicase Biophysical Characterization of Full Length EXOG a Human Mitochon- Michael R. Wasserman, Grant D. Schauer, Michael E. O’Donnell, drial Inner Membrane Nuclease Shixin Liu. Andrzej B. Dubiel, Michal R. Szymanski. Rockefeller University, New York, NY, USA. Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical The eukaryotic replicative helicase CMG is assembled at replication origins University of Gdansk, Gdansk, Poland. and is thought to remain topologically closed until termination. Upon encoun- Human mitochondrial nuclease, EXOG (hEXOG), is a part of mtDNA repair tering a lesion, CMG must vacate a stalled fork to allow DNA repair. How- machinery. It is anchored to the inner mitochondrial membrane via N-terminal ever, it remains unclear whether a repaired fork can be reactivated by the transmembrane domain. Besides its endo- and exonucleolytic activities it is same CMG that is retained on DNA, or if fork restart relies on a distinct re- proposed to serve as a scaffold for assembly of the mtDNA repair complex plisome traveling from another origin. Here, using correlative single-molecule on mitochondrial inner membrane. Recently the X-ray crystal structures of fluorescence and force microscopy, we show that when uncoupled from a ND58 hEXOG in apo and DNA-bound forms were solved and activity of trun- DNA polymerase, CMG opens a single-stranded (ss) DNA gate to traverse cated mutant, without transmembrane part of the protein, was determined. Here a forked junction and reside on double-stranded (ds) DNA. Surprisingly, we show that full-length hEXOG can be purified and refolded in the presence of CMG undergoes rapid diffusion on dsDNA and can transition back onto different detergents and model membranes. We show that the nature of these ssDNA for continued fork progression. The accessory protein Mcm10 is biomimetic systems has a significant effect on the stability, structure and func- required for ssDNA gating. These results reveal an Mcm10-induced pathway tion of hEXOG. that preserves CMG on DNA and allows it to access an empty fork for swift replication recovery. 385-Pos Role of Mis Localization of DNA Repair Factors in Cell Cycle Arrest 382-Pos Manasvita Vashisth, Sangkyun Cho, Dennis Discher. A New DNA Inversion Mechanism: Recombination of the DNA Foldback Chemical and Biomolecular Engineering, University of Pennsylvania, Intercoil Structure Philadelphia, PA, USA. Byung Ho Lee1, Soojin Jo1, Hyunki Kim1, Sung Ha Park2, We propose optimal Lamin A levels in the nuclear membrane are essential to Byung-Dong Kim3, Moon Ki Kim1. prevent membrane rupture through acto-myosin stress on the nucleus and mis 1School of Mechanical Engineering, Sungkyunkwan Univ, Suwon, Republic localization of DNA repair factors like Ku80/Ku70 that results in cell cycle ar- of Korea, 2Department of Physics, Sungkwunkwan Univ, Suwon, Republic of rest. Lamin A knockdown in U2OS and A549 cells results in an increase in Korea, 3Department of Plant Science and Plant Genomics and Breeding gH2AX foci count and % of nuclear membranes that are ruptured (as indicated Institute, Seoul National University, Seoul, Republic of Korea. by mis localization of Ku80 & Ku70) as well as the % of ‘4N’ cells (late cell It is widely known that DNA inversion is performed by serine recombinases cycle / G2), compared to the respective WT cells, which is rescued upon bleb- through site-specific recombination. In detail, the recombinases help recombina- bistatin(myosin inhibitor) treatment. Furthermore, the % of nuclear membranes tion sites undergo exchange between inverted-repeat strands, resulting in the ruptured increases when cells are treated with the nuclear import inhibitor Iver- inversion of target sequences. However, the following questions still need to mectin but not Mifeprestone which is more specific and does not act on the NLS be clarified: (i) it is not clear how synaptic complex can be formed by recombi- of Ku80. The mis localization of Ku80 accompanied by increase in cells in G2 nases, and (ii) the subunit rotation model for strand exchange is unexplained. To phase indicated cell cycle arrest. Some nuclei with mis localized Ku80 were answer these questions, in this study, we propose a new DNA inversion mecha- found to form chromatin bridges through telomeres(as indicated by TRF1 nism based on the DNA foldback intercoil (FBI) structure. As a kind of four- localization). stranded DNA structure, FBI is constructed by folding back a DNA duplex (loop part) and intertwining two inverted repeat sequences in the major groove 386-Pos (stem part). The stem part consists of four DNA strands sharing the same helical Tracking the Acceleration of DNA Repair in X-Ray Irradiated Cells by axis and its diameter is closely in agreement with that of a conventional B-DNA. Interaction with Healthy Cells We suggest that the recombination can be achieved within this stem part by flip- Sha Jin, Nils Cordes. ping base pairs around their N-glycosidic bonds. It implies the recombination is OncoRay, Medizinische Fakult€at Carl Gustav Carus Technische Universit€at performed by not only enzyme-DNA contact but also DNA-DNA interaction. Dresden, Dresden, Germany. Our hypothesis also suggests a spatially reliable pathway for strand exchange Intercellular communication is essential for multicellular tissue vitality and in consideration of geometrical features of the FBI structure. To verify the pro- homeostasis. In spite of the fact that exposure to ionizing radiation can be posed DNA inversion pathway, we evaluate structural stability of intermediate detrimental for a cell, little is known mechanistically about the prosurvival conformations on the pathway in terms of conformational energy and robustness impact of unexposed neighboring cells on genotoxically injured cells. There- of new base pairing by using 3D computer modelling and molecular dynamics fore, we addressed this question using human pancreatic cancer cells (Mia þ þ simulation. In conclusion, through this computational and theoretical approach, PaCa-2), ATM / and ATM-/- fibroblasts, which were co-cultured upon we demonstrate a new possibility of existence of more reasonable and effective X-ray irradiation to identify their role as donor for and acceptor of prosurvival DNA inversion mechanism in aid of the FBI structure. cues. Different markers were employed to visualize the dynamics of double

BPJ 9306_9309 Sunday, March 3, 2019 77a strand break (DSB) repair (53BP1, phospho-ATM and phospho-Histone Myosins are actin-based molecular motor that are involved in various cellular H2A.X), vitality and cell cycling by performing high resolution 3D imaging processes including cell migration and endocytosis. More recently, Myosin VI (in fixed and live cells) using spinning disk microscopy. DSB were produced has been characterised in the nucleus where it has a direct interaction with DNA by X-ray irradiation. X-ray irradiated (6 Gy) Mia PaCa-2 cells in co-culture that is critical for RNA polymerase II transcription. Along with this interaction with unirradiated cells showed highly significantly less DSB relative to irra- it has also been shown that myosin VI stabilises p53 allowing DNA repair to diated cells cultured alone. Intriguingly, the presence of unirradiated Mia occur before the cell cycle continues. However, very little is known about PaCa-2 cells resulted in a rapid DBS repair in irradiated ATM-/- fibroblasts how Myosin VI may function in the DNA damage response. down to levels of ATMþ/þ fibroblasts within 1 d after irradiation. Addition- We have found cisplatin-induced DNA damage causes a large increase in the ally, when irradiated Mia PaCa-2 cells were co-cultured with ATM-/- nuclear localisation of myosin VI and we wish to understand the reason for fibroblasts or ATMþ/þ fibroblasts pretreated with an ATM inhibitor, the accel- this recruitment. We have begun to understand that myosin VI is required for eration of DNA damage foci was completely abrogated similar to non-co- a normal response to DNA damage by analysing the global changes of chro- culture conditions. Our findings suggest that ATM is a key transducer in mosome arrangement and histone modifications. These chromatin markers cell-cell communication eliciting a rescue of neighboring cells from are directly or indirectly linked with the DNA damage response, and the radiogenic DNA damage through a yet to be identified mechanism. Conse- work presented here describes how myosin VI may be involved in not quently, normal cells support each other in surviving radiogenic DNA only signalling but also repair factories themselves. By using techniques damage, while cancer cells might be prone to develop resistances to current such as high content screening, co-localisation studies and clustering analysis radiochemotherapies. we are beginning to characterise a new role for myosin VI within the nucleus. 387-Pos Dual-Colour Live Cell Single Molecule Imaging Reveals the Dynamics of 390-Pos Nucleotide Excision DNA Repair Complexes in E. coli Molecular Dynamics Simulations Reveal Multiple Roles of Polymerase Alexandra M. Moores, Jingyu Wang, Neil M. Kad. Thumb Domain During Primer Strand Translocation in DNA School of Biosciences, University of Kent, Canterbury, United Kingdom. Polymerase III Bacteria harness multiple repair pathways that locate and correct DNA dam- Thomas W. Dodd, Ivaylo Ivanov. age. Swift and accurate repair of a broad range of DNA damage substrates, Chemistry, Georgia State University, Atlanta, GA, USA. such as ultraviolet (UV) light photoproducts is mediated by the nucleotide DNA polymerase III (PolIII) is a high-fidelity enzyme that can synthesize over excision DNA repair (NER) pathway. The first protein in this pathway, 100,000 basepairs (bp) per binding event, with an error rate of 1 per million. UvrA, cooperates with the second, UvrB, to scan and verify damage in the On the rare occasion that a misincorporation does occur, PolIII utilizes a 3’-5’ genome. UvrA is subsequently released and UvrB recruits UvrC; this endo- exonuclease to remove the misincorporated nucleotide so that synthesis may nuclease nicks the DNA backbone on either side of the lesion. Post-incision, continue. Combined with its exonuclease activity, PolIII’s error rate reaches UvrD removes the oligonucleotide allowing DNA polymerase to mediate 1 per billion. Although recent cryo-EM data has elucidated structural infor- repair synthesis. Recent studies (Hughes et al., 2013; Springall et al, 2018) mation on PolIII during DNA synthesis (polymerase mode) and error correction have identified a novel UvrBC-DNA complex that can locate damage in (editing mode), details on the primer strand separation and its translocation to the absence of UvrA. Here we investigate the existence of various NER com- the exonuclease active site remain elusive. Using novel path-sampling method- plexes using a combination of stroboscopic dual-colour imaging in vitro and ologies we have constructed a transition pathway between the polymerase in live E. coli. Stroboscopic imaging enables the use of high laser power mode and editing mode. Evenly spaced configurations along the transition pulses with short exposure times to visualise single NER molecules binding path were further sampled using molecular dynamics (MD). We then utilized to DNA, while reducing the effects of photobleaching and increasing the res- time-independent component analysis (tICA) to distinguish between the slow- olution of the images. By tracking the location and movement of single- est motions in the transition between polymerase and editing modes. Addition- molecules we determine lifetimes, stoichiometry and alternative complex for- ally, we’ve identified several key intermediate states highlighting crucial roles mations of UvrA, UvrB and UvrC proteins in the presence and absence of of the polymerase thumb domain in primer strand separation and stabilization. UV damage to DNA. Our initial results show that global motions coupled with intimate protein- nucleic acid contacts provide PolIII with a powerful mechanism for the correc- 388-Pos tion of misincorporated nucleotides. Untangling DNA Mismatch Repair Complexes with smFRET and Teth- ered Particle Motion Analysis 391-Pos Pengyu Hao1, Sharonda LeBlanc2, Dorothy A. Erie2, Keith R. Weninger1. Binding Specificity of E. coli SSB C-Terminal Tails to SIPs 1Dept Physics, North Carolina State Univ, Raleigh, NC, USA, 2Dept Min Kyung Shinn1,2, Alexander G. Kozlov2, Timothy M. Lohman2. Chemistry, Univ North Carolina, Cary, NC, USA. 1Department of Physics, Washington University in St. Louis, St Louis, MO, DNA is the genetic information carrier in all organisms and therefore must be USA, 2Department of Biochemistry and Molecular Biophysics, Washington replicated and propagated across generations with high fidelity. Several DNA University School of Medicine, St. Louis, MO, USA. repair pathways correct different types of lesions that arise during DNA repli- One of the roles of the E. coli single-stranded DNA binding (SSB) protein is cation or damage. Among these pathways, DNA mismatch repair proteins to recruit at least 15 different proteins for different aspects of genome main- recognize and activate repair of incorrectly incorporated nucleotides that tenance via the last 9 acidic amino acids (tip) of its intrinsically disordered escape polymerase proofreading. Although intensely studied for decades, de- C-terminal tails. A major question is whether SSB shows any specificity tails of the mechanisms within DNA mismatch repair are still controversial. for these 15 SSB interacting proteins (SIPs). While previous studies have Here we present experiments using single molecule Fluorescence Resonance shown specificity of SSB for E. coli PriA vs. the chi subunit of DNA Pol Energy Transfer and tethered particle motion analysis to provide access to III, we re-examined these and two additional SIPs, E. coli RecO, involved the dynamic and transient interactions of MutS and MutL with DNA mis- in recombination and repair, and PriC, involved in replication restart, using matched bases. Using photocleavable end blocking of DNA, we present an isothermal titration calorimetry. We observe that SSB does display binding assay that can report the mobility of these proteins sliding on DNA. These sorts specificity for these four proteins such that equilibrium binding affinity ranks of single molecule experiments provide insight into the mechanisms that cause as follows: RecO > PriA > PriC > chi under identical solution conditions. activation of DNA mismatch repair processes. We also examined the effect of the length of the intrinsically disordered linker that connects the SSB DNA binding domain to the acidic tip on the 389-Pos binding of the tip to these four SIPs in order to probe for any additional in- Myosin VI and its Role in the DNA Damage Response teractions beyond the 9 amino acids that comprise the SIP interacting region. Alexander Cook1, Yukti Hari Gupta1, Toma´s Venit2, Piergiorgio Percipalle2, The three lengths of the tail examined showed similar binding affinities and Christopher P. Toseland1. enthalpies for each protein, indicating that the interactions between the SIPs 1School of Biosciences, University of Kent, Canterbury, United Kingdom, and the SSB tails are defined mainly by the 9 acidic residues. A temperature- 2Experimental Research, New York University Abu Dhabi, Abu Dhabi, dependent series of studies on E. coli RecO showed its interaction with SSB United Arab Emirates. C-terminal tails to be enthalpically driven (supported by NIH GM030498 to TML).

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Posters: Membrane Physical Chemistry I vesicles (AUVs). To do this, AUVs composed of various lipid compositions were prepared using alpha-cyclodextrin-catalyzed exchange. Both Spin- and 392-Pos NBD-labeled lipids were restricted to either only the inner or only the outer A Calorimetric Study of Brain Cerebrosides in Mixtures with Cholesterol, leaflet. Then the quenching was measured versus temperature to study the for- Brain Ceramide and Phospholipids mation of ordered domains and their melting at high temperature. This method Alicia Alonso1, Emilio Gonza´lez-Ramı´rez2, Goni Felix M1. can detect nanoscopic domains, which cannot be detected by microscopy. Pre- 1Instituto Biofisika (UPV/EHU, CSIC), Leioa, Spain, 2Inst Biofisika, Leioa, liminary results suggest that inner leaflet domain formation is affected by Spain. vesicle curvature, osmotic pressure, and lipid composition. Bovine brain cerebroside (z75% galactosyl) has been studied by differential scanning calorimetry (DSC) in mixtures with cholesterol (Chol) and/or brain 395-Pos ceramide, the latter containing mostly C18:0 fatty acid. Complementing pre- The Role of Ceramide Structure in Regulating the Stability of Membrane vious studies we have focused our attention on mixtures in which the cerebro- Domains side is predominant. Pure, fully hydrated cerebroside exhibits an ordered- Frederick A. Heberle1, Mitchell DiPasquale2, Tye Deering3, Mark Kester3, disordered thermotropic transition centred at 64.8C. In binary mixtures John Katsaras4, Drew Marquardt2. with Chol (up to 30 mol% Chol) DSC reveals good mixing in that range of 1Department of Integrative Biology and Pharmacology, University of Texas concentrations, and a cerebroside behaviour similar to that of sphingomyelin Health Science Center, Houston, TX, USA, 2Department of Chemistry and (SM) or DPPC in the presence of Chol: the transition temperature hardly Biochemistry, University of Windsor, Windsor, ON, Canada, 3Department of changes, but the transition widens until it becomes hardly detectable at 30 Pharmacology, Biomedical Engineering, Molecular Physiology and mol% Chol. Mixtures with 0-40 mol% brain ceramide show also good mixing, Biophysics, University of Virginia, Charlottesville, VA, USA, 4Large Scale with a gradual increase in transition temperature as increasing amounts of cer- Structures Group, Oak Ridge National Laboratory, Oak Ridge, TN, USA. amide are added. Mixtures with phospholipids (brain SM, Tm z 35C, and Ceramides are sphingolipid metabolites that are thought to nucleate lipid egg PC, Tm z-15C ) display a rather similar behaviour, with a progressive domains, forming patches of ordered lipids termed signalosomes that are asso- widening of the endotherm and a decrease in the transition temperature of the ciated with pre-assembled protein signaling molecules. Ceramides are chem- mixture. A ternary system composed of cerebroside:ceramide:Chol (54:23:23 ically diverse, comprising multiple species whose acyl chains vary in length mol ratio) appears to be a homogeneous gel phase with a transition to a fluid and degree of unsaturation. Previous studies using fluorescent reporter mole- phase centred at z 67C. The calorimetric data are supported by Laurdan cules found a dramatic influence of ceramide structure on domain stability measurements of bilayer order as a function of T. Partial phase diagrams of in model membranes. Here, we investigated the ability of ceramide to promote cerebroside:Chol and cerebroside:ceramide have been obtained from the domain formation in the well-characterized benchmark system DPPC/DOPC/ above data. cholesterol using small-angle neutron scattering (SANS). SANS is a particu- larly powerful tool for interrogating membrane structure owing primarily to 393-Pos the unique ability to generate internal contrast between different lipid compo- Direct Imaging of Membrane Domains in Sub-Micron Lipid Vesicles by nents through selective deuteration, thereby avoiding the use of bulky and Cryo-EM potentially perturbing probes. By matching the neutron contrast of the aqueous Caitlin E. Cornell1, Alexander Mileant2, Kelly K. Lee2, Sarah L. Keller1. solvent to that of the average hydrocarbon and headgroup composition, the 1Dept Chemistry, Univ of Washington Seattle, Seattle, WA, USA, 2Dept transverse bilayer scattering contribution that would otherwise dominate the Medicinal Chemistry, Univ of Washington Seattle, Seattle, WA, USA. scattering signal is greatly diminished, enabling detection of lipid demixing To date, the only type of membrane in a living cell that is known to phase (i.e., the segregation of chain perdeuterated DPPC from protiated DOPC separate into large (micron-scale) domains is that of the yeast vacuole. In and cholesterol). We found that replacement of 10 mol% DPPC with the satu- contrast, within plasma membranes, the distribution of lipids and proteins ap- rated chain ceramides C16:0 or C18:0, or 16:0 diacylglycerol (DAG), stabi- pears to be heterogeneous on short time and length scales. In model mem- lized domains to higher temperature. In contrast, domains were destabilized branes containing ternary or quaternary mixtures of lipids, the presence of by ceramides with short saturated chains (C6:0 or C12:0) or a long saturated submicron domains has been inferred by indirect methods such as FRET, chain (C24:0), or by ceramides with an unsaturated chain of any length neutron diffraction, and NMR. In cases when indirect methods rely on [C6:1(3), C6:1(5), C16:1, and C24:1], or by C16:1-DAG. These data suggest model-dependent assumptions, it is powerful to directly image the mem- that a narrow set of ceramide structural features confer the ability to stabilize branes. However, direct imaging of submicron domains presents significant lipid rafts, namely, a fully saturated N-acyl chain of 16-18 carbon length, with experimental challenges. Here, we present two novel cryo electron micro- the nature of the backbone (i.e., sphingosine vs. glycerol) being much less scopy methods to directly image domains in submicron vesicles containing important. ternary and quaternary lipid mixtures. First, we use a probe-free method to image domains that are thicker than the surrounding membrane. To do so, 396-Pos we leverage existing ternary phase diagrams (assessed via fluorescence mi- The Role of Ergosterol in Phase Separation of Yeast Vacuole Membranes croscopy) and AFM data to identify lipid types and ratios likely to produce Chantelle L. Leveille1, Caitlin E. Cornell1, Alexey J. Merz2, thickness mismatches = 1 nm. Second, we label domains in quaternary mix- Sarah L. Keller1. tures of lipids with an electron-dense protein probe, and we image the distri- 1Dept. Chemistry, Univ. of Washington, Seattle, WA, USA, 2Dept. bution of this probe. Biochemistry, Univ. of Washington, Seattle, WA, USA. Membranes of vacuoles, the lysosomal organelle in yeast, phase separate into 394-Pos two coexisting liquid phases. This phenomenon provides a physical mechanism The Influence of Lipid Composition Upon Lipid Domain Formation in the for lateral organization of the membrane in vivo [Toulmay & Prinz 2013, Inner Leaflet of Asymmetric Vesicles Using Spin-Labeled Lipids Rayermann et al. 2017]. The yeast growth cycle follows a characteristic Qing Wang1, Erwin London2. sequence of events: a logarithmic phase of rapid growth, followed by a station- 1Dept Chemistry, Stony Brook Univ, Stony Brook, NY, USA, 2Dept ary phase in which nutrients become scarce and vacuole membranes exhibit co- Biochem. and Cell Biol, State Univ New York Stony Brook, Stony Brook, existing liquid domains. Recent studies show that the resulting domains play a NY, USA. key role in a central eukaryotic signaling pathway. However, how the cell Biological membranes are asymmetric such that lipid compositions in the inner adapts and tunes its vacuole membrane in order to invoke phase separation and outer leaflet of the lipid bilayer are different. While the outer leaflet con- in the stationary phase is still largely unknown. Sterols play a significant role tains lipid-mixtures containing high-Tm (lipid gel-to liquid melting tempera- in phase separation within model membranes, and it has been shown that ture) lipids, low-Tm lipids and cholesterol, which allows ordered domain Ltc, a sterol transport protein, is pivotal for the formation of domains in formation, the inner leaflet is predominantly composed of low-Tm lipids and glucose-starved yeast [Murley et al. 2017]. Here, we directly investigate the cholesterol, which do not form ordered lipid domains spontaneously. However, role of ergosterol, the main sterol in yeast, in domain formation in vacuoles. interleaflet coupling between outer and inner leaflet is one possible mechanism We isolate vacuoles from yeast in the logarithmic phase of growth, in which for ordered lipid domain formation in the inner leaflet of asymmetric vesicles. the membranes do not natively exhibit domains. Using fluorescence micro- In another words, lipid domains in the outer leaflet couple with the inner leaflet scopy, we observe changes in vacuole phase separation as a function of lipids to induced domains in the inner leaflet. Herein, we used the quenching of ergosterol content, which we manipulate by using a known sterol exchanger, NBD-labeled lipids by nitroxide (spin)-labeled lipids and to investigate both in- methyl-b-cyclodextrin. We find the surprising result that domains appear in ner leaflet and outer leaflet domain formation in asymmetric large unilamellar vacuole membranes with reduced ergosterol. We conduct control experiments

BPJ 9310_9313 Sunday, March 3, 2019 79a in model lipid membranes in order to confirm that the methyl-b-cyclodextrin hydration than monohydrates and that the broad peak at 93C corresponds to reduces ergosterol content under our conditions. conglomerated smaller hydrated CHCs. When investigating the cholesterol solubility threshold in phospholipid membranes, it is important to prepare 397-Pos membranes with the RSEM to avoid artifacts (anhydrous CHCs form when Complex Coacervate Formation on a Heterogeneous Membrane FDM is used). DSC measurements showed that only monohydrate CHCs Andrew Balchunas, Sarah Veatch. were formed from cholesterol/phospholipid mixtures, although the transition Biophysics, University of Michigan, Ann Arbor, MI, USA. from monohydrate to anhydrous form shifted and broadened (as compared Mixtures of oppositely charged polymers in salt solutions can undergo liquid- with the transition when CHCs were formed from pure cholesterol) depending liquid phase separation through a process called complex coacervation. on the type of phospholipid used. Polyelectrolyte coacervates are liquid droplets enriched in both polymers and Supporting grants: NIH R01 EY015526 and Polish National Science Center stabilized via electrostatic interactions that occur in three dimensions. Multi- 2016/22/M/NZ1/0187. component lipid membranes can also undergo a liquid-liquid phase separation within the two-dimensional plane of the membrane. Phase separated membrane 400-Pos domains are enriched in particular lipid species and are stabilized via interac- DPPC Lipid Melting Transition in Concentrated Sucrose Solutions tions between lipids that are highly temperature dependent. Here we investigate Mattia I. Morandi, Fabrice J. Thalmann, Monika Kluzek, the coupling of these two distinct phase transitions by tethering polyelectrolytes Andre P. Schroder, Carlos M. Marques. directly to a lipid bilayer. We demonstrate that constraining one polymer to CNRS Institut Charles Sadron, Strasbourg, France. a uniform supported membrane supports polymer coacervation at much lower The properties of lipid bilayers in sucrose solutions have been intensely scru- polymer concentrations than are required to form droplets in three-dimensions, tinized over recent decades because of the importance of sugars in the field consistent with previous reports. We are also investigating coacervate forma- of biopreservation. Here, we present a study on the effect of sucrose on 1,2-di- tion on multicomponent membranes capable of forming liquid-ordered and palmitoyl-sn-glycero-3-phos- phocholine bilayers that combines calorimetry, liquid-disordered phases, and can selectively conjugate polyelectrolytes to spectral fluorimetry, and optical microscopy. Our results show a significant lipids that partition strongly into one phase. Our overall goal of this work is decrease in the transition enthalpy but only a minor shift in the transition tem- to investigate regimes where polymer interactions impact the organization of perature. Our observations can be quantitatively accounted for by a thermody- membrane lipids, or where heterogeneity of membrane lipids contributes to namic model that assumes partial delayed melting induced by sucrose the stabilization of coacervates. Analogous coupling of phase transitions is adsorption at the membrane interface [1]. The influence on melting of short hy- hypothesized to occur in a variety of biological contexts, including at sites of drophobic oligomers inserted into the bilayer will also be discussed, and immunoreceptor activation and neuronal synapses. compared with our results for sucrose. [1] Morandi et al., Biophysical Journal 114, 2165-2173, 2018 398-Pos Characterizing Giant Plasma Membrane Vesicles Isolated from Xenopus 401-Pos laevis Oocytes Effects of Passive Flip-Flop Phospholipid and Asymmetric External Fields Eva S. Chakravorty. on Bilayer Phase Equilibria University of Michigan, Ann Arbor, MI, USA. Peter Olmsted1, John Joseph Williamson2. Isolation and investigation of giant plasma membrane vesicles (GPMVs) from 1Dept Physics, Georgetown Univ, Washington, DC, USA, 2Francis Crick mammalian cells has proven a useful tool to characterize the thermodynamic Institute, London, United Kingdom. mixing properties inherent to cellular plasma membranes. This poster will pre- Compositional asymmetry between the leaflets of bilayer membranes mod- sent our recent efforts to isolate and examine giant vesicles from Xenopus lae- ifies their phase behaviour, and is thought to influence other important fea- vis oocytes following a previously published protocol. GPMV isolation from tures such as mechanical properties and protein activity. We address here oocytes requires the removal of the vitelline membrane, which can be accom- how phase behaviour is affected by passive phospholipid flip-flop,such plished using enzymatic or mechanical strategies, and the advantages and dis- that the compositional asymmetry is not fixed. We predict transitions from advantages of both will be discussed. The resulting oocytes are extremely ‘‘pre flip-flop’’ behaviour to a restricted set of phase equilibria that can fragile, and sometimes GPMV preparations are contaminated by oil droplets persist in the presence of passive flip-flop. Surprisingly, such states are not that form after the rupture of oocyte yolks. The overall goal of this work is necessarily symmetric. We further account for external symmetry- breaking, to characterize the mixing behavior of vesicles isolated from Xenopus laevis such as a preferential substrate interaction, and show how this can stabilise oocytes in order to better interpret the impact of membrane domains on chan- strongly asymmetric equilibrium states. Our theory explains several experi- nels expressed and interrogated in these membranes. A second goal of these mental observations of flip-flop mediated changes in phase behaviour, measurements is to develop an experimental system whereby expressed chan- and shows how domain formation and compositional asymmetry can be nels partition into vesicles, since this does not readily occur in mammalian sys- controlled in concert, by manipulating passive flip-flop rates and applying tems for several channels and cell systems previously investigated in the external fields. laboratory. Kinetics of the competition between domain symmetry and asymmetry in a phase-separating bilayer membrane, J. J. Williamson and P. D. Olmsted, Phys- 399-Pos ical Review E 92 (2015) 052721. Phase Behavior of Cholesterol Crystals Formed in Water from Pure Registered and antiregistered phase separation of mixed amphiphilic bila- Cholesterol and from Cholesterol/Phospholipid Mixtures yers, J. J. Williamson and P. D.Olmsted, Biophysical Journal 108 (2015) Laxman Mainali1, Marta Pasenkiewicz-Gierula2, Witold Subczynski1. 1963-1976 1Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA, 2Department of Biophysics, Jagiellonian University, Krakow, Poland. 402-Pos Cholesterol is an indispensable component of animal cell membranes. It also Investigating Single-Cell Variation in Membrane Fluidity and Respiration participates in pathologic processes that involve the formation of cholesterol Rates crystals (CHCs). Cholesterol is an amphiphilic molecule with a very low mono- Krishna Ojha, Sam Blechman, Joshua Kasburg, Michael C. Konopka. meric solubility in water, where it readily self-associates into organized struc- Dept Chemistry, Univ Akron, Akron, OH, USA. tures. There is experimental evidence that the final form of cholesterol Within a bacterial population, there is significant cell-to-cell variation in mem- aggregates in water is monohydrate CHCs. Here, we used differential scanning brane fluidity. Measuring the diffusion of a novel hydroxy benzothiozol pyridi- calorimetry (DSC) to show that the type of CHCs formed in water from pure nium cyanine in the bacteria cytoplasmic membrane by Fluorescence Recovery cholesterol depend on the method of preparation. Both anhydrous and monohy- After Photobleaching (FRAP), we can assess the membrane fluidity in individ- drate CHCs were formed with the film deposition method (FDM), showing ual cells. Using fluorescence reporters of key genes in the type II fatty acid syn- transitions at 41oC (between two anhydrous CHCs) and at 72oC (conversion thesis pathway, like fabA and fabB, and mutants affecting lipid composition of of monohydrate to anhydrous CHCs), respectively. Only one transition was the membrane, we demonstrate that fluidity is affected by the amount of unsat- observed when the rapid solvent exchange method (RSEM) was used, with urated lipids in the membrane. To investigate if membrane fluidity of individual two clear peaks at 93oC and 95oC, i.e., higher than the 72C of monohy- cells is contributing to the large cell-to-cell variation in respiration rates in bac- drate CHCs. The broader peak at 93oC disappeared when the cholesterol teria, we couple these measurements with a microscope-based single-cell respi- concentration was decreased. After the first and second heating, both peaks dis- ration rate measurement. It utilizes a phosphorescent Pt-porphyrin dye as appeared and a pronounced peak corresponding to anhydrous CHCs appeared. the oxygen sensor since the lifetime of the dye’s excited state depends on We hypothesize that peaks at 93C and 95C represent CHCs with higher the oxygen concentration in the sample. Microspheres embedded with the

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Pt-porphyrin are loaded into microwells on a glass chip designed to isolate 406-Pos single cells in diffusionally sealed chambers. Monitoring the consumption Temperature-Controlled Single-Liposome Proton Permeability Assay of of oxygen in the sealed microwells over time allows a respiration rate to be Extremophile-Inspired Lipid Membranes calculated for the individual cells and related back to either gene expression Anirvan Guha1, Melissa McGuire1, Thomas B.H. Schroeder2, in the type II fatty acid synthesis pathway or direct membrane fluidity Geoffray Leriche3, Jerry Yang3, Michael Mayer1. measurements. 1Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland, 2Harvard University, Cambridge, MA, USA, 3University of California, 403-Pos San Diego, San Diego, CA, USA. Preparation of Asymmetric Charged Large Unilamellar Vesicles Contain- Thermoacidophilic archaea are able to survive in low pH environments by ing Both Cationic and Anionic Lipids maintaining large proton gradients across their cell membranes. The unique Bingchen Li1, Erwin London2. structure of the unipolar and bipolar lipids that compose these membranes, 1Department of Chemistry, Stony Brook University, Stony Brook, NY, USA, 2 with branched isoprene chains and ether linkages, may contribute to their Department of Biochemistry and Cell Biology, Stony Brook University, reduced permeability to protons. Here we present a proton permeability assay Stony Brook, NY, USA. that interrogates hundreds of surface-tethered single liposomes in parallel using Liposomes have important applications for drug delivery. Whether asym- total internal reflection fluorescence (TIRF) microscopy and determines a pro- metric large unilamellar vesicles (asymmetric LUVs) with different charges ton permeability value for each. The large sample sizes afforded by this micro- on different leaflets can be efficiently prepared and have practical applications scopy platform allow for the collection of large quantities of data within a have advantages for efficient drug encapsulation and efficiency has not been single experiment, as opposed to the single, averaged result produced by a explored. To examine this we have developed methods to prepare asymmetric bulk fluorimetry assay. In addition, tethering individual liposomes to a surface LUV with opposite charges on the inner and outer leaflet. We investigated li- prevents liposome aggregation in free solution, which is especially prevalent posomes mixtures containing one of three different kinds of negative charged when performing studies at elevated temperatures. Using a temperature- lipids, POPG, POPS and POPA, one of two different kinds of positive charged controlled chamber, we evaluate the temperature-dependence of proton perme- lipids, POePC and DOTAP, and a zwitterionic lipid, POPC. Using the cyclo- ability for both control and archaea-mimetic liposomes. We also discuss at- dextrin (CD) assisted exchange methods developed by our group, we have tempts to study the proton permeability of liposomes made from natural prepared asymmetric LUVs with about 25 % of POPS or POPA in the outer archaea lipid extract. or inner leaflet (monolayer) and about 25 % of POePC or DOTAP on the opposite leaflet, with about 75 % of lipids in each leaflets being POPC. Ex- 407-Pos change with POPG was somewhat less efficient. To investigate asymmetry Application of Long-Term Air-Stable Lipid Bilayers for Waveguide-Based of the LUVs a TMA-DPH binding assay was developed. Future studies will Biosensors investigate drug entrapment ability and the efficiency of drug delivery using Christine Pedersen1, Aaron Anderson1, Harshini Mukundan2, these liposomes. Jessica Kubicek-Sutherland1. 1Chemistry, Los Alamos National Laboratory, Los Alamos, NM, USA. 404-Pos 2 A New Method to Prepare Asymmetric Unilamellar Vesicles: Hemifusion Los Alamos National Laboratory, Los Alamos, NM, USA. Thais A. Enoki, Gerald W. Feigenson. The Los Alamos National Laboratory Biosensor team strives to detect patho- Dept Molec Biol & Gen, Cornell University, Ithaca, NY, USA. gens for rapid diagnosis. Our waveguide based optical biosensor utilizes its sur- We report a new method to make asymmetric giant unilamellar vesicles face coating for allowing specific attachment of various recognition elements. (aGUVs) using hemifusion. Our method does not use intermediate steps that This surface coating is created by a lipid bilayer, which has an essential compo- involve organic solvents or cyclodextrin. We monitor the hemifusion of a sup- nent of fluidity needed for our assays. Lipid bilayers can only tolerate limited ported lipid bilayer (SLB) and GUVs. Hemifusion consists in the fusion of the handling and environmental conditions, thus introducing constrains of struc- SLB and GUV outer leaflets. Here, hemifusion is triggered by calcium. After tural instability and making long-term storage impossible. In order to overcome the chelation of this cation, we detach GUVs from the SLB and study unsup- these limitations, our team is working to create stable surfaces by utilizing suit- ported vesicles. We use different fluorescent probes to label the inner and the able bilayer precursors with phospholipid DOPC and photoactive lipids Diyne- outer leaflet.First, we stablish the procedure using asymmetric labeled GUVs PC and Diyne-PE. In changing different variables such as surface patterning composed of DOPC in both leaflets. Then, we prepare aGUVs from the hemi- with photolithography and addition of cholesterol, enhancing the bilayer stabil- fusion of GUVs composed of DSPC/DOPC/chol = 0.39/0.39/0.22 and a sup- ity may be possible. This presentation will describe some of these efforts and ported DOPC bilayer. We emphasize the importance of distinguishing levels present data to show micelle formation and fluidity using fluorescent recovery of asymmetry. For highly asymmetric GUVs, the outer leaflet forms a single after photobleaching (FRAP) and dynamic light scattering (DLS) for a func- phase. The phase-separated inner leaflet having coexisting liquid-disordered tional comparison of the different preparations of lipids. Further understanding (Ld) and liquid-ordered (Lo) phases induces different lipid packing/ order in of these lipid bilayers will pave a way for an affordable and deployable sensor the outer leaflet. However, the apparent partition coefficient of a probe in element for broadly applicable strategy or biodetection. this order-induced leaflet isfound to be about 1/3 smaller than the partition co- efficient of a that probe between Ld þ Lo phases of a symmetric vesicle. 408-Pos Nesting Lipid Bilayers in Nanopores: Effect of Pore Diameter on Macro- 405-Pos scopic Order and the Layer Count Toward Realistic Cell Membrane Mimics Morteza Jafarabadi, Melanie Chestnut, Antonin Marek, Alexander Nevzorov, Peter Beltramo. Alex I. Smirnov. Chemical Engineering, UMass-Amherst, Amherst, MA, USA. Dept Chemistry, North Carolina State Univ, Raleigh, NC, USA. To understand biological processes, there is a need to develop experimental Nanotubular lipid bilayer membranes formed by self-assembly and confined by techniques to faithfully recreate model cell membranes, measure their physico- macroscopically aligned nanopores exhibit several attractive features for chemical properties, and mimic the essential physics of different phenomena. biophysics and bio-nanotechnology. When fully hydrated such bilayers systems Towards that end, we have developed a microfluidic technique based on a are stable for at least a month and allow for a quick exchange of buffers and thin film balance in order to create large area model biomembranes (LAMBs). other water-soluble molecules. Even more importantly the bilayers have We will present recent experiments developing this platform and illustrate its well-defined curvature and are macroscopically aligned over an exceptionally ability to quantify membrane tension, elasticity, and modulus. Simultaneous broad range of temperature, pH, and ionic strength. The latter feature facilitates capacitance and optical microscopy measurements are used to confirm the for- studies of membrane and membrane proteins under close to native cellular mation of a nearly solvent-free membrane and measure membrane electrome- membrane conditions by solid-state NMR and other spectroscopic methods. chanical properties, with an emphasis on the importance of the choice of oil Here we report on the effect of nanoscale pore diameter on packing of lipid bi- solvent. Since biological membranes have a diverse phospholipid profile and layers and macroscopic alignment of POPC (2-oleoyl-1-palmitoyl-sn-glycero- contain membrane proteins, we show that the technique can successfully recon- 3-phosphocholine) lipids inside the pores. We show that the pores from 36 to stitute membranes with binary composition mixtures, and show the capability 44, 50 and 72 nm in diameter accommodate progressively larger number of for model membrane proteins, specifically a-hemolysin and alamethicin, to be nesting lipid bilayers – from up to two nanotubes for the smallest 36 nm incorporated into the formed bilayers and measure ion transport. With these pore to about four for 50 nm wide pore at 40 oC. This is consistent with capabilities in hand, the platform may now be applied to understand more com- 26-27 nm being the smallest diameter for the POPC nanotubes. Static 31P line- plex membrane transport phenomena, such as membrane fusion and lipid- width at 300 MHz 1H frequency indicated the best macroscopic alignment cor- membrane interactions. responding to just 1-3% of mosaic spread for either one or two nesting lipid

BPJ 9310_9313 Sunday, March 3, 2019 81a nanotubes for all pore sizes. 31P linewidth and bilayer macroscopic alignment directly depend on the separation between both substituent groups. LAURDAN degraded notably for three nesting bilayers formed in the nanopores and even have been used in studies involving lipids in vitro, in vivo, in cuvette and mi- more for four. The latter is consistent with a formation of wavy lipid tubules croscope, where the normal excitation in cuvette studies is in the UV (360nm). previously reported for much larger 140-180 nm nanopores. Supported by For microscopy studies, a two photon excittaion is needed since one photon DE-FG02-02ER15354 to AIS. excitation produce a severe photobleacing of the probe. In order to maintain the environmental sensitivity of LAURDAN but to change the a excitation 409-Pos and emission spectra (bathochromic shift), we designed and sinthetized a Effect of Silica Support on Electrostatics of Lipid Interfaces in Nano-Bio new molecule were the napthalic group of LAURDAN was replaced by an Hybrid Systems anthracene group, increasing the possibility of aromatic delocalization.[2] Erkang Ou, Maxim Voinov, Alex Irving, Alex Smirnov, The photophysical behaviour of Capyrdaa in homogeneous media showed a Tatyana I. Smirnova. dependence with the solvent. The batochromic shift of emission spectra is Department of Chemistry, North Carolina State University, Raleigh, observed with increase in solvent polarity. Moreover, decrease in fluorescence NC, USA. quatum yield with solvent polarity is also observed. However, not significant Design of new bio-nano hybrid systems calls for understanding and accounting changes are observed in absorbance and lifetime with solvent. Fluorescence mi- for the influence of a nanostructured support and nanoconfinement on the struc- croscopy studies with mono and biphotonic excitation were done in synthetic ture and biophysical properties of lipid bilayer hybrid systems and membrane- microheterogeneous systems and in cells, showing that Caprydaa may be protein interface. Here we report on spin-labeling EPR studies of pH-sensitive a good alternative to study membrane fluidity both in vitro and in vivo. lipids and specifically labeled protein side chains to assess effects of solid inor- [1] G. Weber, F.J. Farris, Biochem., 18 (1979) 3075-3078 [2] Z. Lu, et al, J ganic interface, specifically, silica support in a form of monodispersed nanopar- Org Chem, 71 (2006) 9651-9657. ticles ranging from 20 to 300 nm in diameter on the surface electrostatic potential of lipid bilayers associated with the particles and effective pKa of 412-Pos the membrane-burred peptide ionisable sidechains. We have shown that bila- The Role of Packing Density on Fluorescence Intensity Measurements of yers formed from zwitterionic or mixed lipids on silica nanoparticle surfaces Common Fluorophores in Lipid Monolayers possess a higher negative electrostatic potential than the unsupported bilayers Benjamin L. Stottrup1, Dametre Thunberg2, Joan C. Kunz3. with the potential of mixed bilayers containing negatively charged lipids being 1Dept. of Physics, Augsburg Univeristy, Minneapolis, MN, USA, 2Augsburg significantly more sensitive to the silica support. Effect the silica nanoparticle Univeristy, Minneapolis, MN, USA, 3Dept. of Chemistry, Augsburg size on the lipid bilayer surface electrostatic potential was also observed for Univeristy, Minneapolis, MN, USA. particles smaller than 100 nm. pH-sensitive EPR probes were then employed Fluorescent probes are used in a wide range of experimental contexts to eluci- to label model WALP peptide known to form an a-helix when integrated date the behavior of model lipid membranes. These molecular markers have into a lipid bilayer. The silica support exerted pronounced effects on WALP dy- been successfully used to study phase transitions and critical behavior in namics and the effective pKa of the ionizable probe. It was demonstrated that both lipid monolayers and bilayers as well as micron scale domain shape and the silica nanoparticles shift the effective pKa of the ionizable nitroxide probe packing. Most often, in these experiments the fluorophores provide a binary in a membrane depth-dependent manner. Upon protonation of the membrane- contrast between two lipid environments. However, measurements of fluores- burred model ionisable sidechain the silica support caused significant changes cent intensity in itself may provide new insights and thus far has been an in the membrane association of WALP peptide that are not observed when underutilized tool in Langmuir monolayer studies. We present a series of exper- WALP is integrated into unilamellar phospholipid vesicles of similar curvature. iments measuring how fluorophore density in Langmuir monolayers modulates Supported by NSF 1508607. fluorescence intensity. Image analysis correlated to pressure-area isotherms will provide a baseline measurement for future fluorescence intensity studies. 410-Pos Imaging of Lipid Metabolism through a Phasor Analysis of Membrane Mi- 413-Pos cropolarity Itraconazole Perturbs Behavior of Fluorescent Probes in Lipid Bilayer Flavio Di Giacinto, Marco De Spirito, Giuseppe Maulucci. Chetan Poojari1, Natalia Wilkosz2, Piotr Jurkiewicz3, Ilpo Vattulainen1, Istituto di fisica, Universita´ Cattolica del Sacro Cuore, Roma, Italy. Mariusz Kepczynski2, Tomasz Rog1. Lipid metabolism is characterized by a complex set of sequential reactions 1Physics, The University of Helsinki, Helsinki, Finland, 2Chemistry, which underpin many biological processes, including the maintenance of the Jagiellonian University, Krakow, Poland, 3J H Inst Phys Chem, Prague 8, cellular energy balance and the intercellular communication through the so- Czech Republic. called lipid signalling. The lipid composition and distribution within cells 1,6-diphenyl-1,3,5-hexatriene (DPH) is a frequently used fluorescence probe to change following chemical and physical interactions with the surrounding envi- study the ordering and mobility of the lipids bilayer. Here we present our ronment in response to specific needs or conditions, as is the case of the acti- studies combining molecular dynamics (MD) simulations and fluorescence vation of lipolysis or storage pathways. The method here presented allows anisotropy measurements in the lipid bilayer with antifungal drug Itraconazole characterizing some of these processes through the evaluation of the micro- (ITZ) added. ITZ is long, rigid, amphipathic molecule locating in the lipid environment polarity within the cell membranes with a high spatial and tempo- bilayer below the water-membrane interface and adopting orientation parallel ral resolution. This is achieved by using an algorithm based on the phasor to the membrane surface [1]. MD simulations showed that ITZ increases the approach to the spectral imaging. As a result, a metabolic parameter is fur- order of the hydrocarbon chains in their upper part and slightly decrease order nished to enable a quantitative assessment of the storage of fatty acids in tria- of the lower part of the chain while steady-state fluorescence anisotropy mea- cylglycerols in the form of lipid droplets or the triacylglycerols hydrolysis in surement indicated substantial disordering effect of the ITZ. MD simulations of response to energetic demands, as well as changes in the global and local dis- the bilayer with both ITZ and DPH showed that DPH behavior in this bilayer is tribution of polar and non-polar lipids with a sub-micrometre resolution. The determined by ITZ and not by lipids chains as assumed in traditional interpre- method has been applied both to tissues and cell cultures to study the reaction tation - DPH locates below ITZ thus its position is shifted towards bilayer cen- of the systems to lipid metabolism disorders or nutritional overload and starva- ter and adopt orientation parallel to the membrane surface which likely results tion, which is becoming increasingly central in the study of many kinds of from interactions with ITZ. These results show the limitation of the DPH in metabolic, cardiovascular and neurodegenerative diseases. studies of lipid bilayers as DPH might report the behavior of, e.g. ITZ like mol- ecules instead of lipids hydrocarbon chains. 411-Pos Photophysical Characterization and Microscopy Application of an 414-Pos Anthracene Analogous of Laurdan Excitation of Fluorescent Lipid Probes Accelerates Phospholipid Vesicle German Gunther, Javier Gajardo, Vicente Castro, Catalina Sandoval, Rupture and Supported Lipid Bilayer Formation Susana A. Sanchez, Leonel Malacrida. Ashley M. Baxter, Nathan J. Wittenberg. Quimica Organica y Fisico Quim, Univ Chile, Santiago, Chile. Department of Chemistry, Lehigh University, Bethlehem, PA, USA. The series of polarity sensitive fluorescent probes designed and synthesized by Lipid bilayer membranes are readily rendered fluorescent by incorporating Gregorio Weber in the eighties, correspond to a naphthalene aromatic moiety, fluorescently-conjugated phospholipid probes. Previous work has showed that substituted with a dimethyl amino group in position two (donor group) and excitation of fluorescent lipid probes in isolated secretory vesicles resulted in an alkyl carbonyl group in position 6 (acceptor group). In general terms, a fluo- a higher frequency of vesicle rupture on carbon electrodes. Here we examine rescent enviromental sensor must have in its structure two different substitu- the influence of excitation of fluorescently-conjugated phospholipids on the ents, with electron-acceptor an electron-donnor character, respectivelly, both rupture and supported lipid bilayer (SLB) formation processes for phospholipid attached to an aromatic ring.[1]. The magnitude of the sensitive response would vesicles on SiO2. Using quartz crystal microbalance (QCM-D), we find that

BPJ 9310_9313 82a Sunday, March 3, 2019 illumination of vesicles containing BODIPY-conjugated lipid fluorophores spherical aggregates to rod like fibrillar aggregates. In summary, the aggregation significantly accelerates the rupture and SLB formation process. Tail group- of Dox in microheterogeneous medium of C8mimCl SAIL to spherical vesicular conjugated BODIPY groups have a more pronounced effect than head group- aggregates and also the bile salt induced disruption into rod like fibrillar aggre- conjugated BODIPYs. Other commonly used fluorophores, such as NBD and gates were studied in detail. The formation and disruption of Dox forming vesic- Texas Red do not cause illumination-induced acceleration regardless of the fluo- ular aggregates can be modeled as a new generation future delivery system for rescent group position. The accelerating effect of BODIPY is dependent on un- biologically important molecules with smart loading and release phenomena saturation in the acyl chains of the background phospholipid. Vesicles composed where the biomolecule itself aggregates to form the delivery system. of saturated phospholipids (diphytanoyl-PC) are not susceptible to the influence of BODIPY-conjugates in the presence of illumination. This suggests that the ac- 417-Pos celeration of SLB formation in the presence of light may be mediated by lipid Biological Membrane Solubilization by Styrene-Maleic Acid Copolymers: oxidation. To test this, we included an antioxidant (alpha-tocopherol) in the ves- Importance of Polymer Length 1 1 2 1 icles. When alpha-tocopherol is present, the acceleratory effect of BODIPY and Adrian H. Kopf , Min Xie , Randy Cunningham , Martijn C. Koorengevel , 1 1 2 2 light is significantly reduced. Using QCM-D we measured the mass of the SLB Helene Jahn , Jonas M. Do¨rr , Rueben Pfukwa , Bert Klumperman , Antoinette J. Killian1. formed by vesicle rupture. After illumination, SLBs formed from unsaturated 1 2 lipids and BODIPY-conjugated probes are ‘‘lighter’’ than those without illumi- Utrecht University, Utrecht, Netherlands, Stellenbosch University, nation. Zeta potential measurements show that illumination of these vesicles Stellenbosch, South Africa. causes them to become more negatively charged, and DLS measurements Styrene-maleic acid (SMA) copolymers are excellent alternatives to detergents show that illumination causes the vesicles to shrink in size. This suggests photo- for the extraction of membrane proteins from cellular membranes. The copol- chemical reactions are altering the vesicle composition. Together our results ymers are able to solubilize membranes in the form of native nanodiscs which show that excitation of BODIPY-lipid conjugates can significantly alter mem- are stabilized by the copolymer, allowing for the characterization of membrane brane properties, perhaps due to photosensitized lipid oxidation. proteins in their native lipid environment. Commercially available SMA copol- ymers are polydisperse, having large variations in their length distribution. Us- 415-Pos ing synthetic lipid model systems in conjunction with copolymers having more Concentration-Controlled Fascinating Vesicle-Fibril Transformation us- defined length distributions it has been observed that polymer length is an ing Merocyanine 540 and 1-Octyl-3-Methylimidazolium Chloride important parameter in determining both the kinetics of solubilization as well Rupam Dutta, Arghajit Pyne, Sangita Kundu, Pavel Banerjee, as the properties of the resulting nanodiscs [1]. Here we investigated whether Nilmoni Sarkar. the observed role of polymer length also translates to biological membranes, Chemistry, IIT Kharagpur, Kharagpur, India. comprising a rich diversity of lipids as well as proteins. Interestingly, in In this work, we have constructed highly ordered fibrillar and vesicular aggre- contrast to synthetic model lipid systems where the smallest polymer is the gates employing an anionic, lipophilic dye Merocyanine 540 (MC540) and most efficient solubilizer, biomembranes seem to have an optimum length cationic surface active ionic liquid (SAIL), 1-octyl-3-methylimidazolium chlo- that is somewhat larger. In addition to polymer length, several other environ- ride (C8mimCl) with the aid of ionic self-assembly (ISA) strategy. It is impor- mental factors were explored. Different sources of biomembranes were tested tant to note that the concentration of the counterions has predominant role on together with different SMA copolymers having better defined length distribu- the morphology, as on lowering the concentration of both the building blocks tions. The results of these experiments will be shown and the effects will be at stoichiometric ratio 1:1, provides vesicle to fibril transition. Here, we have compared with those of commercially available SMA copolymers. The study shown a concentration-controlled fibril-vesicle transition utilizing the emerging sheds light on the process of biomembrane solubilization and helps in deter- fluorescence lifetime imaging microscopy (FLIM) technique. Moreover to mining optimal copolymer properties and conditions. [1] Juan J. Dominguez gain additional support, we have detected this morphological transformation Pardo et al., Biophysical Journal, 115, 129-138, (2018). by means of other microscopic techniques like field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM) and cryogenic-transmission electron microscopy (cryo-TEM). We have replaced Posters: Membrane Active Peptides and Toxins I the SAIL, C8mimCl by analogous traditional surfactant, n-octyltrimethylam- monium bromide (OTAB) and interestingly it exhibits a discernible change 418-Pos Characterization of Hybrids Made from Two Membrane Translocating in morphology similar to that of C8mimCl, whereas 1-octanol is unable to exhibit any structural aggregate and thus reveals the importance of electrostatic Antimicrobial Peptides Ju Young Kwag1, Hannah Klim2, Donald E. Elmore3. interaction in the supramolecular aggregate formation. On the otherhand, the 1 SAILs having the same imidazolium head group with different chain lengths Department of Chemistry, Wellesley College, Wellesley, MA, USA, 2Biochemistry Program, Wellesley College, Wellesley, MA, USA, other than C8mimCl are unable to display any structural transition and conclude 3 the importance of correct chain length for efficient packing of the counterions Department of Chemistry and Biochemistry Program, Wellesley College, to form a specific self-assembly. Our study reveals the synergic interplay of Wellesley, MA, USA. electrostatic, hydrophobic and p-p stacking interactions in the construction of Antimicrobial peptides (AMPs) are a form of innate immunity in many organ- self-assembly and their concentration dependent morphological conversion. isms. They are promising substitutes for current antibiotics against which an increasing number of bacteria are becoming resistant. AMPs can form hybrids 416-Pos in which sequences of peptides are linked together by one or more amino acids, A Detailed Microscopic Insight into the Aggregation Behaviors of Doxoru- and these hybrids can potentially have increased activity. Most previous work bicin Hydrochloride in Different Microheterogeneous Media on hybrids AMPs has involved peptides that kill bacteria by disrupting the cell Arghajit Pyne, Sangita Kundu, Pavel Banerjee, Nilmoni Sarkar. membrane. However, here we focus on hybrid peptides that are synthesized Chemistry, IIT Kharagpur, Kharagpur, India. from two peptides that translocate across the membrane without causing signif- In recent time periods, a large portion of research interests include the study of icant membrane disruption—the naturally occurring peptide buforin II (BF2) aggregation properties of different amphiphilic molecules and the resulting and the designed peptide DesHDAP1, both of which are derived from the morphological architectures due to their significant applications in diverse core histone H2A. These peptides cross the cell membrane without disruption research fields. The aggregation behaviors of different biomolecules are also and bind to the target’s nucleic acids to inhibit cellular function, killing the bac- very much important and thus recently become the cup of tea to a large number terium. We designed a series of BF2-DesHDAP1 hybrids aimed at systemati- of research communities. In this respect, the aggregation result of potentially cally investigating the role of the sequence order and linkers on activity. important chemotherapeutic molecule, doxorubicin hydrochloride (Dox) in a Radial diffusion assays showed that although hybrid peptides with no linker conventional imidazolium based surface active ionic liquid (SAIL), 1-octyl-3- or an alanine linker show little or no enhancement in activity compared to a methylimidazolium chloride (C8mimCl) was studied in details. The complete mixture of the parent peptides, hybrid peptides with proline linkers were signif- characterizations using dynamic light scattering (DLS), different microscopic icantly more active. Interestingly, other bacterial assays showed that these techniques like fluorescence lifetime imaging microscopy (FLIM), transmission hybrid peptides operate via a membrane-permeabilizing mechanism despite be- electron microscopy (HR-TEM and analytical TEM), field emission scanning ing derived from two peptides that cause little disruption on their own. We have electron microscopy (FESEM) and atomic force microscopy (AFM) with also used circular dichroism (CD) spectroscopy to investigate how these activ- fourier-transform infrared spectroscopy (FTIR) measurements revealed the for- ity trends relate to structure, finding that hybrids linked by proline actually ex- mation of large spherical vesicular Dox/C8mimCl aggregates. The guiding forces hibited increased helical secondary structure. Future work will utilize confocal behind such SAIL induced aggregation of Dox were also explored. Finally a com- microscopy to further investigate mechanisms of the hybrid peptides and test mon bile salt, sodium cholate (NaCh) was observed to transform Dox/C8mimCl their cytotoxicity against human cells.

BPJ 9310_9313 Sunday, March 3, 2019 83a

419-Pos mune signaling proteins able to induce chemotaxis of immune cells to the Mechanism of Action of pH-Triggered, Membrane Active Peptides site of infection. In addition to signaling functions, the chemokine CXCL10 Sarah Y. Kim1, Anna Pittman2, Gavin King3, William C. Wimley4, exhibits direct antimicrobial activity. This project’s goal is to elucidate the Kalina Hristova5. mechanism by which CXCL10 kills bacteria and create one or several new 1Dept Molec Biophys, Johns Hopkins Univ, Baltimore, MD, USA, 2Univ antibiotics derived from CXCL10 by optimizing this antimicrobial behavior. Missouri, Columbia, MO, USA, 3Dept Phys/Biochem, Univ Missouri, To discover the molecular determinants responsible for antimicrobial activity, Columbia, MO, USA, 4Dept Biochem, Tulane Sch Med, New Orleans, our collaborative group synthesized a peptide library derived from CXCL10 LA, USA, 5Dept Matl Sci/Eng, Johns Hopkins Univ, Baltimore, MD, USA. and screened these peptides for bactericidal activity. Peptides found to The plasma membrane insulates a cell from its outside environment, serving as a comprise broad-spectrum antimicrobial activity represented the N- and C-ter- selectively impermeable barrier across which only small or hydrophobic mole- minal regions of CXCL10. These peptides were selected for further studies, cules can pass. Although the membrane is necessary for life, it is also problematic along with a non-antimicrobial peptide as a negative control. As other antimi- when useful macromolecules such as antibodies, peptides, polysaccharides, and crobial peptides are known to form an alpha helix, we used circular dichroism imaging agents are blocked from entry. Most macromolecules can easily be up- spectroscopy to assay the ability of the three CXCL10-derived peptides to taken by the cell throughendocytosis, butremain trapped and eventually degraded adopt a similar conformation in the presence of a Gram-negative bacterial within endosomes, which mature into lysosomes. To promote the escape of mac- membrane mimetic. Surprisingly, while both antimicrobial peptides became romolecules from endosomes prior to their maturation into lysosomes, we used a more ordered in the presence of the bacterial membrane mimetic, only the high throughput screen to discover pH triggered, pore-forming peptides[1]. To spectra of the C-terminal peptide showed features of an alpha helix. To deter- determine their mechanism of action, we measured the peptides’ membrane bind- mine the potential compatibility of these peptides with human cells, in hopes ing affinity, secondary structure, and induction of pore-formation in POPC mem- of developing a novel antimicrobial therapeutic, we assessed the ability of the branes. We identified that at least 5 acidic residues are essential for mediating a peptides to lyse human red blood cells via a hemolysis assay. Even at concen- change from a soluble, predominantly unfolded, and inactive state at pH 7 to a trations many fold higher than those required for bacterial killing, CXCL10- membrane bound, helical, and active state at pH 5. These peptides are highly derived peptides showed minimal, statistically insignificant hemolysis. These potent, with significant macromolecular leakage occurring at concentrations as observations are encouraging for further development of antimicrobial thera- low as 2 peptides per 1000 lipids. We determined that the peptides behave dynam- peutics based on CXCL10. ically, associating with and dissociating from membranes, and consequently form pores on multiple vesicles. By atomic force microscopy imaging, we confirmed 422-Pos that the peptides form macromolecular pores, with diameters as large as 50 nm. Characterization of a Histidine Containing Antimicrobial Peptide with pH Furthermore, we developed a kinetic model to explain how a low net fraction Dependent Activity of bound peptide can lead to significant leakage. This work yields biophysical Luis Santiago-Ortiz, Morgan Hitchner, Thaddeus Palmer, insights that will improve the design of peptides for new biomedical applica- Gregory A. Caputo. tions.[1]Wiedman, G., Kim, S.Y., Zapata-Mercado, E., Wimley, W.C. and Department of Chemistry and Biochemistry, Rowan University, Glassboro, Hristova, K., 2017. J. Am. Chem. Soc., 139(2), pp.937-945. NJ, USA. Antimicrobial peptides (AMPs) have been an area of great interest, due to the 420-Pos high selectivity of these molecules toward bacterial targets over host cells and Discovering Novel Antimicrobial Peptides using High-Throughput the limited development of bacterial resistance to these molecules throughout Screening and Rational Variation evolution. The peptides are known to selectively bind to bacterial cell surfaces Jenisha Ghimire, Charles G. Starr, William C. Wimley. through electrostatic interactions as the cell surface is net negative charged Department of Biochemistry and Molecular Biology, Tulane University while the peptides are positively charged. Once bound, the peptides insert School of Medicine, New Orleans, LA, USA. into the cell membrane and cause local disruptions of membrane integrity lead- Antimicrobial peptides (AMPs) have long been attractive drug candidates for ing to cell death. Previous experiments showed that when Histidine was incor- the next generation of clinical antibiotics due to their potent antimicrobial ac- porated into the peptide C18G it lost all antimicrobial activity. Due to the side tivity and low propensity for inducing resistance in pathogens. However, due in chain pKa is near physiological pH, we wanted to investigate the role of pH on part to toxicity concerns and activity loss in vivo, AMPs have yet to have any the activity of the peptide. MIC results demonstrated that decreased media pH impact clinically. Our lab hypothesized that the presence of host cells could increased antimicrobial activity. Intrinsic Trp fluorescence was used to perform cause depletion of free peptide available to target bacterial cells and showed binding assays to model lipid vesicles under different pH conditions while Trp this to be true for some AMPs using concentrated human red blood cells quenching was used as a reporter of the local environment. TCE quenching was (RBCs) as a model eukaryotic cell. To solve this problem, we synthesized a used to determine peptide aggregation state in solution and showed a clear pH combinatorial peptide library based on the potent AMP, ARVA, and screened dependence on peptide aggregation. Acrylamide quenching demonstrates that the library for activity in the presence of concentrated RBCs. We isolated nine peptides are more deeply buried in the bilayer in PC:PG membranes compared unique, but similar sequences from the screen. We designed a consensus to PC bilayers. Dual Quencher Analysis (DQA) confirmed that the peptides in- sequence based on the nine peptides and synthesized it using only D-isomer serted more deeply in PC;PG membranes overall, but could insert into PC bi- amino acids to form D-NOGCON. D-NOGCON displays excellent antimicro- layers at pH conditions above the pKa of the His. Circular Dichroism (CD) was bial activity against multiple human pathogens in the presence and absence of used to determine the secondary structure and show that in solution the peptide concentrated RBCs, causes very little hemolysis, and is not susceptible to appears to adopt an unstructured conformation while they become helical upon cleavage by cellular or plasma proteases. D-NOGCON also has high activity binding to the bilayer. Experiments are currently being conducted to further against bacterial biofilms and does not readily induce leakage. In this work, investigate aggregation properties in solution. we created rational variants of D-NOGCON with truncations, insertions and mutations to test various hypotheses about the basis for highly potent antimi- 423-Pos crobial activity with low hemolysis and low toxicity against nucleated cells. Membrane Remodeling Induced by a pH Dependant Pore Forming Pep- In the near future, we will use this information to design a next generation tide via Atomic Force Microscopy combinatorial peptide library based on D-NOGCON that will be screened Anna Pittman1, Sarah Y. Kim2, William C. Wimley3, Kalina Hristova4, for i) clinically relevant antimicrobial activity in the presence of serum and Gavin King5. 1Univ Missouri, Columbia, MO, USA, 2Dept Molec Biophys, Johns Hopkins concentrated red blood cells, ii) negligible hemolysis, and iii) negligible 3 toxicity against nucleated cells. Univ, Baltimore, MD, USA, Dept Biochem, Tulane Sch Med, New Orleans, LA, USA, 4Dept Matl Sci/Eng, Johns Hopkins Univ, Baltimore, MD, USA, 421-Pos 5Dept Phys/Biochem, Univ Missouri, Columbia, MO, USA. Toxicity and Structure of Antimicrobial Peptides Derived from the Che- One hallmark of a cancer cell is the slightly acidic environment which is found mokine, CXCL10 in its immediate vicinity. It is of interest, therefore, to design mechanisms Peter Bailer1, Amanda E. Ward2, Matthew Crawford3, Debra Fisher3, which can deliver cargo—drugs, nanoparticles, etc.—across membranes under Lukas K. Tamm2, Molly Hughes3. acidic conditions only. Such delivery would bypass healthy cells in favor of 1University of Maryland Baltimore County, Baltimore, MD, USA, cancerous ones. One method to deliver cargo is to create pores selectively under 2University of Virginia, Charlottesville, VA, USA, 3Medicine, University of acidic conditions. pHD108 is a pore-forming peptide which was synthetically Virginia, Charlottesville, VA, USA. evolved to form pores large enough to pass macromolecules under acidic Antibiotic-resistant bacteria are a rising public health concern. To counter this conditions, but not at neutral pH (J Am Chem Soc. 139: 937 (2017)). Recently, threat, new antibiotics need to be developed. Chemokines are a class of im- we applied single molecule atomic force microscope (AFM) imaging to

BPJ 9310_9313 84a Sunday, March 3, 2019 characterize membrane remodeling induced by other pore forming peptides results show the changes in the crystallographic order of the model systems such as a mutant derivative of the bee venom peptide melittin in supported lipid upon their interaction with the peptides. Complimentary studies on lipid bilayers (Langmuir 34: 28 (2018)). The data revealed time dynamic intercon- bilayer systems are on their way. version between membrane-thinned and pore-like states, as well as colocaliza- tion between these different modes of membrane remodeling. Here, we use 426-Pos AFM imaging to characterize the lipid bilayer remodeling induced by Can Molecular Dynamics Simulations Predict the Effect of Truncating pHD108. Studies of pHD108 conducted on supported POPC bilayers at Histone-Derived Antimicrobial Peptides? differing pH revealed pH-dependent membrane distortions including punctate Kerry Gao1, Donald E. Elmore2. 1 pore-like features. To provide a broader mechanistic picture, single molecule Department of Biological Sciences, Wellesley College, Wellesley, MA, 2 AFM imaging results are placed in context with other measurements (e.g., USA, Department of Chemistry and Biochemistry Program, Wellesley leakage assays) performed on the same peptide/membrane system. College, Wellesley, MA, USA. Antimicrobial peptides (AMPs) represent a potential source of therapeutics to 424-Pos address the continued evolution of bacteria and viruses to resist existing anti- Elastic Behavior of Model Membranes with Antimicrobial Peptides De- biotics. Since the cost of producing AMPs increases for longer peptides, we pends on Lipid Specificity and D-Enantiomers evaluated the effect of removing residues from the N and C termini of the Akari Kumagai1, Fernando G. Dupuy2, Zoran Arsov3, Yasmene Elhady1, histone-derived antimicrobial peptides DesHDAP1 and DesHDAP3. Like Diamond Moody1, Belita Opene4, Robert K. Ernst4, Berthony Deslouches5, many AMPs, DesHDAP1 and DesHDAP3 are cationic peptides that have Ronald C. Montelaro6, Y.P. Peter Di5, Stephanie A. Tristram-Nagle1. electrostatic interactions with the negatively charged head groups of bacteria. 1Dept Physics Biological Physics Group, Carnegie Mellon Univ, Pittsburgh, Thus, we have focused on exploring the effects of removing arginine and PA, USA, 2INSIBIO, CONICET, National University of Tucuma´n, San lysine residues from either end of the peptide sequences. Bacterial assays Miguel de Tucuma´n, Argentina, 3Condensed Matter Physics, Jozef Stefan showed that truncating five amino acids from the N-terminus of DesHDAP1 Inst, Ljubljana, Slovenia, 4Dept Microbiol Pathogenesis, University of had no effect on the activity of the peptide but appeared to alter its mechanism Maryland, Baltimore, MD, USA, 5Dept Environmental and Occupational of action from a membrane translocating to a membrane permeabilizing Health, University of Pittsburgh, Pittsburgh, PA, USA, 6Dept Microbiology peptide. Further N-terminal truncations and any C-terminal truncations and Molecular Genetics, Univ Pittsburgh, Pittsburgh, PA, USA. dramatically decreased DesHDAP1 activity, and DesHDAP3 was also more In an effort to provide new treatments for the global crisis of bacterial resis- sensitive to truncation with only a one residue C-terminal truncation maintain- tance to current antibiotics, we have used a rational approach to design several ing full activity. To provide a molecular-level interpretation for these findings, new antimicrobial peptides. The present study focuses on 24-mer WLBU2 we have performed MD (molecular dynamics) simulations of both full-length and its derivative D8. This amino acid sequence contains only R, W and V: peptides interacting with a mixed POPE-POPG membrane. In particular, we RRWVRRVRRWVRRVVRVVRRWVRR. In D8, all of the valines are the have considered whether one could have predicted the effect of truncations D-enantiomer. WLBU2 and D8 have similar bactericidal activity, with low on DesHDAP1 and DesHDAP3 activity from simulations of the full-length MIC values for both Gram-negative (G(-)) and Gram-positive (G(þ)) bacteria, peptides. To this end, we have measured whether there is a correlation be- however, D8 is considerably less toxic to eukaryotic cells than WLBU2. Us- tween the interactions a particular amino acid has with the lipid membrane ing low- and wide-angle X-ray diffuse scattering, we measure the elastic and whether it that residue must be present for full peptide activity. Future behavior of lipid membrane mimics with increasing concentrations of work will use MD to further investigate the effect of specific truncations on WLBU2 or D8 to probe if membrane elastic behavior and lipid chain order peptide mechanisms and consider how truncations might affect other AMP are correlated with bactericidal activity and toxicity. We employ membrane properties, such as DNA binding. mimics of the outer lipopolysaccharide (LPS) membrane of G(-) bacteria, and the inner membranes of both G(-) and G(þ) bacteria. LPS model mem- 427-Pos branes are very soft with disordered chains, and both WLBU2 and D8 further Effects of Cholesterol on Fengycin, an Antimicrobial Lipopeptide Using increase softness and chain disorder. In both G(-) and G(þ) cell membrane Weighted Ensemble Path Sampling Method mimics, there is a stiffening at low concentrations of both peptides, followed Sreyoshi Sur1, Alan Grossfield2. by a softening at higher concentrations; lipid chain order follows a similar 1Dept Chemistry, Univ Rochester, Rochester, NY, USA, 2Dept of trend. In eukaryotic mimics containing 25 mole% cholesterol, both peptides Biochemistry and Biophysics, Univ Rochester, Rochester, NY, USA. cause a general softening and disordering of chains. However, in mimics con- Fengycin is an antimicrobial lipopeptide synthesized by the bacteria Bacillus taining 50 mole% cholesterol such as in red blood cells, D8 causes a dramatic subtilis and commercially available as an agricultural fungicide. One of the stiffening and increase in chain order, while WLBU2 causes membrane soft- ways fengycin kills fungal cells is by binding and damaging their cell mem- ening and chain disorder. These elastic results suggest that domain formation branes. Previous all-atom simulations suggested aggregation of lipopeptides may play a role in killing G(-) and G(þ) bacteria, and that cholesterol may is the first step that leads to membrane deformation. Here we attempt to play a role in protecting eukaryotic cells from antimicrobial peptides with explain fengycin’s selectivity for fungal over mammalian cells by examining D-enantiomers. the effects of cholesterol on its aggregation. Using weighted ensemble path sampling, we found that fengycin causes membrane demixing to some extent 425-Pos in the membrane consisting of POPC and cholesterol, with the latter being Human Antibacterial Peptides Modify Lateral Structure in Lipid Mono- clustered around the lipopeptides. We also found that the free energy of a layers Upon Interfacial Adsorption single fengycin binding to a membrane is independent of the presence of Thomas Gutsmann1, Beate Klo¨sgen2, Christian Nehls1, Laura Paulowski1, cholesterol. We are currently generalizing these calculations to examine fen- Chen Shen3. gycin aggregates in solution and their affinity for membranes. The results 1 2 Biophysics, Research Center Borstel, Borstel, Germany, Department will help us understand the implications of cholesterol on fengycin’s fungi- of Physics, Chemistry, and Pharmacy / MEMPHYS - PhyLife, University cidal properties and possibly indicate whether fengycin can be a potential 3 of Southern Denmark, Odense, Denmark, Photon Science, Deutsches drug candidate. Elektronen-Synchrotron, Hamburg, Germany. The increasing bacterial resistance against antibiotic treatment is a challenge 428-Pos of high socio-economic impact. An improved insight into the mechanisms of Effect of Biopolymer Tethers on Antimicrobial Peptide Activity in the natural biological antibacterial defense systems may trigger the develop- Biomembranes ment of alternative medical approaches for the treatment of infective dis- Fathima T. Doole1, Abhishek Singharoy2, Michael F. Brown1,3, eases. Here we report results on a study of the interaction of the human Minkyu Kim4,5. antimicrobial peptide LL-37, and its fragments LL-32 and LL-20, with lipid 1Department of Chemistry and Biochemistry, University of Arizona, Tucson, interfaces. As a starting point, lipid monolayers at the air/water interface AZ, USA, 2School of Molecular Sciences, Arizona State University, Tempe, were chosen as a tool to observe the impact of the peptide when adsorbing AZ, USA, 3Department of Physics, University of Arizona, Tucson, AZ, USA, to and into the lipid film from an aqueous subphase. During interaction, 4Department of Material Science and Engineering, University of Arizona, the surface pressure of the monolayer was monitored, and the initial Lang- Tucson, AZ, USA, 5Department of Biomedical Engineering, University of muir isotherm and its variation upon to the peptide insertion was acquired. Arizona, Tucson, AZ, USA. At selected isotherm points, the lateral structure of (A) neutral monolayers Remedies for health-associated infectious diseases entails innovations in (DPPC) and (B) negatively charged monolayers (90% DPPC, 10% DPPS, cost-effective alternatives, industrial-scale synthesis, non-cytotoxicity, and mol/mol) were studied by grazing incidence X-ray diffraction (GIXD). The non-biodegradability. We propose a smart biopolymer composed of an

BPJ 9310_9313 Sunday, March 3, 2019 85a antimicrobial peptide (AMP) and a scaffold made from elastin-like polypep- is to characterize the biophysical properties of treated M. luteus cells, tide chains. Direct immobilization of AMPs onto a biomaterial surface re- including putative pore formation in the outer membrane, and to deter- duces its bioactivity; in contrast, surface attachment of AMPs via tethers mine to what extent this peptide can function effectively on cells within increases bioactivity and enhances stability. We hypothesize that unstruc- biofilms. tured hydrophilic protein linkers will behave similarly to conventional hy- drophilic polymer tethers. Unstructured hydrophilic protein tethers have 431-Pos been designed using atomistic MD simulations and are being tested exper- Mechanistic Studies on Daptomycin-Induced Phase-Transitions on Model imentally. To obtain insight into how the designed protein tether impacts Lipid Membranes: Effect on Membrane Permeability AMP activity, its molecular mechanism must be established. The AMP hu- Alaina K. Howe, Stavroula Sofou. man cathelicidin LL-37 [1] is known to be in equilibrium between various Chemical and Biomolecular Engineering, Johns Hopkins University, oligomeric states, ranging from monomers to heptamers. Using the visuali- Baltimore, MD, USA. zation tool VMD, these oligomeric states were modeled starting from the For infections caused by multidrug resistant Gram-positive pathogens the available dimeric LL-37 structure (PDB#:5NNM). The structure prediction cyclic-lipopeptide antibiotic Daptomycin is considered a potent treatment op- server Robetta was employed to predict energetically important amino tion. Its bactericidal activity is attributed to a calcium- and PG lipid- depen- acid residues (hot spots) involved in protein-protein interfaces of the dent disruption in the membrane potential resulting in the release of bacterial modeled structures. Molecular Dynamics Flexibility Fitting (MDFF) was contents. Formation of oligomeric-Daptomycin membrane pores has been pro- utilized with the available PDB structure (PDB#:2YMK) of the hexameric posed as an action mechanism. In this study, we propose a different mecha- AMP channel dermcidin to fit the hexameric LL-37 structure. The nism on model lipid membranes.We studied the interactions of Daptomycin CHARMM GUI membrane builder was subsequently utilized to simulate with model lipid bilayers in the form of vesicles with symmetric and asym- the membrane around the hexameric structure. Deducing the orientation metric lipid leaflet compositions. We characterized the release kinetics of flu- of the oligomeric structure in the membrane and adding the protein tether orescently labeled probes encapsulated in vesicles composed of lipids with [2] allows us to observe its impact on the LL-37-membrane interactions. systematically varied headgroups and tails.Our quantitative findings suggest A complete model of this system will allow the design of unstructured hy- that the increase in membrane permeability by Daptomycin is due to the for- drophilic protein tethers, and aid in understanding the effects of these tethers mation of transient, leaky lipid-interfaces that span the bilayer. These inter- on AMP conformation as well as bacterial toxicity. [1] K.A. Henzler- faces originate right on the boundaries of lateral, phase-separated, well- Wildman et al. (2004) Biochemistry 43, 8459. [2] D.J. Callahan et al. packed lipid domains induced by Daptomycin and, importantly, are registered (2012) Nano Lett. 12,2165. across the bilayer. The following two-step process is proposed. In the first step, the binding of Daptomycin on vesicles results in formation of 429-Pos well-packed single-leaflet lipid-domains via its association with the PG- Effect of N-Terminal Metalation and Lipid Composition on the Activity of headgroups only. Interestingly, in this process, a well-packed outer lipid- Antimicrobial Piscidins in Membranes leaflet domain occurs independent of the extent of unsaturation of the tails Ella Mihailescu1, Roderico Acevedo1, Vitalii Silin1, Frank Heinrich2, of PG-lipids that partition in the Daptomycin-induced domain. In turn, for Myriam Cotten3. this initial phase-transition to result in increase of the membrane permeability, 1Institute for Bioscience and Biotechnology Research, Rockville, MD, USA, the outer lipid leaflet domains need to induce cross-registered phase-transi- 2Center for Neutron Research, National Institute for Standards and tions of also well-packed lipids in the inner leaflet. This second phase- Technology, Gaithersburg, MD, USA, 3Department of Applied Science, separated domain occurs only when the tails of the lipids in the inner leaflet College of William and Mary, Williamsburg, VA, USA. are saturated independent of their headgroups. Accordingly, the extent of Host-defense peptides (HDPs) play an integral role in the fight against invading release via the interfacial domain boundaries depends on the relative fluidity pathogens. To fully exploit their potential as prototypes for novel antimicro- of the surrounding bilayer. bials the molecular basis of their mechanism of action, which includes disrupt- ing plasma membranes, must be understood. The host-defense peptides piscidin 432-Pos P1 and piscidin P3, two potent HDPs isolated from striped bass, display strong Correlation of an Antimicrobial Peptide’s Potency and Its Influences on antimicrobial activities against a large number of Gram-positive and -negative Membrane Elasticity bacteria, including methicillin-resistant S. aureus (MRSA), viruses such as Wen-Fang Chang, Si-Han Chen, Yi-Fan Chen. HIV-1, fungi, yeasts, and cancer cells. The two 22-residue, helical, cationic Chemical and Materials Engineering, National Central University, Taoyuan, peptides differ only slightly in amino-acid sequences, but P1 is more potent Taiwan. than P3. Solid state NMR showed that P1 and P3 adopt a very similar helical The killing power of membrane-active antimicrobial peptides (AMPs) lies structure in fluid lipid membranes. However, neutron diffraction and reflectom- in their ability to disrupt the structures of pathogenic membranes. While etry, in conjunction with electrical impedance spectroscopy (EIS) and surface understanding the AMPs’ mechanisms of action is key to realizing their plasmon resonance (SPR) measurements reveal differing conformations of the therapeutic potentials, how the AMPs modulate membrane elasticity and two peptides in membrane, depending on lipid composition and peptide metal- thereby affect membrane structure remains an open question, even though ation state (Ni2þ or Cu2þ bound to the N-terminal ATCUN motif). These find- the AMP-induced variations in membrane curvature are widely considered ings offer new insights on the structural interactions of the peptides with to be crucial. Here, we exploit the x-ray diffraction technique to examine membranes at the molecular level. The contrasting behavior of the two AMP how the dominant bacterial lipid, phosphatidylethanolamine, varies its mono- homologues offer a unique opportunity to examine the structure-function rela- layer elastic properties upon interacting with six artificial peptides mimicking tionship of ATCUN HDPs. the AMPs’ common amino acid content. Remarkably, the monolayer sponta- neous curvature C0 is unaffected by any of the peptides. In contrast, the 430-Pos peptides designated as (K2W)2, K4W2, R6, and R9, mimicking the AMP Biophysical Properties of Magainin-Treated Biofilms mutants with microbicidal potency, are able to modify the monolayer 1 2 2 1 Ryan MacVicar , Thelma Mashaka , Catherine B. Volle , Megan E. Nunez . bending moduli Kcp, while those derived from the impotent mutants cannot. 1Chemistry, Wellesley College, Wellesley, MA, USA, 2Cottey College, The results are consistent with the scenario that the AMPs disrupt the struc- Nevada, MO, USA. ture of pathogenic monolayers by, additionally if not exclusively, modulating In the environment, bacteria may exist as free-swimming planktonic cells their Kcp’s, with stiffening and softening leading to two different modes or within interdependent communities of microorganisms called biofilms. of disruption, that is, toroidal pore formation and membrane micellation, Cells in biofilms excrete mixtures of polysaccharides, nucleic acids, proteins, respectively. and small molecules to create a sticky coating called exopolymeric sub- stances (EPS). Within these complex, three-dimensional communities, 433-Pos some cells grow actively while others are senescent, and the development Measuring the Stoichiometry of Antimicrobial Peptides in Nanodiscs with of antibiotic resistance is common within these communities. To explore Native Mass Spectrometry one alternative to conventional antibiotics, we seek to understand how Michael T. Marty1, Lawrence Walker1, Marius Kostelic1, Elaine Marzluff2. the antimicrobial peptide magainin II (MGII) interacts with Gram positive 1Department of Chemistry and Biochemistry, University of Arizona, Tucson, biofilms. We have determined that the growth of Gram positive Micrococcus AZ, USA, 2Department of Chemistry, Grinnell College, Grinnell, IA, USA. luteus cells is inhibited by this peptide. Using Atomic Force Microscopy, Antimicrobial peptides (AMPs) and other membrane active compounds attack we are measuring nanoscale changes in the morphology and membrane pathogens by targeting the lipid bilayer rather than specific proteins, which may integrity of planktonic M. luteus cells upon the addition of MGII. Our goal have advantages in combating drug resistance. However, the mechanisms of

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AMP toxicity and selectivity are poorly understood due to challenges in residues. These new peptides are used to challenge three cell lines to assess measuring polydisperse interactions of AMPs within lipid environments. their efficacy as potential cancer therapeutics: MCF-10A (human breast Native or noncovalent mass spectrometry (MS) has recently made significant epithelial cell), HMLER (human breast cancer bulk cell), and HMLER- strides in characterizing the stoichiometry of polydisperse protein complexes shEcad (human breast cancer stem cell). Measurement of the half maximal and lipid interactions. Here, we employ lipoprotein nanodiscs as a membrane inhibitory concentrations (IC50) shows that several peptides are effective mimetic to assemble AMP complexes for native MS. The nanodiscs are ionized against breast cancer cells and the mammospheres with low micromolar con- under non-denaturing conditions to introduce the entire nanodisc complex with centration and have selectivity toward cancer cells. Remarkably, D-form pep- embedded AMP into the mass spectrometer. By precisely measuring the mass tides can further reduce the dosage to nanomolar concentration to selectively of the intact nanodisc complexes, we can measure the oligomeric state of the eliminate the cancer cells. We further show that these peptides also can AMP without disrupting the lipid bilayer. As a positive control, we observed enhance other anticancer agents to wipe out cancer cells with a lower overall that gramicidin A forms dimers exclusively, although multiple dimers can be dosage, which can significantly reduce the cytotoxicity of these agents toward inserted into the nanodisc with sufficient concentration. Ongoing work is inves- normal cells. Overall, we have shown that our LDKA peptide libraries are tigating AMP complexes with unknown stoichiometries, including LL-37, ala- promising templates for treatment of drug-resistant bacteria and cancer cells, methicin, and melittin. We are also exploring the kinetics of complex assembly and have a broad spectrum of applications to address different biomedical by monitoring the formation of specific oligomeric species over time. Ulti- needs. mately, our goal is that these novel measurements of AMP complex assembly and stoichiometry will shed light on the mechanism of their selectivity and 436-Pos toxicity. Aurein 1.2, a Short and Potent Antimicrobial Peptide, Changes Charged Lipid Distribution and Lipid Dynamics in Bilayer 434-Pos Shuo Qian1, Veerendra K. Sharma2. Antimicrobial Peptidomimetics with Activity Towards Cancer Cells 1Neutron Scattering Division, Oak Ridge Natl Lab, Oak Ridge, TN, USA, Konstantin Andreev1, Michael W. Martynowycz2, Mahesh Lingaraju2, 2Solid State Physics Division, Bhabha Atomic Research Centre, Mumbai, Christopher Bianchi3, Amram Mor4, David Gidalevitz2. India. 1Molecular BioScis, HHMI/Northwestern Univ, Evanston, IL, USA, 2Dept Aurein 1.2 is a 13-amino acid antimicrobial peptide that was found from the Physics, Illinois Inst Tech, Chicago, IL, USA, 3Illionis Inst Tech, Chicago, Australian frog Litoria genus. It exhibits strong and broad-spectrum anti- IL, USA, 4Dept Biotechnology and Food Engineering, Technion-Israel biotic and anticancer activities, but the mechanism of action is not under- Institute of Technology, Haifa, Israel. stood. It binds spontaneously to membrane surface with and without a Cancer is one of the most serious threats to global public health. Aside from charged lipid but has stronger effect on membranes with charged lipids. the surgical treatment, radiation and immunotherapy, chemotherapeutic ap- To understand how this peptide interacts with the lipid membrane, we proaches remain at the frontline of curative and palliative care for oncology used small-angle neutron scattering (SANS) to study the redistribution of diseases. Due to the current limitations of conventional chemotherapies, charged lipid in the presence of Aurein 1.2. We found that it alters the dis- identifying potential targets for novel anticancer therapeutics is of great tribution of anionic lipids across the bilayer leaflets at different concentra- importance. Antimicrobial peptides (AMPs) have previously demonstrated tions. At a high concentration, it causes significant lateral segregation in a broad-spectrum anticancer activity, yet the proteolytic biodegradation in an initially uniform lipid bilayer made of zwitterionic and anionic lipids. vivo and high manufacturing costs prevent them from being clinically The lateral heterogeneity structure is similar to a domain-like structure applied. Synthetic oligomers of acylated lysines (OAKs) mimicking natural observed in the membrane below the lipid order-disorder phase transition AMPs demonstrate superior selectivity towards the cells with high content temperature. With quasielastic neutron scattering (QENS), we found that of glycosylated molecules on their surface. Melanoma cells displaying malig- Aurein 1.2 significantly reduces the lateral motion of lipid in the fluid phase, nant characteristics are shown to overexpress mono- and disialylated gangli- making the lipid a more ordered arrangement even at low concentrations. osides GM3 and GD3, respectively. Several melanoma, non-malignant This suggests Aurein 1.2 induces ordered domains as observed in our adenocarcinoma, as well as primary cell lines, are tested for their sus- SANS experiment, even above the lipid phase transition temperature. We ceptibility to OAK and magainin II. The anticancer effect by peptide is rela- will discuss the implication of our results on the mechanism of antimicrobial tively weak. In turn, a precipitous drop of cell viability is observed in peptide, especially in the case that involves no pores formation or mem- ganglioside-enriched melanoma at micromolar concentrations, when treated branes disintegration. with OAK. Surface X-ray scattering on Langmuir monolayers composed of phosphatidylcholine with either GM3 or GD3, shows OAK to permeate 437-Pos through the outer carbohydrate region into lipid hydrophobic core, which The Role of Greasy Residues in Teixobactin Derivatives Po-Chao Wen1, Emad Tajkhorshid1, Susan B. Rempe2. is not observed for peptide. Multimodal interactions by OAK with sialic 1 acid groups, but not phosphatidylserine, induce cancer cell death by the de- Department of Biochemistry, Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA, molition of membrane integrity along with the targeting intracellular mech- 2 anisms of nuclear assembly. Our results provide first-time mechanistic Sandia National Laboratories, Albuquerque, NM, USA. insights into the ganglioside-mediated anticancer activity by antimicrobial Teixobactin is a novel antibiotic that inhibits cell wall biosynthesis by bind- peptidomimetics. ing to cell wall precursors, such as lipid II. Previously, we characterized multiple conformations of the teixobactin-lipid II complex in membrane 435-Pos using modeling and molecular dynamics simulations (Chem. Sci., 2018, Rational Design of Polyleucine-Based Antimicrobial Peptides as Promising 9:6997-7008). Common structural features shared by these conformations Agents Against Cancer Cells include pyrophosphate coordination by the C-terminal cyclodepsipeptide Charles H. Chen1, Arvin Eskandari1, Jenisha Ghimire2, ring and membrane anchoring by the mid-chain hydrophobic residues. Since William C. Wimley2, Kogularamanan Suntharalingam1, the cell wall precursors exist in low concentrations at bacterial surfaces, it is Martin B. Ulmschneider1. likely that teixobactin binds and inserts into the membrane before reaching 1Chemistry, King’s College London, London, United Kingdom. its targets. Here, we performed microsecond-scale molecular dynamics sim- 2Dept Biochem, Tulane Sch Med, New Orleans, LA, USA. ulations to investigate the membrane binding and insertion of teixobactin We have previously developed several 15-residue polyleucine-based antimi- and a highly potent derivative (D-Arg4/Ile10-teixobactin) in the absence crobial peptides, called LDKA, using atomic detail structural information of their target molecules. Multiple membrane models were employed in derived from unbiased long-timescale equilibrium folding-partitioning molec- our simulations, including the Highly Mobile Membrane Mimetic model ular dynamics simulations to guide the experimental design of a combinatorial and full-length phospholipid bilayers, consisting of either zwitterionic phos- peptide library (2,916 peptides) from which potent pore forming peptides phatidylethanolamines or anionic phosphatidylglycerols. We found that teix- were selected via high-throughput screening. Several of these peptides are obactin derivatives always anchor to the membrane using residues N-Me-D- promising against drug-resistant e. coli at low micromolar concentration Phe1, Ile2, D-allo-Ile5, Ile6, and Ile11. In particular, the insertion depths of without harming human red blood cells. We found the hydrophobic moment the N-terminal hydrophobic residues are consistently comparable to those of plays a critical role in tuning the cell selectivity between bacteria and the mid-chain hydrophobic anchors, implying they also play a significant mammalian cells; while the net charge did not play a role in cytotoxicity. role in membrane insertion. Additionally, the membrane insertion of Ile11 Here, we further design a small combinatorial peptide library (36 peptides) restricts the orientation of the cyclodepsipeptide ring at the membrane based on our LDKA peptides, fine-tuning the charge distribution on the polar surface, suggesting that one of the lipid II-bound forms characterized face, while maintaining the positions of the previously optimised hydrophobic in our earlier work would be more prominent due to a compatible

BPJ 9310_9313 Sunday, March 3, 2019 87a cyclodepsipeptide ring orientation. In summary, almost half of the side mimics were investigated, consisting of a mixture of POPE, POPG and chains in teixobactin participate in membrane-anchoring and not direct lipid tetraoleoyl-cardiolipin (TOCL). Structural and thermodynamical properties II-binding. These hydrophobic residues appear invariable in several studies of IM and OM mimics were studied using different techniques, including of the structure-activity relationship of teixobactin derivatives, suggesting small angle scattering (SAS), differential scanning calorimetry (DSC) and that membrane binding and insertion act as an essential step in its antibac- dynamic light scattering (DLS). This provides us with a valuable platform terial mechanism. for interrogating specific interactions of antimicrobial peptides with either membrane. 438-Pos Use of a Stereochemical Strategy to Probe the Mechanism of 441-Pos Phenol-Soluble Modulin A3 Toxicity Lipopolysaccharide Simulations Are Highly Sensitive to Ion Parameters Zhihui Yao1, Brian P. Cary2, Craig A. Bingman3, Samuel H. Gellman2. and Phosphate Charge State 1Biophysics Program, University of Wisconsin-Madison, Madison, WI, USA, Amy Rice, Jeffery M. Wereszczynski. 2Department of Chemistry, University of Wisconsin-Madison, Madison, WI, Dept Physics, Illinois Inst Tech, Chicago, IL, USA. USA, 3Department of Biochemistry, University of Wisconsin-Madison, Lipopolysaccharides (LPS), a main constituent of Gram-negative bacterial Madison, WI, USA. outer membranes, are central to the organization and function of the bacte- Phenol-soluble modulin a3(PSMa3) is a cytotoxic peptide secreted by rial outer membrane and are implicated in its low permeability and Staphylococcus aureus. Increased expression of PSMa3 is associated antibiotic resistance. The structure of LPS is different from that of phospho- with the enhanced toxicity of community-associated Methicillin-resistant lipids in that it contains multiple hydrophobic tails covalently linked to a S. aureus (CA-MRSA), a highly virulent strain of MRSA. Though large, highly charged polysaccharide; due to their large number of phos- previous reports suggest membrane disruption, the mechanism of PSMa3- phoryl and carboxyl groups, LPS membranes must be stabilized by a mediated cell death is not fully understood. Here, we used a stereochemical network of divalent cations bridging these moieties. A number of LPS che- strategy to examine the mechanism of cytotoxicity. We found that racemic motypes have recently been parameterized, and outer membrane simulations PSMa3 (L and D-PSMa3 mixed at 1:1 ratio) can form non-toxic and stable with LPS are increasingly more common; however, little work has been fibrils, while chiral PSMa3 remains toxic under the same conditions. We done to evaluate the performance of common ion force fields with these solved the crystal structure of racemic PSMa3at1.4A˚ , which shows a LPS models. Additionally, minimal attention has been paid to the charge cross-a, homo-chiral packing pattern along the crystal growth axis. We state of phosphate groups within LPS, with no consensus on charge assigna- conclude that PSMa3 exerts toxicity primarily through interactions with tion between different parameterizations. In this study, we investigate the the lipid bilayer of a cell, and that fibril formation is not necessary for effects of both ion type and phosphate charge on four distinct chemotypes cytotoxicity. of S. enterica LPS. We report that bilayer properties, such as the area per lipid, tail fluidity, and core hydration are highly influenced by the choice 439-Pos of cation identity, force field parameters, and phosphate group charges. Investigating Intracellular Functions of Antimicrobial Peptides Using an Further experimental and computational work are needed to determine Internal Gene Expression Systems which LPS representations are biologically relevant and to develop more Sattar Taheri-Araghi1, Salimeh Mohammadi1, Federico Prokopczuk2, robust LPS models. Overall, these results highlight the importance of Xintian Li3. choosing appropriate LPS models to better reproduce and understand LPS 1 2 Dept Physics, Calif State Univ Northridge, Northridge, CA, USA, Dept membrane properties. Biology, Calif State Univ Northridge, Northridge, CA, USA, 3National Cancer Institute, Rockville, MD, USA. 442-Pos The field of antimicrobial peptides (AMPs) has been facing the long- Determining Volumes of Lipid Components: Hidden Assumptions Have standing question of whether AMPs only attack the cell membranes or Not-So-Hidden Consequences also interact with intracellular targets. It is intrinsically impossible to John F. Nagle1, Richard M. Venable2, Ezekiel Maroclo-Kemmerling1, decouple the targets because any AMP should translocate through the cell Stephanie A. Tristram-Nagle3, Paul E. Harper4, Richard W. Pastor5. membrane to reach the cytoplasmic space, which inevitably affects the 1Dept Phys, Carnegie Mellon Univ, Pittsburgh, PA, USA, 2NHLBI NIH, cell membrane. To tackle this question, we took a novel approach by utiliz- Bethesda, MD, USA, 3Dept Physics Biol, Carnegie Mellon Univ, Pittsburgh, ing a tunable, internal gene expression system to induce the production of PA, USA, 4Dept Physics and Astronomy, Calvin College, Grand Rapids, MI, the AMP LL-37 inside Escherichia coli cells. We have tracked the growth USA, 5Lab Compu Biol, NHLBI NIH, Bethesda, MD, USA. rate, cell size and mortality rate of thousands of cells in a live, single-cell In addition to obtaining the highly precise volumes of lipids in lipid bilayers, it imaging platform. In this talk, I present our recent findings on how the phys- has been desirable to obtain the volumes of parts of each lipid, such as the head- iological characteristics of single E. coli cells are compromised by the inter- group and the terminal methyls on the hydrocarbon chains. This work re- nal AMP expression, even prior to any cell membrane damage. While a high examines the technique and assumptions undergirding the standard approach level of expression inhibits bacterial growth and lyse the cells, a low level of to determining component volumes and compares the results with the more AMP expression reduces growth rate and increases cell size by delaying cell detailed information derived directly from simulations. This comparison re- divisions. Our results provide clues towards internal functions of AMPs veals a 1% level flaw in the experimental determination of the average methy- which ultimately improve our understanding of the molecular mechanism lene component volume. Literature experimental volume data for unsaturated of action of AMPs. phosphocholines and for alkanes have been used and new data have been acquired for saturated phosphocholines. Data and simulations have covered Posters: Membrane Structure I extended ranges of temperature to assess temperature dependence of the component volumes. Refining the experimental determination of component 440-Pos volumes is discussed. Structural Properties of Inner and Outer Membrane Mimics of Gram-Negative Bacteria 443-Pos Lisa Marx, Enrico Semeraro, Karl Lohner, Georg Pabst. Effect of Alcohol on Water Translocation in All-Atom Simulations Institute of Molecular Biosciences, Biophysics Division, University of Graz, of Osmotic Gradient Across Lipid Membranes Graz, Austria. Robert E. Coffman1, David D. Busath2, Dixon J. Woodbury2. Antimicrobial peptides are well-studied compounds with high potential for 1Neuroscience Center, Brigham Young University, Provo, UT, USA, application against multi-resistant bacterial strains mostly acting on the cell 2Physiology & Dev. Biol., Brigham Young University, Provo, UT, USA. envelope. The majority of peptide/lipid interaction studies have been per- To explore how alcohol changes movement of water molecules through a mem- formed using model membranes with a symmetric lipid distribution. The outer brane, we are using all-atom molecular dynamic simulations with NAMD. One membrane (OM) of Gram-negative bacteria however, is well-known for its challenge of using an osmotic gradient to induce this movement is correctly us- asymmetric placement of lipopolysaccharides (LPS) and phospholipids. Based ing periodic boundary conditions (PBCs). We use a system with two mem- on cyclodextrin-mediated lipid exchange we were able to produce OM mimics branes and three solvent compartments, two of which are connected by in form of asymmetric large unilamellar vesicles (aLUVs) with an outer means of the PBCs. Hong et al. (Langmuir 2014, 30:11993-12001) have shown leaflet enriched in LPS and an inner leaflet composed of a mixture of that higher salt concentrations cause increased lipid packing in simulated palmitoyloleoyl-phosphatidylethanolamine (POPE) and -phosphatidylglycerol membranes. To represent this, we used CHARMM-GUI to generate a 2000 (POPG). To compare the peptide’s mode of action, also inner membrane (IM) and a 150 mM KCl solvated bilayer with lipid numbers that give the same

BPJ 9310_9313 88a Sunday, March 3, 2019 cross-sectional area for the solution they’re exposed to. Using VMD, both sys- 446-Pos tems were split in half at the leaflet-leaflet interface and restacked such that Cholesterol-Dependent Bending Energies in Both Leaves Play a Significant there are two membranes, each with differing number of lipids in their two Role in Determining the Cholesterol Distribution in the Plasma Membrane monolayers. Constant pressure and temperature (NPT) simulations for 0.8 ms David Allender1, Alexander J. Sodt2, Michael Schick3. with this system show 1) stable area/lipid, 2) no lipid flip flop, and 3) tens to 1Physics, Kent State University, Kent, OH, USA, 2National Institute of Child hundreds of water translocations depending on temperature. With this innova- Health and Human Development, National Institutes of Health, Bethesda, tive strategy for simulating osmotic-induced water movement, we hope to pre- MD, USA, 3Physics, University of Washington, Seattle, WA, USA. dict the effects of an osmotic gradient on alcohol partitioning in membranes. We consider a plasma membrane that contains a cholesterol mol fraction This will help us understand how alcohol affects membrane fusion as measured of 0.4, and ask how that cholesterol is distributed between the two experimentally in our lab. leaves. Because of the rapid flip-flop of cholesterol between leaves, we assume that its distribution is determined by the equality of its chemical 444-Pos potentials in the two leaves. When we consider only the contributions of The Effect of Phloretin on the Thermotropic Behavior of Membrane entropy and interactions to the cholesterol chemical potential in our model Forming Lipids system we find, not surprisingly, that due to the strong attraction between Svetlana S. Efimova, Olga S. Ostroumova. cholesterol and sphingomyelin, which is predominantly in the outer Inst of Cytology RAS, St Petersburg, Russian Federation. leaf, the cholesterol is mostly in that leaf. We find 74% of the total choles- Phloretin, a flavonoid belonging to the class of dihydrochalcones, is mainly terol there. The explicit interaction energy does not include the bending found in fruits, leaves, and roots of apple tree. Phloretin exerts antioxidant, energy, which we consider next. Simulations indicate that the bending en- anti-inflammatory, and anti-tumor activities. The focus of the present ergy of cholesterol-sphingomyelin mixtures varies non-linearly with compo- study is the effect of phloretin on the thermotropic behavior of vesicle sition. This energy is obtained from the product of bending modulus and forming lipids. Large unilamellar liposomes were prepared by electroforma- spontaneous curvature that is available from simulation. We find that the tion method from pure DPPC or POPE, or binary mixture of DPPC with addition of cholesterol to the outer leaf reduces the spontaneous curvature, 5 or 10 mol% cholesterol. The lipid:flavonoid ration was equal to 9:1. Using which is initially positive. It passes through zero when the mol fraction of differential scanning microcalorimetry, it was shown that the addition of the cholesterol in the outer leaf is 0.28. Additional cholesterol is driven toward phloretin to the cholesterol-containing liposomes leads to the decrease in the the inner leaf by the sphingomyelin. This is resisted by the bending energy temperature of main phase transition on the 2.1, 2.9 and 3.3 degrees in contribution to the inner leaf. We find, again by simulation, that the addition the case of DPPC, DPPC:cholesterol (95:5) and DPPC:cholesterol (90:10), of cholesterol monotonically increases the magnitude of the spontaneous respectively. The half-width of the peak increased with a growth in the curvature of the inner leaf, which is negative. This increases its bending concentration of cholesterol in DPPC-liposomes on the 2.0, 2.3 and 2.5 energy. We conclude that, as a result of these competing effects, the degrees. The introduction of phloretin to POPE liposomes led to decrease percent of total cholesterol that is in the outer leaf is reduced to about in the temperature on the 0.7 degree and increase in the half-width of 62% 54%. the peak on the 0.9 degree. It could be explained by phloretin integration between the lipid polar "heads". The mobility of the hydrocarbon chains 447-Pos increased as a result of an expansion of the area per lipid molecule. Effect of Sterol Structure on Ordered Lipid Domains in Symmetric and Herewith, the peak corresponding to the inverted hexagonal phase of Asymmetric Model Membranes POPE was not detected in the presence of phloretin. The obtained results Johnna R. St Clair. indicated the ability of phloretin to increase the lateral pressure in the Dept Biochemistry & Cell Biology, Stony Brook University, Stony Brook, region of the hydrophilic lipid heads and to inhibit the formation of NY, USA. the inverted hexagonal lipid phase. The study was supported by RFS The packing of ordered sterol-sphingolipid-rich liquid ordered domains in (#17-74-10137). biomembranes is greatly impacted by sterol structure. For this reason, the dependence of biological functions upon sterol structure can be used to iden- 445-Pos tify processes likely to be dependent upon ordered domain formation. In this Morphology and Dynamic Effect of Ergosterol or Cholesterol on Domains study we compared the effect of sterol structure upon ordered domain for- Present in POPC-Esm-Sterol SLB mation in symmetric vesicles composed of a mixture of sphingomyelin, Arturo Galva´n-Herna´ndez1, Armando Antillo´n2, Jorge Herna´ndez-Cobos1, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol, to that Ivan Ortega-Blake1. in asymmetric vesicles in which sphingomyelin was introduced into only 1Biophysics, Instituto de Ciencias Fı´sicas UNAM, Cuernavaca, Mexico, 2 the outer leaflet of vesicles composed of DOPC and cholesterol. In most Theoretical and Computational Physics, Instituto de Ciencias Fı´sicas cases, sterol behavior was similar in symmetric and asymmetric vesicles, UNAM, Cuernavaca, Mexico. with ordered domains being most strongly stabilized by 7-dehydrocholes- An atomic force microscopy study on the effect of sterol on supported lipid terol and cholesterol, to a moderate degree by lanosterol, epicholesterol bilayers composed of the mixture 1-Palmitoyl-2-oleoyl-sn-glycero-3-phos- and desmosterol, and very little if at all by 4-cholesten-3-one. However, phocholine and egg sphingomyelin in a 1:1 molar ratio is presented. The we found that in asymmetric vesicles desmosterol stabilizes ordered domain effect of two different sterols, cholesterol and ergosterol, was studied. almost as well as cholesterol, and stabilizes ordered domains to a much Ergosterol increases the height difference between the gel and the liquid greater degree than epicholesterol. From previous studies using symmetric phase regions, whereas cholesterol does not. At low concentration (5 mol vesicles and a different lipid composition, we had come to the opposite %) both sterols increase gel-phase area coverage, at medium concentration conclusion: that epicholesterol stabilizes ordered domains similarly to (20 mol%) gel-phase coverage in the ergosterol containing membrane re- cholesterol and to a greater degree than desmosterol. Based on behavior turns to the sterol-free value whereas for cholesterol containing membrane in asymmetric membranes, which mimic cell membranes more closely there is a considerable reduction. At high concentration (40 mol%) both ste- than symmetric membranes, we have re-evaluated our prior endocytosis rols conduce to a homogeneous membrane. Bilayer rupture forces were and bacterial uptake studies and now conclude that sterol raft-forming abil- determined for the different systems. The sterol-free membrane shows a ity may be the sole structural property of sterol that is necessary for these clear difference in rupture force for the liquid (5 nN) and the gel (10 nN) functions. regions. Addition of 5 mol% cholesterol stiffens both regions up to 10 and 12 nN respectively whereas addition of 5 mol% ergosterol has a 448-Pos different behaviour; it stiffens considerably the gel phase region (15 nN) Influence of Sterol in Ternary Mixtures Containing Sphingomyelin: An but has no effect on the liquid region. At 20 mol% sterol content the mem- All-Atom Molecular Dynamics Study brane is completely homogeneous for cholesterol, as it is at 40 mol%, and Fernando Favela-Rosales1, Arturo Galva´n-Herna´ndez2, for ergosterol the difference between the stiffness of the liquid (5 nN) and Jorge Herna´ndez-Cobos2, Iva´n Ortega-Blake2. the gel (10 nN) regions is reduced. At 40 mol% ergosterol content there 1Investigacio´n, Tecnolo´gico Nacional de Mexico - ITSZO, Sombrerete, is a homogeneous membrane. Since raft formation is an important biological Mexico, 2Instituto de Ciencias Fı´sicas, Universidad Nacional Auto´noma de process, the distinct effect between ergosterol and cholesterol on the prop- Mexico, Cuernavaca, Mexico. erties of these structures is important, and could be affecting the expression Model membranes have been widely used to study the phase behavior of of membrane active molecules, particularly the observed selectivity of ternary mixtures having liquid-ordered and liquid-disordered phase coexis- polyenes towards ergosterol containing membranes. Funding: DGAPA- tence. One of these models is composed of Sterol, Sphingomyelin and PAPIIT-IG100416 POPC. Despite of many different experiments that propose lipid-phase

BPJ 9310_9313 Sunday, March 3, 2019 89a diagrams, we lack full understanding at molecular level of the influence of ste- Lipidleafletasymmetryisanessentialaspectofbiomembranesthatleads rol on these systems. Here, we have simulated ternary mixtures of POPC, to numerous novel properties not present in ordinary symmetric membranes. sphingomyelin and either cholesterol or ergosterol in order to understand the To investigate these systems through molecular dynamics simulation changes in bilayer properties with different sterols at different concentrations. over large length and time scales usually necessitates coarse-grained models To quantitatively access the structure and dynamics of membranes, deuterium to reduce computational cost. However, highly coarse-grained models, order parameters, lateral distribution of thickness, thickness correlation and such as the Cooke model, exhibit artificially accelerated dynamics, in partic- radial distribution functions are calculated. The results are compared to ular lipid flip-flop. This may cause a membrane’s asymmetry to decay Atomic-Force Microscopy (AFM) images from studies of the same systems. before statistically significant measurements of its consequences can The combined approaches allowed us to advance in the understanding of be made. We investigate a remedy for this problem proposed by Wang the molecular structure from coexisting phases. Funding: DGAPA-PAPIIT- et al. (Commun. Comput. Phys. 13), which energetically discourages lipids IG100416. from entering the opposite leaflet. We measure the efficiency of this modi- fication in maintaining asymmetry in normal unstressed membranes as well 449-Pos as bilayers with varying degrees of stress imbalance between leaflets. We Does Cholesterol Matter in the Lung Surfactant? A Biophysical Study on propose further changes to improve the remedy under extenuating circum- Realistic Lung Surfactant Lipid Mixtures stances, and we report changes to basic membrane characteristics caused Agnieszka Olzynska, Pauline Delcroix, Lukasz Cwiklik. by the modification as well as possible ways by which one can compensate Department of Biophysical Chemistry, J. Heyrovsky´ Institute of Physical for them. Chemistry of the CAS v. v. i., Prague, Czech Republic. Lung surfactant (LS), secreted by type II cells in the alveolar epithelium, 452-Pos is a complex lipid-protein mixture that spreads as a surface active film All Atom Simulations of the Inner and Outer Leaflet of the Erythrocyte at the air-water interface of alveoli. Its primary role is a reduction of Plasma Membrane surface tension at the alveoli-air interface during inspiration. Additionally, Edward R. Lyman1, Kandice R. Levental2, Joseph Lorent2, Ilya Levental3. 1 2 the film protects the alveoli from collapse when they reach the lowest Dept Phys/Astron/Chem/Biochem, Univ Delaware, Newark, DE, USA, UT 3 volume. Hlth Sci Ctr-Houston, Houston, TX, USA, Univ Texas Houston, Houston, LS deficiency or alterations in its composition consequence in such condi- TX, USA. tions as the neonatal respiratory distress syndrome (RDS) or the acute By combining enzymatic degradation of specific lipids with shotgun lipido- respiratory distress syndrome (ARDS). Although most of pharmaceutical- mics, the composition of the inner and outer leaflet of the human erythrocyte grade LS supplements applied in RDS are on purpose depleted in choles- were determined. The inner and outer leaflet mixtures are broadly consistent terol, the physiological amount of cholesterol (10%) is suggested to play with expectations (negative charge mainly on the inner leaflet, which tends an essential role for LS stability. LS acts under persistent oxidative stress to also include more unsaturation), but offer the most detailed look at mem- and hence contains products of lipid oxidation, including those from the brane asymmetry to date. Measurements of lipids packing were obtained oxidation of phospholipids and cholesterol. In ARDS the number of separately for the inner and outer leaflet using di-4-ANEPPHQ, microinject- oxidized lipids in LS was found to be elevated. To address the issue of ing the dye into cells to assess the inner leaflet. The lifetime of the dye is the role of cholesterol in LS under oxidative stress, we apply combined consistent with a more ordered lipid environment on the outer leaflet. The experimental and molecular simulations approach. We experimentally char- biophysical ramifications of these two lipidomes were studied further by acterize LS-mimicking systems by Langmuir balance combined with fluo- all-atom simulations reduced complexity lipidomes. These ‘‘minimal mix- rescence microscopy to probe the macro-scale behavior of the surface tures’’ were obtained by projecting onto a representative set of 20 lipids, films. We combine this with classical molecular dynamics simulations to which includes three sphingolipids, three plasmalogens, PIP2, cholesterol, obtain molecular-level information about the role of individual film compo- and 11 different glycero-phospholipids with varying degrees of unsaturation nents at the nanoscale. We observe that under physiological temperature the and headgroup chemistry. The outer leaflet is significantly more ordered presence of both cholesterol and oxidized lipids alters phase behavior and than the inner leaflet, reflecting the increased concentration of sphingolipids stability of the LS model. While these effects are somewhat competing at in the outer leaflet, vs. an increased concentration of poly unsaturated fatty the macroscopic scale, we observe some synergy between oxidized phos- acids on the inner leaflet. This yields in turn a thicker outer leaflet with a pholipids and cholesterol at the microscale. smaller area per chain, and an inner leaflet with a higher density of hydro- phobic chain exposure to solvent. Analysis of the area density of chain unsaturation suggests local clustering of lipids with similar hydrocarbon 450-Pos tails. Rigidity of Asymmetric and Asymmetrically Stressed Membranes Amirali Hossein, Markus Deserno. 453-Pos Department of Physics, Carnegie Mellon University, Pittsburgh, PA, USA. Surface Roughness and Palmitoylation of Transmembrane Helices Lipid membranes play a central role in many biological phenomena at the Influence Membrane Structure and Dynamics cellular level. Much work over the past decades has been devoted to symmet- Adela Melcrova´1, Marie Olsinova´2, Marek Cebecauer3, Lukasz Cwiklik1. ric membranes, but it is known that in living matter membranes are almost 1Inst. of Phys. Chemistry CAS, Prague, Czech Republic, 2Inst Phys Chem, always asymmetric, meaning that they consist of leaflets that differ in their BIOCEV, Vestec, Czech Republic, 3Biophysical, Inst. of Phys. Chemistry compositions. Recent experiments have found that the bending rigidity of CAS, Prague, Czech Republic. compositionally asymmetric membranes can be more than twice that of sym- It is widely accepted that large multi-pass proteins can influence membrane metric membranes. This conflicts with the expectation that the rigidity of a structure and dynamics. On the other hand, biological membranes are densely compositionally asymmetric membrane should be the average rigidity of populated by small single-pass proteins. While such bitopic species may consti- the two corresponding symmetric bilayers. We argue that while asymmetry tute up to half of all transmembrane proteins, their influence on membrane between the leaflets can indeed increase a bilayer’s rigidity, the observed ef- properties is not well understood. Furthermore, transmembrane proteins are fect should be attributed to stress asymmetry and not a lack of additivity of commonly post-translationally palmitoylated. It is postulated that palmitoyla- monolayer rigidities. Using coarse-grained molecular dynamics simulations, tion plays a key role in helix-membrane interactions, yet the details are still we show that membranes begin to stiffen when this differential stress ex- elusive. To address these issues, we study small transmembrane helical pep- ceeds some minimum threshold. These findings also show that it is insuffi- tides in model lipid bilayers using combined experimental and computer simu- cient to classify the state of an asymmetric membrane via the composition lation approaches. We demonstrate that synthetic single-pass transmembrane alone without also specifying its differential stress. Our results have impor- helices interfere with the mobility of membrane components via trapping of tant implications for biological membranes, for which the effects of stress lipid acyl chains at their rough surface resulting in increased local membrane asymmetry on elastic properties is yet to be investigated. viscosity. The effect is more pronounced in membranes containing cholesterol which segregates from the rough surface of transbilayer helices. This may 451-Pos contribute to the formation or stabilization of membrane heterogeneities. Extending a Highly Coarse-Grained Lipid Model to Asymmetric Regarding palmitoylation, we reveal that palmitoyl chains of the naturally Membranes for MD Simulations doubly-palmitoylated PAG peptide have a complex influence on the mem- Samuel Foley, Markus Deserno. brane. In particular, they cause local membrane thickening, interleaflet Physics, Carnegie Mellon University, Pittsburgh, PA, USA. coupling and, somewhat counterintuitively, increased membrane hydration.

BPJ 9310_9313 90a Sunday, March 3, 2019

454-Pos ment to optical techniques, as it reveals molecular scale structural details The Role of Hydrophobic Mismatch on Transmembrane Helix without fluorescent probes. On its own, however, SANS is not sufficient to Dimerization in Living Cells determine the organization of the membrane. We therefore take a two- Brayan Grau1, Matti Javanainen2,3, Maria Jesu´s Garcı´a-Murria1, pronged approach, comparing molecular dynamics simulations of ternary Waldemar Kulig3,2, Ilpo Vattulainen2,3, Ismael Mingarro1, mixtures to experimental data obtained for the same mixtures. In-silico Luis Martı´nez-Gil1. neutron scattering experiments on simulated membranes are compared to 1Departamento de Bioquı´mica y Biologı´a Molecular, Universitat de the experimental data, simultaneously validating the simulation data and Vale`ncia, Burjassot, Spain, 2Laboratory of Physics, Tampere University providing a molecular scale model of the experimental system. Our all- of Technology, Tampere, Finland, 3Department of Physics, University of atom molecular dynamics simulations use preexisting software packages Helsinki, Helsinki, Finland. (e.g., Gromacs) to produce atomic trajectories of ten microseconds in length. Lipid content of biological membranes strongly affects the folding and These trajectories are processed and analyzed using custom software to packing of membrane proteins. Here, we explore how the hydrophobic generate scattering intensities for comparison to experiment, with full ac- mismatch (the difference between the hydrophobic span of a transmembrane commodation of lateral inhomogeneities. Simulated scattering intensities protein region and the hydrophobic thickness of the lipid membrane around are fit to experimental data to extract information about the lateral structure. the protein) influences the transmembrane helix dimerization and packing in Our analyses have contributed insights to the design of proposed experi- cellular environment. Using a ToxRED assay in Escherichia coli and a ments, which in turn may be used to further validate the structural signatures Bimolecular Fluorescent Complementation approach in human-derived cells reported by our computational methods. complemented by atomistic molecular dynamics simulations we analysed the dimerization of Glycophorin A derived transmembrane segments. Our 457-Pos data show that biological membranes can accommodate transmembrane Effects of Monovalent Salt on Ether-Linked Phospholipid Bilayers homo-dimers with a wide range of hydrophobic lengths. Interestingly, the Matthew W. Saunders1, Mark Steele2, Wyatt Lavigne2, Sameer Varma3, hydrophobic mismatch are found to be considerably weaker than those pre- Sagar A. Pandit2. viously observed in model membranes, or under in vitro conditions, indi- 1Department of Cell Biology, Microbiology and Molecular Biology, cating the presence of robust compensatory mechanisms able to alleviate University of South Florida, Tampa, FL, USA, 2Department of Physics, membrane stress in biological membranes. Results based on atomistic sim- University of South Florida, Tampa, FL, USA, 3Department of Cell Biology, ulations support this view, as they revealed that Glycophorin A dimers Microbiology and Molecular Biology / Department of Physics, University of remain stable, despite of poor hydrophobic match, using mechanisms based South Florida, Tampa, FL, USA. on dimer tilting or local membrane thickness perturbations. Furthermore, A distinguishing feature of the plasma membranes of Archaea is that cells also tolerate the hetero-dimers with large length disparity between their they are primarily composed of ether-linked phospholipids and not ester- monomers, and the conclusions that one can draw are essentially similar to linked phospholipids that are found in bacteria and eukaryotes. Experiments those found with homo-dimers. However, large differences between trans- on model lipid bilayers have shown that this chemical difference can man- membrane helices length hinder the monomer/dimer equilibrium, confirm- ifest into notable differences in bilayer properties. Here we employ molec- ing that, the hydrophobic mismatch has, nonetheless, biologically relevant ular dynamics simulations to understand the physical basis underlying effects on helix packing in vivo. an experimental observation that ether-linked phospholipids are less permeable to water and salts than their ester-linked chemical analogs. 455-Pos Specifically, here we present a comparison of equilibrium properties of Devlopment and Application of Bicelle Builder in Charmm-Gui two model lipid bilayers in NaCl solution — POPC and its ether-linked Christopher J. Sohn. analog that we refer to as HOPC. We note two key differences between Biological Sciences, Lehigh University, Bethlehem, PA, USA. the two bilayer systems. Firstly, the headgroup region of HOPC adsorbs Bicelles are of much interest in the field of membrane protein structure and fewer ions compared to the headgroup region of POPC. Consistent with function as they are useful models that provide appropriate bilayer environ- this observation, we note that the Debye screening length in the HOPC ments. Experimentally, researcher have used bicelles to characterize membrane system is shorter than that in the POPC system. In fact, we verify that proteins in bicelles with different lipid type. We aim to develop Bicelle Builder the mean-field approximation in Gouy-Chapman theory accurately in CHARMM-GUI (http://www.charmm-gui.org) and to apply it to explore describes the ionic distribution in bulk solvent. Secondly, we note that the various bicelle systems using molecular dynamics simulation. Toward this HOPC bilayer has an additional water density peak that coincides with a aim, we simulated both two-dimensional (2d) and three-dimensional (3d) bi- trough in the electrostatic potential. Waters in this region also have longer celle systems using a q-value (q = lipid/detergent ratio) of 0.5, 0.7, and 0.9 autocorrelation times and smaller lateral diffusion rates compared to the to examine their properties. We intend to use a geometric approach in calcu- corresponding region in the POPC bilayer. This region of tightly coordi- lating the number of detergents to place in both 2d and 3d systems. Addition- nated waters in the HOPC system also has a lower cation density. Taken ally, we simulate the caveolin-1 protein in a DMPC only system and a together, this study attributes the lower permeability of ether-linked DMPC:cholesterol system. Our preliminary simulations of the lower q-value phospholipids to the more rigid organization of water in its headgroup systems proved to be very unstable after a couple nanoseconds of simulation region. time. In addition, previous simulations of bicelle systems by other labs have only been conducted on systems with q values larger than 0.5, so our simula- 458-Pos tions will explore the biological relevance of these smaller systems and Distinct Interactions of Sodium and Calcium Cations and Neutral compare different q -value systems in a consistent manner. We aim to find suit- Phospholipid Membranes and How to Simulate Them able sizes for stable bicelle systems. Hector Martinez-Seara1, Matti Javanainen2,Adela Melcrova´3, Piotr Jurkiewicz3, Pavel Jungwirth1, Aniket Magarkar1, Martin Hof3, 456-Pos Josef Melcr1, Ricky Nencini1, Samuli O. Ollila1. Nanoscale Structure of Lipid Bilayers Revealed by In-Silico and 1Institute of Organic Chemistry and Biochemistry of the Czech Academy of Experimental Small Angle Neutron Scattering Sciences, Prague, Czech Republic, 2Dept Physics, Tampere Univ Tech, Mitchell Dorrell1,2, Frederick A. Heberle3, John Katsaras4,5, Tampere, Finland, 3J. Heyrovsky´ Institute of Physical Chemistry, Czech Edward Lyman2,6, Alexander J. Sodt1. Academy of Sciences, Prague, Czech Republic. 1Eunice Kennedy Shriver National Institute of Child Health and Human Metal cations adsorption to cellular membranes, such as sodium and cal- Development, National Institutes of Health, Bethesda, MD, USA, cium, change many crucial biological functions. Few examples are the inter- 2Department of Physics and Astronomy, University of Delaware, Newark, action with charged moieties, cell membrane potential, membrane fusion or DE, USA, 3University of Texas Health Science Center, Houston, TX, USA, cell volume. They do this by altering membrane ordering, head group region 4Biology and Soft Matter Division, Oak Ridge National Laboratory, Oak presentation and surface charge in different ways. Currently, it is unclear Ridge, TN, USA, 5Department of Physics and Astronomy, The University of how or whether cells regulate cations adsorption and hence the related func- Tennessee, Knoxville, TN, USA, 6Department of Chemistry and tions by adjusting the local lipid composition of their membranes. We have Biochemistry, University of Delaware, Newark, DE, USA. employed both fluorescence techniques and computer simulations to study Through the combination of simulations and numerical analysis with small how the presence of cholesterol - a key molecule in inducing membrane het- angle neutron scattering, we probe the lateral organization of lipid bilayer erogeneity - and temperature can affect the adsorption of sodium and cal- mixtures. Small Angle Neutron Scattering (SANS) is an excellent comple- cium on neutral phosphatidylcholine (PC) bilayers. We find that whereas

BPJ 9310_9313 Sunday, March 3, 2019 91a transient sodium binding is dependent on the number of exposed PC head Posters: Exocytosis and Endocytosis I groups, the stronger adsorption of calcium is determined only by the available surface area of the membrane at a given cation concentration. 461-Pos Notably, cholesterol plays an indirect role in enlarging the total membrane Differential Roles of Pioneer Proteins in Initiation and Stabilization of area, therefore, increasing calcium adsorption, while does not affect the Early Clathrin Coat Unveiled by a Novel Disassembly Bias Score adsorption of sodium. This difference suggests a potential mechanism for Xinxin Wang1, Zhiming Chen2, Sandra L. Schmid2, Gaudenz M. Danuser1. cells to modulate ion-membrane interaction by optimizing their lipid 1Department of Bioinformatics, UT Southwestern Medical Center, Dallas, composition. TX, USA, 2Department of Cell Biology, UT Southwestern Medical Center, Finally, when simulating these membranes, we found substantial over-binding Dallas, TX, USA. of sodium and calcium cations in the slipids and charmm36 simulation force Clathrin-mediated Endocytosis (CME) is a major pathway for cells to inter- fields. This overbinding amplifies the real cations impact to membrane structure nalize molecular cargo and recycle membrane components. Despite the well in simulations. When using non-polarizable force fields, we can attenuate this characterized essentials of CME, like clathrin, AP2 and dynamin, the roles pitfall substantially by introducing the missing electronic polarizability using of other endocytic accessory proteins (EAP) are much less understood, espe- a well-founded mean-field correction, i.e., electronic continuum correction cially the pioneer EAPs. To study their functions during CME, we probe subtle (ECC). This correction improves the behavior of current force fields when shifts of clathrin kinetics in ARPE cells after knock down (KD) of EAPs. We charge-charge interactions are critical with a zero overhead cost in the labeled the clathrin light chain (CLC) in these cells with eGFP and imaged simulations. them on TIRFM. The images are then automatically tracked, providing a statis- tically large population of CLC-eGFP time series. Unfortunately, regardless of the KD perturbation, this population is highly heterogeneous in lifetime and in- 459-Pos tensity, defying conventional analyses for detecting meaningful shifts between Memsurfer: A Tool for Robust Computation and Characterization of KD conditions and wild type. Here, we propose a non-parametric method to Bilayer Membranes classify the population based on a novel disassembly bias score, indicating Harsh Bhatia, Helgi I. Ingolfsson, Timothy S. Carpenter, the statistical bias toward early coat disassembly. The method divides the Felice C. Lightstone, Peer-Timo Bremer. full population of time series into two subgroups. The first subgroup (contain- Biosciences and Biotechnology Division, Lawrence Livermore National ing N time series) prematurely disassembles and reflects nonproductive Laboratory, Livermore, CA, USA. 1 events; the second (containing N time series) is more stable and reflects Advances in simulation methodologies, code efficiency, and computing po- 2 bona fide clathrin coats. We use the sizes and ratio of the two subgroups to wer have enabled larger, longer, and more-complicated biological mem- phenotype ten EAP KD conditions. We find EAP- and isoform-specific reduc- brane simulations. The resulting membranes can be highly complex, and tions in the initiation (N þN ) and stabilization (N / N ) of clathrin coats as- canhavecurvedgeometriesthatgreatly deviate from simple planar state. 1 2 2 1 sembly. Notably, KD of Eps15 and NECAP1 reduce both initiation and Studying such membranes require appropriate characterization of geometric stabilization, KD of Epsin reduces stabilization only, and KD of NECAP2 and topological properties of the membrane surface before any local lipid has no effect. Finally, we are combining a stochastic model of the clathrin properties, such as areas and curvatures, can be computed. We present plus EAPs and a finite element model of the cell membrane to quantitatively MemSurfer, an efficient and versatile tool to robustly compute membrane test and interpret the discovered phenotypes. surfaces for a wide variety of large-scale molecular simulations. MemSurfer works independent of the type of simulation, directly on the 3D point coor- 462-Pos dinates. As a result, MemSurfer can handle a variety of membranes, such Cell to Cell Heterogeneity of Clathrin Coat Dynamics Is Cell Cycle Depen- as tethers and vesicles, as well as atomic simulations. Using Delaunay trian- dent gulations and surface parameterizations, MemSurfer not only computes Umidahan Djakbarova1, Nathan Willy1, Shahriar Shamiluulu2, common lipid properties of interest, but also provides direct access to the Comert Kural1. membrane surface itself, allowing the user to potentially conceive and 1Physics, Ohio State University, Columbus, OH, USA, 2Private Practice, compute a variety of nonstandard properties. The software is written in Columbus, OH, USA. Cþþ with a simple-to-use Python interface, and is released open source un- Clathrin-mediated endocytosis (CME) is the most extensively studied trafficking der MIT license. This work has been supported in part by the Joint Design of pathway from the cell surface to membrane-bound organelles. Cells exhibit two Advanced Computing Solutions for Cancer (JDACS4C) program established major modes of endocytic clathrin coat formation: dynamic clathrin-coated pits by the U.S. Department of Energy (DOE) and the National Cancer Institute and large, long-lived clathrin lattices called plaques. Even within monoclonal (NCI) of the National Institutes of Health. This work was performed under cell populations, there is significant cell-to-cell heterogeneity in the relative den- the auspices of the U.S. Department of Energy by Lawrence Livermore sity of these distinct classes of clathrin coats and overall dynamics of CME. Us- National Laboratory under Contract DE-AC52 07NA27344. LLNL-ABS- ing fluorescence live cell imaging and single particle tracking analysis, we 758817. observed significant variation in CME dynamics across different stages of the cell cycle. We also found that cell spreading area swayed significantly during interphase, negatively correlated with plaque density. Taken together with 460-Pos recently demonstrated cell cycle dependent alterations in directed migration, LDB: Lipid Databank from the NMRlipids Project cytoskeletal rearrangement, adhesion remodeling and cell traction forces, these Markus S. Miettinen1, NMRlipids Collaboration2, O.H. Samuli Ollila3. 1 results provide important insights into the non-canonical functions of clathrin- Theory & Bio-Systems, Max Planck Institute of Colloids and Interfaces, coats. Our findings also suggest that conflicting observations regarding CME dy- Potsdam, Germany, 2The NMRlipids Project, nmrlipids.blogspot.fi, Finland, 3 namics can be minimized by characterizing dynamic and structural properties of Institute of Biotechnology, University of Helsinki, Helsinki, Finland. endocytic coats at different stages of the cell cycle. We present an interface for easy access to the world’s largest fully open re- pository of lipid bilayer simulation data. The contents of the LDB databank 463-Pos have been collected during the open NMRlipids project by the collective Curvature Generation by Endocytic Clathrin Coats effort of the NMRlipids participants; it currently contains PC, PS, PE, PG Joshua Ferguson1, Cemal Cakez2, Farah Hasan1, Emanuele Cocucci1, phospholipids and cholesterol. The interface available at www.nmrlipids.fi Comert Kural1. provides easy to access pointers to the datafiles stored at the public Zenodo 1Ohio State University, Columbus, OH, USA, 2University of New Mexico, server, and combines these with the available experimental data. The quality Albuquerque, NM, USA. of different simulation models (force fields) in various situations are Sculpting a flat patch of membrane into an endocytic vesicle requires curva- described using a standard report; this eases the task of choosing the most ture formation on the cell surface, which is the primary function of endocytic appropriate force field for each research question, as well as eases the com- protein complexes. The mechanism through which membrane curvature is parison of different force fields, and allows the force field developers to imposed during formation of clathrin-coated vesicles is an ongoing contro- easily see where force field improvement effort would most crucially be versy. Using electron micrographs, it was originally proposed that clathrin needed. We note that our standard metric (currently comprising the C-H initially grows into a flat array on the plasma membrane prior to transitioning order parameters and scattering form factors) used for the assessment of into a curved coat. However, flat-to-curved transition model was rejected force fields also allows automatic approaches for optimizing force field by others as it requires a substantial structural rearrangement, which is parameters. energetically unfavorable. As an alternative, it was suggested that curved

BPJ 9310_9313 92a Sunday, March 3, 2019 clathrin-coated structures form gradually without a major structural rearrange- of dynamin assembled on membranes show that nucleotide binding and hydro- ment. Currently, the endocytosis field is literally split between these two lysis leads to membrane constriction, which suggests that membrane fission models due to the lack of experimental and analytical approaches that allow proceeds via membrane constriction. We expect that further insight into the real time detection of conformational changes in clathrin coats with high res- mechanism of dynamin will be obtained from structural studies of disease- olution. In this study, using structured illumination microscopy in the total in- associated dynamin mutants. Also, structural and biophysical studies of dyna- ternal reflection mode, we demonstrate that curvature generation by clathrin- min mutants will elucidate the structural basis of disease. Consequently, we coated pits can be detected in real time within cultured cells and and tissues of have initiated structural studies of classical dynamins and disease-associated developing fruit fly embryos. We found that the footprint of clathrin coats in- mutants. Preliminary cryo-EM studies of a dynamin mutant associated with a crease monotonically until the formation of curved pits. These results show neuropathy, reveal the extent of membrane constriction may differ. This that the proposed flat-to-curved transition is not the mechanism through which work will address outstanding questions in the dynamin field and is significant clathrin pits invaginate. On the contrary, clathrin coats gain curvature at very and impactful because: (1) the structure of dynamin mutants will inform the early stages of their formation. Therefore, curvature generation by clathrin molecular basis of dynamin-associated diseases; and (2) structures of classical coats does not necessitate a dynamically unstable clathrin lattice. dynamins will enable the identification of structural features of dynamins necessary for membrane fission. 464-Pos High-Speed Atomic Force Microscopy (HS-AFM) of Clathrin-Coated Pits 467-Pos Grigory Tagiltsev1, Simon Scheuring2. GTP Concentration Bursts Locally at Endocytic Sites for Dynamin- 1Physiology and Biophysics, Weill Cornell Medical College, New York, NY, Dependent Membrane Fisson USA, 2Dept Anesthesiology, Weill Cornell Med, New York, NY, USA. Aisha Azhar, Yuuta Imoto, Shigeki Watanabe. Clathrin-mediated endocytosis is one of the major endocytosis pathways. Cla- Johns Hopkins, Baltimore, MD, USA. thrin molecules bind to the clathrin-coated vesicle (CCV) budding site, Endocytosis is a basic property of cells and is required for a variety of cellular assemble into a lattice, cover growing vesicle that finally detaches from the processes including migration, hormone signaling, and neurotransmission. plasma membrane through the action of dynamin. However, the mechanism During endocytosis, a part of plasma membrane is buckled inwards to form a by which CCVs maturate is still heavily debated. Currently, two models of vesicle. These vesicles are severed from the plasma membrane by dynamin CCV budding have been proposed. The first model states that clathrin forms GTPases. However, this reaction requires a large amount of GTP. The global initially flat lattices that transform into closed CCVs through clathrin lattice re- elevation in the GTP level within cytoplasm would be toxic to the cells, and arrangements where the curvature gradually increases as a function of matura- thus, how its level is regulated for dynamin activity during endocytosis remains tion. The second model postulates that clathrin assembles directly into curved enigmatic. Here, we measure the GTP concentrations around endocytic sites us- lattices and that the curvature stays constant while the assembly grows. Atomic ing GTP sensors and show that the GTP concentration increases acutely around force microscopy (AFM) permits observation of biological samples in 3D with endocytic pits to provide a sufficient amount of GTP for the dynamin activity. high spatial resolution, 1nm in the lateral dimensions and 0.1nm in the ver- This local enrichment of GTP precedes dynamin recruitment by 500 ms, sug- tical dimension. AFM imaging together with specifically developed image pro- gesting that GTP bursting is prerequisite for dynamin assembly. The generated cessing procedures allows us to correlate clathrin assembly size and shape GTP does not diffuse out from endocytic sites, indicating that GTP is constantly distribution on plasma membrane patches and compare the major characteris- consumed by dynamin. GTP bursting is most likely regulated by nucleoside- tics with predictions from the proposed models. diphosphate kinases, which convert ATP to GTP. Our results demonstrate that GTP concentration is spatiotemporally regulated to provide sufficient en- 465-Pos ergy for dynamin-mediated membrane fission. PIP2 Lipids as Regulators of Membrane Curvature Sensing by Enth Do- mains 468-Pos Alexis Belessiotis-Richards1, Molly M. Stevens1, Alfredo Alexander-Katz2. Dynamin-1 Drives Fission of Vesicles within 100 MS during Synaptic 1Departments of Materials, Bioengineering & Institute of Biomedical Vesicle Endocytosis Engineering, Imperial College London, London, United Kingdom, Yuuta Imoto1, Sumana Raychaudhuri2, Aisha Azhar2, Shigeki Watanabe2. 2Department of Materials Science and Engineering, Massachusetts Institute 1Johns Hopkins University School of Medicine, Baltimore, MD, USA, of Technology, Cambridge, MA, USA. 2Cell Biology, Johns Hopkins University School of Medicine, Baltimore, The ENTH (Epsin N-Terminal Homology) domain is an amino-acid sequence MD, USA. found in multiple proteins within the body. This repeated motif is understood To sustain neurotransmission, synaptic vesicles need to be recycled locally to play a crucial role in connecting proteins to cellular membranes. Indeed, the within synaptic terminals. Under physiological condition, the vesicles are ENTH domain binds phosphatidylinositol-4,5-bisphosphate (PIP2) lipids in retrieved from plasma membrane within 100 ms after fusion in hippocampal the membrane allowing it to act as an ‘anchor’ for protein attachment. The neurons via clathrin-independent ultrafast endocytosis. However, constriction localization and binding of this domain to the cell membrane, as part of the at the base of typical endocytic pits has been proposed to take at least 5s, larger Epsin-1 protein, is an essential step during clathrin-mediated endocy- hence molecular mechanism underlying fission during ultrafast endocytosis tosis. The cascade of events, however, involved in ENTH binding to PIP remains elusive. Here, we demonstrate that dynamin-1 coordinates with syn- lipids in the cell membrane is still far from understood. Furthermore, aptojanin and endophilin to constrict the neck of endocytic pits. To reveal ENTH domains localize to regions of membrane curvature and the role of molecular mechanisms, we coupled genetic depletion of these proteins such curvature in endocytosis is not clear. Here, using coarse-grained molec- with the flash-and-freeze approach, which visualizes membrane dynamics ular dynamics simulations, we investigate the relationship between the struc- of synapses in electron micrographs with millisecond temporal resolution. ture of the ENTH domain and its spontaneous membrane-curvature sensing. Our data have shown that synaptojanin and endophilin forms steep negative Interestingly, we observe switchable protein behaviour depending on the pres- curvature at the base of the endocytic pits prior to the fission by dynamin. ence of PIP2 lipids in the membrane and the presence of the terminal H0 Dynamin-1, but not dynamin-2 or -3, specifically binds this curvature amphiphilic helix within the domain. Our model shows that PIP2 lipids and severs the membrane. Phosphorylation state of dynamin-1 within the play a key role in the spontaneous localization of ENTH domains to mem- PRD domain is critical for the kinetics; only dephosphorylated state of brane curvature. Furthermore, our model upholds that the H0 helix of the dynamin-1 can mediate fission within 100 ms. Thus, despite its speed, ultra- domain forms post-PIP2 binding. This suggests a potential mechanism fast endocytosis is mediated through its coordinated actions of synaptojanin, for initiating endocytosis whereby membrane curvature coupled with PIP2- endophilin and dynamin. enrichment at curved membrane regions can localize Epsin-1 and subse- quently kick-off the endocytotic process. 469-Pos Mechanochemical Feedback Control of Dynamin Independent Endocy- 466-Pos tosis Modulates Membrane Tension in Adherent Cells Cryo-EM Studies of Classical Dynamins to Reveal the Mechanism of Mem- Joseph Jose Thottacherry1, Anita Joanna Kosmalska2, Susav Pradhan3, brane Fission Parvinder Pal Singh4, Xavier Trepat2, Ram Vishwakarma4, John Jimah1, Abigail Stanton1, Huaibin Wang2, Jenny E. Hinshaw1. Pramod Pullarkat3, Pere Roca-Cusachs2, Satyajit Mayor1. 1Structural Cell Biology Section, Laboratory of Cell and Molecular Biology, 1National Centre for Biological Sciences, Bengaluru, India, 2IBEC, NIDDK, NIH, Bethesda, MD, USA, 2MICEF, NIDDK, NIH, Bethesda, Barcelona, Spain, 3Raman Research Institute, Bengaluru, India, 4Indian MD, USA. Institute of Integrative Medicine, Jammu, India. The classical dynamin GTPases (dynamin 1, 2 and 3) mediate membrane Plasma membrane separates the cells from its extracellular environment and fission necessary for vesicle scission during endocytosis. Cryo-EM structures modulates bidirectional signalling across it. The regulation of the chemical

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(composition) and physical (tension) properties of the membrane tunes the through CEACAM, can be used to induce uptake of proteoliposomes into hu- nature of the information transmitted across the bilayer. Membrane traf- man cells through Opa-CEACAM interactions. We present a method to mea- ficking via endo-exocytic processes can modulate the chemical and physical sure the cellular binding and uptake of Opa-liposomes using imaging flow (membrane tension) properties of the membrane and is in turn regulated by it. cytometry. Compared to control liposomes, Opa60 on liposomes was found However, the specific cellular processes and molecular mechanisms behind to promote energy-dependent uptake of liposomes into CEACAM-expressing this regulation are poorly understood. We have used multiple ways to modu- HeLa cells. Increased Opa proteoliposome uptake is correlated with higher late membrane tension, including a custom designed cell stretching device, to CEACAM expression on cells. Following internalization, liposomes may be understand role of endocytic processes in membrane homeostasis. In parallel, processed through the classical endocytic pathway. Given the overexpression we have utilized optical tweezers to measure membrane tension on modu- of CEACAM proteins on several distinct cancers and the strong interest in us- lating endocytosis. We find that lowering of membrane tension results in a ing CEACAMs as a component in treatment strategies, these results support passive membrane response and the excess membrane forms membrane tu- further investigation of Opa-dependent nanoparticle internalization in thera- bules or invaginations that is subsequently resorbed by an active endocytic peutic delivery. process. Although multiple endocytic pathways operate simultaneously at the cell surface, we find that a specific endocytic pathway, the dynamin- 472-Pos independent CLIC/GEEC (CG) endocytic pathway is transiently up regulated 3D Trafficking of Epidermal Growth Factor Receptor in Live Cells 1 2 2 during change in membrane tension. By contrast, clathrin or caveolar path- Marco A. Alfonzo Mendez , Harshad Viswasrao , Hari Shroff , Justin W. Taraska1. ways are not modulated. However, alteration in membrane tension does 1 2 not directly modulate CG endocytosis. This modulation requires vinculin, NHLBI, NIH, Bethesda, MD, USA, NIBIB, NIH, Bethesda, MD, USA. a mechano-transducer recruited to focal adhesion in adherent cells. Vinculin Epidermal Growth Factor Receptor (EGFR) controls key cellular functions acts by controlling the levels of a key regulator of the CG pathway, GBF1, at such as proliferation, migration, differentiation and survival. The removal the plasma membrane. On the other hand, perturbing the CG pathway of the receptor from the plasma membrane is triggered by the binding directly modulates membrane tension, suggesting that this cellular mecha- of EGF and mostly occurs through clathrin-mediated endocytosis. The nism is likely to be involved in homeostatic control of membrane tension. latter mechanism has been previously studied using total internal reflection These features suggest that the CG pathway could be a vital component of fluorescence and confocal microscopy in live cells. However, these ap- the cellular machinery that sets resting membrane tension and possibly main- proaches are limited to image specific cell regions or provide low temporal tain area homeostasis. resolution and high photobleaching. Here, we use dual inverted selective plane illumination microscopy (diSPIM) to image the recruitment and 470-Pos intracellular trafficking of EGFR into clathrin-coated vesicles in live cells. The Effect of Acute ATP Depletion on Synaptic Vesicle Endocytosis at the We track the movement and distribution of EGFR and clathrin across entire Ultrastructural Level cells to determine the global trafficking patterns of these receptors in live Quan Gan, Shigeki Watanabe. cells. Department of Cell Biology, Johns Hopkins University, Baltimore, MD, USA. 473-Pos Following synaptic vesicle fusion at the presynaptic terminal, vesicular mem- Phosphatidylserine (PS) Externalization Facilitates Membrane Vesicula- brane and proteins must be retrieved via endocytosis to replenish the vesicle tion through Decreasing Membrane Stiffness pool. A recent study revealed using the pHluorin assay that local depletion Hongyin Wang, Joseph H. Lorent, Lakshmi Ganesan, of ATP at presynaptic terminals impairs the rate of vesicle cycling during sus- Blanca B. Diaz-Rohrer, Kandice R. Levental, Eric Malmberg, Ilya Levental. tained synaptic activity, possibly by blocking endocytosis. However, due to The University of Texas Health Science Center at Houston, Houston, TX, limitations inherent to the pHluorin approach, it is unknown which exact USA. step in the vesicle cycling process is most sensitive to the availability of Extracellular vesicles (EVs) including exosomes and microvesicles play ATP. In the present study, we used time-resolved electron microscopy to cap- critical roles in intercellular communication by exchanging proteins, lipids, ture rapid ultrastructural changes in the presynaptic membrane of cultured hip- and genetic materials between cells. However, the origin and biogenesis of pocampal neurons at various time points following stimulation. We combined these vesicles remain unclear. It has been noted that almost all EVs present this method with drug treatments that acutely block ATP synthesis, and found the anionic membrane lipid phosphatidylserine (PS) on their outer leaflet, in that acute ATP depletion leads to the arrest of spontaneous as well as activity- opposition to their cells of origin, which almost exclusively retain PS in the induced synaptic vesicle endocytosis at early stages. In addition, blocking cytoplasmic leaflet. The "scrambling’’ of PS from the inner to the outer oxidative phosphorylation alone has a weaker effect than blocking both oxida- leaflet of the bilayer is facilitated by the lipid channel Ano6. This process tive phosphorylation and glycolysis. This suggests that anaerobic glycolysis of lipid scrambling has been implicated in a variety of physiological con- alone might be able to partially meet the energy demand of vesicle cycling. texts, including apoptotic cell removal, bone mineralization, viral infection Our results shed new light on the energetics of synaptic vesicle endocytosis, and blood coagulation. However, the biophysical role of PS externalization and may have important implications for the pathophysiology of brain in the formation of extracellular vesicles is still a mystery. Using ischemia. micrometer-sized giant plasma membrane vesicles (GPMVs) that are pro- duced by membrane blebbing from mouse B-cells (BaF3), we studied the 471-Pos role of PS externalization in membrane vesiculation. We first found that a Quantifying CEACAM Targeted Liposome Delivery Using Imaging Flow scramblase (i.e. Ano6) knockout BaF3 cell line was deficient in producing Cytometry large vesicles compared with the wild type cell line, indicating that PS Jason P. Kuhn1, Asya Smirnov2, Alison Criss2, Linda Columbus1. externalization is necessary for membrane budding. Importantly, through 1Chemistry, University of Virginia, Charlottesville, VA, USA, fluorescence lifetime imaging microscopy (FLIM) using a reporter of lipid 2Microbiology, Immunology, and Cancer Biology, University of Virginia, packing (Di4), we found that PS exposure lead to a decrease of plasma Charlottesville, VA, USA. membrane packing, potentially make the membrane softer for bending and Carcinoembryonic antigen-like cell adhesion molecules (CEACAMs) are thus facilitating membrane budding. To confirm this assumption, we artifi- cell-surface proteins that are widely-expressed in the human body. Several cially decreased the lipid order on the plasma membrane by increasing CEACAM proteins exhibit increased expression on tumour cells. Because of the incubation temperature or treating the cells with methyl-b-cyclodextrin the overexpression of CEACAM proteins on malignant cells, there is interest (MbCD). Under all conditions which reduced membrane stiffness, large ves- in developing strategies to target therapeutic delivery to cells overexpressing icles were efficiently produced from the PS externalization-deficient Ano6 CEACAM proteins. We investigate whether Opacity-associated proteins knockout cell line, confirming the crucial role of membrane stiffness in from Neisseria gonorrhoeae, which promote bacterial uptake into human cells cell vesiculation.

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Posters: Excitation-Contraction Coupling I 476-Pos Imperacalcin’s Antiarrhythmic Effects are Preserved at 370C in Whole 474-Pos Hearts from a CPVT Mouse Model Chronic Effects of Aldosterone on Cardiac EC Coupling and Oxidant Rachael N. Thorson1, Jordan Price1, Yuriana Aguilar2, Carmen R. Valdivia3, Hector Valdivia3, Rafael Mejı´a-Alvarez1. Stress 1 Marı´a Guadalupe Montiel-Jaen, Adrian Monsalvo-Villegas, Physiology, Midwestern University, Downers Grove, IL, USA, 2Physiology & Biophysics, Rush University Medical Center, Chicago, IL, Guillermo Avila. 3 Dept Biochemistry, CINVESTAV- IPN, Mexico City, Mexico. USA, Medicine, University of Wisconsin, Madison, WI, USA. Sarcoplasmic reticulum (SR) Ca2þ overload during b-adrenergic stimulation Our previous work investigated the role of aldosterone in the function of 2þ atrial myocytes. Among other effects, aldosterone promoted a striking precipitates arrhythmogenic Ca release in cardiomyocytes expressing 2þ RYR2 mutations associated with catecholaminergic polymorphic ventricular 2-fold increase in the density of Ca current (ICa) and a moderated tachycardia (CPVT). We previously observed in whole hearts at 23C, that increase of 30% in cell membrane capacitance (Cm, suggesting hypertrophy). 2þ Ca2þ and ROS were identified to participate but only on the effect on C . Imperacalcin (IpCa) prevents/reverts CPVT by partially depleting SR [Ca ] dur- m b 2þ The objective of this work was twofold: (a) to determine whether aldoste- ing -adrenergic stimulation. Since SR [Ca ] load is highly temperature depen- dent, the IpCa effects need to be defined at physiological temperatures. If at higher rone regulates ROS generation; and (b) to reexamine the potential of þ 2þ temperature, SR Ca2 flux through IpCa-modified RyR2 is not increased at com- aldosterone to increase ICa,usingalowCa buffering internal solution— þ þ 2þ parable levels than the larger SR Ca2 content due to enlarged Ca2 reuptake, the this allows recording Ca transients as well. Adult rat cardiomyocytes m antiarrhythmic effects of IpCa at 37 C will not be equally effective to those at were chronically incubated with 1 M aldosterone. Subsequently, ICa 2þ and Ca2þ transients were recorded under whole-cell patch clamp. lower temperature. To investigate this possibility, the IpCa effects on Ca tran- sients and action potentials (AP) were studied at 23,31,and37C, in whole CM-H2CDFDA was used to monitor ROS. Under the present conditions, hearts from wild type (WT; n = 49) and RyR2-R4496Cþ/- (n = 6) mice. Ca2þ tran- aldosterone did not alter the peak amplitude of ICa. However, it promoted 2þ sients were recorded with pulsed local field fluorescence microscopy and AP with a significative increase in the magnitude of both Cm and Ca transients þ þ (20-50%). The caffeine-sensitive Ca2 store was not significantly altered, microelectrodes. An arrhythmogenic cocktail (dobutamine caffeine) was used to induce CPVT. When this cocktail was perfused to WT hearts, no arrhythmias indicating an enhancement of the gain of excitation-contraction coupling þ/- at constant SR Ca2þ load. As suspected, aldosterone promoted a 75% were observed; however, RyR2-R4496C hearts showed significant variability 5 increase in ROS production, which: became evident after 2-d of treatment, in the Gaussian-fitted frequency distribution of AP intervals (APint;mean SD). þ/- 5 reaches a maximum in 4-6d, and presents an EC of 1.2 nM (Hill Before CPVT, RyR2-R4496C hearts exhibited one mean APint (751.3 50 5 coefficient of 0.9). This effect may be explained by a potential regulation 123 ms at 31 C; 250.5 60 ms at 37 C), but during CPVT they had two APint, 5 5 of the activity/expression of NADPH oxidase (NOX) and aldehyde one long (1,135.3 402 ms at 31 C; 530 20 ms at 37 C) and one short (350.1 5 49.9 ms at 31C; 310 5 25.9 ms at 37C). Addition of IpCa restored dehydrogenase (ALDH). Thus, we used activators (Alda-1) and inhibitors one APint (1,047 5 120 ms at 31 C; 290 5 12 ms at 37 C). Our results demon- (daidzin) of ALDH and found that they can either, prevent (Alda-1) or 2þ reproduce (daidzin) the stimulated ROS production. The possible contribu- strate that Ca flux through IpCa-modified RyR2 is enhanced at physiological tion of NOX has yet to be examined along with the potential of these en- temperatures. zymes to mediate hypertrophy. These results may contribute to explaining 477-Pos a previously observed correlation between aldosterone levels and atrial Impaired b-Adrenergic Responsiveness in HFpEF Rats dysfunction. Peter J. Kilfoil, Daniel Soetkamp, Rui Zhang, Jae Cho, Stephan Aynaszyan, Eugenio Cingolani, Eduardo Marba´n, Joshua I. Goldhaber. 475-Pos Smidt Heart Inst, Cedars-Sinai, Los Angeles, CA, USA. Underlying Physiological Condition Act as a Critical Factor in Phenotypic Most patients suffering from heart failure with preserved ejection fraction Realizations of Excitation Coupling (Ec) Abnormalities in Targeted (HFpEF) only become symptomatic upon exertion, which could be a sign of Cancer Therapeutic Treated Hipsc Cardiomyocytes abnormal b-adrenergic signaling. To test this hypothesis, we used the Dahl Jaehee Shim. salt-sensitive (DS) rat model of HFpEF to assess the response of systolic and dia- Dept Syst Biol, Mt Sinai, New York, NY, USA. stolic function to adrenergic stress. We generated HFpEF by feeding DS rats a Tyrosine kinase inhibitors (TKIs) are a class of cancer therapeutics that have high salt diet (8% Na) for 11 weeks, and then confirmed abnormal diastolic relax- been implicated for serious cardiotoxicity. Unlike arrhythmias caused by ion ation indicative of HFpEF using mitral inflow and tissue Doppler echocardiog- channel block, TKI-induced toxicity develops over weeks to months of ther- raphy. We also documented clinical signs of heart failure. In patch clamped apy. We propose a ‘‘two-hit hypothesis’’ for revealing TKI-induced cardio- left ventricular cardiomyocytes isolated from HFpEF hearts and depolarized toxicity. Specifically we propose that TKIs induce early changes in gene from 50 to 10 mV, isoproterenol (ISO) increased ICa in HFpEF by only expression that may make cardiomyocytes more susceptible to secondary in- 4258%, compared to control cells where there was a 98515% increase sults. We tested this idea by combining mathematical modeling with tran- (p<0.01). Similarly, Ca transient (CaT) amplitude in field-stimulated HFpEF scriptomic and physiological measurements. TKI-induced changes in gene myocytes loaded with the Ca-indicator fluo-4 AM increased 59511% in expression, measured in stem cell-derived myocytes (iPSC-CMs) were inte- HFpEF, compared to 2157% in control myocytes, p<0.01. ISO caused CaT grated into a mechanistic model that simulated EC coupling of these cells. relaxation rate (t1/2) to speed up by only 13% in HFpEF whereas there was a Simulations assessed drug effects on contractile strength, as well as suscep- much greater increase in relaxation rate (34%) in control, p<0.01. In vivo exper- tibility to the secondary insult of hypokalemia. Specifically, based on tran- iments with tail vein infusion of dobutamine (2.5-20mg/kg/min) elicited a blunt- scriptomic measurements after treatment with 24 TKIs in two iPSC-CM ed contractile (as measured by ejection fraction, p<0.001 by ANOVA) and lines, our simulations predicted the degree of hypokalemia necessary to relaxation (as measured by E/e’, p<0.001) response in HFpEF vs Control. Pro- induce arrhythmic dynamics. We found that the two iPSC-CM lines pro- teomic analysis of HFpEF rats revealed reduced expression of PKA catalytic duced dramatically different results, with the three most dangerous TKIs subunits Prkaca (26.6%, p=0.0001), and the regulatory subunits Prkar1a showing no overlap between the two groups. For instance, trastuzumab- (19.6%, p=0.0001) and Prka1b (30.4%, p=0.0002). Type II regulatory sub- induced changes in gene expression predisposed one cell line to arrhythmias unit Prkar2b was increased 86%, p=0.002. Strikingly, the abundance of PKI, a whereas trametinib caused the greatest increase in susceptibility in the other powerful inhibitor of the PKA catalytic subunit, was increased 1443% in HFpEF line. These predictions are being experimentally tested using optical (p=0.000002). These changes may account for the blunted adrenergic respon- measurements of action potentials (FluoVolt) and intracellular calcium siveness we observe in the HFpEF model. (fluo-3) in iPSC-CMs. Preliminary results are consistent with predictions: untreated cells display arrhythmic dynamics at extracellular [K] = 2.9 mM 478-Pos whereas arrhythmic dynamics occur at [K] = 4.1 mM in trastuzumab- Quantitative In Silico Analysis of the Arrhythmogenic CaMKII-Sodium- treated cells. Calcium-CaMKII Feedback in the Failing Rabbit Ventricular Myocyte These results support the ‘‘two-hit hypothesis’’ that TKIs are not strong Caroline Liu, Bence Hegyi, Haibo Ni, Donald M. Bers, Eleonora Grandi, enough insult to induce EC abnormalities by themselves but the presence of Stefano Morotti. even a mild level of secondary insult can drastically augment the potency Department of Pharmacology, University of California Davis, Davis, of TKI toxicity. CA, USA.

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Heart failure (HF) is a complex disease characterized by abnormal con- myocytes exhibit a limited life span and immortalized cell lines lack traction, metabolic imbalance, and increased propensity for arrhythmias. Dys- physiologically relevant automaticity. Pediatric animal models include regulation of intracellular Naþ handling is a major (yet understudied) aspect caveats related to action potential morphology, ion channel expression, and of HF-induced remodeling of cardiac myocytes. Elevated late Naþ current excitation-contraction coupling. Changes that occur in these parameters as (INaL) in HF prolongs the action potential (AP), thereby facilitating the devel- the animal transition from neonate to adult are relatively unknown, especially opment of arrhythmogenic early afterdepolarizations. Increased Naþ loading as it may be translated to a human model. In quantifying the toxicological limits the ability of the Naþ/Ca2þ exchanger to remove Ca2þ, which along effect of plasticizer exposure in a pediatric population, we first sought to with the reduced sarcoplasmic reticulum (SR) Ca2þ uptake and increased dia- establish the limits of using hearts from developing neonatal rats as a viable stolic SR Ca2þ leak leads to Ca2þ overload, thus contributing to diastolic model. dysfunction and triggered arrhythmias (i.e., via delayed afterdepolarizations). This study aimed to quantify the advantages and limitations associated with Increased Ca2þ signals enhance the activity of the Ca2þ/calmodulin-depen- using neonatal rat hearts as a model for pharmacology screening. dent protein kinase II (CaMKII), which is upregulated and chronically active The heart from rats ranging in age from 2 days old, up to adult were excised, þ 2þ in HF and directly promotes INaL, diastolic Na influx and SR Ca leak. To and the aorta was cannulated. It was placed on a Langendorff system and investigate quantitatively this vicious cycle of positive feedback in HF, we retrograde perfused. Calcium and voltage sensitive dyes were used to stain updated our computational model of the failing rabbit ventricular myocyte. the heart for imaging via a single sensor, single excitation, dual emission sys- We modified the main repolarizing and depolarizing currents to reproduce tem. It was mechanically uncoupled with blebbistatin to eliminate motion the HF-induced changes measured during AP-clamp experiments performed artifacts. with physiologic Ca2þ handling 5 CaMKII inhibition. We validated the Compared with adults, isolated neonatal rat hearts displayed a longer action cellular model using data describing the frequency-dependence of AP and potential duration (APD80: adult= 61.28ms n=9, neonatal=109.4ms Ca2þ transient properties assessed in normal condition and when various n=34, P<0.05) associated with delayed repolarization as also evidenced branches of the feedback loop are blocked. This updated model serves as a by an increased in triangulation. Calcium handling was also slower in framework to investigate the role of the CaMKII-Naþ-Ca2þ-CaMKII feed- the neonatal heart (Cad80: Adults: 126.8ms n=12, neonatal=170.1 n=30, back in promoting Ca2þ and AP instabilities. Analysis of the relative roles P<0.05), likely due to immature expression and localization of key of the interacting components that form the feedback loop within the inte- calcium handling proteins. The developing excitation-contraction coupling grated AP-Ca2þ cycling-signaling model will allow the identification of the machinery will be further probed using pharmacological tools, and key relationships in the signaling network that could be targeted therapeuti- this newly established pediatric model will be used for toxicological cally to limit arrhythmias in HF. screening.

479-Pos 481-Pos Loss of Dystrophin Alters Calcium-Handling Maturation in Response to In Vivo Gene Delivery of R-Cepia1Er: A New Approach to Study [Ca]Sr Microenvironment in Hipsc-Cardiomyocytes from Duchenne Muscular Handling in Cardiomyocytes Dystrophy Patients Elisa Bovo1, Quan Cao1, Daniel Kahn1, Roman Nikolaienko1, J. Manuel Pioner1, Raffaele Coppini1, Lorenzo Santini1, Chiara Palandri1, Jody L. Martin2, Ivana Y. Kuo1, Aleksey V. Zima1. Flavia Lupi1, Marianna Langione1, Patrizia Benzoni2, Sara Landi2, 1Cell and Molecular Physiology, Loyola University Chicago, Maywood, IL, Andrea Barbuti2, Chiara Tesi1, David L. Mack3, Michael Regnier3, USA, 2Dept Physiology/CCVR, Univ of Illinois Chicago, Chicago, IL, USA. Camilla Parmeggiani1, Corrado Poggesi1, Cecilia Ferrantini1. In cardiomyocytes, [Ca] within the sarcoplasmic reticulum (SR; [Ca] ) deter- 1 2 SR University of Florence, Florence, Italy, University of Milan, Milan, Italy, mines the amplitude of SR Ca release which, in turn, governs myocardial 3 University of Washington, Seattle, WA, USA. contraction. Therefore, it is critical to understand the molecular mechanisms Cardiomyopathy invariably affects teenage patients with Duchenne Muscular that regulate SR Ca handling. Until recently, the best approach available to Dystrophy (DMD). In humans, developmental alterations due to loss of dys- directly measure [Ca]SR was using low affinity Ca indicators, such as Fluo- trophin are poorly understood. We analyzed maturation of human induced 5N. However, this method presents several limitations, including non- pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to provide in- specific cellular localization and dye extrusion. This approach also does not sights into early-stage of disease mechanisms. We combined nanopatterned work in myocytes isolated from small rodents that are commonly used for substrates with long-term culture to improve maturation of hiPSC-CMs. We transgenic studies. Recently, we designed an approach to selectively measure studied hiPSC-CM Ca-handling at multiple time points (60-75-90 days). To [Ca]SR in cardiomyocytes using the genetically encoded Ca indicator, model the consequences of full-length dystrophin loss, we used a CRISPR- R-CEPIA1er. However, expression of R-CEPIA1er requires >48 hours of Cas9 genome edited cell line targeting wild-type dystrophin locus in the culturing, which significantly alters myocyte structure and function. To over- control-hiPSC line (DMD-DExon1) and compared with hiPSC-CMs derived come this limitation, we used in vivo gene delivery of the R-CEPIA1er from a DMD patient (DMD-DExon50). In Control- and DMD-CMs, we cDNA with adenovirus (AV) in mice hearts. Six days after AV injection in 2þ simultaneously measured action potentials (AP) and Ca transients (Ca-T), the left ventricular wall, myocytes were isolated. Confocal microscopy was with Fluovolt and Cal630 dyes, respectively. In control CMs we observed used to analyze [Ca]SR dynamics in freshly isolated myocytes. Myocytes ex- over time a prolongation of action potential duration (APD), increased Ca-T pressing R-CEPIA1er showed periodic peaks of fluorescence at SR junctions amplitude, and faster Ca-T rise (time to peak) and Ca-T decay (RT50). Inter- and in the nuclear envelope. We characterized the effect of different pacing fre- estingly, APD and CaT-RT50 were positively correlated, suggesting that quencies on key parameters of SR Ca regulation such as diastolic SR Ca load, larger sarcolemmal calcium entry underlies longer duration of CaT. The systolic [Ca]SR transient and Ca fractional release. These parameters were contribution of the SR to Ca release (as estimated with a post-rest potentiation analyzed under control conditions and after adrenergic receptor stimulation protocol or Caffeine-induced Ca-T) increased over time and appeared as the with isoproterenol. We also used this approach to study SERCA-mediated main determinant of the progressive rise of CaT amplitude. Compared to con- Ca uptake and ryanodine receptor-mediated Ca leak in permeabilized ventric- trols, at all stages of maturation the DMD-CMs showed: shorter APD, reduced ular myocytes. The results of this study demonstrate that in vivo gene delivery CaT amplitude and faster Ca-T rise and RT50. The positive-correlation be- of R-CEPIA1er can be used as a sensitive assay to study [Ca]SR regulation in tween APD and CaT was preserved, while, no increased post rest potentiation control and transgenic rodents. was observed over time, suggesting impaired maturation of SR function. We then tested different surfaces, with various stiffness during maturation. Increased stiffness associated with larger CaT amplitude. Notably, DMD- 482-Pos CMs had less adptiveness to changes in the substrate stiffness. Acknowledg- An Autosomal Dominant Mutation in Calsequestrin 2 Causes CPVT ment: Telethon Italy grant (GGP16191). Without Changing Protein Levels Matthew Wleklinski, Shan Parikh, Bjorn C. Knollmann. 480-Pos Vanderbilt University Medical Center, Nashville, TN, USA. Dual Calcium and Voltage Mapping Reveals Differences in Maturity of Calsequestrin-2 (Casq2) is a high capacity, low affinity calcium (Ca) binding Excitation-Contraction Coupling in Young Rat Hearts protein located in the junctional sarcoplasmic reticulum (SR) of cardiac my- Luke Swift, Rafael Jaimes, Nikki G. Posnack. ocytes. As the major SR Ca buffer, Casq2 interacts with the ryanodine recep- Children’s National Health System, Washington, DC, USA. tor (RyR2), a Ca release channel, to regulate the amount of Ca that is released In the broad field of cardiology, pediatric research has lagged behind in es- during the excitation-contraction (EC) coupling cycle, a process that couples tablishing appropriate models to study the developing heart. Human cardio- electrical activation to mechanical force (i.e. a heartbeat). Alterations in EC

BPJ 9314_9317 96a Sunday, March 3, 2019 coupling can cause both contractile dysfunction and cardiac arrhythmias. littermates. Using whole-cell patch clamp, we studied how the absence of Reduction or loss of Casq2 due to mutations causes a severe genetic CK system influences calcium handling in cardiomyocytes of AGAT KO arrhythmia syndrome known as catecholaminergic polymorphic ventricular and WT littermates. Voltage dependence of L-type calcium channel tachycardia (CPVT). Casq2-linked CPVT is usually autosomal-recessive, (LTCC) current was significantly different in KO and WT animals with with mutations resulting in either a severe reduction or complete loss of the significant differences between sexes within groups. LTCC amplitude Casq2 protein. As a result, SR Ca buffering is reduced, which leads to spon- was influenced by the absence of active CK system only in female mice taneous Ca release and arrhythmias. In 2016, a genetic analysis conducted in in control conditions but was found to be the same in KO and WT male a family that had an autosomal dominant inheritance of CPVT uncovered a mice and mice of both sexes after adrenergic stimulation. The absence of novel missense mutation (K180R) within Casq2. This was the first autosomal active CK system had a major impact on calcium uptake, with the KO dominant mutation found in Casq2. It was proposed that this mutation created mice requiring more time for reducing calcium concentration to the resting a dominant negative mutation in Casq2, which would lead to degradation and level at the lower uptake rates. The effects of the active CK system absence loss of Casq2. The goal of our study was to generate a heterozygous K180R on LTCC and calcium uptake, presumingly mainly by SR calcium ATPase, knock-in mouse model to evaluate the levels Casq2 and test whether the were fully reversed by creatine feeding to the KO animals. Our results sug- K180R mutation causes CPVT. Our results show that K180R knock-in gest that there is a major role of the CK system in providing energy to mice have normal Casq2 protein levels but exhibit CPVT when stressed. excitation-contraction machinery in the heart to maintain normal calcium This suggests that Casq2-K180R causes CPVT by a different mechanism homeostasis. than previously reported autosomal-recessive Casq2 mutations. Future studies will investigate the ability for Casq2-K180R to regulate RyR2 Ca 485-Pos release channels and how this could alter SR Ca buffering, leading to spon- Functional Impact of Cell Culture on Excitation-Contraction Coupling taneous Ca release. in Canine Myocytes Alida Cooke1, Zachary Williams1, Samuel Olczyk1, Robert J. Goodrow1, 483-Pos Jonathan A. Cordeiro1, Jacqueline A. Treat1, Gary L. Aistrup1, Bridging HTS Ion Channel and Myocyte Data Jonathan M. Cordeiro2. George O. Okeyo1, Sonja Stoelzle-Feix2, Timothy Strassmaier1, 1Masonic Medical Research Lab, Utica, NY, USA, 2Dept Exper Cardiol, Krisztina Juhasz2, Nadine Becker2, Ulrich Thomas2, Leo Doerr2, Masonic Med Res Lab, Utica, Markus Rapedius2, Nina Brinkwirth2, Claudia Haarmann2, Tom Goetze2, NY, USA. Matthias Beckler2, Michael George2, Andrea Bruggemann€ 2, Niels Fertig2. Downregulation of ion currents as well as a loss of T-tubules is documented 1Nanion Technologies Inc., Livingston, NJ, USA, 2Nanion Technologies during heart failure. A similar reduction of both has been observed in cultured GmbH, Ganghoferstrasse 70A, 80339 Munich, Germany. ventricular myocytes. We further assessed the functional impact of cell-culture Human induced pluripotent stem cells (hiPSCs) are relevant for cardiac on excitation-contraction (EC) coupling in dog ventricular, atrial and Purkinje safety testing due to their validated predictivity as described in recent pub- myocytes. lications derived from the comprehensive in vitro proarrhythmia assay Ventricular, atrial and Purkinje myocytes were isolated and cultured for (CiPA) study. We combined automated patch clamp (APC), impedance up to 48 hours. Myocytes were stained with di-8-ANEPPS to visualize and extracellular field potential (EFP) measurements to study cardiac ion ultrastructure and Ca2þ transients (CaTs) were recorded by confocal micro- channels in cell lines and hiPSC-CMs. Cell lines expressing different cardiac scopy. Ion channel currents were assessed via patch electrode voltage ion channels were recorded on two different APC instruments (8 or 384 clamp. wells simultaneously) at room and physiological temperature, at 4 different Membrane staining with di-8-ANEPPS indicated dog ventricular myocytes sites. Within the myocyte validation study of the CiPA initiative, having an extensive T-tubular network; atrial myocytes having a rudimentary hiPSC-CMs from different providers were used on a device combining T-tubule system at best, and Purkinje myocytes having no T-tubules. Trans- impedance-based contractility and extracellular field potential (EFP) record- verse X-t lines scans of electrically stimulated ventricular myocytes showed ings. Within the ion channels working group of the CiPA initiative, ion CaTs rising synchronously across the cell, whereas in Purkinje myocytes the channel data on 7 cardiac ion channel currents were measured. The effects CaT rise showed a U-shaped profile. CaT rise in atrial myocytes typically of >20 CiPA reference compounds deemed low, intermediate and high showed a U-shaped profile, but less pronounced than that in Purkinje myocytes. risk by the FDA were investigated. In parallel to APC investigations, ar- Ventricular myocytes cultured for 48 hours indicated a dramatic loss of rhythmogenicity of this and other compounds were investigated via contrac- T-tubules and a switch from a synchronous CaT rise to a U-shaped profile. tility and field potential recordings in 2D monolayers of iPSC-CM. Our In cultured atrial cells, no loss of T-tubules nor alterations in CaTs was noted. results show that high risk compounds such as dofetilide prolong field poten- ICa in ventricular myocytes cultured for 48 hours was reduced by 21%. tial duration which resulted in the detection of arrhythmic events in both Comparative assessment of culture-induced changes in ion channel currents impedance and EFP recordings. Datasets from multiple sites and cell types in atrial myocytes is ongoing. will be shown and compared. Cross-site/cell comparisons of APC ion chan- While cultured ventricular myocytes show a loss of T-tubules and phenotypic nel data and myocyte repolarization data was combined in order to investi- switch from a homogeneous CaT rise to a ‘U’-shaped profile, atrial myocytes gate the mode of action of reference compounds. This promotes a good cultured identically show little change in ultrastructure and CaT profile. understanding of drug-induced arrhythmia and thus represents a valuable These results suggest that character and degree of change in EC coupling approach in drug development efforts. induced by similar pathological insults markedly depends on cardiac myocyte type. 484-Pos The Absence of Active Creatine Kinase System Influences Cardiac 486-Pos Calcium Handling Double Regulation of Cardiac Excitation-Contraction Coupling and Martin Laasmaa, Jelena Branovets, Niina Karro, Rikke Birkedal, Oxidant Stress by Pirfenidone Marko Vendelin. Adrian Monsalvo-Villegas, Guillermo Avila. Department of Cybernetics, Tallinn University of Technology, Tallinn, Dept Bioquimica, Cinvestav-IPN, Mexico City, Mexico. Estonia. Pirfenidone (PFD) is well-known for improving cardiac performance in Creatine kinase (CK) system is known as a phosphoryl buffering and trans- models of disease. Thus, here we have investigated its functional effects on fer system between mitochondria and ATPases. Functional coupling be- cardiomyocytes. Cells were cultured under control culture conditions or the tween CK and adenine nucleotide translocase, ATPases, and ATP-sensing presence of PFD (1 mM, 1-2d). Subsequently, they were subjected to electri- potassium channel, has been demonstrated suggesting a major role of the cal stimulation to examine the levels of intracellular Ca2þ and contractility. CK system in the heart. To study CK system role, we used two lines of Remarkably, PFD improved both peak contraction and kinetics of shortening creatine-deficient mice, which lack either guanidinoacetate methyltransfer- and relaxation. Moreover, the amplitude and kinetics of Ca2þ transients were ase (GAMT) or arginine-glycine amidinotransferase (AGAT) on creatine also enhanced. Excitation-contraction coupling (ECC) was also investigated, synthesis pathway. Intriguingly, there have been no significant adaptations under whole-cell patch-clamp. In keeping with a previous report, PFD to the absence of active CK system in GAMT and AGAT mice cardiomyo- elevated twofold the activity of CaV1.2. Besides, a similar increase in the cytes from bioenergetics perspective: mitochondrial arrangement, respira- magnitude of Ca2þ transients was also observed. Thus the gain of ECC was tion kinetics, activities of alternative pathway enzymes, intracellular unaltered. Likewise, PFD did not alter the caffeine-sensitive Ca2þ store, indi- compartmentation are the same in wild-type (WT) and knock-out (KO) cating stimulation of Ca2þ-induced Ca2þ-release at constant sarcoplasmic

BPJ 9314_9317 Sunday, March 3, 2019 97a reticulum Ca2þ load. Interestingly, although the expression of the Naþ/Ca2þ cellular response to change in preload we developed mathematical models of exchanger (NCX) was unaffected, the decay of Ca2þ signal during caffeine the mouse ENDO and EPI cells. applications was 50% slower in PFD-treated cells (compared with controls), In consistency with the previous results, under auxotonic contractions at low indicating that PFD downregulates the NCX activity. PFD also inhibited preload (unstretched cells), time to peak contraction (Tmax) was significantly the production of reactive oxygen species, under both, basal conditions and longer in ENDO cells than in EPI cells. An increase in preload (stretch) pro- the presence of oxidant agents (acetaldehyde and peroxide hydrogen). longed Tmax in both cell types, however, the slope of ‘‘Tmax - sarcomere Conversely, the amount of nitric oxide (NO) was either, increased (in atrial stretch’’ relationship was significantly higher in EPI cells, demonstrating myocytes) or unchanged (in ventricular myocytes). Western-blot analysis greater contraction sensitivity of EPI cells to the stretch. Our mathematical showed that endothelial nitric oxide synthase expression remains unaffected, cellular models reproduced the experimental results when we accounted for while a dual regulation was observed for that of neuronal isoform (nNOS) the ENDO/EPI differences in the parameters of cooperativity of calcium acti- which consisted of inhibition and stimulation, in ventricular and atrial vation of myofilaments. The models predicted greater prolongation of action myocytes, respectively. Thus, in atrial cardiomyocytes, an up-regulation potential in EPI cells under high preload. of nNOS was enough to augment the synthesis of NO. Apart from improving Our results suggest that mechanical load changes transmural gradient in our knowledge of the molecular mechanisms of PFD action, these contraction of ENDO and EPI cardiomyocytes that in turn affect the gradient findings may also help in explaining the corresponding cardioprotective in electrophysiological function via mechano-calcium-electric feedback. Sup- effects. ported by The Russian Science Foundation (#18-74-10059) and JSPS KA- KENHI (#16K12878). 487-Pos 489-Pos Mechanical Load Effects on Cardiac Action Potential and Arrhythmo- D Functional Connectome of the Mechanically Loaded Cardiomyocyte II: genic Ca2 Activitiesrevealed by a Novel Patch-Clamp-In-Gel Technology 1 1 1 1 Coordinated Changes of Subsystems Zhong Jian , Bence Hegyi , Mark Jaradeh , Zana A. Coulibaly , 1 1 2 1 1 2 1 1,3 Zana Coulibaly , Zhong Jian , Rafael Shimkunas , Ye Chen-Izu , Yi-je Chen , Kit S. Lam , Leighton T. Izu , Ye Chen-Izu . 1 1 Leighton T. Izu . Department of Pharmacology, University of California, Davis, Davis, CA, 1Dept Pharmacology, Univ Calif Davis, Davis, CA, USA, 2MyoKardia, South USA, 2Department of Biochemistry and Molecular Medicine, University of San Francisco, CA, USA. California, Davis, Davis, CA, USA, 3Department of Biomedical Engineering, An isolated cardiomyocyte responds to mechanical load in a manner that sug- University of California, Davis, Davis, CA, USA. gests an inherent ability to adapt to an increase in mechanical load. In our other Cardiomyocytes are under mechanical load when the heart pumps blood abstract, we have shown that this inherent ability is possible through multiple against peripheral resistance. However, the mechanical load effects on cells changes in the calcium handling subsystems. We found that the changes in the have been missed in previous patch-clamp experiments because the cells calcium subsystems are not unique. Here we take advantage of the non- were bathed in solution under load-free condition. uniqueness of the solutions to identify the underlying structure and patterns We have developed an innovative Patch-Clamp-in-Gel technique to embed sin- in the acceptable parameter sets. These patterns describe the coordination of gle cells in a 3-D hydrogel made of viscoelastic polymer matrix. Experiments the calcium handling subsystems that underlie the adaptation to mechanical were performed when mouse ventricular myocytes were contracting in-gel loading in cardiomyocytes. under mechanical load. To identify the underlying structure and patterns in the acceptable parameter (1) Compared to load-free cells, the myocytes in-gel under mechanical sets, we use a singular value decomposition wherein the parameter sets in load showed prolonged action potential duration (long APD), early the acceptable modulations are projected onto the most dominant singular afterdepolarization (EAD), delayed afterdepolarization (DAD), and trig- vectors. This method allows us to classify the data in an unbiased way as gered AP, indicating that mechanical load increases arrhythmogenic AP 2þ the dimensionality of the parameter sets is reduced. Additionally, pattern activities. (2) Simultaneous triple-signal recordings of AP, Ca ,and and grouping of the data naturally arises from the projected data. contraction revealed that load-induced DAD occurs in conjunction with þ þ Our result shows that although mechanically-activated messengers can affect spontaneous Ca2 wave. (3) The EADs, DADs, and Ca2 tides were abol- þ þ calcium subsystems in many ways, there exists an underlying structure to the ished by blocking the late Na current, suggesting changes in the Na 2þ þ modulation. Specifically, the L-type Ca current (LTCC) subsystem needs and Ca2 homeodynamics. (4) The load-induced arrhythmogenic activities to be upregulated; the modulations of ryanodine receptor (RyR) and SERCA were abolished by specific inhibition of nitric oxide synthase 1 (NOS1 or subsystems are independent of one another; the LTCC and RyR parameters nNOS), indicating a critical role of nNOS in the mechano-chemo-electro- must change together in the same direction; and the NCX subsystem parameters transduction. 2þ are positively but weakly correlated with the RyR and LTCC subsystems. We Our data show that mechanical load significantly affects action potential, Ca call the connections between subsystems that enable the myocyte to respond to signaling, and myocyte contraction. The Patch-Clamp-in-Gel technology pro- mechanical load its ‘‘functional connectome’’. vides a new tool to control mechanical load on single myocytes, which enables studying mechanotransduction effects on the three dynamic systems - electri- cal, Ca2þ signaling, and contractile systems - that control cardiac function Posters: Cardiac Smooth and Skeletal Muscle and arrhythmogenesis. Electrophysiology I

488-Pos 490-Pos The Effects of Mechanical Load on Transmural Differences in Mechano- Pharmacological and Ischemic Preconditioning Up-Regulate the Expres- Electric Feedback in Single Cardiomyocytes sion of Socs in Adult Cardiac Myocytes: Its Physiological Significance Anastasia Khokhlova1,2, Gentaro Iribe3, Pavel Konovalov1, Rau´l Sampieri, Joice Thomas, Maria C. Garcia, Elba D. Carrillo, Leonid Katsnelson1,2, Olga Solovyova1,2. Eridani Fuentes, Wilibaldo Orea, Jorge A. Sanchez. 1Institute of Immunology and Physiology, Yekaterinburg, Russian Dept Pharmacology, Cinvestav IPN, Mexico City, Mexico. Federation, 2Ural Federal University, Yekaterinburg, Russian Federation, Voltage- and SOCS channels are the major routes of Ca(2þ) entry in mamma- 3Okayama University, Okayama, Japan. lian cells and a negative reciprocal interaction between these channels has been We have shown previously that subendocardial (ENDO) and subepicardial described (Doltmetsch et al., 2010). We have previously reported (Gonzalez et (EPI) cardiomyocytes isolated from the mouse left ventricle (LV) have al., 2010) that preconditioning in heart leads to down-regulation of Cav1.2 different mechanical responses to changes in preload (stretch) to the channels. In the present experiments we examined the changes that take place cells (Khokhlova et al., 2018). However, the main limitation of previous by preconditioning in the expression and function of STIM1 and Orai1, major study was small amount of stretch (about 3.5%) applied. Here we components of SOCS in heart. resolved the limitation applying up to 20% of stretch and used our new We measured membrane currents with the whole-cell patch-clamp technique. electromechanical ENDO and EPI mathematical models to predict To assess the effect of preconditioning on SOCS, protein expression levels effects of mechanical load on electrophysiological function of ENDO and of STIM1 and Orai1 were measured by Western blotting and confocal micro- EPI cells. scopy, and intracellular Ca(2þ) measurements was quantitated by Fura-2. Di- Various preloads imposed via axial stretch were applied to the mouse LV cells azoxide (100 uM), was used to induce pharmacological preconditioning. using carbon fiber technique for single myocytes (Iribe et al., 2014). To simu- Ischemic preconditioning was produced by brief periods of ischemia. Depletion late experimentally obtained results and to predict mechanisms underlying the of internal stores was achieved with thapsigargin.

BPJ 9314_9317 98a Sunday, March 3, 2019

Both forms of preconditioning greatly reduced infaction areas relative to con- subcellular localization. Cardiac tissue is modeled by a 50-cell strand of trols. Store depletion generated Ca(2þ) selective currents with strong inward electrically and ephaptically coupled cardiomyocytes, with each cell discre- rectification. Preconditioning almost completely suppressed these currents, an tized into axial and ID patches to account for nonuniform Naþ channel effect that was almost blocked by 5HD a selective mitoKATP channel blocker subcellular localization. Consistent with our prior work, across all age- and by intracellular BAPTA. And as assessed by Fura-2 this influx was also dependent conditions, wider cleft width prolonged APD due to enhanced blocked by preconditioning. In contrast, the expression at the protein level of late Naþ current. We find that properties increasing total cellular Naþ chan- both STIM1 and Orai1 was strongly up-regulated by preconditioning and nels expression (increased Naþ channel density, increased cell size) promoted confocal microscopy revealed a higher density of Orai1 channels at the surface longer APDs, but with a complex dependence on Naþ channel localization membrane. and gap junctional coupling. Our works predicts that intercellular cleft Naþ Our results indicate that while the expression of protein components of SOCS is a key regulator of cardiac repolarization that is further modulated by age- in heart cells (STIM1 and Orai1) are up-regulated by preconditioning, influx of dependent factors. Ongoing ex-vivo imaging and optical mapping studies Ca(2þ) through SOCS is severely impaired by preconditioning, probably by a will be compared with model predictions. slow Ca(2þ)-dependent inactivation process. Supported by CONACYT, grant 284053 to JS. 493-Pos Inhibiton of Protein Kinase G Preserves Prolonged Ventricular Action 491-Pos Potentials via Improvement of Slow-Activated Voltage-Dependent KD- Diabetic Hyperglycemia Regulates Potassium Channels and Arrhythmias Channel Currents in Aged Rat Cardiomyocytes in the Heart via Autonomous CaMKII Activation by O-Linked Belma Turan, Yusuf Olgar, Erkan Tuncay. Glycosylation Dept Biophysics, Ankara Univ., Faculty of Medicine, Ankara, Turkey. Bence Hegyi, Johanna M. Borst, Austen J. Lucena, Logan R.J. Bailey, Aging, being a complex process, is the most important risk factor for most Julie Bossuyt, Donald M. Bers. diseases in humans giving a dominant risk factor for cardiovascularchanges. Department of Pharmacology, University of California, Davis, Davis, Aging, itself, does not cause heart failure among human, however it results in CA, USA. progressive deteriorations in the structure and function of the heart. Studies Diabetic cardiomyopathy is associated with altered cardiac repolarization, Kþ mentioned that carbohydrate intolerance develops as part of the aging pro- current density and arrhythmia risk. However, little is known about how acute cess, being carbohydrate intolerance associated with the consequence of hyperglycemia regulates cardiac Kþ channels and arrhythmias. We measured peripheral insulin resistance. However, there is an age-associated increase action potentials (APs) and Kþ currents in isolated mouse ventricular myocytes in the prevalence of abdominal obesity and insulin resistance in elderly exposed to high glucose (30 mM) versus control (5.5 mM glucoseþ24.5 mM subjects, with a clear relationship between metabolic syndrome and insulin mannitol to match osmolarity). Acute hyperglycemia reduced transient outward resistance, while the percentage of QT-prolongation is high between þ K current (Ito) amplitude but enhanced Ito recovery from inactivation and elderly-subject comparison to adults at most due to depressed slow- þ þ increased inward rectifier K current (IK1). These changes were prevented by in- activated voltage-dependent K -channel currents (IKs), which is an indicator hibitors of O-linked N-acetylglucosamine modification (O-GlcNAcylation), of an increased risk for potentially fatal cardiac arrhythmias. In the present Osmi-I and DON. Conversely, O-GlcNAcase inhibitor thiamet-G produced study, we aimed to examine the underlying mechanism of aging-associated the same Kþ current effects as hyperglycemia. Thus O-GlcNAcylation is neces- QT-prolongation in aged- and insulin resistant-rats. We emanined action þ sary and sufficient for these K channel effects. Additionally, IK1 amplitude potential parameters in left ventricular cardiomyocytes, under either insulin and Ito recovery effects (but not Ito amplitude) were prevented by CaMKII application or a IKs activator. Since we observed significantly activated inhibitor AIP, in CaMKIId-knockout myocytes and in mice in which a known PKG and positive response to insulin application in aged-rat cardiomyocy- O-GlcNAcylated Serine on CaMKII was mutated (S280A knock-in). Acute tes,we also tested whether a PKG inhibitor can also induce recovery in the hyperglycemia slightly prolonged AP duration, increased delayed afterdepolari- prolonged action potentials. Our all data demonstrated that the depressed zations and inducibility of AP-duration alternans during tachypacing in WT only IKs can play important role in aged-rat electrical activity via a activation of (not in CaMKIId-KO or S280A-KI). Thus, acute hyperglycemia-induced insulin resistant dependent activation of PKG. Theerfore, PKG seems to be O-GlcNAcylation of CaMKII and/or Kþ channels may contribute to arrhythmo- a new target for the improvement of cardiac disorders during aging heart. genesis. In chronic diabetic models (streptozotocin & db/db)Ito and IK1 were (Supported by TUBITAK SBAG-216S979). both downregulated (as in heart failure and chronic CaMKII activation). AP duration was significantly prolonged in diabetes, and delayed afterdepolariza- 494-Pos The Effects of Pinacidil, an ATP Sensitive KD Channel Opener on Cardiac tions and alternans were enhanced at normoglycemia and worsened by hypergly- D 2D cemia. We conclude that Kþ channels are differently modulated by acute versus Na /Ca Exchanger Function in Guinea Pig Cardiomyocytes 1 1 2 1 chronic hyperglycemia and O-GlycNAcylation-dependent CaMKII activation. Keisuke Iguchi , Masao Saotome , Kanna Yamashita , Takenori Ikoma , þ O Prottoy Hasan1, Yuichiro Maekawa1, Yasuhide Watanabe2. Acute hyperglycemia enhances K channel function via -GlcNAcylation of 1 2þ Internal Medicine III, Hamamatsu University School of Medicine, CaMKII at Ser280, which may also promote arrhythmogenic SR Ca leak. 2 However, chronic hyperglycemia and CaMKII activation appear to downregu- Hamamatsu, Japan, Health Science, Hamamatsu University School of late Kþ channel expression, which also promotes arrhythmia susceptibility. Medicine, Hamamatsu, Japan. Pinacidil, a nonspecific ATP sensitive Kþ (KATP) channel opener, inhibited 492-Pos delayed afterdepolarizations (DADs) in dog heart (Spinelli et al., 1991). Age Dependent Regulation of Cardiac Sodium Channel Gain of Function Although it is well known that DADs are caused by the inward Naþ/Ca2þ ex- 1 2 2 Madison B. Nowak , David Ryan King , Steven Poelzing , change current (INCX), the relationship between pinacidil and INCX remains still þ þ Seth H. Weinberg1. elusive. Thus, we here investigated the effects of pinacidil on cardiac Na /Ca2 1Biomedical Engineering, Virginia Commonwealth Univ, Richmond, VA, exchanger (NCX1) by whole-cell patch clamp technique and Fura-2/AM anal- USA, 2Dept Biomed Eng, Virginia Tech, Roanoke, VA, USA. ysis using isolated guinea pig cardiac ventricular myocytes. Pinacidil enhanced Action potentials (AP) in the heart are initiated by sodium (Naþ) ion influx via INCX in a dose dependent manner (3 mM to 100 mM), and the EC50 values were voltage-gated Naþ channels. Gain-of-function mutations in the gene encoding 23.5 mM for the outward (Ca2þ entry) and 23.0 mM for the inward (Ca2þ exit) the Naþ channel associated with the long QT type-3 (LQT3) syndrome can components with Hill coefficients of approximately 1, respectively. The pina- produce a ‘‘late’’ Naþ current, producing pro-arrhythmic early-afterdepolari- cidil induced INCX increase was inhibited by L-NAME (an endothelial NO syn- zations (EADs), and prolonging AP duration (APD). High-resolution imaging thase inhibitor), ODQ (a soluble guanylyl cyclase inhibitor), KT-5823 (a cGMP has shown that Naþ channels are clustered at the intercalated disc (ID), facil- dependent protein kinase inhibitor), suggesting that the pinacidil increases INCX itating the formation of Naþ nanodomains in the intercellular cleft. We through NO/cGMP/PKG signaling pathway. Glibenclamide, a nonselective recently showed in simulations and ex-vivo guinea pig experiments that nar- KATP channel inhibitor, also suppressed the pinacidil-induced INCX increase, row clefts suppressed APD prolongation in LQT3-associated models, which whereas 5-HD, a selective mitochondrial KATP channel inhibitor, did not. In simulations predict is mediated by changes in cleft Naþ concentration Fura-2/AM analysis, pinacidil (30mM) also enhanced the intracellular Ca2þ (Greer-Short, et al., Circ AE, 2017). LQT3 is a cardiac disease that often concentration increase through Ca2þ entry mode of NCX1. Pinacidil-induced does not manifest symptoms until later in life, suggesting age-dependent fac- intracellular Ca2þ concentration increase was inhibited by L-NAME, ODQ, tors influence the formation of EADs and APD prolongation. Here, we per- KT-5823, and glibenclamide, respectively. Pinacidil increased nitric oxide formed new simulations to predict how intercellular cleft width regulation (NO) production, and the pinacidil-induced NO production increase was depends on key cellular and tissue properties that vary with age, specifically attenuated by glibenclamide and L-NAME in our experimental condition. In cell size, gap junctional coupling, and Naþ channel expression density and conclusion, pinacidil may enhance NCX1 function by activating cGMP/PKG

BPJ 9314_9317 Sunday, March 3, 2019 99a signaling pathway with NO production through plasma membrane KATP Dynamic clamp is a powerful tool involving injection of real-time simulated channel opening. currents into patch-clamped cells, and has been employed in conventional patch-clamp to inject IK1 current into human induced pluripotent stem cell- 495-Pos derived cardiomyocytes (hiPSC-CMs). Extracellular pH But Not Osmotic Pressure Modulates Cl Current in hiPSC-CMs are attractive cell types because of their unlimited availability and Freshly-Isolated Guinea Pig Detrusor Smooth Muscle Cells human origin. However, IK1 is expressed at low levels in those cells, hence Viktor Yarotskyy, Georgi V. Petkov. they display a more depolarized membrane potential than adult cardiomyocytes Department of Pharmaceutical Sciences, University of Tennessee Health (CM). Introducing simulated IK1 into hiPSC-CMs improves resting potential Science Center, Memphis, TN, USA. and makes them a viable alternative to the scarcely available adult human We recently characterized Cl currents in freshly isolated guinea pig detrusor - CMs. Another limitation of hiPSC-CMs is that the ratio of seal resistance smooth muscle (DSM) cells and hypothesized that Cl conductance plays a crit- (Rseal) to membrane resistance (Rm) is small due to smaller capacitance and ical role in regulation of urinary bladder excitability and contractility. A robust typically slightly lower Rseal in automated patch clamp (APC). Thus, mem- voltage-dependence with midpoint of activation at positive voltages and out- brane potential dissipation is more pronounced compared to patch-clamp ward rectification, insensitivity to a physiological intracellular Ca2þ concentra- - recordings of adult CMs. To correct for that, we implemented a Rseal compen- tion, and Cl selectivity suggested that ClC channels/antiporters activation sation mechanism to inject current compensating for the potential dissipation underlies Cl- current in freshly-isolated guinea pig DSM cells. Depending on - through Rseal. In this study, we combined dynamic clamp with APC to demon- the type, ClC Cl current can be modulated by an extra- and/or intra-cellular strate IK1 conductance can be added to hiPSC-CMs, and applying automatic pH. Here we used the whole-cell patch-clamp technique at conditions opti- Rseal compensation simultaneously. Our results show virtual IK1 can be suc- mized for Cl- conductance to examine the modulatory effect of external pH - cessfully injected into hiPSC-CMs in up to 4 cells simultaneously and Rseal is on Cl current in freshly-isolated guinea pig DSM cells. Elevating extracellular correctly compensated avoiding overcompensation. Our approach results in - 5 pH from 7.4 to 8.5 caused a reversible reduction in Cl current of 22.4 4.9 % more stable resting membrane potentials and improved action potential (AP) þ < at 100 mV (number of cells n=11, number of animals N=3, p 0.05). shape. Increased IK1 resulted in AP shortening and acceleration of the up- Decreasing pH from 7.4 to 6.0 had no effect (n=8, N=3, p>0.05) and further þ - stroke. We measured native, but small, Ba2 -sensitive IK1 in voltage-clamp decrease of pH to 5.0 caused a reversible increase in the Cl current of mode in approximately 50% of these cells. Adding a Ca2þ channel activator 5 < 32.4 10.9 % (n=11, N=3, p 0.05). ClC-3 or ClC-7 channels/antiporters fit (BayK 8644), or blocker (Nifedipine) caused an increase and decrease of the this pH sensitivity profile in guinea pig DSM cells. ClC-3 is thought to be a AP duration, respectively. In conclusion, combining dynamic clamp and Rseal volume-regulated channel. However, a replacement of control extracellular compensation with APC resulted in an enhanced, medium-throughput platform solution (347 mOms) with hypotonic solution (182 mOsm) mimicking a cell for safety pharmacology. volume increase via osmotic pressure reduction caused no change in Cl- current (n=5, N=3, p>0.05). The data suggest that ClC-7 rather than ClC-3 is the most 498-Pos likely candidate for Cl- conductance in freshly-isolated guinea pig DSM cells. Establishing Pathogenicity of Novel LQTS8 Variant via Genomic Editing Supported by NIH R01-DK106964 grant to Georgi V. Petkov. of Human iPSC Dmytro O. Kryshtal1, Nikhil V. Chavali2, Shan S. Parikh2, Lili Wang1, 496-Pos Andrew M. Glazer1, Moore B. Shoemaker1, Bjorn C. Knollmann1. Engineering an Optogenetic System for Point-Pacing Cardiomyocytes 1Medicine, Vanderbilt University Medical Center, Nashville, TN, USA, in Culture 2Vanderbilt University, Nashville, TN, USA. 1 1 1 2 Geran Kostecki , Shivani Pandey , Renjun Zhu , Emilia Entcheva , Long QT syndrome (LQTS) is a major cause for sudden cardiac death (SCD) in Leslie Tung1. 1 2 the young. The use of commercial genetic testing for LQTS has rapidly Dept Biomed Eng, Johns Hopkins Univ, Baltimore, MD, USA, Dept expanded, but inability to predict whether a rare variant is pathogenic has Biomed Eng, George Washington Univ, Washington, DC, USA. limited clinical benefit. Novel missense variants are routinely reported as Maturation of cardiomyocytes remains a challenge in engineered cardiac tis- ‘‘Variants of Unknown Significance (VUS)’’, which cannot be used to screen sues, and can be facilitated by electrical pacing during culture. Until now, family members at-risk for SCD. The development of new approaches to deter- this has been done directly with electrodes, which requires a specialized set- mine pathogenicity of rare variants is a major unmet need. Here, we studied up and immersion of the electrodes into the bath, hazarding contamination. a novel heterozygous missense variant (p.N639T) detected in CACNA1C In this study we develop a contactless point-pacing system by placing clusters 2þ (encodes CaV1.2 Ca channel) by commercial genetic testing and reported of ChR2-transduced HEK (‘‘spark’’) cells onto monolayers of cardiac cells and as a VUS in a family with LQTS. We first studied electrophysiological charac- pacing with light, obviating any further handling. Furthermore, by point pacing, teristics of L-type Ca2þ current (LTCC) using HEK293 cells expressing wild this system creates an electromechanical delay, as exists in vivo, which is not type and mutant (N639T) channel. We found significant decrease in the rate reproduced by field pacing. of voltage-dependent inactivation (using Ba2þ as a charge carrier) in N639T m 5 Spark clusters (500 m-diameter) were fabricated by plating 1x10 spark cells/ cells and right shift in steady-state inactivation, but current density was also well in a 0.5% agarose-coated 96-well V-bottom plate and centrifuging at 800g significantly reduced in N639T. We then used CRISPR/Cas9 to introduce het- for 10 min. The next day, a single cluster was plated onto individual mono- erozygous N639T into control hiPSC from a healthy volunteer, which were layers of neonatal rat ventricular myocytes. The monolayers could be acutely 2 then differentiated into cardiomyocytes (CM). N639T hiPSC-CM were paced by pulses of blue light (0.05 mW/mm , 100 ms duration), although min- compared against isogenic (iCTRL) and population control (pCTRL) hiPSC- imum capture cycle length varied from 3 s to 500 ms. Using optical mapping CM expressing WT CACNA1C, by measuring the extracellular field potential with voltage-sensitive dye, we confirmed pacing originated from the spark clus- (EFP) of paced hiPSC-CM monolayers, and by standard single-cell patch- ter. Using a customized LED set-up, cells were chronically paced in culture, clamp. EFP was significantly prolonged in N639T hiPSC-CM. Verifying our and videos of cell beating were recorded every 15 minutes over 24 hours. data from HEK293 experiments, patch-clamp studies showed that N639T Some monolayers could be continuously captured at 2 Hz pacing rate. To prolongs ventricular action potential (APD 50=131527 ms (iCTRL) vs. improve the efficacy of spark cluster pacing, we transduced the spark cells 254553 ms (N639T), p<0.05) by slowing voltage-dependent LTCC inactiva- with Cx43, selected transduced cells using FACS, and confirmed upregulation tion (t=74516 ms (iCTRL) vs. 150529 ms (N639T), p<0.01), without of Cx43 by qPCR. affecting current density. We conclude that genetic editing of hiPSC-CM can In conclusion, we have engineered an optogenetic pacing system that provides rapidly and efficiently establish pathogenicity of a VUS in LQTS such as the point stimulation using spark clusters excited by light pulses. This lays the CACNA1C-p.N639T mutation. groundwork for scalable, contactless point pacing of engineered cardiac tissues in culture to increase maturation, as well as studies of effects of point versus 499-Pos field pacing. Effect of a Small Molecule Activator of Potassium Currents on Repolarization Reserve in hiPSC-Cardiomyocytes 497-Pos Jacqueline A. Treat, Robert J. Goodrow, Gary L. Aistrup, Corina T. Bot, Introducing Simulated IK1 into Human iPSC-Cardiomyocytes using Jonathan M. Cordeiro. Dynamic Clamp on an Automated Patch Clamp System Masonic Medical Res Inst, Utica, NY, USA. 1 1 1 2 1 Gang Lu , Andra´s Horva´th , Nadine Becker , Alan Fabbr , Christian Grad , Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) Michael George1, Niels Fertig1, Teun P. de Boer2. 1 2 have been used for safety pharmacology and to investigate genetic diseases Nanion Technologies, Munich, Germany, University Medical Center affecting cardiac ion channels. We have previously shown that the maximum Utrecht, Utrecht, Netherlands. diastolic potential (MDP) in hiPSC-CMs depends on the rapid delayed rectifier

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þ K current (IKr). We examined the effect of a dual Ito and IKr agonist (NS3623) Additionally, tissue can be partially depolarized, resulting in an increase in on hiPSC-CMs and determined its contribution to action potentials (APs), conduction velocity in either the annulus or the second excitation pathway. extracellular field potentials (EFPs) and ionic currents. Our experiments demonstrate that asymmetries in simulation location or local hiPSC-CM monolayers were used to record EFPs using CardioExcyte 96, and conduction velocity are key factors in arrhythmia induction. AP recordings were made using high resistance electrodes. Whole cell patch clamp was used to record IKr in single hiPSC-CMs. All measurements were 502-Pos made at 36 C. Effect of Real-Time Leak Current Correction on Action Potentials EFP signals from hiPSC-CMs monolayers showed a pseudo-QRS complex and Recorded from Induced Pluripotent Stem Cell-Derived Cardiac Myocytes T-wave similar to that observed in native ventricular tissue. Application of Brian Panama1, Mark Nowak2, Brandon Franks2, Leigh Korbel2, NS3623 (5 mM) resulted in a small shortening of the QT interval. Similarly, Glenna Bett1, Randall Rasmusson1. 1 AP recordings in the presence of NS3623 resulted in hyperpolarization of the Dept Physiol/Biophys, State Univ New York Buffalo, Buffalo, NY, USA, 2 MDP (from 70.451.9 to 73.951.9 mV) and shortening of APD (from Cytocybernetics, Buffalo, NY, USA. 196.6528.0 to 176.6525.7 ms). Voltage clamp analysis of IKr tail currents re- Accurate measurement of the effect of drugs on the action potential (AP) is vealed a 4759% increase in IKr following application of NS3623. A minor critical for drug safety screening and research. Cellular leak currents increase inhibition of Ito was observed following NS3623. AP clamp experiments re- during voltage-clamp recordings, and can lead to instability and errors. We vealed the IKr transient was largest during repolarization with some contribu- determined the effect of leak current on resting membrane potential (RMP) tion during phase 4 depolarization. and AP characteristics, as well as how changes in leak affects observed APs A robust IKr is present in hiPSC-CMs which can be augmented by the agonist during drug-cell interactions. We recorded APs from human induced pluripo- NS3623. Application of the agonist resulted a slowing of the spontaneous tent stem cell-derived cardiac myocytes (hiPSC-CMs) and used the Cybercyte rate and shortening of the APD but minimal effect on MDP. Although we dynamic clamp system to electronically express IK1, which gives hiPSC-CMs have previously shown that the MDP in hiPSC-CMs critically depends on physiological RMP and enhanced AP stability. Additionally, we used the IKr, increasing the magnitude of IKr resulted only in a small hyperpolarization Cybercyte to express real-time electronic leak current correction (ILCC). As of the MDP. leak current increases, RMP depolarizes, AP morphology changes and, eventu- ally, APs cannot be evoked. Electronic expression of leak current correction, 500-Pos ILCC, restored RMP to -84 mV and resulted in stable APs. Dofetilide, which Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes as Early- blocks IKr and increases AP duration (APD), and nifedipine, which blocks ICa, Screening Platform of Anti-Arrhythmic Effects by Pufas and decreases APD, were applied to cells with and without leak current correc- Alicia de la Cruz1, Rene Barro-Soria2, Sara I. Liin3, H. Peter Larsson1. tion. 30nM dofetilide prolonged APD, but the effect was exaggerated with leak 1Physiology and Biophysics, University of Miami, Miami, FL, USA, 2Dept of current (51525%, n=6), compared with cells with leak current correction, 3 Med, University of Miami, Miami, FL, USA, Clin Experiment M, Linko¨ping (ILCC:34512%, n=8). Conversely, 100 nM nifedipine shortened APD, but Univiversity, Linkoping, Sweden. the effect was blunted with cell leak (11528%, n=4), compared with leak Long QT syndrome (LQTS) is a congenital cardiac disorder defined by the pro- current corrected cells (þILCC:4559%, n=5). Leak current correction also longation of the ventricular action potential and the QT interval of the ECG significantly reduced APD measurement variability. Our data demonstrate (electrocardiogram). QT interval prolongation might lead to Torsade des that real-time leak current correction combined with electronic addition of pointes and sudden cardiac death. The voltage-gated potassium channel IK1 improves the accuracy of measuring the effects of dofetilide and nifedipine Kv7.1 co-assembled with the KCNE1 regulatory subunit generates the slow de- on AP morphology and improves the efficiency by reducing measurement þ layed rectifier K current (IKs). IKs is one of the most important repolarizing variability and the number of data points required to determine the effect of currents of the ventricular action potential (AP). Loss-of-function mutations drugs on the AP. in Kv7.1 channels and KCNE1 are associated with LQTS (LQT1 and LQT5). Polyunsaturated fatty acids (PUFAs) have been proposed as anti- 503-Pos arrhythmic drugs due to their ability to modulate NaV,CaV and KV channels. The Effects of Noise in Biological Excitable Media Different types of PUFAs, such as u-3, u-6 and u-9, and PUFA analogues, J. Miguel Romero Sepu´lveda, B. Adrienne Caldwell, Alvin Shrier, Gil Bub. such as N-arachidonoyl taurine (N-AT), have been described as Kv7.1/ Physiology, McGill University, Montreal, QC, Canada. KCNE1 channel activators and, therefore, could possible function as anti- Spiral waves can form in cardiac tissue when a wave of excitation breaks and arrhythmic drugs (especially for LQT1 and LQT5). In order to study the effects the edge of the wave curves inward around a pivot point. Once initiated, a spiral of PUFAs, not only on IKs current but also on all currents involved in the can persist, break up into multiple waves and/or wavelets, or disappear when it generation, maintenance and resolution of the cardiac AP, we have optimized reaches an unexcitable border. One of the most important aspects of this mech- an all-optical electrophysiology system. Human induced pluripotent stem cell- anism is the presence of functional heterogeneities that support and spiral wave derived cardiomyocytes (iPS-CMs) has been described as a useful drug-testing formation. platform. Indeed, it has been shown that iPS-CMs are sensitive to ion channels The simplest cardiac model system that displays re-entrant activity are cardiac inhibitors in terms of cardiac AP modulation. We here use a combination of monolayers, which display undamped wave propagation, wave breaks and spi- electrically coupled iPS-CMs monolayers with a high resolution all-optical ral wave formation. Additionally, their relative thinness allows for the use of electrophysiology system for early screening of the anti-arrhythmic properties optogenic techniques to control activity in every cell in the monolayer using of diverse PUFAs. projected patterned blue light. Studies by our research group and others have used optogenetic techniques to 501-Pos terminate spiral waves with light, control cardiac conduction velocity as well as Optogenetic Control of Re-Entrant Waves Demonstrated in Human chirality (rotation direction) of spiral waves. However, the effect of noise in Induced Stem Cell Derived Cardiomyocytes (hiPSC-CMs) heart tissue has yet to be studied. B. Adrienne Caldwell, Miguel Romero Seplvuda, Gil Bub, Alvin Shrier. The presence of noise is intrinsic and universal in nature where dynamical sys- McGill University, Montreal, QC, Canada. tems interact with external perturbations. These interactions are well studied in Anatomical cardiac re-entry occurs when an impulse propagates in a circuit light sensitive chemical systems like the Belousov-Zhabotinsky reaction. around an inexcitable obstacle instead of terminating at the heart’s apex. Sim- Here, we introduce noise to our cultured cardiac monolayer using a projector ulations using the FitzHugh-Nagumo model suggest that a previously unre- to shine subthreshold patterned blue light with different spatial frequencies ported mechanism may be responsible for arrhythmia generation around on the tissue in order to determine how spatial variations in conduction velocity obstacles with a second excitable pathway. Rapid pacing of the theta ‘(q)’ promote wave-breaks and spiral wave formation. like geometries, which consists of an annulus with a second conducting pathway through the center, can result in a unidirectional block and sustained 504-Pos re-entrant propagation. Based on simulations, we propose that asymmetry in the Longitudinal Cardiotoxic Effect of Doxorubicin in a Multicellular Cardiac location of the pacemaking site leads to a higher propensity for arrhythmia gen- Model eration. We investigate the robustness of this mechanism in a bioengineered hu- Viviana Zlochiver, Stacie Edwards, Rosy Joshi-Mukherjee. man tissue model system. The model system used was a monolayer of human Aurora Research Institute, Milwaukee, WI, USA. induced pluripotent stem cells (iPSCs) that had been differentiated into cardi- Doxorubicin (Dox), a widely effective chemotherapeutic agent, causes QT pro- omyocytes, plated in theta geometries and sensitized to light by expression of longation and dose-dependent cardiotoxicity often resulting in heart failure. Channelrhodopsin-2 (ChR2). By inscribing light sensitivity into tissue, precise Here we examine the progression of Dox cardiotoxicity on human induced stimulations of tissue could be applied in various locations around the ring. pluripotent stem cell derived cardiomyocytes on m-gold multielectrode arrays.

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We recorded the time and dose dependent differential drug response on field plateau-phase of CAP (gain-of-function). This results in the long-QT syndrome, potentials (FPs) from 23 independent constructs simultaneously before and af- which increases the risk of developing cardiac-arrhythmias. To determine the ter exposure to the Dox concentration range of 0.1 to 10 mM. We observed a molecular mechanisms responsible for the different gating and selectivity prop- time and dose dependent effect on FP amplitude and cell viability. At 1 h erties for wild-type (WT) and mutant (N629D) hERG1A-variants, we used post-treatment there was no significant relative FP amplitude change at any computational-electrophysiology approaches and all-atom molecular dynamics concentration when compared to pre-treatment indicating minimally altered (MD) simulations to study cation permeation and selectivity-filter (SF) dy- electrical properties of the network. However, a time and dose dependent atten- namics. Starting from the open-state channel structure (Cryo-EM, 3.8 A˚ resolu- uation of FP amplitude ensued at 24 h relative to control (15526% for 0.1 mM tion), we generated several 2 to 4 ms trajectories for each system. We observed and 45512% for 1 mM) and gradually increased by 48 h (33521% for 0.1 mM only Kþ permeation through WT, while both Kþ and Naþ were found to and 8856% for 1 mM). Viability measured by percentage of active electrodes permeate in the N629D mutant albeit with fewer permeation events. In good also decreased in a dose and time dependent fashion. Exposure to the highest agreement with experimental observations of small, flickering conductive states concentration of 10 mM abolished electrical activity in all 5 constructs probed of hERG, we observe that SF of WT hERG spontaneously dehydrates and col- by 24 h while the same occurred in the 6 constructs exposed to 1 mM after lapses, shifting Kþ permeation events from ns to ms timescales. The collapsed 72 hours. The lowest concentration of 0.1 mM had the least cardiotoxic effect states result from a complex reordering in the intra- and inter-chain hydrogen- with minimal effect on viability thus the cumulative effect on the FP properties bonds involving residues from the SF, outer mouth vestibule and S5P-linkers. could be tracked on the same constructs over 72 h. At this concentration the N629D mutation resulted in the significant widening at the binding sites normalized RR interval was significantly prolonged at all time points (8% in- S1-S2, reminiscent of the selectivity filter structure observed in a bacterial chan- crease at 1 h, 17% at 24 h, 88% at 48 h and 52% at 72 h) when compared to nel NaK. Unlike WT hERG, N629D mutant displays intact and dynamically control. These results strongly suggest that the proposed model has the capa- maintained geometry of the SF through stable intra-chain hydrogen-bonds. bility to mimic the Dox induced cardiotoxicity. These results agree with previous mutagenesis reports, suggesting that the fast C-type inactivation in hERG1-WT results from meta-stable structure of the SF. 505-Pos A Computational Approach to Predict Mechanisms of Phenotypic 507-Pos Variability in Induced Pluripotent Stem Cell-Derived Cardiomyocytes Dynamics of Pore Domain Affected by Single Mutations in S4 Segment of Divya C. Kernik1, Stefano Morotti2, Garg Priyanka3, Joseph C. Wu3, Shaker Potassium Channel Jose Jalife4, Eleonora Grandi2, Colleen E. Clancy1. Carlos Alberto, Z. Bassetto Jr, Joao Luis Carvalho-de-Souza, 1Dept Physiology and Membrane Biology, Univ Calif Davis, Davis, CA, Francisco Bezanilla. USA, 2Dept Pharmacology, Univ Calif Davis, Davis, CA, USA, 3Stanford Biochemistry and Mol Biology, Univ Chicago, Chicago, IL, USA. Cardiovascular Institute and Department of Medicine, Stanford University, C-type inactivation is a process thought to occur in the pore domain (PD) of Stanford, CA, USA, 4Center for Arrhythmia Research, University of ion channels whereby prolonged depolarizations decrease ionic conduction by Michigan, Ann Arbor, MI, USA. disruption of the selectivity filter. Here, we show that biases in voltage sensor Patient-specific induced pluripotent stem-cell derived cardiomyocytes (iPSC- domain (VSD) voltage dependence induced by mutations in S4 segment CMs) have the potential to provide a method for study of cardiac disease and (L366H and V367H) affect differently C-type inactivation. In L366H but not drug effects in individuals. However, several factors have limited the use of in V367H, the peak of conductance decreases as the depolarizing voltage pulse iPSC-CMs, including their immature cardiac phenotype and variability in increases indicating, after ruling out other unspecific mechanism, that inactiva- behavior among cells derived from the same source. We have developed a tion is enhanced in this channel. The more depolarized the V1/2 of G-V the slower computational framework which utilizes experimentally observed variability is the Kþ current inactivation time constant (V367H: 8.2 s, WT: 5.9 s, and in underlying ionic currents to predict whole-cell phenotypic variability in a L366H: 4.5 s). Compared to WT (V1/2= 30 mV), G-V curve of L366H is left- population of models. We will use this computational framework to: (1) Study ward shifted (20 mV) whereas V367H is rightward shifted (25 mV). The the mechanisms of immature cardiac behavior in iPSC-CMs and (2) Explore voltage-dependent inactivation after 10 s inactivating periods at different volt- patient-specific susceptibility to long QT syndrome (LQTS). To study the ages showed V1/2 of 65 mV for L366H, 31 mV for WT and 4 mV for mechanisms of immaturity in iPSC-CMs, we will exploit the inherent vari- V367H, showing linear correlation with V1/2 of G-V curves. Differences in inac- ability of whole-cell behavior in the population of models, which includes tivation entry kinetics accompanied by no change in voltage-dependence of the immature and mature cardiac phenotypes. Experimentally, iPSC-CMs are char- same process suggest at least some independency between inactivation and open acterized as immature based on their depolarized resting potential and low up- probability. The S4 L366H mutation made the pore W434F mutation a partially stroke velocity. However, with new experimental preparations, iPSC-CMs can conducting channel showing an increase in the open probability by 200 times more closely resemble adult human cardiomyocytes. This spectrum of maturity compared to the W434F mutant in the WT-IR channel background (which has is captured across our model population. A comparison of immature and mature an open probability of 50000th of WT-IR channel). This dramatically shows a subpopulations will be used to analyze contributing factors for each phenotype, direct effect of VSD into the PD. Our results strongly suggest a non-canonical including differences in IK1,INa, and If. Cell-to-cell variability, which has been functional connection between VSD and PD in Shaker. This data contributes a limiting factor for iPSC-CMs experimentally, can be a strength when appro- to a more complete understanding of sensor to pore coupling in voltage depen- priately utilized in this computational framework to identify phenotypic mech- dent ion channels. Support:NIHGM030376. anisms and mechanisms of rare events. In addition, we will show an application of the computational population-based modeling approach to predict the impact 508-Pos 2D of genetic mutations. The same mutations have vastly different cardiac effects Screening of Negative Charges by Ca in the Turret Region Controls in different patients, and these phenotypic differences are recapitulated in our Kv7.1 Inactivation Gating and is Regulated by PIP2 and Calmodulin model population. We will present population predictions showing the variable Bernard Attali1, William S. Tobelaim2, Maya Lipinsky1, Asher S. Peretz3, effect of KCNH2 mutations that are causally linked to LQT2. Daniel Yakubovich4, Yoav Paas5. 1Sackler Fac Med, Tel Aviv Univ, Tel Aviv, Israel, 2Dept Physiol Pharmacol, Posters: Voltage-gated K Channels I Tel Aviv Univ, Tel Aviv, Israel, 3Dept Physiology, Tel Aviv Univ, Tel Aviv, Israel, 4Dept Physiol/Pharmacol, Tel Aviv Univ, Tel Aviv, Israel, 5Fac Life 506-Pos Sci, Bar Ilan Univ, Ramat Gan, Israel. þ Molecular Mechanisms of Filter-Level Gating and Loss of Selectivity in Inactivation is an intrinsic property of numerous voltage-gated K (Kv) chan- hERG1 N629D Mutant from Microseconds MD Simulations nels and can occur by N-type or/and C-type mechanisms. While fast N-type Williams E. Miranda1, Henry J. Duff2, Jiqing Guo2, Igor V. Vorobyov3, inactivation involves the inner pore occlusion by N-terminal peptide domains Kevin R. DeMarco3, Colleen E. Clancy3, Sergei Yu Noskov1. of a and b subunits, C-type inactivation is suggested to involve structural rear- þ 1Department of Biological Sciences, Univ Calgary, Calgary, AB, Canada, rangements in the outer pore leading to a loss of K coordination sites in the 2Libin Cardiovascular Institute of Alberta, Cumming School of Medicine, selectivity filter. In Kv7.1 channels, inactivation is invisible macroscopically Univ Calgary, Calgary, AB, Canada, 3Department of Physiology and and does not exhibit the hallmarks of N- and C-type mechanisms. However, Membrane Biology, Univ Calif Davis, Davis, CA, USA. Kv7.1 inactivation is revealed by hooked tail currents, which reflect the recov- þ The hERG1 human potassium-selective channel displays fast C-type inactiva- ery from an inactivation state. We show that removal of external Ca2 tion during cardiomyocyte-membrane depolarization, enabling the long increased the activation kinetics and produced a large voltage-dependent inac- þ plateau-phase of the cardiac action-potential (CAP). The mutation N629D tivation of Kv7.1 channels. Increasing external Ca2 suppresses inactivation 2þ 2þ adjacent to the selectivity filter (SF) signature-sequence 626GFG628 produces gating with an EC50 of 1.5 mM. While Sr and Cd mimicked the effects þ þ þ a non-inactivating/non-selective channel (Kþ:Naþ 3:2) that prolongs the of Ca2 , other divalent cations like Mg2 and Mn2 were ineffective. External

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Kþ (50 mM) did not prevent the inactivation evoked in Ca2þ-free external so- 511-Pos lutions suggesting a mechanism different from C-type inactivation. External Voltage Dependent Gating of BK Channels - Where is the Spring? acidification or introduction in the pipet solution of calcified calmodulin Karl L. Magleby, Yanyan Geng. or PIP2 slowed down the activation kinetics and precluded inactivation gating Dept Physiol/Biophys, Univ Miami Miller Sch Med, Miami, FL, USA. evoked in Ca2þ-free external solutions. Experimental data and kinetic BK channels are activated synergistically by voltage and Ca2þ. BK channels modeling indicated that Kv7.1 channels exhibit two distinct inactivation states. include a central pore-gate domain (S5-S6) surrounded by four voltage sensor do- Mutagenesis studies and structural modeling suggest that external Ca2þ ions act mains (S0-S4), with each S6 attached to a large cytoplasmic gating ring via a pep- to screen the negative charges of neighboring glutamate and aspartate residues tide linker. The Ca2þ transduction pathway may involve gating ring expansion located respectively, in the turret and filter entrance of the channel pore. Our upon binding Ca2þ to open the pore by pulling on S6 through the linkers; the results reveal a new mechanism whereby external Ca2þ exquisitely controls voltage transduction pathway may involve voltage sensor movement transferred inactivation gating of a Kv channel that is allosterically modulated by PIP2 to S5 and then to S6; and both transduction pathways interact and may use one and calcified calmodulin at the inner face of the channel transmembrane core. or more common elements (Horrigan and Aldrich, 2002; Lee and Cui, 2010; Pan- tazis and Olcese, 2016; Hite, Tau and MacKinnon, 2016). Niu, Qian and Magleby 509-Pos (2004) found in 0 Ca2þ that the voltage for half activation, V1/2, increased linearly Quantum Calculations on Proton Transport in the Kv1 Channel Voltage with linker length, suggesting that the linker-gating ring complex acts as a Hoo- Sensing Domain, with Comparison to Analogs in Bacteriorhodopsin, kean spring in 0 Ca2þ. Hite et al. (2016) found that linker length was unchanged Cytochrome C, and the HV1 Proton Channel for open and closed states induced by changing Ca2þ, excluding that the linker Alisher M. Kariev1, Michael E. Green2. þ 1 2 acts as a spring during Ca2 activation. We now use a theoretical approach to Dept Chemistry, City College New York, New York, NY, USA, Dept explore if the voltage dependent spring in 0 Ca2þ could arise from indirect volta- Chemistry and Biochemistry, City College New York, New York, NY, USA. ge induced expansion of the gating ring, from spring properties of the linker Quantum calculations on 976 atoms (including 24 water molecules) of the Kv1.2 for activation by voltage but not Ca2þ, or from other structures in the channel. voltage sensing domain (VSD) show a triad of amino acids that can transfer a We consider different possible configurations of the Ca2þ and voltage transduc- proton in the presence of an external electric field: in the pdb 3Lut numbering, tion pathways, ranging from independent to highly coupled, including sharing these are Y266,R300,E183. The effective pKa changes by enough to transfer a common elements, such as the linker being a series component in the voltage proton from the tyrosine to the arginine; the lowest energy conformation crosses transduction pathway. Depending on the assumed configurations of the gating þ from the closed (all neutral) to open state (tyrosine(-), arginine( ), glutamate(0)) pathways, we find that the spring could reflect apparent spring properties of near 20 mV, close to the potential at which the membrane opens (reasonable the gating ring, linkers, and voltage sensors, singly or in various combinations. errors would not change the overall conclusion). The proton path could continue NIH GM114694. through R303 and E226. S176, which does not ionize, rotates its –OH group from having the H oriented toward R303 when it is neutral (open state), to oriented 512-Pos away when it is positively charged (closed), thus altering the local electric field. Influence of Dimeric Interactions on Voltage Sensing Phosphatase Activity The path is hypothesized to continue through H310 and on to the gate along the Vamseedhar Rayaprolu1, Perrine Royal2, Guillaume Sandoz2, intracellular membrane boundary. Other membrane proteins transmit protons, Susy C. Kohout1. and they show similar amino acid triads which would be candidates for being 1Dept Cell Biol & NeuroScience, Montana State University, Bozeman, MT, 2 part of the proton path: channel Hv1 (3WKV numbering): H136,D119,E115; USA, CNRS, INSERM, iBV, Universite Cote D’Azur, Nice, France. R201,D108,R204; Y157,D170,R207. For cytochrome c (3S8F): Y237, via a far- Multimeric interactions in proteins are a very common and extremely impor- nesyl to Y244,Y248. Bacteriorhodopsin (1FBB) shows similar candidates: tant phenomenon. Signaling cascades depend on delicate balances of homo- Y57,R82,E194, W189,Y83, and Y185,W86,D212 (postulating that tryptophan or heteromeric protein interactions to execute cellular responses to stimuli. accepts a proton on its nitrogen). Such triads of amino acids are apparently com- We recently showed that such homomeric interactions exist in the voltage mon features of proton transmitting membrane proteins, and the Kv1.2 VSD fits sensing phosphatase (VSP) from Ciona intestinalis. VSP bridges two funda- this group’s characteristics. Also, arginine apparently accepts a proton on its NE mental cell signals, voltage and phosphatidylinositol phosphate (PIPs) nitrogen, making its side chain amphoteric. Calculations on additional parts of concentrations, through voltage regulated enzyme catalysis. The voltage the Kv1.2 VSD (up to H310), and the Hv1 pathways, are in progress. sensing domain (VSD) from VSP senses membrane voltage changes trig- gering the cytosolic phosphatase domain (PD) to dephosphorylate PIPs. 510-Pos VSP activity directly links voltage to PIP concentrations, both of which Mobility of S3-S4 Linker Modulates Activation Process in Shaker are key components in cell excitability, migration and immune response. Potassium Channels Hence, understanding the biophysics of VSP will play a critical role in iden- Joao L. Carvalho-de-Souza, Carlos Bassetto Jr, Elizabeth E.L. Lee, tifying the biological context. Our recent study showed that VSP dimerizes Francisco Bezanilla. predominantly through the VSD and that VSP dimers can act in a Biochemistry and Molecular Biology, The University of Chicago, Chicago, dominant-negative fashion (Rayaprolu et al, JGP, 2018). To further probe IL, USA. þ those dimeric interactions, we identified potential interacting residues from In Shaker, A355R mutation in S3-S4 linker makes gating currents (Ig) and K the crystal structures of both the VSD and PD of Ci-VSP. We made muta- conductance activation two-to-six times slower. A355R slightly alters Q-V tions targeting those residues and tested them for their ability to co- curve consistent with a small right shift in G-V curve of the channel. Possible immunoprecipitate. Some appear to break the dimer while others may salt bridges between A355R and a negative charge nearby is a possible mech- enhance the interactions. Next, we will confirm the presence or absence of anism. We ruled out E422 in S5 segment as a possible partner. Other options, a dimeric unit with these mutants using single molecule pulldown (SiMPull) being tested, are several acidic residues in S1-S2 and S3-S4 linkers, and residue experiments and subsequently characterize the voltage dependence and E283 in S2. Tetramethylrhodamine-labelable cysteine residues in S2 (T276C or enzymatic activity using electrophysiology. These results may shed light P278C) to probe movements in this region via fluorescence during slow gating on the function of the two human VSP homologs since one of them is endog- show that TMR fluorescence change upon voltage-sensor activation follows Ig, enously inactive and suggests a physiologically relevant dominant-negative in contrast of what happens with A355 (WT residue) when Ig move faster than behavior. The functional impact of dimerization could ultimately reveal fluorescence changes. We also studied the mutant A355C which we found to how VSPs may be involved in voltage dependent PIP signaling in cells. form bridges with constitutive residues nearby. Results with 355C show Ig kinetically unaltered but half of the total charge movement is blocked by bridge 513-Pos formation. Most likely, disulfide bridges make part of gating too slow to be de- Nature Utilization of the Slow Inactivation Mechanism in Voltage Gated D tected therefore only the detectable part was used for calculating kinetics. This K Channels interpretation is consistent with Kþ currents recordings, showing only a 15% Izhar Karbat1, Hagit Gueta2, Tibor Szanto3, Shelly Hamer-Rogotner1, decrease in amplitude but activating with two kinetic components in bridged Orly Dym1, Felix Frolow2, Dalia Gordon1, Gyorgy Panyi3, channels: one fast, consistent with Ig, and another much slower, likely related Michael Gurevitz4, Eitan Reuveny1. to the very slow (undetected) charge movement. For 355R, weaker salt bridges 1Biomolecular Science, Weizmann Inst, Rehovot, Israel, 2Molecular Biology also make gating slow but it can still be fully recorded and studied. This data and Ecology of Plants, Tel Aviv University, Tel Aviv, Israel, 3Biophys/Cell strongly suggest that gating in Shaker channels is extremely dependent on Biol, Debrecen, Hungary, 4Dept Plant Sci, Tel Aviv Univ, Tel Aviv, Israel. þ the mobility of the S3-S4 linker and makes possible to study kinetic details Voltage gated K channels gate in response to changes in the electrical mem- þ of intermediate states of voltage sensor movement and its coupling to the brane potential by the coupling of a voltage sensing module with a K -selective pore domain. Support: NIHGM030376. pore. During prolonged depolarizations, these channels enter a non-conductive

BPJ 9314_9317 Sunday, March 3, 2019 103a slow-inactivated state that involves constriction of the channel pore. Recent genesis and structural prediction data from previous studies to generate a hERG studies suggest that the slow inactivated state is triggered by the penetration inactivated state model, demonstrating a SF constriction and widened pore of water molecules into well-defined peripheral cavities surrounding the selec- suggesting a plausible inactivated state model. We will apply these methods tivity filter. Here we describe Cs1, a cone-snail toxin that blocks the drosophila to check for ion conduction in other mutants with different inactivation procliv- shaker channel using a novel mode of action that shares common features with ities, as well as at different voltages, ion concentrations, and force field its inherent slow inactivation. Using double-mutant cycle analysis comple- parameters. mented by unconstrained docking and refinement by molecular-dynamics we obtained a docked toxin model, in which the toxin is bound off the pore axis. 516-Pos Analysis of the MD data suggested that Cs1 does not block the access of water The Role of HCN Domain in the Function of HCN Channels molecules to the pore, but greatly modifies the access of water to the peripheral Zejun Wang1, Sebastien Hayoz1, Tinatin I. Brelidze2. 1 cavities. In concert with recent studies, we demonstrate that water permeation Department of Pharmacology & Physiology, Georgetown University 2 into these cavities is governed by a network of hydrogen-bonds formed at the Medical Center, Washington, DC, USA, Dept Pharma/Physio, Georgetown extracellular face of the channel. The toxin specifically target these hydrogen University, Washington, DC, USA. bonds in three different channel subunits, by either eliminating or stabilizing Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels them. The resulting imbalanced movement of water molecules around the generate funny currents (If) in the heart and hyperpolarization-activated cur- selectivity filter triggers a series of molecular events culminating in an asym- rents (Ih) in the brain. HCN channels are assembled from four subunits, with metric collapse of the selectivity filter. A series of electrophysiological exper- each subunit containing six transmembrane segments (S1-S6) and a cyclic- iments utilizing D2O and various toxin/channel mutants that support the nucleotide binding (CNB) domain in the C-terminal region linked to the proposed mechanism will be described. pore-forming transmembrane segment with a C-linker. Recent cryo-EM struc- ture of HCN1 channels (Lee et al., Cell, 2017, 168:111-120) identified a stretch 514-Pos of 45 amino acids directly preceding the S1 transmembrane segment as a Calcium Regulation of Kv4-Kchip Ion Channel Complexes 3-alpha-helical structural motif conserved in all four mammalian HCN channel Jonathan G. Murphy, Dax A. Hoffman. isoforms. The 3-alpha-helical domain, dubbed HCN domain, forms direct inter- NICHD, National Institutes of Health, Bethesda, MD, USA. actions with the S4 transmembrane segment in the voltage sensor from the þ The Kv4 family of A-type voltage-gated K channels play a unique role in the same subunit and the C-linker/CNB domain from the adjacent subunit. Here dendrites of hippocampal pyramidal neurons. In particular, Kv4.2 channels we explored the role of the HCN domain in the function of HCN channels. are key determinants of dendritic excitability and integration, spike timing- Deletion of the HCN domain abolished currents from HCN channels expressed dependent plasticity, long-term potentiation, and learning. Downregulation of in Xenopus laevis oocytes. Mutant HCN channels with alanines substituted for Kv4.2 channel expression occurs following hippocampal seizures and in epi- the interacting residues between the HCN domain and the C-linker/CNB lepsy suggesting A-type currents as targets for novel therapeutics. In addition domain gave rise to hyperpolarization-activated currents with the half- to pore forming Kv4 subunits, native hippocampal A-type currents require maximal activation voltage (V1/2) similar to the V1/2 of the wild-type HCN modulatory auxiliary subunits known as K-channel interacting proteins channels. Taken together our observations point towards the role of the HCN (KChIPs). KChIPs function to enhance Kv4 expression and activity. KChIP domain in the functional assembly of HCN channels. auxiliary proteins may convey Ca2þ sensitivity upon Kv4 channels as they are members of the EF-hand domain containing neuronal Ca2þ sensor protein 517-Pos D family; however, it is currently unclear to what degree intracellular Ca2þ Trapping the 2,4-K -Ion Bound Configuration of KcsA’s Selectivity Filter regulates Kv4.2-KChIP complexes. To assess this possibility, we expressed Cholpon Tilegenova, D. Marien Cortes, Nermina Jahovic, Emily Hardy, KChIP2 with Kv4.2 in HEK293T cells and performed whole-cell patch clamp Parameswaran Hariharan, Lan Guan, Luis G. Cuello. recordings in either nominal or 10 mMCa2þ. In the presence of 10 mMCa2þ, Dept. Cell Physiology and Molecular Biophysics., TTUHSC, Lubbock, we measured a 1.5 fold increase in Kv4.2 current density. Intriguingly, Ca2þ TX, USA. þ regulation was specific to short splice isoforms of KChIP2, KChIP2b and 2c. In all of the crystal structures of K channels solved to date, an aggregate of þ KChIP mRNAs contain multiple start sites and alternative exons that generate four K ions has been observed bound to the selectivity filter (SF) (i.e., ion considerable N-terminal variation and functional diversity in shaping Kv4 binding sites S1, S2, S3 and S4 counting from the extracellular side)[1]. It is þ currents. Sequence alignment of the variable N-termini of KChIP2 isoforms believed that the K channel’s SF exists in two different isoenergetic arrange- suggest a novel domain limits Ca2þ sensitivity of the longer KChIP2 isoforms, ments known as the S1/S3 and the S2/S4 configurations. This observation and þ KChIP2a1 and KChIP2a. electrostatic considerations form the basis of the canonical model for K -chan- nel ion conduction[2]. The interconversion between these configurations is 515-Pos driven by the electrostatic repulsion between Kþions separated by water mol- In Silico Determination of Open Conducting and Inactivated Atomistic ecules. Water transport during ion permeation was clearly evidenced by stream- þ KV11.1 Channel Models ing potential measurements in Calcium-activated K -channels[3]. Here, we John R.D. Dawson1, Kevin R. DeMarco2, Borislava Bekker3, report two KcsA mutant channels (G77A and G77C) that trap KcsA’s SF in Sergei Y. Noskov4, Colleen E. Clancy2, Igor V. Vorobyov2. the 2,4-ion bound configuration by precluding the binding of an ion at the S3 1 Biophysics Graduate Group, University of California Davis, Davis, CA, site. Interestingly, in agreement with the canonical model, a concomitant ion 2 USA, Department of Physiology and Membrane Biology, University of vacancy was observed at the S1 site, as if the S1 and S3 sites were energetically 3 California Davis, Davis, CA, USA, American River College, Sacramento, coupled. Trapping a Kþchannel’s SF in the 2,4-ion bound configuration 4 CA, USA, Centre for Molecular Simulation, Department of Biological provides tangible and undisputable evidence to the canonical model for ion Sciences, University of Calgary, Calgary, AB, Canada. permeation and decisively argues against the new alternative model of direct The voltage gated potassium channel KV11.1, encoded by the human Ether- knock-on[4]. Finally, the G77A mutant, although having caused a marked a´-go-go related gene (hERG), is responsible for the delayed rectifier potas- reduction in the ion transport rate, retained wild-type-like selectivity in ion I sium current ( Kr) during repolarization of the cardiac action potential (AP). binding and permeation properties. Delayed rectification in hERG results from unique gating properties: upon Support: NIH award (2R01GM097159-06) and Welch Foundation Award membrane depolarization, hERG opens but rapidly inactivates, ceasing con- (BI-1949) to L.G. Cuello and NIH award (R01GM122759) to L. Guan. duction. hERG then quickly recovers from inactivation during AP repolariza- 1. Zhou, Y., et al.Nature, 2001. 414(6859): p. 43-8. 2. Morais-Cabral, tion, resulting in larger current and hastened repolarization. We have J.H.,et al.Nature, 2001. 414(6859): p. 37-42. 3. Alcayaga, C., et al.Biophys J, developed open and inactivated state models of hERG based upon a recently 1989. 55(2): p. 367-71. 4. Kopfer, D.A., et al.Science, 2014. 346(6207): p. 352-5. published Cryo-EM structure (PDB: 5VA2) and used molecular dynamics (MD) simulations to characterize their stabilities and ion conduction at 518-Pos D multi-microsecond timescales. In MD simulations of wild-type hERG with Quantum Calculations on the K Ion in the KV1.2 Channel Pore: and without applied voltage, substantial selectivity filter (SF) distortions Hydration and Cosolvation were observed, likely preventing ion conduction. However, under a high Alisher M. Kariev1, Michael E. Green2. (500 mV) applied voltage and high (0.5M) KCl several slow Kþ conduction 1Dept Chemistry, City College New York, New York, NY, USA, 2Dept events were observed for WT and the inactivation-deficient S641T mutant. Chemistry and Biochemistr, CCNY, New York, NY, USA. þ þ These results were validated through simulations of KV1.2/2.1 Paddle Chimera The progress of the K ion through the pore in a K channel from the gate to channel (PDB: 2R9R) under identical conditions, which yielded multiple fast the selectivity filter has been extensively studied, but uncertainties remain. In Kþ permeation events along with a more stable SF. We also incorporated muta- these calculations, the ion was placed in each of five locations (calculations

BPJ 9314_9317 104a Sunday, March 3, 2019 are ongoing at two more locations); for an 871 atom (protein, 50 water mole- Hax-1. Inhibition of TBK1 in the cell lines prevents the increase in CD63 levels cules, and the ion) pore section, quantum calculations optimized (energy mini- produced by the Kv3.3 mutation. Our findings suggest that Kv3.3 channels are mized) the section, and the energy and ion hydration at each ion position were directly coupled to pathways that regulate the trafficking of proteins into multi- determined. The protein structures the water column. It appears that hydration vesicular bodies and that loss of the Hax-1 may contribute to the neurodegen- may explain the reason for the strong conservation of the threonine at the bot- eration caused by SCA13. tom of the selectivity filter, which interacts with water in a characteristic way, producing a ‘‘basket’’ of four water molecules H-bonded to the threonines. One 521-Pos remaining problem is determining how the ion exchanges its own hydration Modulation of Kv7.1/KCNE1 Channel Activity by Navb1 shell to be cosolvated by the four threonine hydroxyl groups, breaking the water Spencer Mason Webber, Carlos Villalba-Galea. ‘‘basket’’. Once that is accomplished, the ion can proceed through the selec- Physiology and Pharmacology, University of the Pacific, Stockton, CA, USA. tivity filter. At the entrance to the pore at the bottom of the water column KV7.1 is a voltage-gated potassium channel encoded by the gene KCNQ1. (i.e., the gate), hydration is strongly rearranged when an ion arrives. A small This channel is found in several organs, including the heart and the brain. increase in diameter from the closed state, approximately 5 A˚ , is adequate to In the mammalian heart, KV7.1 assembles with KCNE1 forming a channel admit the ion and hold it by a local increase in the density of the water hydrating complex that is critically important for ventricular repolarization. The activity it; a wide open gate would often lose the ion back to solution, greatly diminish- of the KV7.1/KCNE1 complex results in the delayed rectifier current known as b b ing the current. The entering ion remains held as the ion above it moves; IKs. The voltage-gated sodium channel type 1 subunit (NaV 1) is also ex- following this, the gate ion can move up to the center of the pore pressed in the mammalian heart. Mutations in this protein have been impli- cavity. Optimizations were done using HF/6-31G*, while energy calculations cated in disorders such as epilepsy and cardiopathies, including Brugada b used B3LYP/6-31G** on the optimized structures. syndrome and atrial fibrillation. The NaV 1 subunit modulates the activity, trafficking, and expression of NaV channels. It has been shown that NaVb1 519-Pos also modulates channels from the KV1 and KV4 families, and KV7.2 channels. Nobiletin Inhibition of BK Channels Preliminary data from our laboratory show that NaVb1 modulates the activity Liang Sun, Lorie Ann Gonzalez, Frank T. Horrigan. of KV7.1. When expressed in Xenopus oocytes, KV7.1 channel-mediated Baylor College of Medicine, Houston, TX, USA. Kþ-currents display a mild slow inactivation. These channels recover from Nobiletin, a flavone found in citrus peels, is a component in traditional Chinese inactivation during deactivation driven at negative voltage, displaying a medicine and has a number of beneficial effects that have been studied in an- ‘‘hook’’ like kinetics. In the presence of NaVb1, the inactivation is remarkably imals and humans. It improves memory in various animal models of dementia enhanced, producing a strong resurgent conductance during deactivation at including Alzheimer’s disease, and has neurotrophic effects as well as effects negative potentials. Furthermore, the deactivation rate is decreased, while on catecholamine synthesis and secretion. Using a high-throughput screen, activation remains seemingly unaltered. Since KV7.1, KCNE1 and NaVb1 we discovered that nobiletin inhibits BK channels in a manner that is sensitive are expressed in the heart, we decided to investigate whether NaVb1 could to both voltage-sensor activation and beta subunit expression. Patch clamp modulate KV7.1/KCNE1 activity. We found that expressing KCNE1 subunit 2þ studies further indicated that nobiletin inhibits BK channels in a Ca - and seems to override the NaVb1 subunit effect on KV7.1 channels. However, voltage- dependent manner. At 80 mV, 30 mM nobiletin has no significant decreasing the relative expression of KCNE1 resulted in Kþ-currents that dis- 2þ effect on Slo1 current in 0 Ca , but reduces channel activity (NPO)by played features of the activity of both type of complexes. We therefore m 2þ 90% in saturating 100 MCa with no effect on single channel conductance. conclude that NaVb1 can modulate the KV7.1/KCNE1 complex activity. m 2þ In 100 MCa , nobiletin reduces channel activity at all voltages tested ( 200 Our work suggests that, in the heart, the interaction of KCNE1 with KV7.1 þ m 2þ to 200 mV ), with an IC50 of 10.4 Mat 80 mV . However, in 0 Ca , no- has evolved to avoid the effect of NaVb1 on the channel, so preventing its biletin reduces channel activity only at voltages above 40 mV. Nobiletin is also inactivation. less effective on channels composed of Slo1 and b2 subunits, but not when the N-terminal residues FIW are deleted, suggesting that the b2 inactivation ‘ball’ 522-Pos protects the channel from nobiletin inhibition. b4 subunits alter the nobiletin Structural Modeling of the HERG Channel in an Inactivated State and its inhibition as well. The state-dependence and subunit-dependence of nobiletin Drug Interactions action, suggest it could be a potentially tissue-selective inhibitor of BK Jan Maly, Aiyana M. Emigh, Kevin R. DeMarco, Jon T. Sack, channels, conferring sensitivity to voltage and Ca2þconditions in different Igor Vorobyov, Colleen E. Clancy, Vladimir Yarov-Yarovoy. tissues, and accessory BK channel subunits which are expressed in a tissue- University of California Davis, Davis, CA, USA. dependent manner. Defining the action of nobiletin on BK channels could be The voltage gated potassium channel, KV11.1, is encoded by the human ether-a- relevant to understanding its therapeutic mechanism of action. go-go related gene (hERG) and expressed in cardiac myocytes, where it is crucial for the membrane repolarization of the action potential. hERG is implicated in a 520-Pos number of drug-induced arrhythmias, caused by long QT syndrome. Gating of Loss of HAX-1 May Contribute to the Neurodegeneration Caused by a hERG is characterized by rapid, voltage-dependent, C-type inactivation, which Kv3.3 Mutation blocks ion conduction and is suggested to involve constriction of the selectivity Yalan Zhang, Luis Varela, Klara Szigeti-Buck, Tamas L. Horvath, filter. Mutations S641A/C, G648A, and F627Y in the selectivity filter region of Leonard K. Kaczmarek. hERG have been shown to alter the voltage-dependence of channel inactivation. Yale, New Haven, CT, USA. To explore conformational changes associated with hERG inactivation, we used Spinocerebellar Ataxia type 13 (SCA13) is caused by mutations in the KCNC3 RosettaRelax to simulate the effects of those mutations, as well as a potential gene, which encodes the voltage-dependent potassium channel Kv3.3. These hydrogen bond between residues N629 and S620, adjacent to the selectivity filter. channels are expressed at particularly high levels in cerebellum Purkinje cells. Preliminary modeling results show that the S641A mutation is sufficient to SCA13 is an autosomal dominant disease characterized primarily by degener- constrict the top of the selectivity filter, but the G648A and F627Y mutations ation of the cerebellum. We have found the Kv3.3 channel differs from other require formation of the N629-S620 hydrogen bond. The N629-S620 hydrogen potassium channels in that it binds Hax-1, an anti-apoptotic protein that is abso- bond is also necessary to cause pinching of the selectivity filter in the WT channel, lutely required for the survival of cerebellar neurons. We have also found that simulating C-type inactivation. Furthermore, we used RosettaLigand to explore depolarization of Kv3.3 channels directly activates Tank Binding Kinase 1 the binding mechanism of dofetilide, an antiarrhythmic agent that is a highly (TBK1), an enzyme that plays a key role in the formation of multivesicular selective and potent hERG blocker. Structural modeling of hERG interaction bodies, autophagy and mitophagy. A disease-causing mutation, G592R with drugs will lead to a better understanding of molecular mechanisms Kv3.3, produces enhanced TBK1 activation both in cell lines and in the cere- of state-dependent drug action and could enable rational design of safer bellum of knock-in mice bearing this mutation. By electron microscopy, we therapeutics. find the enhanced activation of TBK1 in G592R Kv3.3 knock-in mice is asso- ciated with increased numbers of intracellular multivesicular bodies containing 523-Pos Hax-1, and increased levels of CD63, a molecular marker for these structures. Differential Regulation of BK Channels by Fragile X Mental Retardation We also found that the expression level of Hax-1 is reduced in G592R Kv3.3 Protein knock-in mice by Western blotting. Mitochondrial function may also be Aravind Kshatri, Alejandro Cerrada, Roger Gimeno, Teresa Giraldez. impacted by the mutation because levels of mitofusin-1 and mitofusin-2, pro- Department of Biomedical Sciences, Universidad de la Laguna, Tenerife, teins that are required for fusion and maintenance of mitochondrial structure, Spain. are altered in the mutant mice. Using cell lines expressing the mutant channel, Fragile mental retardation protein (FMRP) is an RNA-binding protein prom- we have shown that the G592R Kv3.3-induced multivesicular bodies contain inently expressed in neurons. Missense mutations or complete loss of FMRP

BPJ 9314_9317 Sunday, March 3, 2019 105a can potentially lead to fragile X syndrome (FXS), a common form of in- Naþ ions. The kainate receptor agonist-binding domain dimer interface has herited intellectual disability (1, 2). In addition to RNA regulation, FMRP been shown to be involved in mediating activation, desensitization, and was also proposed to modulate neuronal function by direct interaction involved in ion modulation. Here, we have characterized the conformational 2þ with the large conductance Ca activated potassium (BK) b4 regulatory and energetic landscape at this interface associated with the resting, active, subunits (BKb4) (3). However, the molecular mechanisms underlying desensitized, and ion-modulated states of the receptor by utilizing two FMRP regulation of BK channels were not studied in detail. We have fluorescence-based methods. These data show that the agonist-binding used electrophysiology and super-resolution stochastic optical reconstruction domain dimer interface exists primarily in the coupled state in both the unli- microscopy (STORM) to characterize the effects of FMRP on pore-forming ganded resting and the glutamate-bound open states in the presence of Naþ BKa subunits, as well as their interactions with regulatory subunits BKb4 ions. In contrast, the receptor occupies multiple conformational states with and BKg1. STORM experiments revealed clustered multi-protein complexes varying degrees of decoupling under desensitizing conditions. Replacing þ þ containing BKa or BKb4 and FMRP, consistent with FMRP binding not only Na with Cs ions leads to a shift in the population to more decoupled- to BKab4 (in agreement with previously published data (3)), but also to dimer state, and decreases transition energy barriers, in the resting and desen- BKa. Interestingly, our data indicate that FMRP alters the steady state prop- sitized states. Additionally, when Naþ is replaced with Csþ,ashifttoamore erties of BKa channels by increasing their activation and deactivation rates. decoupled dimer interface is seen in both the resting and desensitized states. Analysis using Horrigan-Aldrich model revealed alterations in the parame- With our data confirming that the activated state requires a coupled dimer þ ters associated with voltage sensor function (J0) and channel opening (L0). interface, the lower activation in the presence of Cs can be attributed to However, no significant effects of FMRP were observed on the biophysical changes in both the resting and desensitized states. These are the first data properties of BKab4 channels. Conversely, STORM experiments showed characterizing the conformational and energetic landscape of kainate that, in the presence of FMRP, interactions between BKa and BKg1 subunits receptors. are significantly diminished. Consistently, electrophysiological recordings of 526-Pos cells co-expressing FMRP, BKa and BKg1 subunits, did not show BKag1 currents. In summary, our data show that FMRP regulates BKa channels, Single-Molecule FRET Investigations of Negative Cooperativity in the NMDA Receptor does not affect BKab4 channel function, and inhibits modulation by BKg1 1,2 1,2 1 1 subunits. Ryan J. Durham , Nabina Paudyal , Elisa Carrillo , Vladimir Berka , 1 1. Myrick LK et al. PNAS 2015 112:949. Vasanthi Jayaraman . 1Biochemistry and Molecular Biology, Center for Membrane Biology, 2. Santoro MR et al. Ann Rev Pathol. 2012;7:219-45. 2 3. Deng PY et al. Neuron. 2013 77:696-711. UTHealth, Houston, TX, USA, Biochemistry and Cell Biology Program, MD Anderson Cancer Center UTHealth Graduate School of Biomedical Posters: Ligand-gated Channels I Sciences, Houston, TX, USA. Ionotropic glutamate receptors mediate the majority of excitatory neuro- 524-Pos transmission in the central nervous system. One subtype of ionotropic gluta- Fo¨rster Resonance Energy Transfer (FRET) Analysis of the C-Terminal mate receptor, the N-methyl-D-aspartate receptor, is of note due to its Domain of Fp-Tagged Homomeric and Heteromeric AMPARs Upon Phos- requirement for not only glutamate, but also glycine, to bind to the receptor phorylation for activation to occur. The NMDA receptor achieves this co-agonist Linda G. Zachariassen1, Anne-Sophie Hafner2, Daniel Choquet2, requirement due to its nature as an obligate heteromer consisting of two Anders S. Kristensen1. glycine-binding N1 subunits and two glutamate-binding N2 subunits, which 1Department of Drug Design and Pharmacology, University Copenhagen, are arranged in an alternating pattern. Glutamate and glycine exhibit Copenhagen, Denmark, 2Bordeaux Imaging Center, UMS 3420 Centre negative cooperativity in the NMDA receptor, where the binding of one National de la Recherche Scientifique, University of Bordeaux, Bordeaux, co-agonist causes a reduction in the affinity of the receptor for the other France. co-agonist. Herein we have utilized single-molecule fluorescence resonance AMPA receptors (AMPARs) are glutamate-gated cation channels that energy transfer to study the negative cooperativity exhibited by the NMDA mediate the majority of fast excitatory neurotransmission in the central ner- receptor. Our studies have shown that the glycine-binding domain in the apo vous system. AMPARs are tetrameric assemblies of GluA1 to GluA4 sub- state probes primarily an open conformation while occasionally also probing units. Recently Cryo-EM and X-ray crystal structures of homomeric and a closed conformation. The equilibrium between these open and closed heteromeric AMPARs has been provided; showing a highly modular archi- conformations is shifted upon the binding of glycine, which indicates a tecture with an extracellular domain containing four glutamate binding conformational selection mechanism of receptor control. Furthermore, sites coupled to a transmembrane domain containing the central ion channel. upon the binding of glutamate to the adjacent subunit, the glycine-binding AMPARs also contain an intracellular domain (ICD) formed of C-terminals domain will occasionally probe an open state similar to the one seen from each subunit. The ICD is important for interactions with intracellular in the apo state. This shift in conformational stability would favor the proteins and contains multiple regulatory phosphorylation sites, but its unbinding of glycine from the receptor and could therefore be directly structural and mechanistic role is poorly understood as the ICD is not related to the negative cooperativity observed between the NMDA receptor resolved in present structures. We have used a Fo¨rster Resonance Energy co-agonists. In addition, the binding of glycine to the receptor causes the Transfer (FRET) approach to study intramolecular distances and dynamics transmembrane segments to adopt a more tightly packed conformation of the ICD in homomeric and heteromeric AMPARs by insertion of than what is observed when the receptor is in the apo state. These results fluorescent proteins (FPs) at various intracellular positions in the GluA1 serve to reveal the conformational shifts that underlie various aspects of and GluA2 subunits. Using fluorescence life-time imaging (FLIM) to NMDA receptor behavior. determine FRET efficiencies between FPs or a membrane dye, we determine relative distances within the ICD and to the membrane. Furthermore, we 527-Pos investigate the effect of phosphorylation mimicking mutations in the Study of a Heteromeric Kainate Receptor Gluk2/K5 by Probing Single- GluA1 subunit on FRET and identify mutation-induced changes that indicate Molecule FRET phosphorylation state of the ICD can change its structure. Our preliminary Nabina Paudyal, Douglas B. Litwin, Vladmir Berka, Elisa Carrillo Flores, data provides new insight into the structural architecture and potential role Vasanthi Jayaraman. of the ICD. Univ Texas Health Science Center Houston, Houston, TX, USA. Kainate receptors are ligand-gated ion channels that belong to the ionotropic 525-Pos glutamate receptor family. They are unique among glutamate receptors in being The Structural Arrangement and Dynamics of Homomeric Kainate involved in both excitatory and inhibitory synaptic transmission. These recep- Receptors Determined by smFRET tors are homomeric and heteromeric assemblies of subunits GluK1-5 arranged Douglas B. Litwin1, Elisa Carrillo1, Sana Shaikh2, Vladimir Berka2, as dimer of dimers. Each subunit consists of an extracellular amino-terminal Vasanthi Jayaraman1. domain, a ligand binding domain, a transmembrane domain and an intracellular 1Biochemistry and Cellular Biology, Univ Texas Health Science Center C-terminal domain. Despite heteromeric GluK2/K5 receptors being the most Houston, Houston, TX, USA, 2Univ Texas Hlth Houston, Houston, TX, USA. predominantly expressed kainate receptor in the brain, there are currently no Kainate receptors are glutamate-gated cation-selective channels that partici- structures of the full length GluK2/K5. We have used smFRET measurements pate in both the reception and regulation of excitatory synaptic signaling, to determine the specific organization of the GluK2/GluK5 within the tetramer and are unique from other ligand-gated ion channels by being modulated by and additionally studied the structural changes associated with agonist binding,

BPJ 9314_9317 106a Sunday, March 3, 2019 activation, and desensitization of the receptor. These results of the heteromeric However, our ability to map structural changes to each of these functional con- receptors are compared to the homomeric receptors for which there are full figurations has been limited. Here, we integrate single-molecule recordings of length structures available. GluN1-1a/GluN2A receptors with computational modeling of receptor activa- tion to determine structural elements necessary for receptor activation. We 528-Pos found using single-channel recordings of disease-associated mutation, I642L Computing Free Energy of the Magnesium Block in N-Methyl-D- GluN1-1a I642L (GluN1-1a ) reduced receptor open probability within I642L Aspartate Receptors bursts (wild-type: 0.72 5 0.04; GluN1-1a : 0.04 5 0.06), mean open dura- I642L Christopher Kottke, Samaneh Mesbahi-Vasey, Maria G. Kurnikova. tion (wild-type: 4.34 5 0.11 msec; GluN1-1a : 1.10 5 0.09 msec), and Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA. mean closed duration within bursts (wild-type: 3.21 5 0.51 msec; GluN1- N-methyl-D-aspartate receptors (NMDARs) are heterotetrameric membrane 1aI642L: 11.74 5 1.20 msec). To determine the structural mechanism by which proteins vitally important to the conduction of excitatory signals between this disease mutation perturbs channel function, we used GluN1/GluN2B crys- neurons, synaptic plasticity, and cellular ion regulation. NMDARs play sig- tal structures to build a homology model of GluN1/GluN2A lacking cyto- nificant role in neuronal plasticity and thus, memory and learning. The plasmic and amino terminal domains. Using this model, we performed dysfunction of this ion channel has been implicated in many neurodegener- targeted molecular dynamics simulations of the closed receptor into a putative ative processes including Alzheimer’s, Huntington’s, epilepsy, and ischemic ‘‘open’’ conformation. We found that residue GluN1 I642 forms additional cell death following stroke. Unfortunately, developing drugs which may atomic contacts with L550 on the neighboring GluN2A during this transition. adequately control NMDAR behavior has proven to be a challenge. For We performed mutant cycle analysis on these pair of residues by recording I642L this reason, accurate methods for determining the binding affinity of mole- single-channel currents through GluN1-1a/GluN2A, GluN1-1A /GluN2A, L550I I642L L550I cules blocking the NMDAR selectivity filter need to be developed. GluN1-1a/GluN2A , or GluN1-1A /GluN2A . Using kinetic NMDARs are activated by co-binding of glutamate and glycine or D-serine, modeling of these currents to measure the free energy change associated and if the extracellular concentration of either of these neurotransmitters with each transition step in the receptor activation pathway, we found that rises too high, the protein will remain in its active state; allowing the the strongest coupling between GluN1 I642 and GluN2A L550 occurs transi- continual passage of calcium ions through the membrane. This can result tions between states C3 to C2 and O1 to O2. Our approach provides a tool by in the initiation an apoptosis mechanism. At resting membrane potential, which we can begin to map precise structural elements necessary for each tran- NMDARs are blocked by magnesium: an important protection mechanism sition step in receptor function. again calcium exotoxicity. The mechanism of ion selectivity and block is poorly understood. To model the NMDAR ion channel, we use molecular 531-Pos dynamics simulations. Because of the computational cost of simulating Gating of Single AMPA Receptors Cross-Linked at the Ligand Binding this system and the slow exchange rate of magnesium, we explore novel Domain Layer reaction coordinates to give finer energy surfaces of interaction and Sebastian Opfermann1, Jelena Baranovic2, Andrew J.R. Plested1,3. reduce necessary computation time. Specifically, this work focuses on eluci- 1Leibniz-Forschungsinstitut fur€ Molekulare Pharmakologie, Berlin, dating the energy surface of magnesium, a potential of mean force, and Germany, 2University of Edinburgh, Edinburgh, United Kingdom, 3Institut honing the techniques necessary to precisely map the energy surface of the fur€ Biologie, Humboldt-Universita´t zu Berlin, Berlin, Germany. system. AMPARs (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors) are ionotropic glutamate receptors that regulate fast excitatory neurotrans- 529-Pos mission at the postsynaptic membrane. In their tetrameric structure, the Mechanism of AMPA Receptor Modulation by Gamma-8 extracellular amino terminal domain and the ligand binding domain (LBD) Elisa Carrillo Flores, Sana A. Shaikh, Vasanthi Jayaraman. layers are arranged as dimer of dimers. The LBD layer has multiple degrees Biochemistry and Molecular Biology, The University of Texas Health of freedom, from closure of individual LBDs to the number of LBDs occu- Science Center at Houston, Houston, TX, USA. pied by glutamate and their conformational arrangement within the dimers Fast excitatory neurotransmission is mediated by ionotropic glutamate recep- and the tetramer. Glutamate binding within the clamshell-shaped LBDs in- tor (iGluR). AMPA-subtype ionotropic glutamate receptors (AMPARs) are duces clamshell closure resulting in channel gating. However, the extent associated with auxiliary subunits (transmembrane AMPA receptor regulato- of tetramer reorganization during activation and desensitization is not ry proteins (TARPs), cornichons, cysteine knot proteins and GSG1); fully understood. Here, we recorded single channel currents from GluA2 which regulate AMPAR assembly, trafficking, gating, and pharmacology. AMPARs restricted by an interdimer metal bridge between the LBD dimers. Of particular interest is the interactions with gamma-8 which results in re- With the bridge formed, receptors activated with a considerable latency of sensitization of the currents where the transmembrane channel in the recep- 2.8 5 0.7 ms (n = 5 channels), about 10-fold slower than wild-type tor initially undergoes closure due to desensitization but reopens at longer GluA2, and we identified three time components describing the first latency time and remain open in the continued presence of agonist. Here we have to opening. This observation is consistent with multiple pre-open states in investigated the mechanism underlying this resensitization process using sin- a gating pathway. We provide evidence that cross-linking works at the gle molecule FRET and single channel functional recordings. Specifically we single-channel level and a potential daisy chain effect by inter-cross- have determined changes at the AMPA receptor ligand binding domain linking multiple receptors on the macroscopic scale is rather unlikely. We dimer interface in presence of gamma-8 and compared it to the changes at further show that the restraint of the interdimer bridge reduces the open this site observed in the presence of cyclothiazide, a drug that stabilizes probability of individual receptors. We conclude that the LBD layer is highly the open channel state of the receptor. These studies show that while the dynamic, and that both the proper posture of the two LBD dimers and transit overall states populated are similar in the two cases, the kinetics of transi- through an ensemble of states are crucial for rapid and complete AMPAR tions between states are different, with activation barrier between transitions activation. being higher in the presence of gamma-8 . The single channel current record- ings reflected these slower kinetics in the presence of gamma-8 and also 532-Pos showed very rapid channel openings and closing which were no reflected Computational Characterization of the Binding of Non-Competitive at the dimer interface suggesting that these may be localized changes at Inhibitors to AMPA Receptors the transmembrane segments. Chamali Narangoda, Serzhan Sakipov, Maria G. Kurnikova. Chemistry, Carnegie Mellon University, Pittsburgh, PA, USA. 530-Pos Non-competitive inhibitors of AMPA receptors have attracted significant inter- Mapping Structural Elements to NMDA Receptor Activation Steps est in the recent years as potential antiepileptic drugs. While a considerable Gary J. Iacobucci1, Han Wen2, Matthew Helou1, Wenjun Zheng2, number of small molecules have been identified and tested as AMPA receptor Gabriela K. Popescu1. non-competitive antagonists, only pyridone perampanel (PMP) has been 1Biochemistry, State Univ New York Buffalo, Buffalo, NY, USA, 2Physics, approved for clinical use as an antiepileptic drug. PMP, however, still causes State Univ New York Buffalo, Buffalo, NY, USA. side effects at higher doses, indicating the need for safer and more effective NMDA receptors are heterotetrameric glutamate-activated ionotropic receptors drugs. Recently resolved crystal structures of AMPA receptor complexes of that mediate excitatory synaptic transmission in the brain. Numerous NMDA GYKI 53655 (GYKI), CP 465022 (CP), and PMP provided new structural in- receptor subunit mutations are associated with epilepsy and several have formation on non-competitive inhibitor binding to AMPA receptors. While been identified as causal factors. Single-molecule electrophysiological record- these structurally dissimilar inhibitors share a common binding site, each ligand ings reveal that NMDA receptors explore many functional configurations. interacts differently with individual amino acid residues in the binding pocket.

BPJ 9314_9317 Sunday, March 3, 2019 107a

However, due to the limited resolution of the crystal structures they do not pro- modulation of GluA2 homomers by TARP g-2 and its related isoform g-8. In or- vide a comprehensive atomistic picture of specific protein-ligand interactions der to determine the possibility of GluA-subunit-dependent TARP modulation, in that are important in inhibitor binding. In this study, molecular docking along HEK 293 cells we overexpressed g-2 mutants together with homomeric GluA1 or with molecular dynamics (MD) simulations and binding energy calculations GluA2(R) channels. Furthermore, since a majority of AMPARs throughout the were carried out to assess the stability of crystallographically determined bind- brain consist of GluA1 and GluA2(R) heteromers rather than homomers, we ing modes and explore potential alternative binding modes of inhibitors GYKI, analyzed the effect of the TARP mutants on GluA1/A2(R) heteromeric receptors. CP, and PMP. Our MD simulation results provide structural insights into inter- We performed fast perfusion electrophysiology on outside-out patches to assess actions of these inhibitors with AMPA receptors and highlight the features of modulation of gating. Surprisingly, GluA1 homomers were refractory to potenti- the AMPA receptor non-competitive inhibitor binding pocket that are impor- ation of the steady-state current, superactivation or a slow tail current by the g-2 tant in accommodating structurally different inhibitors. This information will positive-modulatory mutant. The cotransfection of GluA1, GluA2(R) and a g-2 aid in structure-based design of new non-competitive inhibitors that target mutant allows a minimum of 4 species resistant to polyamine block to form at AMPA receptors. the plasma membrane: GluA1_g-2, GluA2(R)_g-2, GluA1_GluA2(R) and the in- tended tripartite complex GluA1_GluA2(R)_g-2. In this condition, potentiation 533-Pos was as great as in GluA2 homomeric forms. We reasoned that this might due to Life in the Fast Lane: Binding to Glutamate Receptors the GluA2(R) subunit playing a dominant role in the gating of the heteromer. A Alvin Yu1, Hector P. Salazar2, Andrew J. Plested3, Albert Y. Lau4. model incorporating gating and trafficking effects of g-2 mutants on homomeric 1 2 Chemistry, University of Chicago, Chicago, IL, USA, FMP Leibniz Institut forms illustrates that the GluA2(R) dominance of gating in heteromeric forms is 3 fur€ Molek, Berlin, Germany, Cellular Biophysi, FMP Berlin & HU-Berlin, robust to asymmetries in trafficking. Our study shows that TARP modulation 4 Berlin, Germany, Dept Biophys/Biophys Chem, Johns Hopkins Univ Sch takes place through different molecular mechanisms depending on AMPAR sub- Med, Baltimore, MD, USA. unit composition. Ionotropic glutamate receptors (iGluRs) mediate neurotransmission at the ma- jority of excitatory synapses in the brain. Little is known, however, about how 536-Pos the neurotransmitter glutamate reaches the recessed binding pocket in iGluR Signal Peptide Represses Kainate Receptor GluK1 Surface and Synaptic ligand-binding domains (LBDs). Here we report the process of glutamate bind- Trafficking through Direct Interaction with Amino-Terminal Domain ing to a prototypical iGluR, GluA2, in atomistic detail using both enhanced Guifang Duan. sampling and equilibrium molecular dynamics simulations. Charged residues Nanjing University, Nanjing, China. on the LBD surface are found to form pathways that facilitate glutamate bind- Kainate-type glutamate receptors play critical roles in excitatory synaptic ing by effectively reducing a three-dimensional diffusion process to a spatially- transmission and synaptic plasticity. GluK1 and GluK2 are two major sub- constrained two-dimensional one. Free energy calculations identify residues units of kainate receptors expressed in brain. Previously we have found that metastably interact with glutamate and help guide it into the binding that they possess fundamentally different capabilities in surface trafficking pocket. These simulations also reveal that glutamate can bind in an inverted as well as synaptic targeting in hippocampal CA1 neurons. To pinpoint the conformation and also reorient while in its pocket. Electrophysiological record- underlying molecular mechanism, we construct chimeric receptors by swap- ings demonstrate that eliminating these transient binding sites slows activation ping different domains between GluK1 and GluK2. Intriguingly, we find that and deactivation, consistent with slower glutamate binding and unbinding. the EPSCs are increased significantly by the chimera GluK1(SPGluK2), in These results suggest that binding pathways have evolved to optimize rapid which the GluK1 signal peptide is replaced with that of GluK2. Coexpression responses of GluA-type iGluRs at synapses. of GluK1 signal peptide completely suppresses the gained trafficking ability of GluK1(SPGluK2), indicating the signal peptide represses the receptor 534-Pos trafficking in a trans manner. Furthermore, we demonstrate that the signal Agonist and Inhibitor Binding Effects on AMPA Receptor Internal peptide interplays with the amino-terminal domain (ATD) to inhibit GluK1 Structure and Dynamics receptor synaptic and surface expression and these two functional domains Serzhan Sakipov1, Chamali m. Narangoda1, Samaneh Mesbahi2, interact directly. Thus, we have uncovered a novel trafficking mechanism Jose C. Flores-Canales3, Christopher Kottke2, Maria G. Kurnikova1. for kainate receptors and propose that the cleaved signal peptide behaves 1Dept Chemistry, Carnegie Mellon Univ, Pittsburgh, PA, USA, 2Carnegie as a ligand of GluK1, through binding with the ATD, to repress GluK1 Mellon Univ, Pittsburgh, PA, USA, 3Department of Chemistry, Aarhus forward trafficking. University, Aarhus C, Denmark. AMPA subtype ionotropic glutamate receptor (iGluR) belongs to a family of 537-Pos 2D receptors that mediate a majority of excitatory neurotransmission, and its Development of a High-Throughput Ca Flux Screening Assay to dysfunction is associated with numerous neurological diseases. Hence it is Monitor Cyclic Nucleotide-Gated Channel Activity and Evaluate Achro- crucial to understand regulation of the receptor functional states and intercon- matopsia Disease Mutant Channel Function version between them by agonist and inhibitor binding. We present detailed Jacqueline Tanaka1, Cristy Almonte1, Elizabeth McDuffie1, Laura Jones1, atomistic dynamics level insight into binding modes of agonists and inhibi- Dennis Colussi2, Marlene Jacobson2. 1 2 tors of AMPA based on extensive molecular modeling of the receptor in Dept Biology, Temple Univ, Philadelphia, PA, USA, Temple Univ, lipid bilayers and water. We have discovered a dynamic correlation between Philadelphia, PA, USA. structure of the transmembrane domain ion channel and the ligand binding to Cone photoreceptor cyclic-nucleotide gated channels (CNG) are tetrameric the ligand binding domain that provide insights into AMPA receptor func- proteins composed of subunits from CNGA3 and CNGB3 genes. Cone CNG tional mechanisms. Our analysis will also shed light onto how competitive channels transduce light information into electrical signals conducting both þ 2þ (antagonists) and non-competitive inhibitors modulate channel kinetic prop- Na and Ca ions. More than 100 mutations in the CNGA3 gene are associated erties. AMPA receptor is a target for drug design, especially for epilepsy with the inherited retinal disorder, achromatopsia 2 (ACHM2) which results in therapy. Promising AMPA receptor inhibitors act via non-competitive mech- attenuation or loss of color vision. Classical techniques to measure CNG chan- anism. We present a study of interaction of the non-competitive inhibitors nel function use patch clamp electrophysiology providing high temporal and with the channel. spatial resolution. Due to the low throughput of patch clamp electrophysiology, analysis of the functional properties of mutants by this method is challenging 535-Pos and time intensive. As a functional macroscopic screen, we developed a Subunit-Dependent Modulation of AMPAR Gating by Auxiliary Proteins fluorescence-based, high-throughput Ca2þ flux assay of CNGA3 channels ex- Irene Riva1,2, Jelena Baranovic3, Anna L. Carbone4, Andrew J. Plested1,2. pressed in HEK293 cells. The 384-well microplate assay uses Rhod-4 AM to 1Leibniz-Forschungsinstitut fur€ Molekulare Pharmakologie (FMP), Berlin, monitor Ca2þ influx and a yellow fluorescent tag to monitor cellular localiza- Germany, 2Institut fur€ Biologie, Humboldt-Universita´t zu Berlin, Berlin, tion of the mutant protein subunits. 8-(4-chlorophenylthio)-cGMP (CPT- Germany, 3University of Edinburgh, Edinburgh, United Kingdom, 4Evotec, cGMP) activates channel openings in the assay. Here we demonstrate Ca2þ Hamburg, Germany. levels of wild-type CNGA3 channels along with three ACHM2-associated Understanding the mechanisms of the AMPA receptor (AMPAR) modulation by channel mutants, CNGA3-L633P, CNGA3-R427C, and CNGA3-T291R.The 2þ its auxiliary proteins is fundamental to explain the function of the AMPAR com- K0.5 values for CPT-cGMP activation in the Ca flux assay were 9.4 mM plex at synapses. Here we examined the effect of structural mutations of the extra- for the wild-type CNGA3, 10.9 mM for the L633P mutant and 3.3 mM for cellular loops of the transmembrane AMPAR regulatory protein (TARP) g-2 on the gain-of-function mutant R427C; the Nh values for each were 2.3. A the gating of the GluA1 and RNA-edited GluA2(R) AMPAR subunits. From our CNGA3-T291R mutant had no activated flux activity. These values are in previous work, we knew that these mutations lead to either a loss or an increase in reasonable agreement with cGMP channel activation from patch data. The

BPJ 9314_9317 108a Sunday, March 3, 2019 overall goal of developing this high throughput assay is to identify ACHM2- HCN channels are gated by hyperpolarizing voltages and cyclic-nucleotide associated mutants that are misfolded. Candidates will be expressed with the (CN) binding to intracellular CN binding domains (CNBD), which are con- CNGB3 subunit and screened for compounds that assist folding to recover nected to the gate-forming transmembranal S6 domain via a C-linker (CL). channel function. The CL-CNBD portion exerts an autoinhibiting effect on channel gating which is relieved by CN binding. Based on the hHCN1 structure (Lee and 538-Pos MacKinnon, Cell, 168, 111-120, 2017), we hypothesized that a highly Allosteric Gating Rearrangements of a Prokaryotic Cyclic Nucleotide- conserved lysine, located in the loop linking the A’-helix and B’-helix Gated Ion Channel Revealed with Pulsed Dipolar Spectroscopy (K464 in mHCN2) of the CL, plays an important role for enabling opposing 1 1 2 1 Eric G.B. Evans , Jacob L.W. Morgan , Stefan Stoll , William N. Zagotta . subunits to interact, thereby stabilizing the autoinhibited state of the channel. 1Department of Physiology & Biophysics, University of Washington, Seattle, 2 Wildtype mHCN2 (WT) channels and mutant channels were studied in inside- WA, USA, Department of Chemistry, University of Washington, Seattle, out macropatches excised from Xenopus laevis oocytes. For two mutations, WA, USA. K464A and K464E, we analyzed the steady-state activation relationships Cyclic nucleotide-gated (CNG) ion channels generate the primary electrical re- and voltage-dependent activation kinetics in the absence of cAMP and at a sponses in the visual and olfactory signal transduction pathways of vertebrates. saturating cAMP concentration. The data showed that (1) in K464A the closed CNG channels are tetramers of the ‘‘Kv’’ superfamily, but are distinct in that conformation is destabilized in the absence of cAMP leading to a less pro- they are essentially voltage-independent and are activated by direct binding nounced autoinhibition and consequently to a reduced effect of cAMP, and of cyclic nucleotides (cAMP/cGMP) to a cytoplasmic cyclic nucleotide- that (2) in K464E the closed conformation is even more destabilized leading binding domain (CNBD). Cyclic nucleotide-dependent conformational to a total loss of autoinhibition and, consequently, to an ineffectiveness of changes in the CNBD are thought to be allosterically coupled to the pore by cAMP. Using confocal patch-clamp fluorometry and a fluorescently tagged the so-called C-linker domain, but the molecular mechanism by which this is cAMP-derivative, we observed further that in non-activated K464E channels achieved is not understood. Here we employ double electron-electron reso- the cAMP-affinity is as high as in voltage-activated WT channels and that nance (DEER) spectroscopy to measure select intersubunit distance distribu- this affinity cannot be further increased by activation. This suggests that the Spirochaeta thermophila tions in a CNG channel from , termed SthK. DEER CL-CNBD adopts a similar conformation as in activated WT channels. distributions recorded both in detergent micelles and in lipid nanodiscs reveal Together these data led us to conclude that K464 is involved in closed-state a rearrangement of the C-linker in the presence of activating cAMP, resulting in stabilizing interactions and that these interactions break due to cAMP- an outward movement the C-linkers relative to the channel pore. The amplitude binding, enabling the CL-CNBD portion to adopt a conformation which re- of this radial movement is particularly large at the C-terminal segment of the leases the autoinhibition and promotes channel activation. B’-helix. In the presence of saturating amounts of cGMP, very small relative populations of the dilated - or ‘‘active’’ - conformation of the C-linker can 541-Pos be observed in some distributions, consistent with its classification as a weak Uncoupling the cAMP Binding Domain from the Channel Gate in HCN2 partial agonist. Our structural results are linked with functional states of the Channels E. coli channel through electrophysiological recordings from giant spheroplasts Sezin Yuksel€ 1, Mahesh Kondapuram1, Tina Schwabe1, Michele Bonus2, expressing our DEER constructs. Finally, we employ constrained molecular Holger Gohlke2, Ralf Schmauder1, Jana Kusch1, Klaus Benndorf1. modeling to predict the structure of the activated C-linker domain and discuss 1Institute of Physiology II, University Hospital Jena, Jena, Germany, these results in terms of current hypotheses for cyclic nucleotide-dependent 2Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine gating. Together our results reveal an agonist-dependent structural rearrange- University Dusseldorf,€ Dusseldorf,€ Germany. ment of the C-linker domain of a CNG channel and may provide new insight HCN pacemaker channels are dually gated by hyperpolarizing voltage and into the complex gating mechanisms in this important class of channels. cyclic-nucleotide (CN) binding to intracellular cyclic nucleotide-binding do- 539-Pos mains (CNBD). Each CNBD is connected to the gate, a right-handed S6 helix Functional Characterization of Gating in a Bacterial Cyclic Nucleotide- bundle, via a C-linker (CL). Movement of the voltage sensor causes a leftward Gated Channel unwinding of the helix bundle to open the pore. Functional and structural data Jacob Morgan, Eric Evans, William Zagotta. suggest that this unwinding is supported by cAMP binding, promoting a left- Physiology & Biophysics, University of Washington, Seattle, WA, USA. ward rotation of the CL disk located beneath the S6 gate. In the absence of Cyclic nucleotide-gated (CNG) channels produce the initial electrical signal in cAMP this CL disk autoinhibits channel opening. To study the autoinhibition mammalian vision and olfaction. The transmembrane (TM) region contains a and its cAMP-induced release in more detail, we constructed HCN2 channels voltage-sensing domain and a pore domain. The cytoplasmic region is carrying an additional amino acid sequence (AAAS) between S6 and the first comprised of a C-linker domain followed by a cyclic nucleotide-binding helix (A’) of the CL to uncouple pore and CNBD-CL portion. The AAAS was domain (CNBD). CNG channels assemble as a tetramer with a central pore. formed by either one, two, three or four glycines following S441: 441-G, Xenopus laevis cAMP and cGMP bind to the CNBD and produce an allosteric conformational 441-2G, 441-3G, 441-4G. Recordings were performed in rearrangement that opens the pore. The rearrangement occurring at the CNBD inside-out macropatches. The results of patch-clamp and confocal patch- is well characterized due in large part to structural and biochemical experi- clamp fluorometry experiments showed: (1) The S6 helix bundle is still a ments conducted on the purified C-linker/CNBD fragment from the related functional gate. (2) Autoinhibition was reduced in proportion to the number hyperpolarization-activated, cyclic nucleotide-gated channel, HCN2. However, of inserted glycines, but completely abolished only for the longest AAAS the rearrangement occurring in the rest of the channel (C-linker and TM region) of four glycines (441-4G). (3) cAMP did not release the remaining autoinhi- is not understood. Therefore, we are interested in developing a model system bition in 441-G, 441-2G, and 441-3G. (4) The cAMP binding affinity became for structural and functional studies of gating in full-length CNG channels. independent of activation by voltage. We conclude that the autoinhibitory SthK is a bacterial CNG channel that has the potential to serve as an ideal effect of the CL-CNBD portion is at least partially determined in channel model system for studying CNG channel gating. It can be expressed in regions remote from the S6 helix bundle gate. However, a tight coupling E. coli and purified in large quantities, and its resting-state structure has between this region and the first helix (A’) of the C-linker is essential for recently been published. However, SthK is reported to exhibit a low open prob- releasing the autoinhibition by cAMP-induced conformational changes and, ability (Po) even in saturating cAMP. This property limits its usefulness in following the principle of reciprocity, for transferring the effect of voltage studying the allosteric opening transition. Here, we express SthK in giant activation to the CNBD. E. coli spheroplasts. We patch-clamped these spheroplasts and recorded both single-channel and macroscopic currents to characterize SthK gating in a bac- 542-Pos terial membrane. Furthermore, we introduced mutations and show that they Nucleotide Modulation of KATP Channels Disentangled with FRET increased the Po while preserving SthK’s biochemical tractability. Michael C. Puljung, Samuel Usher, Natascia Vedovato, Frances Ashcroft. Physiology, Anatomy & Genetics, University of Oxford, Oxford, United 540-Pos Kingdom. þ Opposing Subunits Interact to Stabilize the Closed State in HCN2 ATP-sensitive K channel (KATP) gating is modulated via adenine nucleotide Channels (ATP or ADP) binding to three classes of nucleotide binding site (NBS). The 1 1 1 2 Mahesh Kondapuram , Sezin Yuksel€ , Tina Schwabe , Benedikt Frieg , first site is located on the pore-forming Kir subunit of KATP (Kir6.2 in pancre- Holger Gohlke2, Ralf Schmauder1, Klaus Benndorf1, Jana Kusch1. atic b-cells); binding to this NBS is inhibitory. Two stimulatory NBSs are 1Institute of Physiology II, University Hospital Jena, Jena, Germany, located on the cytoplasmic nucleotide binding domains of the accessory sulfo- 2Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine nylurea receptor subunit (SUR1 in b-cells), part of the ABC transporter family. University Dusseldorf,€ Dusseldorf,€ Germany. Despite decades of investigation, the details of nucleotide modulation, the

BPJ 9314_9317 Sunday, March 3, 2019 109a affinity and specificity for each NBS, the interactions between the various site. In the structure, the R249A mutant binds ATP with decreased phosphate NBSs, and the mechanism whereby nucleotide binding events are transmitted coordination, and was not stabilized in the four-fold symmetric conformation to the channel pore are largely unknown. To fully understand the nucleotide ef- observed with the wild-type domain. These data indicate that the mycobacterial fects on KATP, we have labelled each NBS (one at a time) with the fluorescent, K channel can distinguish among ATP, ADP, and AMP to sense the cell’s non-canonical amino acid ANAP and measured nucleotide binding at each site metabolic state and potentially transduce nucleotide binding to control K using FRET between ANAP and trinitrophenyl (TNP) nucleotide derivatives in conduction. unroofed membrane fragments. This has allowed us to dissect nucleotide bind- ing to each site, evaluate mechanistically mutations expected to affect nucleo- 545-Pos tide binding (e.g. SUR1-K1384A, Kir6.2-G334D) or channel gating (e.g. Rck Domains can Assemble as Hetero-Octamers and Control Different Kir6.2-C166S), and investigate the action of clinically important drugs that Ligand-Gated Channels þ inhibit or potentiate KATP (sulfonylureas and K channel openers). We have Rita Rocha, Celso Teixeira Duarte, Joao M.P. Jorge, Joao H. Morais Cabral. I3S - Instituto de Investigacao e Inovacao em Saude, Porto, Portugal. also combined this method with patch-clamp electrophysiology (either in sepa- þ rate experiments or simultaneously with patch-clamp fluorometry) to determine Potassium (K ) uptake is essential in all living cells. In non-animal organisms Kþ transport assumes a major role in adaptation to osmotic stress and high the functional consequences of binding at each NBS. This method will not only þ yield insights into KATP channel activity, but is readily extended to other chan- salinity. RCK (regulating conductance of K ) domains are widespread in na- ture, where they regulate the activity of eukaryotic potassium channels and pro- nels, ABC transporters, and virtually any protein with a suitable fluorescent þ ligand. karyotic K channels and transporters. Bacteria in particular express different families of Kþ transporters and channels, 543-Pos some of which are regulated by RCK domains, that allow growth under different Single Molecule FRET Reveals Lipid Induced Conformational Changes in Kþ limiting concentrations. Two or more different Kþ uptake systems can be Cytoplasmic Domain of Kir2.1 present in a bacterial cell and, some species contain more than one member of Joshua B. Brettmann1, Sun Joo Lee1, Shizhen Wang2, Colin G. Nichols1. the same family. We are exploring the biological role of this functional redun- 1Dept Cell Biol, Washington Univ St Louis, St Louis, MO, USA, 2School of dancy in Bacillus subtilis Ktr Kþ transporters, which are regulated by RCK do- Biological Sciences, University of Missouri-Kansas City, Kansas City, mains. B. subtilis genome codes for two membrane proteins (KtrB and KtrD) MO, USA. with structural similarities to Kþ channels and two cytosolic regulatory RCK Anionic phospholipids are common regulators of ion channel activity, domain proteins (KtrA and KtrC). Since KtrA and KtrB form an operon, while including Kir2.1, an inward rectifying potassium channel crucial for setting KtrC and KtrD are located in different loci of the chromosome, the KtrAB and and maintaining resting membrane potential in many tissues, which is activated KtrCD complexes have been proposed as two independent Ktr transporters. specifically by PIP2 and phosphatidyl glycerol (PG). Crystal structures indicate We have observed complex formation between the different subunits by size- that a simple upward translocation of the cytoplasmic Kir domain is induced by exclusion chromatography and functional complementation assay. We observed PIP2 and PG binding. However, recent computational and single-molecule that the two RCK domain proteins are able to interact and activate both KtrB and FRET experiments show more complex intra-domain movements and rotation KtrD; moreover, when incubated together, KtrA and KtrC can also assemble as in the Kir domain of prokaryotic KirBac1.1 upon PIP2 binding. Single mole- mixed octameric rings. These results unveil a novel feature of RCK domains with cule FRET measurements on eukaryotic human Kir2.1 indicate similar com- implications on the mechanisms of regulation of Kþ transport. plex motions that are not detected in static crystal structures. Our measurements indicate dynamic conformational ‘breathing’ throughout the 546-Pos Kir domain, suggesting greater flexibility in the intracellular domain of ion Characterizing P2X2 Mutants Associated with Progressive Sensorineural channels than may be apparent in structural studies. Specifically, our data indi- Hearing Loss (DFNA41) cate a PIP2 dependent shifts in average single molecule FRET (smFRET) dis- Benjamin I. George, Mufeng Li, Kenton J. Swartz. tributions, with outward motions seen prior to the slide helix (residue 59) and NINDS NIH, Bethesda, MD, USA. an inward motion seen at the base (residues 286 and 288) consistent with a P2X receptors are trimeric ion channels that open in response to extracellular twisting motion of the Kir domain that was previously shown to be associated ATP. Each subunit is comprised of intracellular N- and C-termini, a large extra- with opening in KirBac1.1. In the absence of PIP2, smFRET signals are very cellular domain containing the ATP binding site and two transmembrane (TM) dynamic with relatively large (>20A) motions at specific residues. PIP2 stabi- helices that form a pore that is permeable to cations. P2X2 receptors are hypoth- lizes the conformational dynamics of the Kir domain, with less transitions be- esized to play a role in cochlear adaptation to elevated sound levels and protec- tween structural states, and reduction in the occurrence of large conformational tion from overstimulation. Whole-exome sequencing and linkage analysis of transitions. individuals with dominantly inherited progressive sensorineural hearing loss (DFNA41) revealed three different mutations of human P2X2 (hP2X2) recep- 544-Pos tor: V60L, D201Y, and G353R. To investigate the effects of mutations, recom- Multiple Nucleotide-Dependent Conformations of a Mycobacterial RCK binant hP2X2 receptors were expressed in HEK 293 cells and macroscopic Domain currents were measured with the whole-cell patch clamp technique. We find Alexandre G. Vouga1, Katia K. Matychak1, Michael E. Rockman1, that the G353R mutation lowers the apparent affinity for activation by ATP Lisandra Flores2, Sebastian Brauchi2, Brad S. Rothberg1. and produces a pronounced inward rectification, with large inward currents be- 1 Dept Med Genetics Mol Biochem, Temple Univ Sch Med, Philadelphia, PA, ing observed only below 80 mV. In contrast, expression of the V60L mutation 2 USA, Dept Physiology, Universidad Austral de Chile, Valdivia, Chile. produces measurable constitutive currents with little rectification. To verify Potassium (K) channels play a critical role in bacterial electrolyte homeostasis that the constitutive currents observed with V60L arise from the mutant recep- and control of membrane potential, and are thus important in bacterial physi- tor, we inserted a Cys into the pore-lining TM2 helix and observed robust and ology. Here we present X-ray crystal structures of the ligand-binding RCK irreversible inhibition by thiol-reactive methanethiosulfonates (MTS). Interest- domain from the nucleotide-sensing Mycobacterium intracellulare K channel, ingly, larger MTS reagents reacted more slowly with the V60L construct in complex with ATP, ADP, and AMP. In this RCK domain, ATP is coordi- compared to control, suggesting that the mutation produces a structural alter- nated by a hydrogen bond between N6 (on the adenine base) and the side chain ation in the pore. Finally, the D201Y mutant channel does not respond to of Asp-200, a pair of hydrogen bonds between O2’ and O3’ (on the ribose ATP and appears to be non-functional. Taken together, our results suggest sugar) and the two side chain oxygens on of Asp-180. In addition, an intersu- that all three mutations greatly diminish or ablate ATP-activated currents at bunit salt-bridge is formed between the gamma phosphate of ATP and Arg-249, physiological voltages, and that the V60L also produces constitutively active and an intersubunit H-bond is also formed between the gamma phosphate and channels. It will be interesting to further explore how these perturbations Ser-260. The ATP-bound RCK complex forms a four-fold symmetric ring, give rise to severe progression of hearing loss. in which each ATP-bound subunit is in an identical conformation. Co- crystallization with either ADP or ATP, however, yields a two-fold symmetric 547-Pos ring in which the Ser-260 H-bond is absent in alternating subunits. To deter- Elucidating the Function and Cell-Specific Interactions of P2X7 Receptor mine the energetic roles of these residues in nucleotide coordination, we esti- Variants Linked to Mental Disorders mated ATP affinities of WT and mutant RCK domains using isothermal Mette Homann Poulsen, Jamie Fang, Stephan A. Pless. titration calorimetry. We observed that the wild-type RCK domain bound Department of Drug Design and Pharmacology, University of Copenhagen, ATP with a Kd of 4.1 þ/- 0.2 mM, whereas mutation of Arg-249 to disrupt Copenhagen, Denmark. the salt-bridge (R249A) yielded a Kd of approximately 270 mM. This corre- With a quarter of the world’s population affected by neurological or mental sponded to a free energy change of approximately 2.5 kcal/mole per binding disorders at some stage of life, there is great demand for understanding the

BPJ 9314_9317 110a Sunday, March 3, 2019 molecular basis of these disorders. In the CNS, activation of microglial pore. Members of this family include the acid-sensing ion channels (ASICs), P2X7 receptors (P2X7Rs) triggers neuroinflammation, which is intricately formed by identical or homologous subunits that mediate excitatory Naþ linked to some mental disorders. P2X7Rs belong to the family of adenosine currents in the nervous system (relative Naþ/Kþ permeability of approx. triphosphate (ATP)-gated ion channels, which are trimeric receptor assem- 10/1). The epithelial sodium channels (ENaCs) are obligate heterotrimers blies. Interestingly, the P2X7R displays an unusually high number of single and display a 10-fold higher Naþ selectivity than ASICs. The most recent nucleotide polymorphisms (SNPs) across the population, resulting in findings indicate that this discrepancy might be due to different selectivity numerous SNP-containing receptor variants, some of which are linked to dis- filter (SF) locations; while the ENaC SF is likely formed by the conserved eases, such as mental disorders. Notably, the functional and pharmacological G/S-X-S motif in the center of the pore, we have recently shown the mouse consequences of P2X7R SNPs remain unexplored. To gain a comprehensive ASIC1a SF to be composed of two acidic side chains in the lower part of understanding of the function of pathophysiologically relevant P2X7R SNP M2, namely E18’ and D21’. In order to elucidate if other parts of the variants, we study halotypes of the human P2X7R found in patients suffering channel contribute to the stark differences in Naþ selectivity between ASICs from mental disorders. The functional and pharmacological characterization and ENaCs, we used conventional and non-canonical amino acid substitu- of these halotypes is performed both in human microglia and HEK cells, tions to probe the contribution of M1 residues to ion selectivity. Our results using patch-clamp and high-throughput fluorescence assays. Additionally, show that aromatic residues in ASIC M1 are important for ion selectivity. both wild type P2X7Rs and SNP variants have been suggested to play ENaC contains additional aromatic residues in M1. We hypothesize that diverse and sometimes contradictory roles in different cell types. We hy- these aromatics are similarly important for ion selectivity and that pore pothesize that this differential role is caused by cell-specific protein-protein diameter plays an important role in both channels. Furthermore, the intracel- interactions (PPIs) between the P2X7R N- and/or C-terminal domains and lular domains have previously been suggested to contribute to ion selec- intracellular proteins, which we aim to identify and characterize using tivity. Using a novel split intein-based approach we fuse partial ASIC1a mass spectrometry. Together, this starts to disentangle the molecular basis constructs with recombinant or synthetic peptides corresponding to the of disease-linked microglial P2X7R variants, leading to a better understand- N- or C-terminus of the full length protein. This enables us to introduce ing of mental disorders. non-canonical amino acid substitutions, including post-translational modifi- cations, into these less-studied regions of the channel. 548-Pos Stomatin Dependent Regulation of the Acid Sensing Ion Channels 550-Pos Robert C. Klipp, John Bankston. Molecular Basis for Ion Selectivity in Heteromeric Acid-Sensing Ion Physiology and Biophysics, University of Colorado Anschutz Medical Channels Campus, Aurora, CO, USA. Zeshan P. Sheikh1, Timothy Lynagh2, Emelie Flood3, Celine Boiteux4, In the peripheral and central nervous systems, Acid Sensing Ion Channels Toby W. Allen5, Stephan A. Pless1. (ASICs) are the primary mediators of enhanced neuronal activity due to 1Drug Design and Pharmacology, Univ Copenhagen, Copenhagen, Denmark, extracellular acidification. Despite ASICs importance in regulating patho- 2Univ Copenhagen, Copenhagen, Denmark, 3RMIT Univ, Brunswick, physiological conditions of tissue acidosis, many of the molecular mecha- Australia, 4Hlth Innovat Res Inst, RMIT Univ, Melbourne, Australia, 5Sch of nisms underlying ASIC function, such as its incorporation into higher Sci, RMIT Univ, Melbourne, Australia. order ion channel complexes, remain poorly elucidated. Stomatin, a member Acid-sensing ion channels (ASICs) occur throughout the nervous system and of the SPFH family of integral membrane proteins has previously been open in response to proton binding. Most ASICs are 10-fold selective for shown to complex with ASIC isoforms 1, 2 and 3, while having differential Naþ over Kþ, and we recently showed that in homomeric ASIC1a channels, regulation of all 3. Stomatin shows a near complete inhibition of ASIC3 preferential Naþ conduction is primarily controlled by glutamate (E18’) and function when recombinantly expressed in mammalian cells. However, in aspartate (D21’) side chains at the intracellular end of the pore. Additionally, DRG sensory neurons, where Stomatin and ASIC3 are known to interact, two leucine residues (L7’ and L14’) and a constriction termed the GAS belt ASIC3 activates to moderate acidifications. Here, we present our preliminary (G10’-S12’) also contribute to ion selectivity. However, it remained unclear findings investigating several aspects of the molecular mechanisms underly- if this mechanism extends to other ASIC isoforms, including heteromeric ing Stomatin’s regulation of ASIC3. First, we seek to localize both regula- channels. Here, we investigated the molecular determinants of ion selectivity tory and non-regulatory Stomatin binding sites, creating a systematic in homomeric ASIC2a and heteromeric ASIC1a/ASIC2a channels, using series of truncated, chimeric, and point mutant channels. Mutant channels site-directed mutagenesis, electrophysiology and molecular dynamics simu- are recombinantly expressed into mammalian cells and patch clamp electro- lations. In contrast to ASIC1a, L7’A mutation had no effect on ion selectivity physiology is utilized to examine channel function in the presence and in ASIC2a. L14’ and, especially E18’ mutations, however, substantially absence of Stomatin. We use FRET to pair mutant channel function with decreased selectivity of ASIC2a, while G10’ and S12’ mutations rendered Stomatin binding using the respective fluorescently labelled constructs. channels non-functional, as in ASIC1a. Coexpression of mutant ASIC2a Finally, we investigate the role that domains on Stomatin play in the dy- with WT ASIC1a (and vice versa) led to functional heteromeric channels namic regulation of ASIC3. containing 1-2 mutated subunits. In these heteromers, E18’ mutations had stronger effects on ion selectivity than any other position tested here. This 549-Pos was consistent with simulations, showing favorable interactions of E18’ Indirect Determinants of Ion Selectivity in Acid-Sensing Ion Channels and side chains with Naþ ions in both homomeric ASIC2a and heteromeric Epithelial Sodium Channels ASIC1a/ASIC2a channels. Furthermore, simulations provided an explanation Zeshan P. Sheikh1, Timothy Lynagh2, Anders S. Kristensen1, for the reduced role of L7’ in ASIC2a channels, revealing a distinct free en- Stephan A. Pless1. ergy profile above the central GAS region, due to attractive interactions with 1Drug Design and Pharmacology, Univ Copenhagen, Copenhagen, Denmark, carboxylate-containing residues at the upper end of the pore, rendering the 2Univ Copenhagen, Copenhagen, Denmark. L7’ site non-rate determining. These results suggest a more pronounced Members of the ENaC/DEG superfamily of ion channels are Naþ-selective role of the external mouth of the pore and the GAS belt in ASIC2a compared ion channels with a common trimeric architecture. Each of the three sub- to ASIC1a, but confirm that E18’ is crucial to ion selectivity in various ASIC units has two transmembrane helices (M1 and M2), of which M2 lines the isoforms.

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Posters: Voltage-gated Ca Channels cells. Of these, Ca2þ-dependent inactivation (CDI) plays a prominent role in shaping cardiac action potential. This important form of regulation is mediated 2þ 551-Pos by a resident Ca sensor, calmodulin (CaM), which is comprised of two lobes, each capable of responding to spatially distinct Ca2þ sources. Disrup- A Mutation Linked to Malignant Hyperthermia in the Skeletal CaV1.1 Channel Stabilizes the Resting State of Voltage Sensor I and Impairs tion of CaM-mediated CDI leads to severe forms of long-QT syndrome Channel Activation (LQTS). Thus, a model capable of capturing the nuances of CaM-mediated Nicoletta Savalli1, Fenfen Wu2, Marbella Quinonez2, Stephen C. Cannon2, CDI would facilitate increased understanding of cardiac (patho)physiology. 1,2 However, a critical barrier to achieving a detailed kinetic model of CDI has Riccardo Olcese . 2þ 1Department of Anesthesiology & Perioperative Medicine, UCLA, Los been the lack of quantitative data characterizing CDI as a function of Ca . 2 This deficit in data stems from the experimental challenge of uncoupling chan- Angeles, CA, USA, Department of Physiology, UCLA, Los Angeles, CA, 2þ USA. nel gating from the Ca initiating CDI. To overcome this obstacle, we use Ca2þ photo-uncaging to deliver a measurable Ca2þ input to CaM/LTCCs, Recent studies report that a naturally-occurring mutation in the a1S subunit of 2þ while simultaneously recording CDI. Moreover, we utilize engineered voltage-gated, skeletal Ca channels CaV1.1 is linked to malignant hyper- 2þ thermia and prevents Ca2þ influx in myotubes, leaving excitation- CaMs with Ca binding restricted to a single lobe, so as to isolate the kinetic contraction coupling largely unaffected (Eltit et al., 2012, PNAS; Bannister response of each lobe. These high-resolution measurements enable us to build & Beam, 2013, Biophys J). The mutation is located in the voltage sensing mathematical models for each lobe of CaM, which become the building blocks domain (VSD) of Repeat I (IS4) and neutralizes its innermost basic amino of a full scale bilobal model of CDI. Finally, we utilize this model to probe the acid (R174W). To gain a mechanistic understanding of how this mutation al- pathogenesis of LQTS associated with mutations in CaM (calmodulinopa- ters channel function, we optically tracked the activation of VSDI of human thies). Each of these models accurately captures the kinetics and steady- state properties of CDI in both physiologic and pathologic states, thus offering CaV1.1 channels using the voltage-clamp fluorometry technique. We ex- powerful new insights into the mechanistic alterations underlying cardiac pressed CaV1.1 channel complexes formed by a1s (WT or R174W), STAC3 arrhythmias. and b1a subunits in Xenopus oocytes and labeled VSD I with thiol-reactive flu- orophores at a strategically-introduced Cysteine (L159C) located at the extra- cellular flank of IS4. The mutant channel exhibited a right-shifted voltage 554-Pos 2þ Cardiac O2 Sensor: A Trio of Hemeoxygenase, CaMKII & Carboxyl Tail dependence of channel activation by 60mV, explaining the lack of Ca D influx at physiological membrane potentials in myotubes experiments (Eltit of Ca2 Channel et al., 2012, PNAS). Simultaneous to ionic current recordings, we resolved Jose Carlos Fernandez Morales1, Naohiro Yamaguchi2, Martin Morad1. voltage-dependent fluorescence changes from VSD I, which report the move- 1Cardiac Signaling Center of USC, MUSC, and Clemson University, ment of this voltage sensor. We found that the malignant-hyperthermia-linked University of South Carolina, Charleston, SC, USA, 2Department of mutation compromised VSD I voltage dependent activation, causing a right Regenerative Medicine and Cell Biology, Medical University of South shift (40mV) of the F(V) curve and a decrease of its effective valence by Carolina, Charleston, SC, USA. 50%. That is, the R174W mutation stabilizes the resting state of VSD I, Acute hypoxia modulates cardiac ICa and calcium signaling in adult and neonatal rats and human cardiomyocytes derived from stem cells (hiPSC- thus impairing channel opening. This finding suggests a contribution of þ VSD I in channel activation in CaV1.1 channels in this specific subunit CMs). Our studies suggest that Ca2 -dependent inactivation (CDI) of cal- composition. cium channels serves as physiological protective mechanism against hypoxia where O2 is sensed by hemoxygenase-2 (HO-2). Mutation studies of the 552-Pos carboxyl tail of a1C subunit of the calcium channel have shown this region 2þ Design and Applications of the New Calcium Sensor GCaMP-X modulating critically CDI, Ca -dependent facilitation and beta-adrenergic Yaxiong Yang1, Yuanyuan He2, Xiaodong Liu1. regulation. Other reports suggest that the carboxyl tail plays a role in 1 a School of Bio. Sci. and Med. Engr, Beihang University, Beijing, China, hypoxia-induced suppression of ICa. Here we report on three mutant 1C- 2Tsinghua University, Beijing, China. 77 subunit harboring point mutations on: (1) calmodulin kinase (CaMKII) GCaMP, one popular type of genetically-encoded Ca2þ indicators, has been phosphorylation sites (S1487A/S1545A), (2) calmodulin (CaM) binding associated with various side-effects. Here we unveil the intrinsic problem pre- site (I1624A/Q1625A) and (3) PKA phosphorylation site (S1898A), ex- vailing over different versions and applications, showing that GCaMP contain- pressed in HEK293T cells. Our data show that mutation preventing CaMKII ing CaM (calmodulin) interferes with both gating and signaling of L-type phosphorylation of a1C (S1487A/S1545A mutation), render cells more 2þ calcium channels (CaV1). GCaMP acts as impaired apoCaM and Ca /CaM vulnerable to 100s hypoxia suppressive effect ( 72% Vs 33% ICa blockade 2þ 2þ both critical to CaV1, which disrupts Ca dynamics and gene expression. in the WT recombinant Ca channel). Surprisingly, preventing CaM binding We then design and implement GCaMP-X, by incorporating an extra to the IQ motif and thereby suppressing CDI (I1624A/Q1625A) attenuated apoCaM-binding motif, effectively protecting CaV1-dependent excitation- the hypoxia ICa blockade ( 8% suppression of ICa). S1898A mutant showed transcription coupling from perturbations. GCaMP-X resolves the problems the same hypoxia suppressive profile as WT. HEK293T cells dialyzed with of detrimental nuclear accumulation, acute and chronic Ca2þ dysregulation, 100 mM cAMP reduced the suppressive effects of hypoxia on the WT and and aberrant transcription signaling and cell morphogenesis, while still demon- also on S1487A/S1545A mutant channels. We found in adult rat myocytes strating excellent Ca2þ-sensing characteristics partly inherited from GCaMP. and hiPSC-CMs that CaMKII (CK59) and HO-2 (Zinc-protoporphyrin-IX) In summary, CaM/CaV1 gating and signaling mechanisms are elucidated for inhibitors, rapidly suppressed ICa at normoxia condition with similar degree GCaMP side-effects, while allowing the development of GCaMP-X to appro- and kinetics that hypoxia did. Co-application of hypoxia with these inhibitors 2þ priately monitor cytosolic, submembrane or nuclear Ca (Yang et al. Nature did not exhibited additive suppressive effects on ICa suppression. Taken 2þ Communications 2018), which is also expected to guide the future design of together, data suggests that HO-2 complexing with C-terminal tail of Ca CaM-based molecular tools. Some recent progress in GCaMP-X applications channel may serve as the O2 sensor through the CaM/CaMKII signaling will also be discussed for the biophysical and physiological mechanisms in pathway. neurons. 555-Pos 553-Pos Treatment of CaV1.2 Channelopathies May Be Complicated by Altered Probing L-Type Channel Calcium-Dependent Inactivation – a Bilobal Channel Inactivation Model of Calmodulation Moradeke A. Bamgboye1, Maria Traficante2, David T. Yue3, Ivy E. Dick1. Worawan B. Limpitikul1, Joseph L. Greenstein2, David T. Yue3, 1Dept Physiology, Univ Maryland, Baltimore, MD, USA, 2Univ Maryland Ivy E. Dick4, Raimond L. Winslow5. Baltimore, Baltimore, MD, USA, 3Dept Biomed Eng/Neuro, Johns Hopkins 1University of California, San Francisco, San Francisco, CA, USA, 2Johns Univ, Baltimore, MD, USA. Hopkins Univ, Baltimore, MD, USA, 3Dept Biomed Eng/Neuro, Johns Timothy Syndrome (TS) is a multisystem disorder, featuring neurological Hopkins Univ, Baltimore, MD, USA, 4Dept Physiology, Univ Maryland, impairment, autism, and cardiac action-potential prolongation (long QT) Baltimore, MD, USA, 5Inst Comp Med, Johns Hopkins Univ, Baltimore, MD, with life-threatening arrhythmias. The underlying cause of TS was first USA. described as a single point mutation (G406R) in CaV1.2 channels, which re- L-type calcium channels (LTCCs) play a major role in orchestrating cardiac sulted in a disruption of both calcium- and voltage- dependent inactivation of electrical activity and initiating downstream signaling processes. As such, the channel. Since this first discovery, a growing number of potentially ar- 2þ LTCCs employ feedback mechanisms to precisely control Ca entry into rhythmogenic CaV1.2 mutations have been found, increasing the need for

BPJ 9318_9321 112a Sunday, March 3, 2019 targeted therapies. As many of these mutations have been shown to impair ically disrupts b2AR/Cav1.2 regulation (Myr-Pep2). As S1928 is an impor- channel inactivation, calcium channel blockers (CCBs) such as verapamil tant phosphorylation site for channel potentiation through b2AR in neurons, and dihydropyridines (DHPs) represent a promising option for treatment, the b2AR/Cav1.2 role contribution was further tested in Serine-1928 however, these CCBs often prove only partially effective for managing Alanine (S1928A) mice as well as immunoblotting with S1928 phospho- symptoms in these patients. We propose that this lack of efficacy is caused specific antibodies on WT mouse brain slices. CAPCE recordings were by attenuated channel inactivation in the mutant channels. In particular, quantified as the sum of open probabilities of the channels (NPo). NPo verapamil and DHPs are known to preferentially block open and inactivated increased when exposed to bA42, but not when b2AR was blocked. Prein- channels such that loss of entry into the inactivated state would decrease the cubation with 11R-PKI and Myr-Pep2 also reduced the effect of bA42 on efficacy of these CCBs on the mutant channels. Here, we demonstrate that NPo, though not providing a full block like ICI118,551 suggesting that mutations in CaV1.2 which diminish channel inactivation also decrease the Cav1.2 activities are partially regulated by the b2AR/Cav1.2 mechanism. extent of channel block by CCBs. Furthermore, we show that amount of Immunoblotting for S1928 phosphorylation normalized to the total channel channel block by these CCBs is strongly correlated to the fraction of inacti- content supported that bA42 increases Cav1.2 phosphorylation through vation in the channel, whereby efficacy of block is lowest in mutant channels b2AR. Together these results support our hypothesis by showing that with the lowest fraction of inactivation. This decreased efficacy of CCBs on bA42 acts through b2AR to partially upregulate Cav1.2 activities in neurons mutant channels represents a major impediment to the treatment of TS and and that such upregulation can be inhibited by blocking b2AR. Future work cardiac arrhythmias caused by mutations in CaV1.2. These results emphasize will exemplify this upregulation mechanism by examining its contributions the need to tailor patient therapies to the specific channel deficit produced by to cytotoxicity and AMPAR expression. each mutation. 558-Pos 556-Pos Structure Modelling of CaV1.1 Reveals Functional Trans-Domain Interac- Possible Mechanism of Calcium-Dependent Block of L-Type Calcium tions Involved in Voltage Sensing Channel by Glacontryphan-M Monica L. Ferna´ndez-Quintero1, Yousra El Ghaleb2, Petronel Tuluc3, Vyacheslav S. Korkosh1, Denis B. Tikhonov1, Boris S. Zhorov1,2. Campiglio Campiglio2, Klaus R. Liedl4, Bernhard E. Flucher5. 1Sechenov Institute of Evolutionary Physiology and Biochemistry, RAS, St. 1Physiology, University of Innsbruck, Innsbruck, Austria, 2Dept. Physiology, Petersburg, Russian Federation, 2Dept Biochem/Biomed Sci, McMaster Medical University Innsbruck, Innsbruck, Austria, 3Dept Pharmacology & Univ, Hamilton, ON, Canada. Toxicology, Univ Innsbruck, Innsbruck, Austria, 4Institute of Calcium channel-blocking peptide toxins have a large therapeutic potential. General, Inorganic and Theoretical Chemistry, Univ Innsbruck, Electrostatic interactions play a major role in peptide-channel recognition. Innsbruck, Austria, 5Dept Physiol/Med Phys, Med Univ Innsbruck, For example, homology models are proposed with cationic conotoxins Innsbruck, Austria. blocking the cation-attractive outer vestibule of Cav2.2 (Lewis et al., Voltage-gated calcium channels (CaV) consist of four homologous but non- 2012; Wang et al., 2016) and Nav1.4 (Korkosh et al, 2014) channels. Para- identical repeats (I, II, III, IV), each containing a separate voltage-sensing doxically, the Cav1.2 channel is blocked by an anionic peptide, 11-residue domain (VSD) arranged around the common channel pore. Within each glacontryphan-M containing two g-carboxyglutamates, and the block is VSD the positive gating charges (R1, R2, R3, R4) in the transmembrane he- calcium-dependent (Hansson et al., 2004; Grant et al., 2004). Here we em- lix S4 sequentially interact with negative counter-charges in helices S2 and ployed Monte-Carlo energy minimizations to explore how glacontryphan-M S3 to support the movement of the gating charges across the electrical field can block Cav1.2. The cryo-EM structure of Cav1.1 and the X-ray structure of the membrane and thus to activate or deactivate the channel. Previously of contryphan-Vn were used to model Cav1.2 and glacontryphan-M. Com- we identified an interaction between R1 and R2 with aspartate 1196 (D4) in putations predicted that the selectivity-filter region can accommodate S3 of the VSD-IV of CaV1.1 that is critical for modulation of voltage- glacontryphan-M. Two calcium ions bound to g-carboxyglutamates bridged sensitivity and current-density by alternative splicing in the IVS3-S4 linker. the toxin to selectivity-filter carboxylates. We further computed energy pro- Using molecular structure modeling we now identified an additional interac- files of glacontryphan-M pulled through the channel to the selectivity-filter tion with a glutamate (E216) in S5 of VSD-I that participates in the R1/ region. The only pathway where the toxin did not encounter sterical or elec- R2-D4 interaction of VSD-IV. Charge-neutralization (E216Q) or substitu- trostatic repulsion from the channel was that through the fenestration be- tion with alanine (E216A) in CaV1.1e caused a 7mV and 16mV, respec- tween extracellular loops in domains III and IV. Energetically preferable tively, right-shift of voltage-dependence of activation and a 30 % pathway for a calcium ion from the extracellular space to the selectivity fil- reduction in current density. This effect is specific to the splice variant ter glutamates in the toxin-free channel involved several calcium binding lacking exon 29 and is quantitatively in the same range as previously sites where the ion is coordinated by at least two carboxylates. In the observed when R1, R2, or D4 were mutated; indicating that this trans- toxin-bound channel, calcium ions immobilized between g-carboxygluta- domain interaction between repeats I and IV participates in the voltage- mates and the outer carboxylates repelled the incoming calcium ion, but dependent gating and its modulation by alternative splicing. Structure did not move towards the selectivity-filer glutamates. Thus, the pore of models of CaV1.1 in the activated and pre-open state show that the four cationic channels can be blocked by permeant ions bound to either small- VSD differ greatly regarding their intra-domain interactions, consistent molecule cation-attractive blockers (e.g. Tikhonov & Zhorov, 2017) or to with their specific contributions to CaV1.1 gating properties. The newly anionic peptide toxins. Supported by RFBR (17-04-00549) and NSERC discovered inter-domain interaction is unique for VSD-I and IV. Together (RGPIN-2014-04894). in silico structure modelling of this pseudo-tetrameric ion channel revealed hitherto not appreciated differences between the VSDs and a functional 557-Pos inter-domain interaction. Support: Austrian Science Fund (FWF) grant Beta Amyloid Peptide (1-42) Mediated Dysregulation of L-Typed Voltage P30402. Gated Calcium Channel 1.2 through the Beta-2 Adrenergic Receptor Liangying Li1, Jennifer Price2, Boram Lee2, Johannes Hell3. 559-Pos 1 Department of Biomedical Engineering, University of California Davis, Skeletal g1 Subunit Modulation of Human CaV1.1 and CaV1.2 Channels Davis, CA, USA, 2Biomedical Engineering Graduate Group, University of Marina Angelini1, Nicoletta Savalli1, Taleh Yusifov1, Riccardo Olcese1,2. California Davis, Davis, CA, USA, 3Department of Pharmacology and 1Department of Anesthesiology & Perioperative Medicine, UCLA, Los Toxicology, University of California Davis, Davis, CA, USA. Angeles, CA, USA, 2Department of Physiology, UCLA, Los Angeles, CA, The beta amyloid peptide 1-42 (bA42) can independently cause many hall- USA. 2þ marks associated with Alzheimer’s Disease (AD). The beta 2 adrenergic re- Skeletal muscle L-type Ca channels (CaV1.1) are multimeric proteins ceptor (b2AR) has been identified as a potential mediator for this - a composed of a pore-forming a1s subunit and auxiliary a2d-1, b1a and g1 receptor that our lab has shown tightly regulates the activity of the L-type subunits. A cryoEM structure of this protein complex at 3.6 A˚ resolution re- voltage-gated calcium channel 1.2 (Cav1.2). We hypothesize that bA42 veals an interaction of the g1 subunit with S3-S4 helices of a1s repeat IV acts through b2AR to dysregulate Cav1.2 activity. To test effects of acute (Wu et al., Nature 2016). The g1 subunit has been shown to modify bA42 exposure on L-type calcium channel (LTCC) activity, cell-attached CaV1.1 voltage dependence of inactivation, current time dependent proper- patch clamp electrophysiology (CAPCE)wasusedtorecordLTCCactiv- ties and support channel trafficking to the membrane. Taking advantage of ities in rat hippocampal neurons acutely exposed to bA42. To test this stac3 adapter protein essential for skeletal muscle function and surface pathway, we disrupted it with b2AR antagonist ICI118,551, PKA inhibitor expression of CaV1.1 (Polster et al., PNAS 2015), we investigated the bio- 11R-PKI, b1AR antagonist CGP20712, and a unique peptide which specif- physical consequences of g1 association with human CaV1.1 channels.

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Using the cut-open oocyte voltage clamp technique, we recorded ionic cur- 27.052.4mV (þa2d-1) (n=5). The time- and voltage-dependent properties rents from channel complexes formed by a1S/b1a/stac3 with or without g1 of VSD-III remained mostly unperturbed. As VSD-IV was previously found subunit, expressedin Xenopus oocytes. g1 modified CaV1.1 voltage depen- to be important for CaV1.1 channel activation, the observed a2d-1-induced dence of inactivation by decreasing the effective valence from 5.351.1e0 shift of VSD-IV voltage dependence without consequence on the GV curve 0 (N=4) to 1.8750.2e (N=4) and reducing the non-inactivating component of the channel would suggest that a2d-1 causes a concurrent modulation of of the ionic current following 10s pulses from 50% to 30%. In addition, VSDs I and/or II in the opposite direction (under investigation), counterbal- we observed that g1 coexpression did not affect voltage dependence of ancing VSD-IV effect. It is intriguing that the a2d-1 subunit, which dramati- CaV1.1 activation (No g1:Vhalf= 37.151.2mV, N=5, and with g1:Vhalf= cally facilitates the human CaV1.2 voltage activation causing 50 mV 40.051.5mV, N=4). However, when g1 and a2d-1 were coexpressed, the hyperpolarizing shift of the GV, leaves skeletal CaV1.1 activation curve voltage dependence of the CaV1.1 complex (a1S/b1a/a2d-1/g1/stac3) was largely unperturbed. We are using our CaV channel allosteric model to esti- facilitated. g1 also strongly modulated the CaV1.2 channel isoform (a1C/ mate the energetic contribution of each VSD to channel opening in the pres- b2b/a2d-1) by shifting the voltage dependence of channel inactivation by ence of a2d-1. 20mV toward negative potentials. In conclusion, these studies have g revealed the biophysical consequences of 1 subunit association with 562-Pos two distinct human L-type channel isoforms in heterologous expression Auxiliary Beta Subunits are not Obligatory for CaV1.3 Function systems. Sharen Rivas1, Johanna Diaz2, Henry M. Colecraft2, Manu Ben Johny2. 1Vassar College, New York, NY, USA, 2Physiology and Cellular Biophysics, 560-Pos Columbia University Medical Center, New York, NY, USA. 2þ 2þ A Local-Control Model of the Guinea Pig Ventricular Myocyte Allows Un- High voltage-gated Ca channels (CaV1-2) convey vital Ca influx that derstanding of Force-Interval Relations at the Calcium Spark Level triggers muscle contraction, vesicle secretion, and gene transcription. Beyond Roshan Paudel. the pore-forming a-subunit of the CaV channel complex, the auxiliary b-sub- Dept Bioinformatics and Computational Biology, George Mason Univ, unit ranks amongst the strongest modulators of channel properties. For nearly Fairfax, VA, USA. all CaV1-2 channels, b-subunits tune kinetics of voltage-dependent inactiva- A local control model of the guinea pig ventricular myocytes was developed tion and impart unique single-channel gating behaviors. Moreover, in heter- to explore the mechanisms governing Ca2þ dynamics. This dynamic balance ologous expression systems such as HEK293 cells, b-subunits are thought to 2þ is controlled by the model features including L-type Ca channels (LCC), be necessary for surface-membrane expression. Yet, CaV1.3 appears to be sarcolemmal sodium calcium exchangers (NCX) and ryanodine receptors, unusual in this context, with distinct b-subunits only minimally altering type 2 (RyR2s). We developed a new three state RyR2 model that simulates kinetics of voltage-dependent inactivation. Here, we show that CaV1.3 ex- RyR2 adaptation in which the channels accumulate in a closed state when hibits robust currents in HEK293 cells without the co-expression of exoge- exposed to prolonged elevated subspace [Ca2þ] (Gyorke and Fill Science. nous b-subunits, suggesting that b-subunits are not necessary for reliable 1993 260:807-9). The model was tested for agreement with previous experi- surface-membrane expression. In addition, extensive single-channel analysis mental and modeling studies on force-interval relations, i.e. the changes in comparing the open probability of CaV1.3 in the presence of b1 - b4 subunits calcium transient and action potential morphology at different pacing fre- or without exogenous b-subunit expression reveals only minimal differences. quencies. Our local control model displays stable action potential trains at Together, these findings highlight the surprisingly modest modulatory effect 2þ 7 Hz, unlike previous common pool models. The duration of the [Ca ]i tran- of b-subunits on CaV1.3 and raise the possibility that under certain physio- sient and the action potential (AP) decrease with increased pacing rates logical contexts, these channels could potentially operate independent of 2þ consistent with experiment. With increased pacing rate, the [Ca ]i an auxiliary b-subunit. Furthermore, the differential modulation of CaV1.3 transient peak values increases up to 3Hz and decreases afterward, consistent versus other CaV1-2 channels by b-subunits may help identify novel struc- with experimental force-frequency curves. The model predicts, in agreement tural determinants. with our previous modeling studies (Jafri et al., Biophys J. 1998 þ Mar;74(3):1149-68), that diastolic sarcoplasmic reticulum (SR) [Ca2 ] and 563-Pos RyR2 adaptation increases with increased stimulation frequency giving rise þ Pore-Blocking Effect of Isoindoline MDIMP on Voltage-Gated Calcium to rising than falling amplitude of the myoplasmic [Ca2 ] transients. The Channels model also allows us to dissect these frequency dependent changes down to Juan Antonio M. De La Rosa1, Maricela Garcı´a-Castan˜eda1, the spark level giving new insight into mechanism governing cardiac calcium Takuya Nishigaki2, Juan Carlos Gomora3, Teresa Mancilla-Percino4, dynamics. Guillermo Avila1. 1Bioquı´mica, Cinvestav-IPN, Mexico, Mexico, 2Instituto de Biotecnologia 561-Pos UNAM, Cuernavaca, Mexico, 3Instituto d Fisiologı´a, Universidad Nacional 4 How Does the a2d-1 Subunit Modulate Skeletal CaV1.1 Channels? Autonoma de Mexico, Mexico, Mexico, Quı´mica, Cinvestav-IPN, Mexico, Federica Steccanella1,2, Nicoletta Savalli1, Taleh Yusifov1, Mexico. Giovanni Battista Luciani2, Alan Neely3,4, Riccardo Olcese1,5. MDIMP is an isoindoline derived of leucine containing an ester group. mo- 1Department of Anesthesiology & Perioperative Medicine, UCLA, Los lecular docking studies suggest that mdimp interacts with voltage-gated cal- Angeles, CA, USA, 2Department of Surgery, Dentistry, Paediatrics and cium channels (vgccs). In theory, the molecule binds to pore-leaning Gynaecology, University of Verona, Verona, Italy, 3Anesthesiology, segments and interacts with one of the glutamates belonging to the selec- 4 Universidad de Valparaı´so, Valparaı´so, Chile, Centro Interdisciplinario de tivity filter (e736, located in domain ii of cav1.2). Accordingly, mdimp in- Neurociencia de Valparaı´so, Valparaı´so, Chile, 5Department of Physiology, hibits vgccs in cardiac myocytes, and the effect is more potent on low- UCLA, Los Angeles, CA, USA. threshold than on high-threshold currents. Here, we have reexamined the The pore-forming subunit a1S of voltage-gated L-type calcium channels possibility that mdimp preferentially blocks a specific type of calcium chan- CaV1.1 forms macromolecular complexes in skeletal muscles by associating nel isoform by using heterologous expression systems. As previously shown with auxiliary subunits a2d-1, b1a and g1. To gain insights on the molecular in cardiac myocytes, cav3.1 and cav3.2 (low-threshold activated channels, mechanisms of CaV1.1 regulation by the a2d-1 subunit, known to slow lva) showed a higher affinity for mdimp, compared with cav1.2 and cav2.3 down current activation kinetics (Obermair et al., 2005, J Biol Chem.), we (high-threshold activated channels, hva). the ic50 values were (mm): 130 took advantage of the adaptor protein STAC3, which allows for the heterolo- (cav3.1), 220 (cav3.2), 450 (cav1.2), and 560 (cav2.3). hva and lva channels gous expression of CaV1.1 channels (Polster et al., 2015, PNAS). We ex- differ in the residues providing electronegativity to the four homologous do- pressed CaV1.1 complexes (a1S, b1a and STAC3) with or without a2d-1 mains of their selectivity filter. The former contain four glutamates (eeee), subunit in Xenopus oocytes. Using the voltage clamp fluorometry technique, whereas the latter have two glutamates and two aspartates (eedd). Thus, we confirmed that the presence of a2d-1 slowed current activation kinetic, we hypothesized that these structural differences could explain the prefer- while leaving channel voltage dependent activation (GV) largely unaffected: ence of mdimp for lva channels. If so, changing the eedd configuration Vhalf= 32.950.5mV (no a2d-1), n=6; 32.850.3mV (þa2d-1), n=5. By opti- should lead to substantial alterations in the potency of inhibition. Hence, cally tracking the molecular rearrangements of individual voltage-sensing we also investigated whether two pore mutations of cav3.1 (dedd and domain (VSD), we found that the auxiliary subunit a2d-1 modified the dddd) interfere with the effect of mdimp. The ic50 values for mutant proteins voltage-dependent activation of VSD-IV, shifting the F(V) curve by were 390 and 540 mm (respectively), indicating that point mutations of one 20mV towards more negative potentials: Vhalf= 7.451.9mV (no a2d-1); or two pore residues diminish the potency by 3- and 5-fold. Thus, structural

BPJ 9318_9321 114a Sunday, March 3, 2019 properties of the selectivity filter seem to be essential for the blocking effect 566-Pos of mdimp. Interestingly, the glutamate of domain ii only slightly improved Diabetes with Heart Failure Increases Methylglyoxal Modifications in the the blocking effect (i.e., dedd vs. dddd), suggesting that this residue is not Sarcomere Which Inhibit Function critical for affinity. Maria Papadaki1, Ronald Holewisnki2, Sammantha Previs3, Thomas Martin1, Marisa Stachowski1, Amy Li3, Cheavar Blair4, Kenneth Campbell4, Moravec Christine5, Jennifer Van Eyk2, Posters: Cardiac Muscle Regulation Virginie Aubert1, David Warshaw3, Jonathan Kirk6. 1Dept Cell & Molec Physiol, Loyola Univ Chicago, Maywood, IL, USA, 564-Pos 2Cedars Sinai Medical Center, Los Angeles, CA, USA, 3University of Troponin-T Cardiomyopathy Mutations Depress its Inhibitory Properties, 4 In Vitro In Vivo Vermont, Burlington, VT, USA, University of Kentucky, Lexington, KY, , and Stimulate Myocardial Dysfunction, USA, 5The Cleveland Clinic, Cleveland, OH, USA, 6Dept Cell & Molec 1 1 2 Aditi Madan , Meera C. Viswanathan , Georg Vogler , Physiol, Loyola Univ Chicago, Chicago, IL, USA. 3 1 2 4 Kathleen C. Woulfe , William Schmidt , Bosco Trinh , Sineej Madathil , Methylglyoxal (MG), a glycolysis byproduct, is significantly elevated in dia- Cortney Wilson3, Larry S. Tobacman4, Anthony Cammarato1. 1 2 betes and modifies proteins by reacting with arginine and lysine residues to Medicine, Johns Hopkins University, Baltimore, MD, USA, Sanford form irreversible carbonyl adducts. The effect of MG on the cardiac myofil- Burnham Medical Research Institute, San Diego, CA, USA, 3Medicine, 4 ament is explored here. Myofilament was enriched from LV tissue from pa- University of Colorado, Denver, CO, USA, Medicine, University of Illinois tients who had diabetes and heart failure (dbHF), dilated cardiomyopathy at Chicago, Chicago, IL, USA. (DCM), and non-failing (NF) control hearts. dbHF patients exhibited a Gain-of-function mutations in sarcomeric genes often manifest as 40% increase in MG-modifications on myofilament proteins compared to hypercontractility-associated myopathies. Two such diseases are Hypertro- NF and DCM hearts. Skinned myocytes from NF hearts exhibited decreased phic (HCM) and Restrictive cardiomyopathy (RCM). These are clinically Ca2þ sensitivity when exposed to MG, while myocytes from dbHF patients heterogeneous, autosomal dominant disorders characterized by hyperdy- were resistant, suggesting dbHF myocytes were already affected by MG- namic myocardial contraction and impaired relaxation, with (HCM) or induced decreased myofilament function. Mass spectrometry on LV tissue without (RCM) hypertrophy. The current study focuses on charge-altering identified novel MG-modifications on actin and myosin that suggested MG HCM/RCM-causing troponin-T (TNNT2) missense mutations, K124N, might depress actin-myosin binding. Cosedimentation of myosin with bare R130C, E136K, in the highly conserved N-terminal (TnT1) tropomyosin actin showed MG had no effect on their direct binding. However, myosin co- (Tm)-binding element. We have conducted in vitro, in situ and in vivo ex- sedimentation with native thin filaments (NTF) was decreased with MG, sug- periments using human TnT1 peptides in a minimalistic four-element gesting that actin-myosin binding is depressed only when tropomyosin and motility assay, physiological assessment of surgically exposed transgenic the troponin complex are present. Tropomyosin/troponin were directly assed Drosophila hearts by SOHA (Semi-automated Optical Heartbeat Analysis) for MG-modifications by MS and none were found. The in vitro motility and live fluorescence imaging of intact fly cardiac tubes, respectively. assay showed MG depressed Ca2þ-sensitivity when either NTF or myosin This is the first report of an in vivo/in situ studyonthesemutations.We were independently exposed. These data suggest MG-modifications on either tested the hypothesis that these mutations limit TnT1’s ability to help actin or myosin depress function by interfering with how these proteins confine Tm to the ‘‘B-state’’, where it sterically blocks acto-myosin binding interact with tropomyosin or the troponin complex. Given the increased during diastole. We observed increased F-actin-Tm-TnT1 sliding velocities MG-modifications in dbHF patients, we hypothesized they may be patho- with all three mutant peptides in the in vitro motility assay, implying disin- genic. Indeed, in a pre-diabetic mouse model (high fat diet for 12 weeks), hibited acto-myosin interactions. Transgenic Drosophila hearts over- while there were no overt health issues, there was a 17% increase in myofil- expressing mutant TnT’s showed cardiac restriction, impaired relaxation ament MG-modifications. Overall, these results show a potential underlying and reduced cardiac output. To elucidate underlying mechanisms, we treated 2þ mechanism by which diabetes causes heart failure through MG-modifications the heart tubes sequentially with EGTA-AM (to chelate intracellular Ca ) of the myofilament, having important implications for treatment and/or and blebbistatin (to inhibit myosin), and measured live-cell responses prevention. consistent with impaired Ca2þ homeostasis, as well as Ca2þ-independent cross-bridge cycling at rest. Additionally, we isolated single myofibrils 567-Pos from a novel CRISPR-Cas9 fly line, engineered to express the E136K Troponin I Tyrosine Phosphorylation: Novel Regulator of Cardiac RCM mutation, and observed elevated resting tension relative to controls. Function Our results confirm our hypothesis that these cardiomyopathy mutations Elizabeth A. Brundage1, Vikram Shettigar1, Ying-Hsi Lin2, Brendan Agatisa- disrupt TnT’s normal contribution to myocardial relaxation, which likely Boyle1, Mark Jeong2, Mark T. Ziolo1, Brandon J. Biesiadecki1. serves as the mechanistic basis of pathology in our unique fly models and, 1Dept Physiol/Cell Biol, The Ohio State Univ, Columbus, OH, USA, 2Dept potentially, in human patients. Med, Univ Colorado Denver, Aurora, CO, USA. 565-Pos Serine/threonine kinase mediated phosphorylation of the contractile regula- The Effect of Phosphorylation on the Structure and Topology of the tory protein troponin I (TnI) represents a fundamental mechanism of the SERCA-PLN Complex heart to modulate cardiac contractile function in response to altered physi- Daniel Weber, Songlin Wang, Erik Larsen, Tata Gopinath, Gianluigi Veglia. ological or pathological demand. While non-receptor tyrosine specific University of Minnesota, Minneapolis, MN, USA. kinasesareexpressedintheheartandtheiractivityisalteredindisease, Phospholamban (PLN) is a small membrane protein that binds as a regulatory the role of tyrosine kinase mediated phosphorylation to directly modulate subunit to the sarcoplasmic reticulum Ca2þ-ATPase (SERCA) responsible cardiac contraction is unknown. Previous work demonstrated TnI is phos- for pumping Ca2þ back into the SR lumen following cardiac muscle contrac- phorylated at tyrosine 26 (Tyr26). We subsequently demonstrated that tion. PLN’s interaction is inhibitory towards the SERCA pump but reversed TnI Tyr26 phosphorylation alters myofilament function thereby identifying upon phosphorylation at serine 16 (PLNpS16) by protein kinase A (PKA), the first tyrosine phosphorylation to directly modulate cardiac contractile which essentially couples SERCA activity to b-adrenergic regulation. The function. We now demonstrate that TnI Tyr26 phosphorylation is structural differences between the inhibited SERCA-PLN and non-inhibited increased during in vivo myocardial ischemia implicating its role in this SERCA-PLNpS16 complexes, however, have been unresolvable by conven- pathology, yet the role of TnI Tyr26 phosphorylation in the normal or tional structural methods primarily due to the dynamic nature of PLN. diseased heart is unknown. To determine the effect of TnI Tyr26 phosphor- Here we utilize developing solid-state NMR (ssNMR) techniques to ylation on heart function, we generated a novel TnI Tyr26 phosphorylation construct a complete structural model of the SERCA-PLN complex in lipid (Tg Y26E) mouse. At the muscle level, myofibrils from Tg Y26E mice bilayers. High-resolution oriented ssNMR of the 116 kDa complex reconsti- exhibit decreased calcium sensitivity and faster myofibril relaxation. tuted into magnetically-aligned bicelles obtained topological information Hemodynamic evaluation demonstrates these muscle level effects result in about the transmembrane domain of PLN bound to SERCA, while magic- altered in vivo heart function, accelerating relaxation without decreasing angle-spinning (MAS) ssNMR allowed structural determination of PLN’s contractility. As a whole these data support that the phosphorylation of highly-dynamic regulatory cytoplasmic domain in the complex. Overall, TnI at Tyr26 is a novel myofilament phosphorylation to enhance diastolic our results provide critical structural insights into how SERCA activity is function without depressing systole. Ongoing experiments are focused allosterically regulated by b-adrenergic-induced phosphorylation of the on the beneficial effects of TnI Tyr26 phosphorylation during cardiac PLN subunit. disease.

BPJ 9318_9321 Sunday, March 3, 2019 115a

568-Pos Ig10 domains of titin and a unique 198-amino acid sequence of novex-3. Predicting Effects of Tropomyosin Stiffness on Cardiac Muscle Contrac- Although the direct binding between obscurin and titin has been known tion Using Coarse-Grained Stochastic Modeling for over 15 years, the physiological significance of their interaction has Yasser Aboelkassem1, Kimberly J. McCabe2, Michael Regnier3, remained elusive.To assess the effects of the obscurin/titin complex in James B. Bassingthwaighte3, Andrew D. McCulloch1. vivo, we generated a constitutive deletion mouse model, Obscn-DIg58/59, 1Bioengineering, Univ Calif San Diego, La Jolla, CA, USA, 2UC San Diego, that expresses truncated obscurin lacking Ig58/59. Transthoracic echocardi- San Diego, CA, USA, 3Dept Bioengineering, Univ Washington, Seattle, ography of homozygous Obscn-DIg58/59 animals demonstrated that males WA, USA. develop compensatory left ventricular hypertrophy at 6-months that pro- During the cardiac thin filament activation process, Tropomyosin (Tm) oscil- gresses to dilation and contractile impairment by 1-year, whereas females lates over the surface of actin filament in the azimuthal direction to regulate do not exhibit any significant alterations. Moreover, electrocardiography the access of Myosin heads to actin binding sites. These dynamical modes of revealed the presence of arrhythmia in both genders, with males exhibiting oscillation and the resultant cooperative activation effects depend critically more severe episodes. The distinct phenotypes of the female and male on the stiffness characteristics and the mobility of Tm molecules. In this populations indicate the presence of sex dimorphism in the Obscn-DIg58/ study, we developed a stochastic coarse-grained computational model to 59 model at least in response to aging. Given that exertion of stress can un- describe the Tm motions over the surface of actin filament using mask the consequences of molecular alterations, we examined the effects Langevin-Brownian dynamics. The model represents the structural arrange- of pathological stress on cardiac function in young Obscn-DIg58/59 mice, ment of Tm molecules as a flexible chain with variable torsional stiffness us- which do not yet show signs of disease. Subjecting 4-month old animals ing a crystal elastic network approach. The model accounts for the spatial to b-adrenergic stress revealed severe electrical abnormalities in both interactions among nearest-neighbor regulatory units (RUs), which are genders. Mutations in the genes encoding obscurin and titin, including thought to appear from the structural coupling of adjacent Tropomyosins. several that disrupt their binding, have been linked to cardiac and skeletal This elastic coupling between RUs is accomplished by assigning a multi- myopathies in humans. It is therefore apparent that the obscurin/titin com- well potential energy for each RU. The model is then used to study the ef- plex is essential for normal muscle structure and function, and that disrup- fects of Tm torsional stiffness variations on the cooperative activation of tion of their binding is associated with muscle pathogenicity and disease the thin filament. The results suggest that small perturbations in Tm torsional development. Our findings using the Obscn-DIg58/59 model corroborate þ stiffness can lead to a significant effect on force-Ca2 sensitivity, the rate of this notion. tension redevelopment, relaxation rate, and other contraction characteristics. The present stochastic computational model draws for the first time a more 571-Pos detailed molecular connection between Tm torsional stiffness, Tm modes Characterization of the Alpha-Kap FRET Biosensor to Determine Com- of oscillations over actin surface, cooperativity among RUs, and dynamic partmentalized Beta-Adrenergic Receptor Camp Signaling in Distinct muscle twitches. Thus, this coarse-graining approach may be useful in ex- Intracellular Locations plaining many cardiomyopathies induced by structural remodeling and stiff- Michael W. Rudokas, John P. Post, Alejandra Sataray-Rodriguez, ening in Tm molecules as a result of point mutations in the human gene Chase M. Fiore, Shailesh R. Agarwal, Robert D. Harvey. TPM1. Pharmacology, University of Nevada, Reno, Reno, NV, USA. The sympathetic nervous system helps maintain cardiac homeostasis primar- 569-Pos ily through the activation of b1 and b2-adrenergic receptors (b-ARs). Both Molecular Mechanism of a Mutation Implicated in Pediatric-Onset Heart types of receptors rely on the production of the diffusible second messenger Disesase 3’,5’-cyclic adenosine monophosphate (cAMP) to mediate a variety of Samantha K. Barrick, Michael J. Greenberg. cellular functions. However, both receptors do not produce the same Biochemistry and Molecular Biophysics, Washington University in St. Louis, cAMP-dependent responses. One explanation for these differences is that St. Louis, MO, USA. cAMP is compartmentalized, but what is responsible for this compartmenta- Familial cardiomyopathies are the leading cause of sudden cardiac death tion is not well understood. FRET-based biosensors targeted to different sub- among young people, and pediatric-onset disease is particularly devastating. cellular locations were used to test the hypothesis that variation in the Dilated cardiomyopathy (DCM) is a familial cardiomyopathy characterized distribution of b1- and b2-ARs in the plasma membrane contribute to differ- by dilation of the left ventricular chamber of the heart. DCM can be caused ences in the subcellular pattern of cAMP production. The novel a-Kinase by mutations in proteins involved in cardiac muscle contraction, including Anchoring Protein (EPAC2-a-KAP) FRET biosensor was created and char- troponin T; however, it is not well understood how these mutations affect car- acterized to determine cAMP production in proximity to the free sarco- diac contraction and contribute to the disease phenotype. To address this crit- plasmic reticulum, an intracellular site hypothetically devoid of b2-AR ical gap in our knowledge, we examined the molecular-level impact of a cAMP production. In combination with other targeted FRET biosensors, troponin T mutation implicated in pediatric-onset DCM, R134G. In vitro distinct cAMP signals were observed in response to selective b1- and b2- motility assays, in which troponin/tropomyosin-regulated thin filaments are ARs stimulation. All the FRET probes demonstrated cAMP production in propelled across a myosin-coated surface, were carried out over a range of response to b1-AR specific stimulation but the a-KAP probe did not respond physiologically relevant calcium concentrations. These measurements re- to b2-AR specific stimulation as predicted. The results of this study should vealed decreased calcium sensitivity in the troponin complex containing the provide a better understanding of the differences in b1- and b2-AR regula- R134G mutant. To elucidate the molecular mechanism underlying the altered tion of cAMP signaling and the importance these differences plays in sym- calcium sensitivity conferred by the R134G mutation, we used stopped-flow pathetic functional responses. and steady-state fluorescence measurements to determine the equilibrium con- stants that define binding of myosin to regulated thin filaments. By inputting 572-Pos these equilibrium constants into a computational model of the sarcomeric Engineered Thin Filament Mutation to Increase Calcium Sensitivity of contraction, we were able to explain the observed changes in calcium-based Force in Tropomyosin Mutation of Dilated Cardiomyopathy myosin regulation and make testable predictions about the effect of the muta- Kristina B. Kooiker1, Joseph D. Powers2, Jil Tardiff3, Michael Regnier2, Jennifer Davis2, Farid Moussavi-Harami1. tion on cardiac contractility. Taken together, our results provide new insights 1 into the mechanism of pediatric heart disease. Medicine (Cardiology), University of Washington, Seattle, WA, USA, 2Bioengineering, University of Washington, Seattle, WA, 570-Pos USA, 3School of Medicine, University of Arizona, Tucson, AZ, Loss of Binding between Giant Obscurin and Titin Results in Cardiac Mal- USA. adaptation Dilated cardiomyopathy (DCM) is a leading cause of heart failure, which is Alyssa Grogan1, Li-Yen R. Hu1, Christopher Ward1, quickly rising in prevalence in the United States. In roughly 20% of DCM Aikaterini Kontrogianni-Konstantopou2. cases, mutations in sarcomeric proteins, such as troponin (Tn), tropomyosin 1University of Maryland Baltimore, Baltimore, MD, USA, 2Dept (Tm) and titin, are considered the cause. Decreased tension and calcium sensi- Biochemistry and Molecular Biology, Univ Maryland Baltimore, Baltimore, tivity of force are characteristics of sarcomere-linked DCM. Currently there are MD, USA. no therapies that directly target the underlying condition and typically treat- Giant obscurin is a modular protein that plays key structural and regulatory ments target only the symptoms. Here we are exploring treatment of DCM roles in striated muscles. It was originally discovered as a binding partner by genetically altering Ca2þ binding in the sarcomere. Specifically, we coun- of canonical titin and a smaller splice variant of titin, novex-3. Immu- tered a DCM mutation in cardiac Tm, D230N, by introducing an engineered noglobulin (Ig) domains Ig58/Ig59 of obscurin mediate binding to Ig9/ mutation in cardiac TnC (cTnC), L48Q, that binds Ca2þ more strongly than

BPJ 9318_9321 116a Sunday, March 3, 2019 native cTnC. Mice with the D230N-Tm mutation were crossed with minus were imaged within this lattice in cardiac muscle preparations using L48Q-cTnC mice (DTG). At 4-6 months of age, when DCM is well established Stochastic Optical Reconstruction Microscopy (STORM) and single particle in D230N mice, triton X-100 skinned left ventricular trabeculae were mounted averaging. Hearts from transgenic mice expressing MyBP-C with an N-termi- 2þ between a force transducer and motor. Ca sensitivity (pCa50) of force was nal Myc tag were chemically fixed, frozen in liquid nitrogen with sucrose as a determined at two sarcomere lengths (SL), 2.0mm and 2.3mm. As expected, cryoprotectant and thinly sliced to produce 400 nm-thick cross-sections. pCa50 is lower in D230N-Tm versus WT mice at both SL 2.0mm (5.373 5 MyBP-C and actin were labeled with anti-Myc-Alexa647 and phalloidin- 0.033 vs 5.655 5 0.088) and 2.3mm (5.476 5 0.037 vs 5.751 5 0.079), and Alexa488 respectively. Two-color STORM movies were collected using total L48Q-cTnC mice show increased pCa50 (2.0mm: 5.784 5 0.055; 2.3mm: internal reflection microscopy, and images of actin and MyBP-C were recon- 5.962 5 0.095). Importantly, trabeculae from DTG mice showed increased structed. The actin lattice was identified from the actin reconstructions pCa50 (2.0mm: 5.486 5 0.034; 2.3mm: 5.582 5 0.022) compared to D230N- by cross-correlation with a hexagonal template. Individual actin hexagons Tm mice. No changes were seen in maximal Ca2þ-activated force. We will were cropped from the images and averaged to improve the resolution to 4 next examine Ca2þ handling in isolated cardiomyocytes. In conclusion, intro- nm. The average distance between two adjacent actin filaments within the ducing a mutation with stronger Ca2þ binding can successfully overcome the hexagon was 28 5 1 nm, in agreement with the 25 5 4 nm distance measured decrease in Ca2þ sensitivity at SL 2.0 and 2.3mm caused by the D230N-Tm by negative stain EM. To identify the position of MyBP-C’s Myc-tagged DCM mutation. N-terminus, the signal from the second color channel was averaged and over- laid on the actin hexagon. The signal from MyBP-C’s N-terminus was diffuse 573-Pos but had strong colocalization with actin. These data support a model The Role of Cardiac MYBPC in Regulating Frank Starling Relationships where MyBP-C’s N-terminal domains are flexible but interact with actin 1 2 3 Laurin M. Hanft , Daniel P. Fitzsimons , Timothy A. Hacker , in situ. Richard L. Moss2, Kerry S. McDonald1. 1Department of Medical Pharmacology and Physiology, University 575-Pos of Missouri, Columbia, MO, USA, 2Department of Cell and Modulation of Calcium Sensitivity and Twitch Contractions in Cardiac Regenerative Biology, University of Wisconsin, Madison, WI, Muscle with Troponin-C Mutations: Simulations and Experiments USA, 3Department of Medicine, University of Wisconsin, Madison, WI, Srboljub M. Mijailovich1, Momcilo Prodanovic2, Lazar Vasovic2,3, USA. Boban Stojanovic2,3, Mladen Maric2,3, Danica Prodanovic2,3, According to the Frank-Starling Relationship, elevations in end-diastolic vol- Joseph D. Powers4, Jennifer Davis5, Michael A. Geeves6, Michael Regnier4. ume progressively increase peak ventricular pressure and stroke volume in 1Department of Biology, Illinois Institute of Technology, Chicago, IL, healthy hearts. This relationship is modulated by a number of physiological USA, 2Bioengineering Research and Development Center (BioIRC), inputs and is often depressed in human heart failure. Emerging evidence sug- Kragujevac, Serbia, 3Faculty of Science, University of Kragujevac, gests cardiac Myosin Binding Protein-C (cMyBP-C) may be intricate in regu- Kragujevac, Serbia, 4Department of Bioengineering, University lating the Frank-Starling Relationship. For instance, transgenic animal studies of Washington, Seattle, WA, USA, 5Department of Pathology, University have implicated both length dependence of Ca2þ sensitivity of force (Cazorla of Washington, Seattle, WA, USA, 6School of Biosciences, University of et al., 2006; Kumar et. al., 2015) and stretch activation (Mamidi et. al., 2016) Kent, Canterbury, United Kingdom. are modulated by the phosphorylation state of cMyBP-C. We measured con- The effects of mutations in cardiac muscle sarcomere proteins is often as- tractile properties across multiple spatial organization levels to discern the role sessed from force-pCa curves of demembraned muscles or transient twitch of cMyBP-C and its phosphorylation in determining i) the sarcomere length contractions in intact muscles. Relaxation abnormalities are rarely studied, dependence of force in cardiac myofilaments and ii) Frank-Starling relation- though this can strongly affect early phase diastole and, perhaps, diastolic ships. For this study, we utilized WT mice, which are thought to have performance (filling) via resting ventricular wall tension. Using MUSICO >50% phosphorylated cMyBP-C (Controls), as well as transgenic mice lack- simulations, tightly coupled with experiments, we quantitatively estimated ing cMyBP-C (KO), and transgenic mice expressing cMyBP-C having serine- the contributions of three mechanisms to contraction and relaxation kinetics: 273, 282, 302 mutated to aspartate on the null background to mimic consti- (1) Tm azimuthal movement as a continuous flexible chain (CFC); (2) tutive PKA phosphorylation (cMyBP-C CT-t3SD mice). We observed reduced reduction of cTn calcium affinity resulting from crossbridge detachment slopes and greater variability in sarcomere length-tension relationships in per- to create a negative feedback effect that reduces myosin binding sites; meabilized cardiac myocyte preparations from cMyBP-KO mice. In contrast, and (3) inclusion of a super-relaxed myosin state (SRS) to reduce the length-tension relationships were steep and highly tuned in the myocyte prep- pool of myosins that can rebind during relaxation. Simulations provided arations from cMyBP-C CT-t3SD animals. Also, the pre-load dependence of force-pCa relationships and force transient responses where native cTnC left ventricular power output was greatest in isolated hearts from cMyBP-C was replaced with either cTnC L48Q (decreased Ca2þ off rate) or cTnC CT-t3SD mice. Furthermore, step-wise volume expansion during continuous I61Q (increased Ca2þ off rate). These simulations demonstrated that echocardiographic imaging revealed Frank-Starling Relationships were force-pCa relationships with cTnC mutants require not only changes in diminished in cMyBP-C KO mice and steepest in cMyBP-C CT-t3SD animals. Ca2þ affinity, but also CFC elasticity and its interaction with actin. In addi- These results support the hypothesis that cMyBP-C and its post-translational tion, I61Q vs. WT cTnC resulted in about three-fold lower tension, suggest- modifications precisely tune sarcomere length dependence of myofibrillar ing this mutation also affects TnI interaction with actin. To simulate twitch force and power, and these regulatory processes translate across all spatial contraction of transgenic mice with these mutations we included in the levels of organization to optimize beat-to-beat regulation of ventricular model observed penetrance of 30% cTnC L48Q or 40% cTnC I61Q and performance. estimated increased or decreased Ca2þ affinity, respectively. We fitted simulated twitches in trabeculae with these mutations and estimated the 574-Pos shift toward higher Ca2þ sensitivity in intact vs. demembraned muscles. Resolving the Actin Lattice and Identifying the Relative Position of MYBP- These estimates were compared with force-pCa relations observed in de- C’s N-Terminus in Cardiac Muscle using Storm Microscopy membraned trabeculae. The predicted cardiomyocyte performance showed 1 2 3 Sheema Rahmanseresht , Kyounghwan Lee , Jeffrey Robbins , how the severity of contractile abnormality in cardiac muscle disease is David M. Warshaw1, Roger Craig2, Michael J. Previs1. 1 affected by the mutant penetrance. Supported by: H2020 777204- Dept Molecular Physiology and Biophysics, Univ Vermont, Burlington, SILICOFCM. VT, USA, 2Division of Cell Biology and Imaging, Department of Radiology, University of Massachusetts Medical School, Worcester, 576-Pos MA, USA, 3Department of Pediatrics and the Heart Institute, C0 Ig-Domain of Cardiac Myosin Binding Protein-C Interacts with the Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, Regulatory Light Chain of Myosin-S1 Bound to the Native Cardiac Thin USA. Filament Cardiac myosin-binding protein C (MyBP-C) is a critical modulator of cardiac Cristina Risi1, Betty Belknap1, Samantha Harris2, Howard White1, muscle contractility. MyBP-C is localized to the C-zone’s in the sarcomere Vitold E. Galkin1. through the binding of its C-terminal domains to the myosin rods that form 1Dept Physiol Sci, Eastern Virginia Med Sch, Norfolk, VA, USA, 2University the filament backbone. In vitro studies suggest MyBP-C’s N-terminal domains of Arizona, Tucson, AZ, USA. modulate actomyosin activity through binding to actin and/or the myosin Muscle contraction, relies on sliding of myosin-based thick filaments and heads. Direct evidence for these potential binding partner interactions in thin filaments comprised of actin, tropomyosin and troponin. Cardiac myosin vivo is lacking. In cross-sectional view of muscle, actin and myosin filaments binding protein-C (cMyBP-C), an intrinsic protein of the thick filament, is a form a hexagonal lattice. The relative positions of actin and MyBP-C’s N-ter- key regulator of actomyosin interactions, essential for both normal cardiac

BPJ 9318_9321 Sunday, March 3, 2019 117a contraction and for increased cardiac contractility in response to inotropic consistent with the hypothesis that DWORF competes effectively with PLB stimuli. Mutations in MYBPC3, the gene encoding cMyBP-C, are the single for binding to SERCA. Site-directed mutagenesis of DWORF identifies spe- most common genetic cause of hypertrophic cardiomyopathy (HCM). It has cific residues in DWORF that are important for this competitive binding. A been proposed that the interaction of the N-terminal domains (NTDs) of central factor in heart failure is an increase in basal cytosolic [Ca2þ], resulting cMyBP-C (e.g. C0, C1, M and C2) with myosin heads may stabilize the in decreased cardiac contractility. Therefore, augmentation of SERCA2a ac- super-relaxed state (SRX-state) of the thick filament, while NMR experi- tivity by overexpression of DWORF is a promising therapeutic approach ments directly confirmed the binding of the C0 Ig-domain of cMyBP-C to (gene therapy), and these SERCA2a/DWORF and SERCA2a/PLB biosensors the isolated regulatory light chain (RLC) of myosin. We recently demon- could be helpful for screening small molecules for potential heart failure drug strated that C1 can activate the thin filament to the same extent as rigor therapy. We thank Benjamin Grant (Fluorescence Innovations, Inc.) for myosin-S1, while C0 significantly enhances the activating effect of C1. assistance with instrumentation in early stages of this work. This work Our data also show that C0 competes with myosin-S1 for binding to the was supported by NIH grants (GM27906, HL129814, AR07612, and thin filament. In order to understand how C0 interacts with the thick and DA037622). thin filaments upon active cross-bridge formation we used cryo electron mi- croscopy and image analysis of native cardiac thin filaments decorated with 579-Pos rigor myosin-S1 and the C0-domain of cMyBP-C. We show that C0 readily Cardiac Overexpression of Human Adenylyl Cyclase Type 8 in Mice Elicits binds to the RLC of myosin-S1 bound to the thin filament in rigor state. Tak- Phosphorylation- Dependent Mechanisms that Permit Perpetual Heart Ex- ing into account that the C0-domain is specific to the cardiac muscle and is ercise while Conferring Protection Against Excessive Camp-PKA Signaling 1 2 1 1 absent in the skeletal MyBP-C we suggest a model of how the C0-domain of Khalid Chakir , Alexey E. Lyashkov , Kirill V. Tarasov , Ismayil Ahmet , 1 1 1 cMyBP-C participates in the regulation of cardiac contraction. Dongmei Yang , Yelena S. Tarasova , Daniel Riordon , Yevgeniya O. Lukyanenko1, Thanh Huynh3, Karel Pacak3, 577-Pos Edward G. Lakatta1. Obscurin in Heart Failure 1Laboratory of Cardiovascular Science, NIH/NIA, Baltimore, MD, USA, Aidan M. Ex-Willey1, Heather R. Manring1, Ahmet Kilic2, 2Longitudinal Studies Section, Translational Gerontology Branch, NIH/NIA, Paul M.L. Janssen1, Nathan T. Wright3, Maegen A. Ackermann1. Baltimore, MD, USA, 3Section on Medical Neuroendocrinology, NIH/Eunice 1Department of Physiology & Cell Biology, Ohio State University, Kennedy Shriver National Institute of Child Health and Human Columbus, OH, USA, 2Department of Surgery, Ohio State University, Development, Bethesda, MD, USA. Columbus, OH, USA, 3Dept. of Chemistry & Biochemistry, James Madison Overexpression of the Human Adenylyl cyclase 8 (AdCy8) in mouse heart University, Harrisonburg, VA, USA. leads to a marked 40% increase in heart rate in vivo (telemetry), a marked Cardiovascular disease (CVD) is the largest cause of death worldwide. Heart improvement of the left ventricle (LV) cardiac output and ejection fraction failure, a result of CVD, is a multifaceted disease involving numerous com- and a reduced response to external autonomic input compared to the WT plexes in the heart. Many of the molecular mechanisms that are associated littermates. To explore underlying mechanisms for the LV function þ with heart failure are unclear. Cytoskeletal proteins have been implicated in improvement and protection against excessive cAMP-PKA-Ca2 signaling, heart failure and function to provide structural stability for the myocyte. Ob- We assessed the LV proteomics and phospho-proteomics profiles via mass scurins are a family of cytoskeletal proteins highly expressed in the heart. spectrometry. The results were supplemented by immunolabeling or West- They provide structural support and also contain several active signaling do- ern Blot and enzymatic activities. Proteomics identified 5,317 proteins of mains. Previous studies identified altered obscurin protein levels in human pa- which 243 were expressed differentially between genotypes (cutoff tients and mouse models. Our results identify lower levels of giant obscurins in 1.5-fold, p<0.05) and 11781 phosphopeptides were identified. Phosphoryla- end-stage failing human hearts compared to donor controls. Many mechanisms tion state was increased in 450 phosphopeptides and decreased in 335 can affect obscurin levels, including increased protein turnover. Calpain, a phosphopeptides (cutoff 1.5-fold, p<0.05). LV Human AdCy8 protein calcium-dependent protease, is known to have elevated activity in stressed expression and activity were highly upregulated in TGAC8. PKA activity hearts. Herein, we evaluate the relationship between obscurin protein levels In-vitro was also significantly higher. Proteins implicated in high and calpain-mediated cleavage. We show giant obscurins are cleaved via a performance (PKA, P-PKA, AKAP2, AKAP12, AKAP13, CAMKII and P- calpain-dependent mechanism. In human heart lysates, increased calcium- CAMKII, SERCA2, Sodium/calcium exchanger 1) were significantly upre- induced cleavage of giant obscurin results in increased smaller obscurin pep- gulated, but phospholamban and phospho-phospholamban did not differ tides, a phenomenon reversible with a calpain-specific inhibitor. As supported from WT. Although, total RYR2 protein increased in TGAC8, the ratio in the literature and through in silico analysis, a calpain-target site resides of phosphorylated RyR2/total RYR2 at 3 sites (Ser-2030, Ser-2814 and within the RhoGEF domain of obscurin. Biochemical experiments show that Ser-2030 implicated for RYR activation) are reduced. Proteins implicated recombinant obscurin-RhoGEF protein is cleaved by calpain. Using mutational in protection against chronic stress (PDE1c, PDE3a, PDE4a, PDE4d, analysis we confirm the location of this calpain target site. We are currently PDE8a, PDE12, PP1, P-PP1 and PKIa)andb-adrenergic receptor desensiti- testing the hypothesis that calpain targeting of the obscurin-RhoGEF domain zation proteins (GRK2, bARK2, GRK5, b-arrestin1, DAB2, Gai2/3) exists as a mechanism to regulate the activities of giant obscurins and could were highly upregulated. Voltage-dependent calcium channel subunit alter the balance of obscurin proteins. alpha-2/delta-1 is downregulated. Surprisingly, circulating plasma levels of norepinephrine and epinephrine were lower in TGAC8 vs. WT. Thus, 578-Pos AdCy8 overexpression in heart activates numerous phosphorylation- Biophysics of SERCA2a DWORF Complex and Implications for Thera- dependent adaptations that sustain a profile of chronic exercise and peutic Design augmented LV function while protecting against excessive Ca2þ-cAMP- Ang Li, Daniel Stroik, Tory Schaaf, David D. Thomas. PKA signaling. BMBB, Univ Minnesota, Minneapolis, MN, USA. We have developed cell-based FRET biosensors and used time-resolved 580-Pos FRET (TR-FRET) to measure protein-protein interactions and structural Single Molecule Visualization of Cardiac Myosin-Binding Protein C N- changes of the cardiac sarcoplasmic reticulum Ca-ATPase (SERCA2a) in Terminal Fragments Interacting with Regulated Actin Filaments: Mecha- complex with phospholamban (PLB) or dwarf open reading frame (DWORF). nisms of Calcium Sensitization SERCA2a reduces intracellular [Ca2þ] in cardiac myocytes, inducing relax- Alessio V. Inchingolo1, Samantha B. Previs2, Michael J. Previs2, ation. DWORF, a 35-residue transmembrane peptide, has been proposed to David M. Warshaw2, Neil M. Kad1. 1 enhance SR Ca2þ uptake and myocyte contractility through its displacement School of Biosciences, University of Kent, Canterbury, United Kingdom, 2 of the inhibitory peptide PLB from SERCA. FRET between GFP-SERCA2a Dept Molec Physiol/Biophysics, University of Vermont, Burlington, VT, and RFP-DWORF indicates binding of DWORF to SERCA. To test the hy- USA. pothesis that DWORF competes with PLB for SERCA2a binding, we have Tropomyosin movement on actin resulting from calcium binding to troponin transiently transfected unlabeled DWORF into a stable cell line expressing permits myosin binding, filament sliding and force generation in the cardiac GFP-SERCA2a (donor) and RFP-PLB (acceptor). We observed a significant muscle sarcomere. Cardiac myosin-binding protein C (cMyBP-C) plays a decrease of SERCA-PLB FRET efficiency due to co-expression of DWORF. modulatory role in this activation process. One potential mechanism is Detailed analysis of TR-FRET data showed that this is primarily due to a that cMyBP-C binds directly to the regulated actin filament at low calcium decrease in the mole fraction of SERCA2a molecules that are bound to levels to move tropomyosin, thus recruiting myosin binding. To test this, we PLB. The DWORF concentration dependence of FRET and SERCA function directly visualized fluorescently-labelled cMyBP-C N-terminal fragments indicates that SERCA has a comparable affinity for DWORF as for PLB, and GFP-labelled myosin molecules binding to regulated actin filaments

BPJ 9318_9321 118a Sunday, March 3, 2019 in a single molecule assay. Binding of cMyBP-C’s C0C3 N-terminal Measurements of twitch kinetics or action potentials and recording of X-Y-co- domains to regulated actin enhanced myosin association at low calcium ordinates of the individual plated hESC-CMs was followed by fluorescence-in- levels. However, at high calcium levels, C0C3 fragments bound coopera- situ-hybridization against MYH6 and MYH7-mRNAs, and finally by immuno- tively, blocking myosin binding. Dynamic imaging of thin filament-bound fluorescent staining of a and b-MyHC proteins. Cy3-C0C3 molecules demonstrated that these fragments diffuse along the We found that the majority of single hESC-CMs, though expressing pure b- thin filament before statically binding, suggesting a mechanism that utilizes MyHC, had still detectable variable fractions of MYH6-mRNA. The fraction a weak-binding mode to search for access to actin and a tight-binding mode of MYH7-mRNA corresponded only poorly to the b-MyHC protein expression that enhances myosin binding. Although shorter N-terminal fragments in the very same single hESC-CMs. (Cy3-C0C1 and Cy3-C0C1f) bound to regulated actin filaments similarly Interestingly, plated hESC-CMs showed faster upstroke velocities of action to the Cy3-C0C3 fragment, the shorter fragments were unable to potentials compared to the CMs cultivated in suspension cultures, indicating affect myosin binding. Therefore, the longer N-terminal fragment (C0C3) maturation of voltage-dependent sodium channels. Time to peak and half must possess the requisite domains needed to bind specifically to the thin relaxation times of twitches were similar as for adult human CMs. However filament in order for the cMyBP-C N terminus to modulate cardiac time courses of calcium transients of plated hESC-CMs were rather slow in contractility. comparison to the adult human CMs or human trabeculae, presumably due to lack of T-tubules and low expression levels of calcium storing proteins 581-Pos and SERCA. Regulation of Myofilament Contractile Function in Human Donor and Overall, contraction and action potential parameters in hESC-CMs were highly Failing Hearts variable and independent of MyHC-isoform, suggesting that ventricular spec- 1 1 1 2 Kerry S. McDonald , Laurin M. Hanft , Joel C. Robinett , Maya E. Guglin , ificity in hESC-CMs cannot be fully promoted by the ventricular MyHC 3 Kenneth S. Campbell . protein. 1Department of Medical Pharmacology and Physiology, University of 2 Missouri, Columbia, MO, USA, Division of Cardiovascular Medicine, 583-Pos 3 University of Kentucky, Lenxington, KY, USA, Department of Physiology, Adolescent Binge Alcohol Exposure Affects Cardiovascular Function University of Kentucky, Lexington, KY, USA. Lizhuo Ai, Edith Perez, Quan Cao, Maxime Heroux, Andrei Zlobin, Heart failure (HF) often includes changes in myocardial contractile function. AnnaDorothea Asimes, Toni R. Pak, Jonathan A. Kirk. This study addressed the myofibrillar basis for contractile dysfunction in Cell & Molecular Physiology, Loyola Univ Chicago, Maywood, IL, USA. failing human myocardium. Regulation of contractile properties were More than five million underage Americans binge drink, which elevates measured in cardiac myocyte preparations isolated from frozen, left ventricu- blood alcohol concentration (BAC) to 0.08 g/dL within 2 hours, at least lar mid-wall biopsies of donor (n=6) and failing human hearts (n=8). Permea- once per month. However, the consequences of binge drinking on the adoles- bilized cardiac myocyte preparations were attached between a force cent heart are unknown. To study this, outbred rats were gavaged 3 g/kg 2þ transducer and position motor and both Ca and sarcomere length (SL) EtOH for 3 days, H2O for 2 days, and EtOH for additional 3 days between dependence of force, rate of force, loaded shortening, and power output 5 postnatal day 36-44 (corresponding to adolescence). The BAC was 0.08 were measured at 15 5 1 C. Myocyte preparation size was similar between 0.01 g/dL. Control rats were gavaged H2O for the 8-day protocol. Both groups (Donor: length, 148 5 10 mm, width, 21 5 2 mm, n = 13; HF: length, groups had normal food intake and 30% weight gain during the protocol. 2þ 131 5 9 mm, width, 23 5 1 mm, n = 16). Maximal Ca activated isometric Echocardiography showed Control rats experienced a 30% increase in 2 force also was similar between groups (Donor: 44 5 2kN,m ; HF: 42 5 LV end-diastolic diameter (LVEDD) due to normal growth during this period 2 2kN,m ), implicating that previously reported force declines in failing hu- of adolescence along with a parallel increase in end-systolic diameter so that man myocardium (Haynes et. al., J Mol Cell Cardiol, 2014) arose from tissue ejection fraction remained constant while stroke volume increased. remodeling. Maximal force development rates were lower in failing myocytes Conversely, Binge rats experienced only half that increase in LVEDD, and 5 1 5 1 (Donor: ktr =0.59 0.03 s ;HF:ktr =0.51 0.02 s ), however, both a decreased systolic diameter resulting in increased ejection fraction. One 2þ groups exhibited similar Ca activation dependence of ktr values. Human car- explanation is that impaired cardiac growth from binge alcohol exposure is 2þ diac myocyte preparations exhibited a Ca activation dependence of loaded compensated for by increased systolic function to maintain cardiac output. shortening and power output. Peak power output normalized to isometric force Increased systolic function in the Binge rats suggested increased beta- 2þ 2þ (PNPO) decreased 12% from maximal Ca to half-maximal Ca activa- adrenergic activation, which was supported by an increase in troponin I tions in both groups. Interestingly, SL dependence of PNPO was diminished PKA phosphorylation. Furthermore, doppler revealed a trend towards in failing myocyte preparations. A reduction in SL from 2.25 mmto reduced E/A ratio in the Binge group, and single cell force measurements 1.95 mm caused a 26% decline in PNPO in donor myocytes but only a found significantly increased passive stiffness, indicating an impact on dia- 11% change in failing myocytes. These results suggest that altered regula- stolic function as well. Lastly, we performed RNA-seq and identified a tion of myofilament function may impair ventricular performance in failing pattern of altered genes that suggested a switch in substrate utilization human hearts. from fatty acid to glucose metabolism in the Binge rats. In summary, we have determined that adolescent binge alcohol exposure results in slowed 582-Pos normal growth, increased systolic function, depressed diastolic function, In Human Embryonic Stem Cell-Derived Cardiomyocytes Twitch Ki- and changes in metabolic gene expression, revealing a possibly significant netics, Action Potential Parameters and MyH-mRNA Fractions Are Inde- cardiac health risk. pendent of the Expressed Myosin Heavy Chain Isoform Natalie Weber1, Kathrin Kowalski1, Tim Holler1, Ante Radocaj1, 584-Pos Martin Fischer2, Jeanne de la Roche2, Stefan Thiemann2, Kristin Schwanke3, Impact of Hypertrophic Cardiomyopathy Mutations on the Cardiac Alexander Lingk1, Uwe Krumm1, Birgit Piep1, Ullrich Martin3, Myosin Super-Relaxed State Robert Zweigerdt3, Bernhard Brenner1, Theresia Kraft1. Sriya Byrapuneni, Sami Chu, Joseph M. Muretta, David D. Thomas. 1Institute of Molecular & Cell Physiology, Hannover Medical School, Biochemistry, Molecular Biology, and Biophysics, University of Minnesota- Hannover, Germany, 2Institute of Neurophysiology, Hannover Medical Twin Cities, Minneapolis, MN, USA. School, Hannover, Germany, 3Leibniz Research Laboratories for We aim to examine the effects of hypertrophic cardiomyopathy (HCM) associ- Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, ated mutations on the myosin super-relaxed state (SRX) through functional Hannover, Germany. ATPase assays and time-resolved FRET structural measurements. Cardiac Previously we showed that cultivating human embryonic stem cell-derived myosin isomerizes between the relaxed state (RX), which is available for acti- cardiomyocytes (hESC-CMs) plated onto stiff matrix for 35 days induced vation and subsequent force generation with actin, and the kinetically inactive a switch from mixed a/b-myosin heavy chain (MyHC) protein towards SRX, in which myosin is hypothesized to be structurally occluded from the pure ventricular b-MyHC protein in 60-70% of single cardiomyocytes. force-generation cycle by the interacting-heads motif (IHM). We hypothesize Yet, the remaining 1/3 of individual CMs still had mixed a/b-MyHC that myosin regulatory light chain (RLC) mutations associated with HCM, spe- protein expression. Often expression of MYH6/MYH7-mRNAs is used as cifically E22K and R58Q, cause hypercontractility through disruption of the marker for a/b-MyHC protein expression and cardiomyocyte function. To myosin SRX. Based on proposed models of the IHM, these residues form sta- address the question how twitch kinetics, action potentials and MYH- bilizing salt-bridge interactions with the opposite myosin. We hypothesize that mRNA isoforms are related to the actual MyHC protein isoforms, we estab- mutations in the RLC associated with HCM increase myosin’s basal ATPase lished single-cell mapping technique to analyze different parameters for in- activity due to disruption of SRX. To observe the effect of these mutations, dividual hESC-CMs. we have performed ATPase assays to measure changes in myosin function,

BPJ 9318_9321 Sunday, March 3, 2019 119a and time-resolved FRET to directly detect the changes in head-head interac- Posters: Cell Mechanics, Mechanosensing, and tions, due to changes in the population of SRX myosin. These measurements were performed on bovine cardiac myosin containing human cardiac RLC. Motility I This work was supported by NIH grants AR032961 and AR057220 (to DDT), and an American Heart Association Scientist Development Grant (to 587-Pos JMM). Rescue of DNA Damage After Constricted Migration by DNA Repair Fac- tor Overexpression 585-Pos Yuntao Xia, Charlotte Pfeifer, Kuangzheng Zhu, Jerome Irianto, A Novel a-Tropomyosin Mutation (D55N) Associated with Familial Dennis Discher. Dilated Cardiomyopathy Increases Tropomyosin Binding to Actin Univ Pennsylvania, Philadelphia, PA, USA. Xiaomei Yang1, Michelle A. Recto1, Xinyu Zhang1, Yuejin Li1, Migration through constricting pores ruptures the nucleus, causes mis- Genaro A. Ramirez Correa1, William M. Schmidt2, Brittney Murray2, localization of DNA repair factors among other factors, and increases Anne M. Murphy1. DNA damage. Here, rescue of both nuclear rupture and DNA damage 1Dept Pediatrics/Division of Cardiology, Johns Hopkins Sch Med, Baltimore, occurs with myosin-II inhibition, whereas co-overexpression of multiple MD, USA, 2Dept of Cardiology, Johns Hopkins Sch Med, Baltimore, MD, DNA repair factors rescues the DNA damage but not the nuclear USA. rupture. Inhibition of oxidative stress partially rescues the excess DNA Mutations in sarcomeric proteins are heritable causes of dilated cardiomyop- damage, but cell cycle delays persist after constricted migration for athy (DCM). a-Tropomyosin (TPM1) mutations have previously been re- all methods of rescue. Overexpressed TREX1, which is a cytoplasmic ported in both hypertrophic cardiomyopathy and DCM. We report nuclease, does not add to the DNA damage even though it enters the phenotypic features and in vitro functional assays of a TPM1 sequence nuclear bleb at the rupture site as does endogenous cytoplasmic cGAS variant in four-generation family of Mediterranean ancestry associated (cyclic GMP-AMP synthase). Genome variation can increase after with a novel TPM1 variant, D55N. We assessed the functional role of constricted migration, consistent with effects of DNA damage and similar TPM1 D55N variant in association with DCM. Methods: Clinical evaluation in magnitude to in vivo variation. A simple model fits nuclear envelope and family history was obtained on the proband and family members. After rupture trends in terms of pore curvature, myosin-II activity, and lamina genetic counseling, testing was performed in clinical laboratories and pedi- strength. gree analysis was conducted (Progeny software). In vitro functional assess- ment the mutation measured the actin binding capacity of bacterially 588-Pos expressed Tm WT vs Tm D55N using actin co-sedimentation assays. Re- Spatial Segregation and Boundary Formation in Breast Cancer Aggre- sults:The age of diagnosis of clinical DCM in the family ranged from the gates first to the seventh decade of life, including two individuals who underwent Alex Devanny, Daniel Lee, Laura Kaufman. heart transplant in the first and second decades. The D55N sequence variant Columbia University, New York, NY, USA. was identified in 7 living individuals. The pattern of inheritance of DCM in In order to gain a greater understanding of spatial organization in cancerous this family was autosomal dominant with a 71% penetrance of disease. tissues, we analyze the segregation patterns of 6 breast cell lines Actin binding assay shows that the binding ability of a-Tropomyosin to fila- (MDA-MB-231, MDA-MB-468, MDA-MB-436, MDA-MB-157, ZR-75-1, mentous actin (F-actin) filaments increased significantly, an average of and MCF-10A) of varying morphological classes and at varying stages 64.5%55.4 s.e.m at any given concentration (0.25 mMto2mM) (n=3 along the epithelial-to-mesenchymal transition during the formation of ANOVA p<0.05) in the presence of the mutation. Further functional assess- mixed aggregates. Among these cell lines, sorting is generally robust. A ment of the mutants will be undertaken. Conclusions: Genetic sequencing sorting hierarchy is constructed by mixing cells and observing the develop- and initial functional analyses strongly suggest a pathogenic role for ment of the aggregate over time. To rationalize this sorting hierarchy, we TPM1 D55N mutation in DCM which increases the af?nity of a-Tm binding consider sorting in the context of available adhesion proteins and cortex ten- to F-actin. sion, cell properties that are typically considered in the differential adhesion hypothesis (DAH) and extensions of this hypothesis. We characterize the 586-Pos adhesion proteins mediating aggregate formation and find that these proteins Actin-Binding Compounds that Affect the Weak-To-Strong Actin-Myosin play an important role in compaction speed, with high compaction speed Interaction dictating a preference for cell segregation to the inside of the aggregate. Osha Roopnarine, David D. Thomas. Among cell lines that share the same adhesion proteins, actomyosin contrac- Dept Biochem/Molec/Biophys, Univ Minnesota Med Sch, Minneapolis, MN, tility and cortex tension play distinct roles in further influencing sorting USA. hierarchy. In cell lines utilizing classical cadherin-based compaction, We measured the effects of ten actin-binding compounds on the interaction increasing actomyosin contractility disrupts the aggregate, while in of cardiac myosin subfragment 1 (S1) with pyrene-iodoacetamide-actin integrin-based compaction reducing contractility dramatically increases (PIA). These compounds, previously identified from a small-molecule screen the compaction. To supplement the experiment, we perform Cellular involving lifetime-detected FRET from fluorescently-labeled actin to a Potts Model (CPM) simulations of sorting. The simulations replicate the myosin-derived actin-binding peptide, perturb actin-activated myosin sorting patterns observed experimentally both in cellular organization ATPase and microsecond structural dynamics of actin (Guhathakurta et. al. and morphology. Taken together, this suggests the application of the 2018, JBC 293:12288). In the present study, several of these compounds DAH as a minimum effective model for predicting sorting hierarchy among were evaluated by measuring their effects on PIA fluorescence, which is cell lines that may not necessarily conform to the usual tenets of the quenched specifically by the strong binding (post-powerstroke state) of hypothesis. myosin to actin. In the absence of S1, several compounds reduced the steady-state fluorescence of the compound-PIA complex, indicating an in- 589-Pos crease in the compound-induced structural change and/or the compound’s af- Mapping the Biochemical Interactions of the Mechanoresponsive Contrac- finity for actin. Excess MgATP partially restored the fluorescence. In the tility Controller absence of ATP and compound, the addition of S1 to PIA decreased fluores- Priyanka Kothari1, Vasudha Srivastava1, Vasudha Aggarwal2, cence substantially, indicating strong binding, while the addition of MgATP Irina Tchernyshyov3, Jennifer Van Eyk3, Taekjip Ha2, Douglas N. Robinson1. reversed this effect. However, in several cases, the compound partially 1Dept Cell Biol, Johns Hopkins School of Medicine, Baltimore, MD, USA, prevented the restoration of S1-PIA fluorescence by MgATP, suggesting 2Biophysics, Johns Hopkins University, Baltimore, MD, USA, 3Department that a fraction of actin either retained strongly bound S1 or maintained the of Advanced Clinical Biosystems Institute, Cedar-Sinai Medical Center, compound-induced structural change as in the absence of S1. In some cases, Los Angeles, CA, USA. higher [ATP] was required to increase the PIA fluorescence, suggesting that Every biological process, ranging from cell migration to embryogenesis, re- the compound decreased ATP affinity. Transient kinetics measurements lies on the cell’s ability to adapt to changing mechanical environments. By are ongoing to determine the effect of compounds on the ATP-induced studying the model shape change process cytokinesis in Dictyostelium,we release of S1 from the compound-PIA complex, which will provide insight find that the cell is a finely tuned control system, with proteins that modulate on how actin-myosin interactions are perturbed by these compounds, some their behavior in response to mechanical and biochemical signals. Although of which are currently used for therapeutic purposes. This work was we know many of the players involved in the contractility controller, their supported by NIH grants to DDT (R01AR032961, R01HL129814, and biochemical interactions that allow force propagation through the cortical R37AG026160). network are still poorly defined. We use a proteomics approach to identify

BPJ 9318_9321 120a Sunday, March 3, 2019 direct interactors of two key nodes of the control system, actin crosslinker ofibroblast differentiation of human primary AVICs, as confirmed by the cortexillin I and scaffolding protein IQGAP2. This analysis identifies me- increased expression of alpha smooth muscle actin (aSMA) and transgelin, chanoenzyme myosin II as a biochemical interactor of both cortexillin I produced calcification and up-regulated calponin 2. Interestingly, SDS- and IQGAP2. We use quantitative in vivo biochemical measurements, PAGE revealed consistent induction of a protein band at around 30 kDa including fluorescence cross-correlation spectroscopy (FCCS) and Single in murine, ovine, porcine and human AVICs after culturing with TGF-b1. Molecule Pulldown (SiMPull) to measure concentrations, apparent binding Mass spectrometry analysis identified the induced band as 14-3-3 adaptor affinities, and stoichiometries of complexes. We find that the cooperative protein, previously shown to bind calponin 2. To assess calponin 2’s contri- mechanoaccumulation of myosin II and cortexillin I is potentially due to bution to mechanoregulation of myofibroblast differentiation, calponin 2- their direct interaction. IQGAP1, in turn, competes with IQGAP2 to bind null fibroblasts were cultured on silicon substrates of varied stiffness (0.2, myosin II and cortexillin I, thus negatively regulating contractility. 2, 8, 16, and 32 kPa). The results showed that the substrate stiffness- Our data also suggest that multi-protein complexes are pre-formed in the dependent expression of aSMA was lower in calponin 2-null fibroblasts cytoplasm, primed for activation by chemical or mechanical stimuli to compared to control fibroblasts, which indicates a role of calponin 2 in engage with the cytoskeletal network. In addition, we find a few unusual the mechanoregulation of AVIC differentiation and identifies it as a novel interactors by mass spectrometry analysis, such as RNP1, discoidins, target for therapeutic intervention of CAVD and potentially other fibrosis and methylmalonyl semialdehyde dehydrogenase (mmsdh), which we previ- conditions. Further studies are underway to dissect the molecular signaling ously identified in genetic suppressor screens, providing greater evidence for by which calponin 2 mediates myofibroblast differentiation in CAVD and their function in the network. Using the contractility controller as a model, fibrotic diseases. we are identifying a quantitative interaction map, complete with new inter- actions, that is uncovering new biochemistry associated with the 592-Pos mechanobiome. Effects of Opsonin Density on Phagocytic Behavior of Human Neutrophils Emmet A. Francis, Volkmar Heinrich. 590-Pos Dept Biomed Eng, Univ Calif Davis, Davis, CA, USA. Determining Integrin Molecular Tension for the Recruitment and the Acti- Although many components of the molecular machinery driving phagocy- vation of Focal Adhesion Kinase tosis in immune cells have been identified, a detailed understanding of Anwesha Sarkar1, Yingxiao Wang2, Xuefeng Wang3. the underlying biophysical mechanisms remains elusive. Here, we investi- 1Department of Physics & Astronomy, Iowa State University, Ames, gate phagocytic protrusion dynamics in human neutrophils by monitoring IA, USA, 2Department of Bioengineering, University of California, mechanical processes and signaling events during phagocytosis of targets San Diego, San Diego, CA, USA, 3Iowa State Univ, Ames, IA, coated with varying densities of rabbit immunoglobulin G (IgG). We USA. examine effects of the opsonin density in two complementary experiments: Focal adhesion kinase (FAK), a member of protein tyrosine kinase family, is phagocytosis of IgG-coated polystyrene microspheres, and frustrated an important mediator of signals transduced by integrins (major mechano- phagocytic cell spreading on IgG-coated glass coverslips. We facilitate in- sensitive transmembrane receptors transmitting cellular forces at cell matrix teractions between neutrophils and microspheres using two opposing glass interface). FAK regulates various cellular functions such as cell adhesion, micropipettes to hold and manipulate the cell and target particle; this also proliferation, differentiation and spreading. Although the biochemical role allows us to quantify cell morphology and approximate cortical tension dur- of FAK in integrin signaling is relatively more understood, the biomechan- ing phagocytosis. To analyze frustrated phagocytosis, we image the area of ical processes for FAK’s recruitment and activation by integrins remains contact between spreading cells and the coverslip with high resolution using less clear. To study the mechanobiology of FAK, Our group adopted two reflection interference contrast microscopy (RICM). The frustrated molecular tension tools, tension gauge tether (TGT) and integrative tension phagocytosis assay revealed that the overall speed of contact-area growth sensor (ITS), both having a designable tension threshold, selectable in the is largely independent of IgG density. However, cells are more likely to range of 10-60 pN. TGT globally knocks down integrin tensions in live cells spread and reach a larger maximum contact area on higher densities of to a designed force level, while ITS visualizes integrin tension by fluores- IgG. Monitoring also the cytosolic calcium concentration, we found that cence imaging with high resolution. With TGT, ITS and a FRET-based the timing of calcium surges during frustrated phagocytosis depends on biosensor reporting FAK activation, we manipulated and mapped integrin IgG density – at lower densities, the cells reach larger contact areas before tension in live cells, and studied the consequent FAK aggregation and exhibiting global calcium bursts. Overall, these findings indicate that the activation. ITS and FAK live cell imaging revealed that integrin tension speed of cell spreading is determined by the rate of actin protrusion rather higher than 12 pN was produced after FAK aggregation, indicating that than the density of adhesive contacts, while the number of ligands available FAK recruitment to focal adhesions does not require integrin tension higher for binding to Fcg-receptors impacts both calcium signaling dynamics and than 12 pN. TGT and FAK biosensor revealed that FAK was not activated the likelihood and extent of cell spreading. This suggests that initial actin on TGT surfaces with tension tolerance (Ttol) of 12, 23 and 33 pN, but on polymerization is largely independent of calcium signaling, while later the surfaces with Ttol of 43 pN and 54 pN. These results suggest that stages of phagocytosis mechanics are regulated by elevated global calcium FAK activation by integrin signaling requires integrin tension higher than levels. 33 pN. Overall, our study revealed that FAK recruitment and FAK activation have distinct requirement for integrin tensions at separate force 593-Pos levels. Large Scale Simulations of Cell Resolved Tissue by a Cellular Potts Model Jakob Rosenbauer. 591-Pos Forschungszentrum Julich,€ Julich,€ Germany. Calponin 2 Mediates Activation and Myofibroblast-Like Differentiation of Multicellular organisms all face the same fundamental challenge during tis- Human Aortic Valve Interstitial Cells in Calcific Aortic Valve Disease sue development: the generation of distinct cell types, structures and organs Olesya Plazyo1, Xue-Qun Chen2, Kenneth S. Campbell3, Joy Lincoln4, at specific positions in the body. Advanced imaging techniques provide J.-P. Jin1. detailed insight into processes on a single-cell and sub-cell level. Still, a 1Physiology, Wayne State Univ, Detroit, MI, USA, 2Wayne State thorough mathematical model of cellular dynamics and differentiation has Univ, Detroit, MI, USA, 3Dept Physiology, Univ Kentucky, not been identified. Cellular Potts models are a commonly used tool to Lexington, KY, USA, 4The Ohio State University, Columbus, OH, describe the cellular behavior. Due to the large computational effort the USA. scale of those simulations has so far been very limited to small and mostly Calcific aortic valve disease (CAVD) is a common severe clinical condition two-dimensional simulation setups. Here, we developed a framework with involving impaired cellular mechanoregulation. Calponin 2 is a cytoskeleton a fully parallelized implementation of the cellular Potts model, enabling regulatory protein, and its deletion attenuates the development of CAVD in simulations orders of magnitude larger than possible to date and improve- ApoE knock-out mice (Plazyo et al., J Mol Cell Cardiol 121:233-241, 2018). ments on spatial isotropy. Through parallelization our framework is able To further investigate the role of calponin 2 in the pathogenesis and progres- to run effectively on large cluster architectures making the simulation of sion of human CAVD, we studied the myofibroblast differentiation of hu- up to 109 individually resolved cells possible. We present our simulations man aortic valve interstitial cells (AVICs) and the role of mechanical of tumorigenesis and tumor internal signaling with explicit modeling activation. Immunohistochemichal analysis of the aortic valve tissue ob- of the surrounding tissue. Benchmarking with experimental data, such tained from patients confirmed increased expression of calponin 2 when as light sheet microscopy images, becomes possible as the simulated compared to non-affected controls. Stimulation with TGF-b1toinducemy- areas reach experimentally relevant sizes. Therefore, our framework is a

BPJ 9318_9321 Sunday, March 3, 2019 121a promising tool to study tissue development, effects of medication, formation to the actomyosin network on the apical surface exerting non-negligible me- of metastasis and other tissue internal processes to identify underlying base dio-apical tension on the apical surface. We used femto-second laser to ablate principles. the apical surface of MDCK cells. Through quantitative image analysis, we found that cell area increase accompanied with the straightening of the tight 594-Pos junctions. The apical area expansion rate is positively correlated with the cell Versatile and High-Throughput Microfluidics Platform for Dorsal Cell area and tortuosity. Our findings support our hypothesis. Contrary to the con- Mechanics ventional thinking, the mechanism of MDCK apical constriction force is Seungman Park1, Yoon Ki Joo2, Yun Chen3. 1 2 from the actomyosin-network on the apical surface, instead of the circumferen- Johns Hopkins University, Baltimore, MD, USA, Chemical and tial actomysin cable. Our finding is not only important for understanding how Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA, 3 actomyosin contractility regulate paracellular transport but also in tissue Mechanical Engineering, Johns Hopkins University, Baltimore, MD, USA. morphogenesis such as MDCK cystogenesis. [1] J. R. Turner, Semin. Cell We developed a high-throughput microfluidics technique facilitating mea- Dev. Biol., 11, 301 (2000). surements of cell mechanics parameters at the dorsal side of the cell, including molecular binding strengths, local traction forces, and viscoelastic 597-Pos properties. By adjusting the flow rate, the force magnitude exerted on the A Mechanical Cusp Catastrophe Imposes a Universal Developmental cell can be modulated ranging from pN to nN to perturb various force- Constraint on the Shapes of Tip-Growing Cells dependent processes in cells. Timelapse images were acquired to record Enrique R. Rojas1, Jacques Dumais2. events due to such perturbation. The values of various mechanical parame- 1Biology, New York University, New York, NY, USA, 2Bioengineering, ters are subsequently obtained by single particle tracking. Up to 50 events Universidad Adolfo Ibanez, Vina del Mar, Chile. can be measured simultaneously in a single experiment. Integrating the Understanding the mechanistic basis for morphological diversity is a central microfluidic techniques with the analytic framework established in compu- problem in biology. Here, I examine the evolutionary and mechanical tational fluid dynamics, our method is reliable, accurate, economic and basis for the diversity of cell morphologies in tip-growing organisms from efficient. across the tree of life. To investigate the factors that give rise to this diver- 595-Pos sity, I measured the spatial dependence of the cell-wall expansion rate for A Coupled Excitable Network Model Dictates Cortical Wave Patterns and three species that spanned phylogeny (one plant, one fungal, and one protis- Controls Cellular Protrusion Morphology tan species) and shape space. I found that a single constitutive mechanical Sayak Bhattacharya1, Yuchuan Miao2, Peter N. Devreotes2, model relating the mechanical stresses in the wall to the expansion rates Pablo A. Iglesias1. could explain the expansion-rate profiles from each species, which allowed 1Dept Elec/Compu Eng, Johns Hopkins University, Baltimore, MD, USA, us to calculate the spatial dependence of the mechanical extensibility 2Dept Cell Biology, Johns Hopkins University, Baltimore, MD, USA. (inverse viscosity) of the cell wall. We found that the extensibility profiles, Cell migration - a critical component of various physiological processes such as in turn, could be fit well with a simple two-parameter empirical function. wound healing and cancer metastasis - occurs through repetitive extensions of This function, in combination with the mechanical model, provided a the cell cortex. These protrusions are produced by waves of actin polymeriza- basis that spanned a broad ‘‘morphospace’’ for tip-growing organisms tion that push the cell membrane, suggesting that the cortex is an excitable me- that I described computationally, and which moreover could account for dium. The nature and kinetics of these protrusions are crucial in controlling the shapes of a broad diversity of tip-growing organisms. However, cellular morphology and, consequently, the migratory mode of the cell. Besides the experimentally observed shapes were restricted to a narrow region of actin, signaling species such as PIP3, Ras and Rap also display excitable wave this morphospace. Using computational analysis, I discovered that natural propagation. Although each biochemical species within the cellular network is cell morphologies are bounded by a shape instability that had hallmarks associated with a specific function in literature - the fact that these coordinately of a cusp bifurcation, and which separated fast-growing, thin cells organize into waves suggests a common underlying mechanism. from slow-growing, wide cells. That is, our analysis revealed a universal We classified the molecular events within waves into two networks, the signal developmental constraint on cell shape provided by the interplay of transduction excitable network (STEN) and the cytoskeletal excitable network natural selection for fast cell growth and the physical mechanism of cell (CEN), which displayed different wave substructures. Here, we propose an in- growth. terconnected excitable network model, where STEN is modeled as a slow sys- tem capable of spontaneous wave generation, and CEN is modeled to generate 598-Pos faster puncta-like oscillations observed in the absence of signaling. Together Evolution of Stresses at Cell-Gel Interfaces during Confined Interfacial this STEN-CEN model successfully captured the organization of the signaling Migration Abhishek Mukherjee1, Ramesh Singh2, Wenyi Yan3, Shamik Sen4. and actin waves seen in experiments. 1 We predicted specific changes in wave characteristics by altering the strengths Mechanical Engineering, IIT Bombay Monash University, Mumbai, India, 2Mechanical Engineering, IIT Bombay, Mumbai, India, 3Mechanical and of feedback loops within the model. Guided by excitable wave theory, we 4 experimentally perturbed multiple nodes in this network to produce distinct co- Aerospace Engineering, Monash University, Melbourne, Australia, Dept ordinated alterations in wave patterns, serving to verify our model and to shed Biosci/Bioeng, IIT Bombay, Mumbai, India. light on the detailed biochemical architecture involved. This further allowed us Cell migration at the interface of two substrates under confinement holds to control the type of wave generated and, consequently, the protrusion pheno- clinical significance as it is instrumental in morphogenesis and wound heal- type, creating cortical extensions resembling filopodia, ruffles, pseudopodia or ing but has been insufficiently studied. The adhesions at the cell-gel inter- lamellipodia. Our results suggest that theoverall state of the STEN-CEN system face are instrumental in deciding the fate of cell motility, viability and determines cellular morphology. morphology. However, experiments have not been able to shed light into the evolution of stresses within the cells and at the interfaces. Therefore, 596-Pos a better understanding of the mechanics involved in such a process is neces- Investigating Apical Constriction Force of Madin-Darby Canine Kidney sary. The various components of the cell and both the gels were modelled as Cells by Laser Ablation viscoelastic systems. We used finite element modelling to study the Keng-hui Lin. evolution of stresses and deformation within the cell and at the cell-gel in- Inst Physics, Academia Sinica, Taipei, Taiwan. terfaces. The cell is attached to an underlying soft gel substrate by focal ad- Tight junctions locate at the sub-apical region on the lateral plane. It serves a hesions (FAs), whereas, no adhesions are formed at the cell-stiff gel convenient marker to indicate apical area. The study of tight junction functions interface. The asymmetry in adhesion at the two cell-gel interfaces leads is usually performed on confluent Madin-Darby Canine Kidney (MDCK) cells to an asymmetric stress state of the cell with relatively larger stress or Caco cells grown on filter paper. It has been shown that the actomyosin developing at the interface with stiff gel indicating higher actin density contractility is linked to the morphology of tight junctions and its paracellular at this interface than at that with soft gel. This is validated by prior transport. The change is always accompanied with the morphological change of experiments. The location of FAs is found to play a decisive role in mecha- tight junctions. Under high tension, the tight junctions become tortuous. When notransduction as the FAs close to the region of protrusion are maximally actomyosin contractility is disrupted by drug treatment, the tight junctions stressed during motion but those farther away are scarcely engaged, which morphology become straighter. There was proposed mechanism as actomyosin has also been demonstrated in prior experiments. An understanding of cable as a purse-string connecting to the tight junctions [1] but the observed the stress evolution within the cell undergoing interfacial migration and phenomena which can not fully be explained by this model. In this work, we at the interface can be potentially used to design wound healing tissue propose the tight junctions in mature and polarized MDCK cells are linked adhesives.

BPJ 9318_9321 122a Sunday, March 3, 2019

599-Pos ability using a high-throughput microfluidic device which mimics microca- A Computational Model to Unveil the Role of the Nucleus in 2D Cell pillaries. We perfused erythrocytes from ME/CFS patients and from age Migration and sex matched healthy controls (n=14 pairs of donors) through a high- Adrian Mourer Rosende, Hector Gomez. throughput microfluidic platform (5mmx5mm). We recorded cell movement Mechanical Engineering, Purdue University, West Lafayette, IN, USA. at high speed (4000 fps), followed by image analysis to assess the following The role played by the nucleus in cell migration remains poorly understood. parameters: entry time (time required by cells to completely enter the test Recent works have described different ways in which the nucleus controls channels), average transit velocity (velocity of cells inside the test channels) cell migration in 3D environments, such as, e.g., mechanosensing, force and elongation index (ratio of the major diameter before and after deforma- transmission, or restriction of motion in dense extracellular networks. How- tion in the test channel). We observed that erythrocytes from ME/CFS pa- ever, the role of the nucleus in 2D cell migration is more controversial. The tients had higher entry time, lower average transit velocity and lower literature has usually assumed that the nucleus behaves as a passive element elongation index as compared to healthy controls. Taken together, this data without an active role. Hence, most computational models of cell migration shows that erythrocytes from ME/CFS patients have reduced deformability. do not consider the nucleus. Some papers have shown that intact cells are To corroborate our findings, we measured the erythrocyte sedimentation faster than enucleated cells, despite the biological mechanisms driving cell rate for these donors which show that the erythrocytes from ME/CFS patients motion are identical. These results suggest that the presence of the nucleus had lower sedimentation rates. To understand the basis for differences in de- inside the moving cell cannot be neglected. Here, we study the role played formability, we investigated changes in the fluidity of the membrane using by the nucleus in 2D migration of fish keratocytes by way of a computational pyrenedecanoic acid and observed that erythrocytes from ME/CFS patients model. have lower membrane fluidity. Zeta potential measurements showed that Our model incorporates the nucleus dynamics into well established ME/CFS patients had lower net negative surface charge on the erythrocyte computational mechanistic models of keratocyte migration. The model plasma membrane. Higher levels of reactive oxygen species in erythrocytes resorts to two phase-field variables to track the cell and the nucleus. from ME/CFS patients were also observed. Using scanning electron micro- Thus, we can locate the actin and myosin dynamics to the cytosol (i.e., scopy, we also observed changes in erythrocyte morphology between ME/ inside the cell and outside the nucleus). We employ two force balance CFS patients and healthy controls. Finally, preliminary studies show that equations that account for the forces acting on the nucleus and the actin erythrocytes from ‘‘recovering’’ ME/CFS patients do not show such differ- network. ences, suggesting a connection between erythrocyte deformability and dis- Our simulations show a realistic picture of keratocyte migration on planar sub- ease severity. strates. The model results provide insights into the role played by the nucleus in 2D cell migration. 602-Pos Cooperative Transport by Amoeboid Cells: A Cellular Tug-of-War 600-Pos Valentino Lepro1,2, Oliver Nagel2, Stefan Klumpp3, Reinhard Lipowsky1, Spreading Out: Modeling the Physics of Cell-Substrate Interaction in Cell Carsten Beta2. Spreading and Focal Adhesion Evolution 1Theory and Bio-systems, Max Planck Institute of Colloids and Interfaces, Magdalena Stolarska1, Aravind R. Rammohan2. Potsdam, Germany, 2Institute of Physics and Astronomy, University of 1Dept Mathematics, Univ St Thomas, Saint Paul, MN, USA, 2Dept Modeling, Potsdam, Potsdam, Germany, 3Institute for Nonlinear Dynamics, University Corning Inc, Corning, NY, USA. of Go¨ttingen, Go¨ttingen, Germany. Cell spread area and focal adhesion (FA) sizes are known to increase with As society paves its way towards devices miniaturization and precision substrate stiffness (Yeung et al., Cell Motil. Cytoskeleton., 2005). Different medicine, micro-scale actuation and guided transport become increasingly mathematical models have been developed, some of which can predict prominent research fields, with high potential impact in both technological increases in cell spread area with substrate stiffness while others can and clinical contexts. To date, a promising strategy is to exploit living predict increases in only the FA area. Here, we describe a two-dimensional cells as smart, steerable and biochemically powered carriers, developing model of an actively spreading cell interacting with a deformable elastic the so-called bio-hybrid systems: the cargo is linked to swimming cells substrate through spring-like FA complexes. The model couples FA (e.g. bacteria), which act as smart propellers powered by their own evolution, intracellular stress, and active cell spreading. With these three metabolism. Still, in applications like targeted drug delivery, a guided components coupled, we demonstrate the ability of our model to qualita- transport through complex environments as living tissues remains tively reproduce both, the increase in cell spread area and FA area with challenging. substrate stiffness that has been observed experimentally, as well as the Inspired by leukocytes, efficiently moving through our body to reach target temporal evolution of cell spread areas. To predict the stiffness dependence sites, we propose to exploit the amoeboid motion of eukaryotic crawling cells and the temporal evolution of cell spreading we find there are three for transport of drug-loaded microparticles. Preliminary experiments pointed key mechanisms: i) cell substrate attachments at the periphery, ii) attach- out the ability of amoeboid cells to displace cargos in a directed fashion, by ments in the interior generating retrograde flow, and iii) the resistance means of chemotaxis, both across 2D substrates and through 3D matrices. to spreading from the tensile forces at the cell periphery due to the mem- Remarkably, the random walk of cell-cargo systems shows faster dynamics brane. The 2D model allows us to capture the role of spatial localization than the one of lone cells, suggesting cell-particle interaction as stimulus pro- of these different phenomena and the sensitivity of these mechanisms moting cell motility. Further, by comparing experiments with different particle in enabling the observed global behaviors. Using this model, we generate sizes, a non-monotonic behavior emerges for the cargo diffusivity-size relation. insights on the role of both spatial coupling and integration of the By analysing cell-particle relative dynamics, we found reproducible features of signals to generate large scale changes in cellular dynamics. We also the underlying interaction, such as oscillatory dynamics and effective interac- investigate the role of geometric anisotropy through non-circular shapes tion potential shapes. We use these features to develop a physical model of our during spreading. The overarching aim of this work has been to specify bio-hybrid system; we then embed it into stochastic simulations and compare what minimal physical mechanisms are required to recapitulate cellular with experimental findings. spreading and FA evolution and their mechanical response to the underlying When higher cell densities are used, so to emulate crowded environments substrate. as living tissues, particle transport is performed by several cells which, when cooperating, are capable of displacing heavier cargos. Our next goal 601-Pos is to describe such stochastic Tug-of-War emerging at the cellular scale, Altered Erythrocyte Biophysical Properties in Chronic Fatigue Syndrome in order to make such system predictable, thus closer to a biomedical 1 2 1 3 Amit K. Saha , Brendan R. Schmidt , Julie Wilhelmy , Vy Nguyen , application. Justin K. Do3, Vineeth C. Suja4, Mohsen Nemat-Gorgani1, Anand K. Ramasubramanian2, Ronald W. Davis1. 603-Pos 1Biochemistry, Stanford University, Palo Alto, CA, USA, 2Chemical and Scutoids: Understanding the 3D Packing of Curved Epithelia Materials Engineering, San Jose State University, San Jose, CA, USA, Javier Buceta1,Go´mez-Ga´lvez Pedro2, Pablo Vicente-Munuera2, 3Biomedical Engineering, San Jose State University, San Jose, CA, USA, Luis M. Escudero2. 4Chemical Engineering, Stanford University, Palo Alto, CA, USA. 1Bioengineering & Chem/Biomolec Engineering Departments, Lehigh Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multi- University, Bethlehem, PA, USA, 2Cell Biology Dept., Seville University & systemic illness of unknown etiology affecting millions of individuals world- IBiS, Seville, Spain. wide. In this work, we tested the hypothesis that erythrocyte biophysical During development metazoa are shaped into complex 3D structures. This properties are adversely affected in ME/CFS. We tested erythrocyte deform- process is achieved using epithelial cells as building blocks. Thus, driven

BPJ 9318_9321 Sunday, March 3, 2019 123a by internal/external stimuli, epithelial cells adopt a variety of shapes that 606-Pos affect their packing and the properties of the tissue and its morphology. Spatiotemporal Analysis of Integrin Molecular Tension During Cancer In this context, the accepted view was that when tissues bend, cells change Cell Adhesion theirs shape from columnar (prism) to a ‘‘bottle shape’’ (frustum) hence Byoung Choul Kim. assuming that the 3D packing properties remain unchanged along the apico- Division of Nano-Bioengineering, Incheon National University, Incheon, basal axis. In order to characterize and investigate the 3D packing and the Republic of Korea. shapes of cells in curved epithelia we mimicked computationally the archi- Stable adhesion of cancer cells is required for migration or invasion during tecture of tissues using computational geometry methods (Voronoi tessella- cancer metastasis. The mechanical and physical interaction between integrin tion). To confirm the predictions of our model, we have investigated by expressed on the cell and their environment plays a critical role in cancer microscopy means the 3D packing of the salivary gland of Drosophila cell adhesion. In order to characterize the integrin molecular tension of met- (a model extensively used for tubulogenesis) as well as other tissues in astatic cancer cells, we utilized the single molecule tension measurement, different organisms. Finally, we have also developed a theoretical model termed tension gauge tether (TGT) assay. We monitored a change of the based on energetic considerations to understand the biophysical origin of peak single molecular tension via integrins when non-metastatic and meta- the 3D cellular organization. Our computational model predicts, and our ex- static cancer cells adhered, spread, and migrated. Our results showed that periments confirm, that, as the curvature of the tissue increases, prisms and the peak integrin molecular tension of around 40 pN was required for initial frusta are not the only cell shapes that develop and reveals a previously un- adhesion. We also identified that the integrin molecular tensions above 54 described geometrical shape: the scutoid. As a consequence, the apical and pN were applied via focal adhesion and related cytoskeletal protein com- basal surfaces may have different packing topologies showing a neighbor plexes during cell spreading and migration. However, distinct distribution interchange between cells along their apicobasal. Our energetics analysis re- of integrin tension at the sub-cell level was mapped in the non-metastatic veals that scutoids allow tissues to minimize the packing energy and we pro- and metastatic cancer cells. Integrin tension above 54 pN was localized pose that such geometrical shape is nature’s solution to epithelial bending. at the cell periphery in the metastatic cancer cells whereas integrin tension 1) Scutoids are a geometrical solution to three-dimensional packing of below 54 pN was evenly distributed in the non-metastatic cancer cells. epithelia, P. Go´mez-Ghlvez et al., Nature Communications 9, 2960 (2018). Our single molecule technique demonstrated changes of integrin molecular 2) A 3D cell shape that enables tube formation, G. Blanchard, Nature tension of metastatic cancer cells from initial adhesion to migration, 561,182(2018) allowing us to increase our understanding of a mechanism of cancer metastasis. 604-Pos Actin Flow Dependent and Independent Force Transmission in Integrin- 607-Pos Mediated Adhesions From Nuclei to Artificial Cells: Probing the Mechanics of Minimal Systems Tristan P. Driscoll, Billy Huang, Sang Joon Ahn, Abhishek Kumar, Giulia Bergamaschi, Andreas Biebricher, Gijs J.L. Wuite. Martin Schwartz. Physics and Astronomy, Vrije Universiteit, Amsterdam, Netherlands. Cardiovascular Medicine, Yale University, New Haven, CT, USA. The creation and development of a self-sustaining synthetic cell are intimately Integrin-mediated adhesions are essential for multicellular life because of linked to the presence of a membrane cortex and an internal polymeric skel- their ability to both apply force to and sense the mechanical properties of eton, as these are essential for maintenance of its shape as well as sustaining the extracellular matrix. These effects are attributed to the ‘‘focal adhesion external loads. Thus, understanding the mechanics of natural cortex systems clutch’’, in which moving actin filaments transmit force to integrins via dy- will be a fundamental step towards building a functioning synthetic cell. There- namic protein interactions. To understand mechanotransduction through in- fore, we aim to investigate the mechanics of isolated nuclei as simple biological tegrins, we simultaneously measured force on talin together with rates of systems, which can be modelled as a cortical shell enclosing a crosslinked poly- actin flow by combining a previously developed talin FRET tension sensor meric core. with quantitative actin speckle microscopy. While force on talin in small la- To this end, we employ a combination of Optical Tweezers (OT) and Acoustic mellipodial adhesions correlated with actin flow, force on talin in large adhe- Force Spectroscopy (AFS); while the former allows highly controlled manip- sions in the lamella was mainly flow-independent. Stopping actin flow blocked ulation and sensitive force-detection, AFS enables multiplexing and high force transmission in lamellipodia but not lamellar adhesions. This indicates force measurements. In particular, optical tweezers are used to apply pN there are two unique types of force transfer in integrin based adhesions, one forces to the samples by using Poly-L-lysine coated microspheres as handles, that is actin flow-dependent and one that is actin flow-independent. Flow- while AFS enables the application of forces up to 15 nN to surface-attached dependent force transfer required talin’s C-terminal actin binding site, nuclei. The complementarity of the two techniques allows us to explore nu- ABS3, whereas flow-independent force transfer required vinculin and the cen- clear mechanics across a wide range of forces and loading rates, as well as tral actin binding site, ABS2. Mutation of these sites identified distinct func- in different experimental settings (e.g. force ramps, clamps or oscillatory tions in force transmission, cell spreading and mechanosensing. Together, stress measurements). these results revealed that the focal adhesion clutch is a transient structure Upon application of force ramps, nuclei respond viscoelastically and that mediates events at the cell edge prior to arrest of actin filaments and are observed to strain stiffen at large deformations, possibly due to recruit- establishment of stable adhesions that are the primary force-transmitting ment of specific nuclear components (e.g. the nuclear lamina). Moreover, structures. stretched nuclei exhibit hysteresis when returning to the initial length, while subsequent pullings on the same nucleus do not alter its force respon- 605-Pos se.The data thus obtained is then modelled and reveals how the individual Mechanics of Cells - Implications for Adhesion and Motility structural components of nuclei contribute to their mechanical resilience. Andreas Janshoff. As there are numerous examples of pathologies characterised by altered Institute of Physical Chemistry, Goettingen, Germany. nuclear mechanics, our work will help to shed light on how this might Cellular mechanics and adhesion are two related properties that need to be occur. balanced in order to permit cells to migrate on suitably functionalized sub- strates displaying a compatible surface chemistry. Therefore, organisms that 608-Pos cope with varying environmental surface cues require an adaptive strategy. High Troughput Microfluidic Characterization of Erythrocyte Shapes and We found that the model organism Dictyostelium discoideum faces the Mechanical Variability challenge of coping with highly variable chemistry in the search of food. Em- Felix Reichel1, Johannes Mauer2, Ahmad Ahsan Nawaz1,3, ploying AFM-based single cell force spectroscopy we could show that exper- Gerhard Gompper2, Jochen R. Guck1, Dmitry Fedosov2. imental force curves upon retraction of cells from the surface exhibit two 1BIOTEC - CMCB, TU Dresden, Dresden, Germany, 2Institute of regimes. The first part up to the maximum adhesion force can be described Complex Systems and Institute for Advanced Simulation, in terms of a continuum model that permits to relate the mechanical properties Forschungszentrum Julich,€ Julich,€ Germany, 3School of Mechanical of cells to their capability to adhere onto a wide variety of substrates, while and Manufacturing Engineering (SMME), National University the second regime of the curve beyond the critical rupture force is governed of Sciences and Technology (NUST), Islamabad, by stochastic unbinding of individual binding partners and bond clusters ex- Pakistan. tending the life time of the bonded state. This versatile adhesion mechanism, The circulation of red blood cells (RBCs) in microchannels is important in which works on almost all surface chemistries, allows the cells to adapt to a microvascular blood flow and biomedical applications such as blood analysis large variety of natural surfaces and conditions. in microfluidics. Current understanding of the complexity of RBC shapes

BPJ 9318_9321 124a Sunday, March 3, 2019 and dynamical changes in microchannels is mainly formed by a number of molecular mechanisms of signaling and actin dynamics for growth cone simulation studies, but there are few systematic experimental investigations. dynamics. Here, we present a first systematical mapping of experimental RBC shapes and dynamics for a wide range of flow rates and channel sizes. Results are 611-Pos compared with simulations and show good agreement. A key difference to External Hydraulic Resistance Influences Cell Motility simulations is that in experiments there is no single well-defined RBC state Yizeng Li, Debonil Maity, Sean Sun. for fixed flow conditions, but rather a distribution of states. This result Johns Hopkins University, Baltimore, MD, USA. can be attributed to the inherent variability in RBC mechanical properties, Cells in vivo live in diverse physical environments that provide mechanical which is confirmed by a model that takes the variation in RBC shear elasticity cues for cells to deform, migrate, and carry out their biological function. For into account. These results make a significant step toward a quantitative example, cell migration on 2D surfaces is mostly driven by forces from actin connection between cell behavior in microfluidic devices and their mechanical polymerization and focal adhesions, whereas cells in confined geometries properties. can be driven by water permeation. Water flux also plays an important role in epithelial monolayers, where directed transport can generate epithe- 609-Pos lial domes. While the role of the cell substrate properties in directing cell Rho Mediated Mechanical Force Generation Through Dectin-1 movement has been explored, we have recently found that the fluid flow Rohan Choraghe1, Alan Buser2, Aaron Neumann1. in the cell surroundings also can also influence the cell migration speed 1Biomedical Engineering / Pathology, University of New Mexico, and the relative contributions of actin and water to the observed cell speed. Albuquerque, NM, USA, 2Biology / Pathology, University of New Mexico, In our experiments, we expose cells to high viscosity media and find that the Albuquerque, NM, USA. cell spread area increases with viscosity. This morphology change can be Dectin-1 is an innate immune pattern recognition receptor expressed primar- inhibited by an NHE1 inhibitor. Moreover, the velocity of cell migration in- ily on macrophages, dendritic cells and neutrophils. Dectin-1 recognizes a creases with the viscosity of the media. Analyses on the actin flow reveal highly immunogenic carbohydrate polymer known as b-glucan, which forms that viscous medium reduces actin retrograde flow within the lamellipodial, 50-60% of the dry weight of Candida albicans cell wall. Recognition of b- which can be explained by our theoretical model—cells with faster migra- glucan by immune cells contributes to a variety of responses in these cells tion speed have less prominent actin retrograde flow. On the modeling including phagocytosis, oxidative burst, production of inflammatory cyto- side, we have developed a two-phase mathematical model that describes kine and chemokine. Phagocytosis is a mechanism cell uses to engulf parti- the motion of the cytosol and the actin-network components during cell cles. It is a process that involves rapid and dramatic changes in local cell migration. We explore the interplay of actin-driven and water-driven cell morphology mediated through actin reorganization. In opsonic immune migration, and show that the former mechanism is important on 2D sub- phagocytosis, particle is coated by either IgG or complement before being strates while the later mechanism is more important in confined spaces. phagocytosed. In contrast, non-opsonic phagocytosis involves direct recogni- The transition from actin-driven to water-driven cell migration depends on tion of ligand by phagocytic receptor. Recognition of glucan on fungal path- the external hydraulic resistance, which varies with the mechanical proper- ogens by Dectin-1 involve this non-opsonic process leading phagocytosis of ties of the external fluid and the geometry of the cell surroundings. The Candida. Downstream signaling elements activated upon recognition of b- model has implications on early embryonic development, morphogenesis, glucan by Dectin-1 include Src family kinases, Syk, and CARD9 leading and cancer cell metastasis. k to activation of NF- B and subsequent secretion of pro-inflammatory cyto- 612-Pos kines. However specific mechanisms that coordinate phagocytosis down- Developing Nuclease-Resistant DNA-Based Tension Sensor for Cellular stream of Dectin-1 leading to actin reorganization and internalization of Force Imaging phagocytic particle are relatively poorly understood. We found that stimula- Yuanchang Zhao1, Xuefeng Wang1,2. tion of Dectin-1 by MMW (Medium molecular weight) glucan leads to 1Physics and Astronomy, Iowa State Univ, Ames, IA, USA, 2Molecular, mechanical force generation and areal contraction in Dectin-1 transfected Cellular, and Developmental Biology, Iowa State University, Ames, IA, HEK-293 cells and M1-macrophages. Further with inhibitor studies we USA. found that this force generation is a Syk-independent, Src-dependent process Integrin transmits force for cells to interact with extracellular matrix and thus mediated through the Rho-ROCK pathway. We confirmed activation of plays critical roles in many cellular functions, including cell differentiation, RhoA downstream of Dectin-1 using G-LISA and stress fiber formation spreading and migration. As researchers become increasingly interested in though fluorescence microscopy. Through phagocytosis assays, we also integrin-transmitted forces, a variety of methods have been developed for found direct evidence for importance of Rho-ROCK mediated force genera- the cellular force visualization and calibration. Lately, DNA have been tion pathway in process of phagocytosis of TRL035 C. albicans through commonly adopted for the synthesis of cellular force sensors as DNA are pro- Dectin-1. In conclusion, we found evidence for Rho-ROCK mediated grammable biomaterials with well-defined mechanical properties. Based on mechanical force generation in downstream of Dectin-1 for C. albicans double-strand DNA or hairpin DNA labeled with fluorescent dyes, these sen- phagocytosis. sors convert invisible cellular force to fluorescence signal, therefore enabling force imaging at high spatial resolution. However, the DNA in the tension 610-Pos sensors is generally vulnerable to the degradation by nucleases that are A Minimal Mechanochemical Model for Growth Cone Dynamics anchored on cell membrane or secreted to the extracellular environment. Aravind R. Rammohan1, Padmini Rangamani2, Magdalena Stolarska3. The nucleases may damage the sensors, lower signal-to-noise ratio or even 1Dept Modeling, Corning Inc, Corning, NY, USA, 2Univ Calif San Diego, produce false signal in cellular force imaging. To address these issues, we La Jolla, CA, USA, 3Dept Mathematics, Univ St Thomas, Saint Paul, MN, tested chemically modified DNA or peptide nucleic acid as the materials USA. for the synthesis of a force sensor resisting nuclease degradation. Eventually The dynamics of growth cones at the tip of axons results from finely coordi- we developed nuclease-resistant Integrative Tension Sensor (NR-ITS) with nated interplay between chemical and mechanical signals. We now know DNA modifications of 2’-O-Methyl and phosphorothioation on nucleotides. from decades of experimental evidence that this interplay is modulated by We demonstrated that this force sensor is immune to the degradation caused the response of the cytoskeleton and focal adhesions to intracellular signaling by purified nucleases or nucleases secreted in-situ by cells. This tension sensor cascades. Furthermore, the growth cone itself has two distinct regions of also successfully imaged integrin-transmitted cellular forces at submicron res- actin – the peripheral region, which is lamellipodial in structure and the central olution in various cell types such as fibroblasts, keratocytes and platelets. region, which is filopodial in structure. These differences are marked by the spatial differences in the biochemical activity of actin remodeling proteins including Arp2/3 and cofilin. In this work, we present a minimal mechano- Posters: Cytoskeletal-based Intracellular chemical model for the coupled interaction between biochemical signal trans- Transport duction and mechanics of actin rearrangement at the growth cone. We use this model to establish the physical basis of force generation at the growth cone 613-Pos and investigate the conditions under which the transition from peripheral to Kinesin-1 Acts Independently and Also Regulates Kinesin-3-Dependent central region occurs. We then predict how cytoskeletal remodeling and adhe- Transport of Synaptophysin Vesicles in Mammalian Axons sion respond to different actin remodeling proteins. We anticipate that this Sandra E. Encalada. minimal model will set up the basis for further investigation using detailed Dept Molecular Med, Scripps Res Inst, La Jolla, CA, USA.

BPJ 9318_9321 Sunday, March 3, 2019 125a

In highly polarized neurons, the transport of synaptic components to distal sites Dividing eukaryotic cells move their chromosomes using the mitotic spindle, a is critical for neuronal function, including the maintenance of synapses. Regu- dense scaffold of up to 10^5 microtubules and 200 essential microtubule lation of axonal transport involves the tight coordination of multiple families of associated proteins. To apply pulling forces on chromosomes, molecular mo- processive molecular motors, but how this occurs is not well understood. Here, tors disassemble, move, or slide along microtubules bound to the kinetochores we show that the transport of synaptic vesicle precursors (SVPs) toward the of chromosomes. Microtubule dynamics can also generate forces. Though pre- synapse in mammalian neurons depends on the independent coordination of vious studies have investigated chromosome movement in dividing cells, work- Kinesin-1 (KIF5C) and Kinesin-3 (KIF1A). SVPs associate, co-transport, and ing in vivo has limitations. The microtubule density makes it difficult to resolve co-immunoprecipitate simultaneously with KIF5C and KIF1A. We show that single or small bundles of microtubules, and the presence of the cell cortex hin- KIF1A is required for the anterograde transport of fast-moving SVPs, while ders applying forces. Therefore, many molecular and mechanical details about the function of KIF5C is to normally inhibit the anterograde movement of chromosome movement remain unknown. To overcome limitations in vivo,we slower-moving SVPs. Thus, we report that KIF5C acts independently of have designed an assay that reconstitutes mitotic chromosome movement in vi- KIF1A to coordinate the motility of subpopulations of SVPs with different ve- tro. Theassay uses mitotic cytoplasm extracted from Xenopus laevis eggs, pu- locities. Strikingly, direct targeting of KIF5C to SVPs is sufficient to enhance rified fluorescent centrosomes attached to the coverslip of a microscope flow KIF1A transport by increasing velocities and run lengths of SVPs. These find- chamber, and purified chromosomes with fluorescent kinetochores. When a so- ings indicate that Kinesin-1 both coordinates independently of Kinesin-3, but is lution of egg extract and purified chromosomes is introduced into the flow also able to cooperate with Kinesin-3 to drive the transport of SVPs in mamma- chamber, centrosomal microtubule asters can bind to kinetochores and move lian axons. chromosomes ‘poleward’ to the centrosome, as occurs in vivo. Fluorescent tubulin and microtubule plus-end marking proteins are also included, enabling 614-Pos visualization of microtubule dynamics alongside chromosome movements in Late Endosomal Membrane-Lipid Composition Imparts Change in 3D over time using four-color spinning disc microscopy. Furthermore, X. laevis Oxysterol-Binding Protein-Related Protein 1L’s (ORP1L) Nano-Scale Or- egg extract is amenable to a suite of biochemical tools, allowing us to investi- ganization Which Effects Organelle Motility gate molecular players such as dynein and various kinesins. Using this assay, Shreyasi Thakur, Peter Relich, Melike Lakadamyali. we study the mechanisms and mechanics of chromosome movement in mitotic Department of Physiology, University of Pennsylvania, Philadelphia, PA, centrosomal asters, by inhibiting or depleting molecular motors and then USA. observing changes in how often and at what velocities chromosomes move. Intracellular organelle transport along microtubules (MTs) is highly regulated We also use optical trapping to apply forces on chromosomes, in order to spatiotemporally and is orchestrated by motor proteins dynein and kinesins. gain a mechanical understanding of how the spindle moves chromosomes. The directionality and uninterrupted long-range motion of the organelles are achieved by multilevel regulatory mechanisms including motor recruitment and motor activation by adapter and accessory proteins. Recently, the motor or- 616-Pos ganization on organelle membranes, in particular motor clustering, has been Local Actin Filament Geometry Dictates How Myosin Va Molecular Mo- suggested to play an important role in transport regulation and processes tor Teams Transport Liposomes Through 3D Actin Networks in Vitro 1 2 3 including organelle biogenesis and maturation. While the lipid composition Sam Walcott , Andrew T. Lombardo , Kathleen M. Trybus , 3 of the organelle membrane, specifically the cholesterol content, has been sug- David M. Warshaw . 1Dept Mathematics, Univ Calif Davis, Davis, CA, USA, 2Cornell University, gested to play a role in regulating the molecular arrangement or clustered orga- 3 nization of motors, the exact mechanisms are not clear. We hypothesized that Ithaca, NY, USA, Dept Molecular Physiology and Biophysics, Univ clustering/molecular organization of the motor protein dynein is mediated by Vermont, Burlington, VT, USA. the nanoscale organization of Oxysterol-Binding Protein-Related Protein 1L In cells, myosin Va (myoVa) motor teams transport cargo through complex (ORP1L), which is the main cholesterol sensing protein present on late endo- three-dimensional (3D) actin filament networks. To define this process in vitro, somal, lysosomal and phagosomal compartments. To determine the depen- we created two distinct 3D actin networks comprised of: 1) randomly oriented dence of motor and ORP1L nano-organization on cholesterol levels, we have actin filaments; 2) branched Arp2/3 actin filaments. For these unbranched and been using Stochastic Optical Reconstruction Microscopy (STORM). We branched networks, we visualized the 3D orientation and polarity of each actin found that motor proteins like dynein and ORP1L indeed form nano-clusters filament with 3D super-resolution STORM and characterized the 3D trajec- on late endosomal membranes. By coupling super-resolution imaging with tories of fluorescent 350nm fluid-like liposomes transported by 10 myoVa drug treatments that modulate the cholesterol content of organelle membranes, motors. Interestingly, more directed and fewer stationary trajectories were we observed ORP1L nano-organization is sensitive to cholesterol content of the observed in branched versus unbranched networks. To understand this differ- late endosomal membrane. Using quantitative analysis, we showed that ORP1L ence, we employed a mathematical model that is predictive of myoVa liposome is more clustered on late endosomal membranes under high cholesterol condi- transport (Lombardo et al., 2017). Simulating trajectories on 3D actin networks tions compared to low cholesterol conditions. Further, high cholesterol led to a with geometries identical to those in our experimental measurements resulted redistribution and repositioning of late endosomes at the peri-nuclear region, in more directed and fewer stationary trajectories on branched versus un- suggesting that dynein activity and dynein mediated motility are sensitive to branched networks, as experimentally observed. Further analysis of simulated > cholesterol levels. Overall using high resolution microscopy we are dissecting trajectories shows that stationary events (i.e. 1s pauses) occur at the same 3D the link between organelle membrane nano-organization and organelle actin network locations as we observed experimentally. The model suggests motility/positioning. that these pauses result from tug-of-wars between motor teams attached to the same liposome but interacting with different actin filaments. Because of 615-Pos the force-dependence of the motors’ mechanochemistry, the duration of the Reconstituting Mitotic Chromosome Movement In Vitro tug-of-war, and therefore whether or not a trajectory is stationary, is determined Sagar U. Setru1, Joshua W. Shaevitz1,2, Sabine Petry3. by the relative polarity alignment of the actin filaments, with closer to anti par- 1Lewis-Sigler Institute for Integrative Genomics, Princeton University, allel alignments favoring longer tug-of-wars. These results demonstrate that Princeton, NJ, USA, 2Department of Physics, Princeton University, myoVa transport can be controlled by local (sub-micron) actin geometry, sug- Princeton, NJ, USA, 3Department of Molecular Biology, Princeton gesting that branching and cross-linking proteins may regulate intracellular University, Princeton, NJ, USA. transport through actin filament alignment.

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Posters: Membrane Pumps, Transporters,and 619-Pos Conformational Coupling to Asymmetric ATP Hydrolysis in the Trans- Exchangers I port Cycle of P-Glycoprotein Sepehr Dehghani-Ghahnaviyeh1,2, Karan Kapoor2, Emad Tajkhorshid2,3. 617-Pos 1Center for Biophysics and Quantitative Biology, University of Illinois at Drug-Binding to Distinct Site of the Multidrug Exporter P-Glycoprotein Urbana-Champaign, Urbana, IL, USA, 2NIH Center for Macromolecular Monitored by Tryptophan Fluorescence Modeling and Bioinformatics, Beckman Inistitue, University of Illinois at Ina Urbatsch1, Douglas J. Swartz2, Anukriti Singh1, Courtney Katz1, Urbana-Champaign, Urbana, IL, USA, 3Department of Biochemistry, Sakshi Gautam3, Joachim Weber3. University of Illinois at Urbana-Champaign, Urbana, IL, USA. 1 Cell Biology and Biochem, Texas Tech University Health Sciences P-glycoprotein(Pgp) is an ATP-binding cassette exporter that uses ATP hydrolysis 2 Center, Lubbock, TX, USA, Natural Sciences, Lubbock Christian to actively transport diverse substrates across the cellular membrane. ATP hydro- 3 University, Lubbock, TX, USA, Chemistry and Biochemistry, Texas Tech lysis takes place in the nucleotide binding domains (NBDs) resulting in conforma- University, Lubbock, TX, USA. tional transition of the transporter. Many studies suggested two different models P-glycoprotein (Pgp) exports hundreds of chemically unrelated, hydrophobic for ATP hydrolysis in NBDs, symmetric and asymmetric. In the symmetric model, compounds out of cells. Since Pgp can greatly affect bioavailability, pharma- two ATPs are hydrolyzed at the same time, however, asymmetric model suggests a cokinetics and efficacy of therapeutic drugs, there is great interest in under- two-stroke process corresponding to one ATP hydrolysis at a time. Numerous standing the mechanism by which drugs are extruded. Pgp is an ABC studies have revealed the importance of a number of conserved structural motifs transporter with two nucleotide binding domains (NBDs) that bind and hydro- including several loops in ATP hydrolysis. However, the mechanistic role of lyze ATP, leading to extensive conformational changes across the transmem- conserved loop residues and their protonation states in ATP hydrolysis-initiated brane domains (TMDs) that result in substrate translocation across the cell transport cycle remainelusive.Here, we used equilibrium MD simulations to study membrane. Despite decades of biochemical/biophysical studies, the mecha- the interaction between conserved loops in the nucleotide binding sites, ATP mol- nism by which the NBDs control these conformational changes, and how ecules, and catalytic water during the transport cycle in Pgp. The ATP hydrolysis this is linked to drug binding in the TMDs, and ultimately export of the drugs was simulated by using the slow growth method. The system was equilibrated for is still controversial. Our recent X-ray structures of Pgp identified hydrophobic 100 and 200 ns before and after first ATP hydrolysis, respectively. Then, second and aromatic amino acids that contribute to binding of different inhibitors to ATP was hydrolyzed and the system was equilibrated for another 200 ns. Our anal- the drug-binding site. In this study, we test the hypothesis that therapeutic ysis showed that the interactions between the aspartate-loop (D-loop), histidine- drugs bind to different subsets of residues within defined subpockets in the loop (H-loop), and gamma-phosphate of ATP (Pg) trap the catalytic water in TMDs of the protein. Our general approach is to introduce tryptophans NBD1. D-loop is found to interact more closely with Pg in NBD1, when compared (Trps) at strategic positions in order to monitor drug binding. These Trps toNBD2. However, after ATPhydrolysis inNBD1, similarinteractions are formed were introduced on the background of a new fully functional low-Trp Pgp between the conserved loops, catalytic water, and ATP in NBD2 favoring subse- that retains three native conformationally sensitive Trps in the cytoplasmic do- quent ATP hydrolysis. Furthermore, we also observed that protonated histidine in mains, or onto a Trp-less Pgp. Using purified protein reconstituted in a lipid H-loop promotes the formation of favorable interactions enabling ATP hydrolysis. bilayer nanodiscs, and fluorescence changes, such as quenching, and resonance These results support an asymmetric ATP hydrolysis in Pgp where the first ATP energy transfer (FRET), we present first results of Pgp interactions with proto- hydrolysis may be the trigger for the second hydrolysis. typical substrates, such as the cyclopeptides QZ-Val and QZ-Phe that occupy structurally defined and distinct binding sites, as well as nicardipine and zosu- 620-Pos quidar. The impact on the substrate translocation mechanisms of Pgp will be Lipid-Mediated Inhibition Mechanism of P-Glycoprotein discussed. Karan Kapoor1, Shashank Pant1,2, Emad Tajkhorshid1,3. 1NIH Center for Macromolecular Modeling and Bioinformatics, Beckman Institute, University of Illinois Urbana Champaign, Urbana, IL, USA, 2Center 618-Pos for Biophysics and Quantitative Biology, University of Illinois at Urbana- P-Glycoprotein Activity Is Non-Monotonically Modulated by Transmem- Champaign, Urbana, IL, USA, 3Department of Biochemistry, Center for brane Voltage Biophysics and Quantitative Biology, University of Illinois at Urbana- 1 2 2 3 Thomas B.H. Schroeder , Haiyan Liu , David Sept , Khyati Kapoor , Champaign, Urbana, IL, USA. Divya K. Rao2, Suresh V. Ambudkar3, Michael Mayer4. 1 P-glycoprotein (P-gp) is an extensively studied multidrug efflux pump that is School Eng and Applied Sciences, Harvard University, Cambridge, MA, overexpressed in a variety of cancers and associated with the development of USA, 2Biomedical Eng, Univ Michigan, Ann Arbor, MI, USA, 3Lab Cell 4 multi-drug resistance (MDR). New class-III inhibitors targeting P-gp provide Biol, NCI NIH, Bethesda, MD, USA, Dept Biophysics, Univ Fribourg, novel starting points for overcoming MDR in humans, but the mechanism of ac- Fribourg, Switzerland. tion of these molecules remains elusive. Here, we use molecular dynamics sim- P-glycoprotein (Pgp) is a broadly polyspecific exporter of hydrophobic and ulations to study the structure and dynamics of P-gp þ Mg2þ-ATP system in the amphiphilic compounds that plays an important role in the blood-brain barrier presence and absence of one of these inhibitors, Tariquidar (TAR). Simulations and in multidrug resistance in cancer. Several research groups have recently in a multi-component lipid environment in the absence of TAR show consistent made significant strides toward elucidating the mechanism of Pgp’s catalytic dimerization of nucleotide-binding domains (NBDs) of the transporter, with the cycle, but the biophysical properties of the cellular microenvironment can equilibrium shifted towards the conformations feasible for ATP hydrolysis and have strong effects on the function of Pgp and other ATP-binding cassette the subsequent completion of the transport cycle. On the other hand, in the pres- (ABC) transporters in as-yet poorly defined ways. Here we report that Pgp, ence of the inhibitor bound to the drug-binding pocket in the transmembrane MRP1, and BCRP, three of the most clinically relevant multidrug resistance- domain (TMD) of the transporter, stabilization of the open inward-facing con- linked ABC transporters, are strongly and non-monotonically regulated by formations of the transporter is observed, likely inhibiting the ATP hydrolysis the transmembrane electrical potential difference. We confirmed this result and substrate transport. In multiple simulation runs, the inhibitor is seen to re- with several assays, which used fluorescence and electrophysiological methods cruit lipid molecules through the two drug-entry portals in P-gp, where nega- to measure the efflux of transporter substrates from two human cell lines and tively charged head-groups from the lower lipid leaflet translocate inside the giant unilamellar vesicles and controlled the voltage using electrolyte gradients TMD, while the lipid tails remain extended outside into the bulk lipid environ- or the patch clamp technique. At hyperpolarized (highly inside-negative) po- ment. These ‘hinge’ lipid molecules likely cause the conformational changes tentials, each transporter displayed a pronounced reduction in efflux activity observed in TMD leading to the large separation in the NBDs downstream. compared to the activity at the resting potential; Pgp and MRP1 also showed We suggest a novel inhibitory mechanism for TAR and similar P-gp class-III in- reduced activity at inside-positive potentials. The transport data is well- hibitors, where lipids for the first time are seen to play a secondary inhibitory role described by a model in which the transmembrane potential biases the confor- in the catalytic cycle of membrane transporters. mational distribution of the proteins in a manner similar to the mechanism of action of voltage-gated ion channels. In this model, which is compatible with 621-Pos the ‘‘alternating access’’ framing of Pgp function, each catalytic cycle contains Mechanistic Study of a Peptidase Containing ABC-Transporter, Employ- two transitions with an opposite dependence on the transmembrane voltage due ing Microsecond Level Molecular Dynamics Simulations and Enhanced to the movement of an equivalent of 3-4 elementary charges back and forth Sampling Techniques across the membrane. This finding, which points to an additional axis for modu- Dylan S. Ogden, Vivek Govind Kumar, Mahmoud Moradi. lating transporter activity, underscores the importance of electric field effects University of Arkansas, Fayetteville, AR, USA. on the function of all membrane proteins, particularly those that undergo large ATP binding cassette transporters are of the largest and oldest superfamily of conformational changes in the transmembrane region. membrane transporters. ABC transporters function through an alternating access

BPJ 9322_9324 Sunday, March 3, 2019 127a mechanism, in which the protein can switch between the inward- and outward- cytoplasmic leaflet of the membrane. The occlusion of multiple phospholipid facing conformations by utilizing the binding and hydrolysis of ATP. A specific molecules in MsbA suggests that the average transport stoichiometry may not ABC transporter, PCAT1, contains two peptidase domains positioned at the be an integer, as different numbers of lipid molecules can be flipped in each cycle. lateral openings of the transmembrane domain and cleave secretion signals from substrates. Two crystal structures have been reported both in the occluded 624-Pos conformation, one containing the peptidase domains and the other with nucleo- Mapping Membrane Protein Complex Assembly Pathways in Live Cells tides bound to the nucleotide binding domains. In this study, we present micro- with Progressive Acceptor Photobleaching 1 1 1 1 second level all atom equilibrium molecular dynamics simulations and Michael P. Dalton , Ellen E. Cho , Marsha P. Pribadi , Deo R. Singh , Seth L. Robia2. nonequilibrium enhanced sampling techniques to observe large scale conforma- 1 2 tional transitions and evaluate the free energies associated with ATP binding and Loyola University Chicago, Maywood, IL, USA, Dept Physiology, Loyola hydrolysis, ADP release, and nucleotide binding domain (NBD) dissociation. Univ Chicago, Maywood, IL, USA. We have characterized such free energies both for the isolated NBD’s and for We have previously quantified fluorescence resonance energy transfer of the full length transporter. We observe that although the energetics of ATP hy- membrane proteins using progressive acceptor photobleaching. This simple drolysis is dependent on the presence or absence of the transmembrane domains, assay determines protein complex stoichiometry by quantifying the donor there is drastic change in the nucleotide binding domain dissociation energetics fluorescence (FD) enhancement as a function of acceptor fluorescence (FA) between isolated nucleotide binding domains and the full length protein. photobleaching. Dimeric complexes are distinguished from higher order olig- omers by linear and supralinear FD/FA relationships, respectively. Here we 622-Pos investigated homo-oligomerization of micropeptides that regulate cellular cal- Electrostatic Lock in the Transport Cycle of the Multidrug Resistance cium handling: phospholamban (PLB), sarcolipin (SLN), myoregulin (MLN), Transporter EmrE endoregulin (ELN), another-regulin (ALN), and dwarf open reading frame Josh V. Vermaas1, Susan L. Rempe2, Emad Tajkhorshid3. (DWORF). The micropeptides share structural homology and are proposed 1BioScis Ctr, Natl Renewable Energy Lab, Golden, CO, USA, 2Sandia Natl to interact with the SERCA calcium transporter to regulate its activity. We Labs, Albuquerque, NM, USA, 3Biochemistry, University of Illinois, Urbana, observed dimers at low protein expression levels, but higher order oligomer- IL, USA. ization at high concentrations. Some micropeptides (e.g. ELN) transitioned EmrE is a small, homodimeric membrane transporter that exploits the established sharply to high order oligomers as expression was increased, suggesting coop- electrochemical proton gradient across the Escherichia coli inner membrane to erativity of oligomerization. Other micropeptides increased oligomer stoichi- export toxic polyaromatic cations, prototypical of the wider small-multidrug ometry more gradually, consistent with a mixed population of dimers and resistance transporter family. While prior studies have established many funda- higher order species. One micropeptide, DWORF, was observed to have mental aspects of the specificity and rate of substrate transport in EmrE, low res- very weak dimerization with no higher order oligomerization observed at olution of available structures has hampered identification of the transport any level of expression. Overall, the data suggest that various SERCA- coupling mechanism. We developed a refined atomic structure of EmrE by opti- binding micropeptides differ in the relative population of intermediate species, mizing against available cryo-electron microscopy (cryo-EM) data to delineate but they share a common assembly pathway from monomer to dimer to higher the critical interactions by which EmrE regulates its conformation during the order oligomers. transport process. With the model, we conduct molecular dynamics simulations of the transporter in explicit membranes to probe EmrE dynamics under different 625-Pos substrate loading and conformational states, representing different intermediates Photocycle and Abnormal Activity of the Dual Chromophore Proton Pump in the transport cycle. The refined model is stable under extended simulation. The Archaerhodopsin-4 with and Without the Second Chromophore water dynamics in simulation indicate that the hydrogen-bonding networks Xiaoyan Ding1,2, Chao Sun1, Haolin Cui1, Sijin Chen1, Xinyi Dong1, around a pair of solvent-exposed glutamate residues (E14) depend on the loading Xinru Meng1, Ming Wang1, Yanan Yang1, Weimin Liu3, Qixi Mi3, Xiao He1, state of EmrE. One specific hydrogen bond from a tyrosine (Y60) on one mono- Anthony Watts2, Xin Zhao1. mer to a glutamate (E14) on the opposite monomer is especially critical, as it 1East China Normal University, Shanghai, China, 2University of Oxford, locks the protein conformation when the glutamate is deprotonated. The Oxford, United Kingdom, 3ShanghaiTech University, Shanghai, China. hydrogen bond provided by Y60 lowers the pKa of one glutamate relative to While several archaeal ion pumps have been revealed to contain two chromo- the other, suggesting both glutamates should be protonated for the hydrogen phores, retinal and carotenoid bacterioruberin, their photocycle, activity and bond to break and a substrate-free transition to take place. These findings estab- mediation mechanism by the second chromophore bacterioruberin have not lish the molecular mechanism for the coupling between proton transfer reactions been well explored. In order to address these challenges, the recombinant and protein conformation in this proton-coupled secondary transporter. archaerhodopsin-4 (aR4), either with retinal only or with both retinal and bacterioruberin chromophores, was successfully expressed together with 623-Pos endogenous lipids in H. salinarum L33 and MPK409 respectively. In situ Spontaneous Phospholipid Binding to the Bacterial Flippase MsbA light-induced transient absorption change spectroscopy, low temperature Po-Chao Wen1, Pius Padayatti2, Qinghai Zhang2, Emad Tajkhorshid1. 1 FTIR difference spectroscopy and 2D solid-state NMR, together with molecu- Department of Biochemistry, Beckman Institute for Advanced Science and lar dynamic simulation, were employed to study the photocycle, photo- Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA, 2 intermediates and activity of aR4 with and without the second chromophore Department of Integrative Structural and Computational Biology, The bacterioruberin. Our results revealed for the first time that the photocycle of Scripps Research Institute, La Jolla, CA, USA. wild-type aR4 only contains one M state but two N states, and unlike in bacte- MsbA is a lipopolysaccharide (LPS)/lipid A flippase in bacteria that also func- riorhodopsin the accessibility switch of the SB nitrogen in the photocycle of tions as a multidrug exporter. Several structures of MsbA have been resolved aR4 may occur in the N1 / N2 states. Perturbed L, M and N states were in multiple conformational states, providing a prototypical system to study the observed in the bacterioruberin-excluded aR4 indicating that a strong coupling structural dynamics of ABC exporters that include many multidrug-resistant pro- may exist between bacterioruberin and aR4 photocycle. Furthermore, a consti- teins. Notably, a few recent structures have shown the binding of LPS in the cen- tutively activated aR4 was also observed in the bacterioruberin-excluded aR4 tral lumen of MsbA, demonstrating the substrate-recognition mechanism of which can be characterized by the structural instability and conformational combined hydrophobic and hydrophilic interactions. We have performed response to the deletion of the second chromophore. These new insights may microsecond-scale molecular dynamics simulations using an inward-open be generalized to other receptors and proteins in which functions are perturbed MsbA structure in a phospholipid bilayer and captured spontaneous lipid binding by ligand binding. into the central lumen. The recruitment of phospholipids is aided by transient salt bridges formed between the head group phosphate and charged residues at helix 626-Pos TM4, while the fatty acid chains of lipids need to move across a narrow hydro- What is Your Machine Really Doing? Systematic Exploration of Alterna- phobic cleft between helices TM4 and TM6. Interestingly, this narrow cleft tive Mechanisms as Applied to Transport has been recently characterized as a non-competitive binding site for the novel August George1, Michael Grabe2, John M. Rosenberg3, MsbA inhibitor G907, suggesting this inhibitor might prevent the binding of Daniel M. Zuckerman1. transport substrates by obstructing the binding pathway, despite that LPS was 1Biomedical Engineering, Oregon Hlth Sci Univ, Portland, OR, USA, 2Dept co-crystallized in the inhibitor-bound structure. Remarkably, due to the large vol- Pharmaceutical Chemistry, Univ Calif San Francisco, San Francisco, CA, ume of the central lumen of MsbA, it is capable of accommodating more than one USA, 3Dept Biol Sci, Univ Pittsburgh, Allison Park, PA, USA. phospholipid molecule. In our simulations, we have observed up to four phospho- Do complex molecular machines tend to exhibit a single mechanism, as sug- lipid molecules entering the central lumen of the same transporter, and the phos- gested in literature and textbook cartoons, or is a multiplicity of pathways pholipids inside the lumen do not always retain the same orientation as in the possible or even prevalent? Motivated by ‘‘circumstantial evidence’’ for such

BPJ 9322_9324 128a Sunday, March 3, 2019 pathway heterogeneity, we develop a Monte Carlo computational approach for structural dynamics of the SERCA2a/PLB complex. The membrane protein com- investigating the issue using discrete-state (reaction network) models. The cal- plex between the sarcoplasmic reticulum Ca-ATPase 2a (SERCA2a) and its culations yield families of models, some exhibiting multiple pathways, which regulator phospholamban (PLB) is a validated therapeutic target for reversing are consistent with a fitness goal (e.g., driven transport against a concentration contractile dysfunction caused by aberrant calcium handling in the heart. Unfor- gradient). Many of the models would be difficult to find by intuition alone in the tunately, efforts to discover compounds with specificity for this complex have yet complex state-spaces of interest. Our approach can also address the related to yield an efficacious drug. GFP-SERCA2a (donor) was co-expressed in the issue of investigating what functions are possible for molecular machines. As endoplasmic reticulum of HEK293 cells with and without RFP-PLB (acceptor), an example, we study transporters in a complex environment where it is neces- and FRET was measured in a unique fluorescence lifetime microplate reader sary to discriminate against decoy ligands; computations reveal models with (FLT-PR), which increases the throughput of high-precision FLT measurements non-integer stoichiometry that exploit excess free energy from an ion leak to by several orders of magnitude. A triplicate screen against a small-molecule li- perform driven ‘‘proofreading’’ - i.e., enhanced selectivity. brary containing 1500 compounds (LOPAC = Library of Pharmaceutically Active Compounds) identified ten hit compounds that reproducibly changed 627-Pos FRET by >3 SD and alter SERCA’s ATPase activity. One compound increases Comparative Analysis of Pulsed EPR Distance Measurements in an SERCA2a Ca affinity and activity at physiological calcium levels in cardiac SR, E. coli Cobalamin Transporter in Cells Versus Isolated Outer Membranes but not in skeletal SR, suggesting that the compound is acting specifically on the Reveals Novel Conformational Changes Dependent on the Native SERCA2a/PLB complex (Stroik et al., Nature Scientific Reports 8:12560). These Environment properties characterize a drug that could reverse calcium mishandling, as David Nyenhuis, Thushani Nilaweera, David S. Cafiso. required for treatment of heart disease. We will present recent developments in Dept. Chemistry, University of Virginia, Charlottesville, VA, USA. the development and applications of live-cell FRET biosensors. This work was Gram negative bacteria are reliant on TonB dependent transporters for the move- supported by NIH grants (GM27906, HL129814, AR07612, and DA037622). ment of scarce compounds across their outer membranes. These proteins consist of an outer b-barrel surrounding an internal hatch domain. The substrates, 630-Pos including iron siderophores and cobalamin, are large and would necessitate sig- The Phospholamban Pentamer Functionally Interacts with the Sarco- nificant conformational rearrangement of the hatch domain to facilitate their plasmic Reticulum Calcium Pump SERCA movement. In vitro work on the E. coli cobalamin transporter, BtuB, using the John Paul Glaves1, Joseph O. Primeau1, L. Michel Espinoza-Fonseca2, pulsed EPR technique double electron-electron resonance (DEER) in our lab M. Joanne Lemieux1, Howard S. Young1. has thus far failed to produce evidence of such conformational changes within 1Biochemistry, University of Alberta, Edmonton, AB, Canada, 2Internal the hatch domain. Recently, we have focused on the development of techniques Medicine, University of Michigan, Ann Arbor, MI, USA. to study the protein in intact systems, including whole cells and isolated outer The interaction of phospholamban with the sarcoplasmic reticulum calcium membranes. While investigating hatch-barrel mutant pairs in these systems, pump (SERCA) is a major regulatory axis in cardiac muscle contractility. we have seen evidence for conformational changes in the whole cells that are The prevailing model involves reversible inhibition of SERCA by monomeric induced by the presence of substrate, and that are absent or greatly suppressed phospholamban and storage of phospholamban as an inactive pentamer. How- in the isolated membrane alone. These results may reveal elements of the trans- ever, this paradigm has been challenged by studies demonstrating that phospho- port process, as well as highlighting the importance of studying proteins in their lamban remains associated with SERCA and that the phospholamban pentamer native environments. is required for the regulation of cardiac contractility. We have previously used This work was supported by NIGMS, GM035215. two-dimensional crystallization and electron microscopy to study the interac- tion between SERCA and phospholamban. To further understand this interac- 628-Pos tion, we compared small helical crystals and large two-dimensional crystals of An Approach for Exploring Novel Conformational States and Membrane SERCA in the absence and presence of phospholamban. In both crystal forms, Organization of BtuB in Whole Cells using EPR Spectroscopy SERCA molecules are organized into identical anti-parallel dimer ribbons. The Thushani D. Nilaweera, David A. Nyenhuis, Robert K. Nakamoto, dimer ribbons pack together with distinct crystal contacts in the helical versus David S. Cafiso. large two-dimensional crystals, which allow phospholamban differential access Chemistry, University of Virginia, Charlottesville, VA, USA. to potential sites of interaction with SERCA. Nonetheless, we show that a phos- TonB dependent transporters (TBDTs) are a family of outer membrane proteins pholamban oligomer interacts with SERCA in a similar manner in both crystal (OMPs) in Gram-negative bacteria that facilitate the active transport of nutri- forms. In the two-dimensional crystals, a phospholamban pentamer interacts ents across the outer membrane (OM). The energy for active transport is with transmembrane segments M3 of SERCA and participates in a crystal con- derived from inner membrane (IM) proton motive force (pmf) through a revers- tact that bridges neighboring SERCA dimer ribbons. In the helical crystals, an ible coupling to IM protein TonB. Our current understanding of the molecular oligomeric form of phospholamban also interacts with M3 of SERCA, though function of TBDTs is mainly based on high-resolution structural and biophys- the phospholamban oligomer straddles a SERCA-SERCA crystal contact. We ical characterizations in detergent purified and reconstituted lipid vesicles. Un- conclude that the pentameric form of phospholamban interacts with M3 of fortunately, TBDT has never been reconstituted and structural measurements SERCA, and that it plays distinct structural and functional roles in SERCA have never been made on an active transport system. Recent work has shown regulation. The interaction of the pentamer places the cytoplasmic domains that it is possible to spin label the OMP, BtuB in Gram-negative bacteria, of phospholamban at the membrane surface proximal to the calcium entry fun- 1 thereby enabling the study of TBDT systems using EPR spectroscopy. nel of SERCA. This interaction may cause localized perturbation of the mem- In our present work, we altered the labeling strategy to allow attachment of brane bilayer as a mechanism for altering the turnover rate of SERCA. methanethiosulfonate spin label in cells that are optimized for their metabolic activity. In these preparations we find that specific labeling of single cysteine 631-Pos residues on the extracellular surface of BtuB can be achieved under conditions Single-Molecule Studies of ATP Binding to the Sodium Pump where rapid vitamin B transport is taking place. Distance measurements using Sushi Madhira1, Don C. Lamb2, Promod R. Pratap3. 12 1 2 pulse EPR require spin label pairs, and to our surprise many cysteine pairs that Dept Chemistry, LMU, Munich, Germany, Dept Chemistry, Ludwig 3 are expressed in this system fail to label even though the corresponding single Maximilians Univ, Munich, Germany, Dept Phys/Astron, Univ North labels can be efficiently labeled. This problem is due to the redox homeostasis Carolina Greensboro, Greensboro, NC, USA. The sodium pump uses energy from the hydrolysis of 1 ATP to move 3Naþ out of in the periplasm and it can be overcome by the selection of appropriate mutant þ strains and labeling strategy. We have used this strategy to explore the supra- and 2K into the cell via transitions between phosphorylated and unphosphory- molecular organization of OMPs and to identify conformational states of BtuB lated forms of two major conformations (E1 and E2). In earlier experiments, we that have not been seen in purified protein.1. Joseph et al. (2015) Angew. Chem. had found that ATP binds with low and high affinity to the E1 conformation of Int. Ed. 54, 1-5.This work was supported by NIGMS GM-035215. pump isolated from nasal glands of juvenile ducks. We attributed this heteroge- neity to interactions between ATP binding sites on different protomers within 629-Pos multiprotomeric aggregates. We report here on experiments designed to examine Live-Cell FRET Biosensors for High-Throughput Screening Targeting the ATP binding to pump protomers using single-molecule fluorescence measure- SERCA2a/Plb Complex ments. Pump labeled with Cy3-maleimide was solubilized with C12E8. Proto- Dan Stroik, Samantha Yuen, Evan Kleinboehl, Kevyn Janicek, Tory Schaaf, mers were separated from aggregates using FPLC and reconstituted into lipid Razvan Cornea, David Thomas. vesicles at an average concentration of 1 protomer/vesicle. The fluorescence of Dept Biochem Mol Bio & Biophys, Univ Minnesota, Minneapolis, MN, USA. vesicles tethered to a glass cover slip using a biotin-streptavidin linkage was We have engineered cell-based high-throughput screening (HTS) methods using measured as a function of time using single-molecule total internal reflection time-resolved fluorescence energy transfer (TR-FRET) sensitive to binding and fluorescence microscopy under conditions where the pump was in the E1

BPJ 9322_9324 Sunday, March 3, 2019 129a conformation. Vesicles where fluorescence photobleached in a single-step were enzyme population towards the intermediate states, suggesting that the binding identified as containing single protomers. These traces were either dynamic (fluo- of the last Naþ (deep occluded state) is severely affected and confirming that rescence fluctuated between two states) or nondynamic (no fluctuations). The D923N disrupt the pump function by targeting a Naþ binding site. fraction of protomers that showed dynamic behavior increased saturably with 634-Pos ATP from 40% at 0 ATP to 70% at 1 mM (saturating) ATP. This titration D D Expression of the Na /K -ATPase Subunits in Adult Mouse Brain curve could not be fitted with a single binding isotherm. Analyzed by Single-Cell RNA-Seq Profiling A fit to a model that assumed a heterogeneous mixture of pump molecules with Song Jiao, Cristina Moreno Vadillo, Miguel Holmgren. high- and low-affinity ATP binding showed: (a) high- and low-affinity apparent NINDS, Bethesda, MD, USA. dissociation constants of 0.1 and 40 mM; and (b) that the preparation had equal þ þ Mutations in the Na /K -ATPase alpha 2 and alpha 3 subunits have recently amounts of high- and low-affinity forms of the pump. Our data suggest that been identified as the genetic cause of three neurological disorders: familial binding heterogeneity is an intrinsic property of the protomer and is not attrib- hemiplegic migraine, rapid-onset dystonia parkinsonism and alternating hemi- utable to aggregation. plegia of childhood. To fully understand the pathogenesis of these diseases, it is important to know the expression patterns of these different Naþ/Kþ-ATPase 632-Pos subunits within the brain regions and the types of brain cells of each region. The Brine Shrimp’s Fight against Hypersaline Environments Requires a To address this issue, we have begun investigating the mRNA expression pat- Na/K Pump with Reduced Stoichiometry terns at the single cell level using the newly available scRNA-Seq database of 1 1 1 2 Dylan J. Meyer , Victoria C. Young , Jessica Eastman , Jessica Drenth , mouse brain from Dr. McCarroll’s lab (Saunders et al., Cell. 2018 Aug 2 1 2 1 Abigail Benson , Kerri Spontarelli , Craig Gatto , Pablo Artigas . 9;174(4):1015-1030). We are examining the cellular heterogeneity in the 1Cell Physiology and Molecular Biophysics, Texas Tech University Health þ þ 2 expression of the different Na /K -ATPase alpha subunits among brain re- Sciences Center, Lubbock, TX, USA, Biological Sciences, Illinois State gions and local neural networks. This information will allow us to correlate University, Normal, IL, USA. expression patterns with brain circuits to classify and predict relevant cell pop- Brine shrimp (Artemia salina), are osmoregulators that survive in 4.5 M salt. ulations that are potentially important in the pathogenesis of these diseases. They express two Na/K pump isozymes formed by association of a b-subunit either with a ‘‘normal’’ a-subunit (a1NN), or with a special a-subunit (a2KK), 635-Pos which contains two asparagine-to-lysine substitutions in the ion-binding region. The Single Channel Configuration of Na/K Pump We introduced the equivalent substitutions (N333K and N785K) in the Xenopus Pengfei Liang, Jason Mast, Wei Chen. a1-subunit and functionally evaluated the mutants with electrophysiology in Dept Physics, Univ South Florida, Tampa, FL, USA. þ Xenopus oocytes. N785K reduced apparent affinity for K o, Both individual mu- We re-studied the dialyzed Na/K pumps, where both intracellular and extracel- þ tants reduced Na i apparent affinity. The change in ion affinities observed in lular Na were removed. In contrast to the charge-movement-like pump currents N333K/N785K were not the addition of individual mutants’ effects, indicating from previous study, we successfully observed the forward-only pump currents þ þ these residues are coupled. Simultaneous determination of 86Rb (a K in responding to an electric stimulation without the backward component by congener) uptake and charge extruded under two-electrode voltage clamp yielded elevating the external K concentration to five times higher than usual. The uni- þ ratios of Rb uptake/charge extruded of 2.1150.07 (n=40) for WT and directional pump currents imply that ions could still across the cell membrane un- þ þ 1.0750.04 (n=21) for N333K/N785K, indicating stoichiometries of 3Na :2K der dialyzed mode as long as applied energy is large enough to overcome the con- þ þ and 2Na :1K , respectively. We reared Artemia at 0.25 M and 4 M salt and eval- centration gradient. We also observed this uni-directional pump currents in the uated changes in Na/K pump-subunit mRNA expression. Compared to animals in absence of intracellular ATP, which demonstrated that it was not related to 0.25 M, Artemia at 4 M salt increased expression of a2KK (40-fold) and b (3- ATP hydrolysis induced protein conformational changes. A model of modified fold), while a1NN expression remained unaltered. Inhibition of a1NN by ouabain single-channel configuration is introduced to explain both the experimental re- (IC50100 mM) reveals a tenfold lower affinity than a2KK inhibition (IC50% sults: charge-movement-like pump currents and transmembrane pump currents. 10 mM) (Cortas et al. J. Memb. Biol. 108:187-195, 1989). The LD50 for larvae The negatively charged amino acids deeply inside the transmembrane domain (nauplii) incubated for 24-hr in ouabain was LD50300 mM at 0.12 M and could constitute a spatial matrix that function as an energy well. When the mov- LD5030 mM at 2 M salt, indicating that a2KK Na/K pumps are required for ing ions have enough energy that can overcome the ionic concentration gradient, high-salinity adaptation. Immunostaining of the salt-extrusion organs and guts they can pass through the energy well to cross cell membrane showing a single of Artemia show exclusive basolateral Na/K pump localization. Therefore, the channel configuration. If the energy is not enough, the energy well would entrap thermodynamic need for a Na/K pump with reduced stoichiometry must come the cations as if the channel is obstructed showing two separated access channels. from a very negative basolateral membrane potential. Attempts to measure this potential are currently underway. GM061583 & NSF/MCB-1515434. Posters: Cellular Signaling and Metabolic Networks 633-Pos D D D Extracellular Na Interactions in the WT hNa /K ATPase Alpha 3 and 636-Pos Alternating Hemiplegia of Childhood New Tools for Bacterial Biofilm Electrophysiology Cristina Moreno Vadillo, Miguel Holmgren. Alan L. Gillman1, Joseph W. Larkin1, Edgar Gutierrez2, NINDS, NIH, Bethesda, MD, USA. 3 2 1 þ þ þ Jordi Garcia-Ojalvo , Alex Groisman , Gurol M. Suel . The Na /K ATPase uses the energy of ATP hydrolysis to export 3Na from the 1 þ Division of Biological Sciences, Univ Calif San Diego, La Jolla, CA, USA, cell and import 2K . During the transport cycle the ions are temporarily occluded 2Department of Physics, Univ Calif San Diego, La Jolla, CA, USA, within the protein before being release to the opposite site. A functional ATPase 3Department of Experimental and Health Sciences, Universitat Pompeu is composed by the transporter alpha- and the modulatory beta-subunit. Four Fabra, Barcelona, Spain. alpha (A1-4) and three beta (B1-3) isoforms have been found differentially ex- Recent studies have revealed that bacterial biofilms are electrically active tis- pressed across the human body suggesting tissue-specific functions. sues with spatially organized heterogeneous electrical activity. Indeed, biofilms Mutations in ATP1A3 gene, are linked to Alternating Hemiplegia of Childhood rely on ion channel mediated electrical cell-to-cell signaling to coordinate (AHC) a neurological disease in which transient attacks of hemiplegia occur. many critical activities. Electrical perturbation of biofilms would open new The molecular mechanism of AHC is not well understood. Based on the protein windows of understanding into molecular biology and electrical signaling. structure, it was proposed that AHC-mutations alter the ATPase function by However, there is limited availability of tools for this purpose. Here, we present affecting the ion binding site. However, a functional validation is needed. our latest progress in the development of new tools and techniques for highly Here we studied 1)the details of the beta-dependent modulation and 2)the effect localized direct stimulation of bacterial biofilms. of AHC-mutations on the de-occlusion and release of individual Naþ of the hNaþ/KþATPase-A3. For that, Xenopus Oocytes were injected with WT or 637-Pos mutant hNaþ/Kþ ATPase-A3 mRNA plus B1, B2 or B3 in a 1:1 molar ratio Predicting TGF-b-Induced Epithelial-Mesenchymal Transition using Data and the Naþ currents were evaluated by the Cut-Open Vaseline Gap technique. Assimilation A temporal correlation between the three distinct and sequential events repre- Mario J. Mendez1, Matthew J. Hoffman2, Elizabeth M. Cherry3, senting the de-occlusion and release of individual Naþ was found in all A3-B Christopher A. Lemmon1, Seth H. Weinberg1. combinations. Each B-subunit modulated the three distinct release events in a 1Biomedical Engineering, VCU, Richmond, VA, USA, 2School of slightly different way. Interestingly, B1 poisoned the distribution of the Naþ/ Mathematical Sciences, Rochester Institute of Technology, Rochester, NY, KþATPase conformation towards the deep occluded states. On the other end USA, 3College of Science, Rochester Institute of Technology, Rochester, of the spectrum, the AHC-mutation (D923N) shifted the distribution of the NY, USA.

BPJ 9322_9324 130a Sunday, March 3, 2019

Epithelial-mesenchymal transition (EMT) is a fundamental biological process hypertrophy and fibrosis were assessed. With mdivi1, cardiac hypertrophy that plays a central role in embryonic development, tissue regeneration, and can- was suppressed (ratio of heart/body weight (10-3): 4.0 5 0.06 with mdivi1, cer metastasis. The main characteristic of EMT is the transdifferentiation of an P< 0.01 vs 4.8 5 0.15% without mdivi1, intraventricular thickness: 1.42 5 epithelial cell to a mesenchymal cell, which includes losing epithelial-type cell- 0.05mm with mdivi1, P< 0.05 vs 1.93 5 0.05mm without mdivi1), whereas cell adhesion and gaining the mesenchymal-type enhanced cell motility. Trans- the blood pressure was unchanged. Mdivi1 inhibited not only myocytes hyper- forming growth factor-b (TGF-b) is a major and potent inducer of this cellular trophy (HE-staining: 1.1 5 0.05mm2 with mdivi1, P< 0.05 vs 1.6 5 0.04mm2 transition, which is comprised of two state transitions, first from an epithelial without mdivi1) but also cardiac fibrosis (Azan-staining: 3.3 5 0.3% with state to an intermediate or partial EMT state, then from the partial state to a mdivi1, P< 0.05 vs 15.5 5 2.7% without mdivi1). In addition, mdivi1 reduced mesenchymal state. Experimentally, it is typically not possible to observe the Drp1 expression and ROS production (1.1 5 0.1mM with mdivi1, P< 0.01 more than two EMT cell markers at the same time, which makes predicting vs 2.3 5 0.5mM without mdivi1). Mdivi1 can also suppress the Calcineurin, the timing of state transitions inherently difficult. Here, we propose a data- CaMK2, proteins of hypertrophy. H9c2 myocytes were transfected with siRNA assimilation approach, which combines limited noisy observations with predic- Drp1 and treated with 5mM isoprenaline shows the same tendency. Conclusion: tions from a computational model of TGF-b-induced EMT, to reconstruct the To conclude, Drp1 is related to hypertension-induced LVH, myocyte hypertro- full experimental system and predict the timing of the partial-to-mesenchymal phy, and fibrosis. Mdivi1 and siRNA Drp1 suppress them by inhibiting calci- state transition. We tested our approach in proof-of-concept ‘‘synthetic’’ in sil- neurin and CaMKII through ROS reduction without altering the BP. ico experiments, in which experimental observations were produced from a computational model with the addition of noise. We varied several properties 640-Pos of the data-assimilation approach, including which EMT markers were observed Quantification of Dynamic Glucokinase Regulation in Islets using a Homo- and the time interval between observations. We found that under ideal condi- transfer FRET Reporter tions, the partial-to-mesenchymal transition time could be predicted after 1 Shenq Huey Wong. day of observations, approximately 11 days before the transition, and further University of Maryland School of Medicine, Baltimore, MD, USA. that the protein SNAI1, its inhibitor miR34, epithelial-state marker E-cadherin, Conversion of glucose to glucose-6-phosphate by glucokinase is the rate- and mesenchymal-state marker N-cadherin were the most effective markers for limiting step in pancreatic beta-cell glucose metabolism and couples insulin observation. Additionally, we found that decreasing the time interval between release to elevated blood glucose levels. Glucokinase activity is regulated by ni- observations typically reduced prediction error. Future work will include testing tric oxide, which induces a conformational shift in the protein that facilitates and optimizing our approach over a wider range of physiological conditions and glucose phosphorylation. Even so, central questions remain concerning regula- ultimately testing against in vivo experimental data. tion of glucokinase activity in pancreatic islets. Here, we describe quantitative time-lapse imaging of islets obtained from a transgenic mouse that expresses a 638-Pos glucokinase homotransfer Fo¨rster resonance energy transfer (FRET) reporter. Signalling Growth through Lipid Kinases Characterization of FRET anisotropy patterns of the biosensor in the mouse islets Sanjeev Sharma1, Swarna Mathre1,2, Visvanathan Ramya1, shows spatiotemporal dynamics of glucokinase regulation between cells. Imag- Dhananjay Shinde1, Padinjat Raghu1. ing of the isolated islets and single-cell from this mouse line by polarization epi- 1National Centre for Biological Sciences, Bengaluru, India, 2Manipal fluorescence microscopy demonstrates real-time activation of the glucokinase University, Manipal, Karnataka, India. sensor when the cells are stimulated with forskolin and IBMX to increase cellular Phosphoinositides form a group of signalling glycerophospholipids whose phys- cAMP. The glucose-stimulated response of mouse islets and single cells are also iological function in cells is closely linked to their spatial distribution and turn- assessed by NAD(P)H autofluorescence in the cells. Correlation between the over. Cells employ a network of enzymes to robustly maintain basal lipid levels fluorescence glucokinase biosensor activity and responsiveness of the cells to and modulate their activity in response to a multitude of incoming signals. How- glucose reveals cell-to-cell variability in the activation of glucokinase. Differen- ever, the mechanisms governing these molecular interactions are unclear. Our tial stimulation of glucokinase in live cells may suggest the existence of distinct study describes how during growth factor stimulation, the cross-talk between islets population that shows potential for metabolic pacemaking. two enzymes linked to the turnover of two very low abundance lipid species me- diates optimal cellular growth signalling. Upon insulin receptor activation, Class 641-Pos Prediction of Metabolite Concentrations, Rate Constants and Post- I PI3-Kinase (PI3K) generates phosphatidylinositol 3,4,5-trisphosphate (PIP3) at the plasma membrane, which directs key downstream signalling events lead- Translational Regulation of Neurospora Crassa using Maximum Entropy Optimizations and Reinforcement Learning ing to cell growth and proliferation. We found another lipid-kinase PIP4K, 1 2 2 which controls the level of phosphatidylinositol 5-phosphate (PI5P), was linked William R. Cannon , Samuel R. Britton , Mikahl Banwarth-Kuhn , Mark Alber2, Jennifer M. Hurley3, Meaghan S. Jankowski3, to the levels of PIP3. Biochemical studies indicate that PIP4K can use PI5P to 1 1 1 1 produce the substrate (PIP ) for Class I PI3K to produce PIP . However, imaging Jeremy D. Zucker , Douglas J. Baxter , Neeraj Kumar , Scott E. Baker , 2 3 4 using lipid-binding probes and high-sensitivity mass spectrometry measure- Jay C. Dunlap . 1Pacific Northwest National Lab, Richland, WA, USA, 2Department of ments showed inverse correlations of the PIP3 with PIP4K protein levels in cells, 3 suggesting that a simple substrate channelling mechanism was insufficient to Mathematics, University of California, Riverside, CA, USA, Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY, USA, link the activity of these kinases. Using a combination of genetics and micro- 4 scopy in Drosophila cells we found that PIP4K functions along with an addi- Geisel School of Medicine at Dartmouth, Hanover, NH, USA. tional negative feedback loop to control PIP levels. This was further Nature selects those organisms that can reproduce the fastest while using en- 3 ergy from the environment as efficiently as possible. This problem can be corroborated by quantitative live-cell imaging of PIP3 dynamics ex vivo in sali- vary glands where PIP4K regulated class I PI3K activity. Finally, we show that formulated as a maximum entropy production rate problem that includes exper- PIP4K functions at the plasma membrane, proximal to receptor activation. Over- imental and physical constraints. We report the application this approach for all, the study proposes that receptor activation can directly trigger opposing ac- modeling biological systems in lieu of a priori, in vivo rate constants. The tivities to ensure controlled signal transduction at short time scales. Future method is applied in four steps: (1) a new constrained optimization approach studies will involve quantitative modeling and experimental approaches to un- based on Marcelin’s mass action equation is used to obtain the maximum en- derstanding the how these kinases are connected spatio-temporally. tropy distribution, (2) the predicted metabolite concentrations are compared to experimental data using a loss function from which post-translational regu- 639-Pos lation of enzymes is inferred, (3) the system is re-optimized with the inferred Dynamine-Related Protein 1 (Drp1) Contributes to Hypertensive Cardiac regulation from which rate constants are determined from the metabolite con- Hypertrophy and Fibrosis In Vivo and In Vitro Model centrations and reaction fluxes, and finally (4) a full ODE-based, mass action Prottoy Hasan. simulation with allosteric regulation is inferred using reinforcement learning. Cardiology, Internal Medicine 3, Hamamatsu University School of Medicine, The method is applied to the central metabolism of Neurospora crassa and Hamamatsu, Japan. the flow of material through the three competing pathways of upper glycolysis, Purpose: Hypertension can promote the cardiac hypertrophy, which finally the non-oxidative pentose phosphate pathway, and the oxidative pentose phos- leads to heart failure. Aberrant mitochondrial dynamics is known to be relevant phate pathway are evaluated as a function of the NADP/NADPH ratio. The with heart diseases, however little is known about Drp1, a mitochondrial fission simulation results are compared to experimental transcriptomics and prote- protein, on cardiac hypertrophy. We studied the effects of mdivi1 (an inhibitor omics data obtained over the circadian cycle of Neurospora. The experimental of Drp1) on the hypertension-induced cardiac hypertrophy.Methods & Results: data demonstrate that the rhythmic proteins within the Pentose-Phosphate Dahl salt sensitive rats of 7 weeks-old were fed with a chow containing high pathway peak in the circadian morning, while conversely, in glycolysis and salt (8%) with and without administration of mdivi1 (1mg/kg, every alternative the TCA cycle, the rhythmic proteins peak in the circadian evening. That is, day, via an intraperitoneal administration) and at 15 weeks of age the cardiac the rhythmic proteins of glycolysis are in anti-phase to the rhythmic proteins

BPJ 9322_9324 Sunday, March 3, 2019 131a of the Pentose-Phosphate pathway. Furthermore, it is predicted that regulation and chronic cardiac stress. To test these hypotheses, we employed transcriptome of phosphofructokinase (PFK) is essential for preventing an extreme level of (RNASEQ and RT-QPCR) of SAN and LV tissues and proteome analysis (mass fructose 1, 6-bisphophate accumulation. spectrometry) of LV tissue TGAC8 and wild type littermates. We identified 13,536 transcripts in SAN and 11,829 in LV. 542 transcripts were 642-Pos differentially expressed (q<0.05) in SAN and 2334 in LV WT vs TGAC8. Pro- Toward a Multiscale Model of Valvular Interstitial Cells: An Integrin- teomics identified 6,937 LV proteins, 2,190 of which were expressed differen- Mediated Mechanotransduction Module tially (p<0.05) in TG vs. WT. 845 proteins changed their expression by >15%. Daniel P. Howsmon, Michael S. Sacks. There was an 80% concordance in the direction of expression of 596 differen- Willerson Center for Cardiovascular Modeling and Simulation, The tially expressed proteins and transcripts. We found in TGAC8 mice shift in num- University of Texas at Austin, Austin, TX, USA. ber of signaling networks including increased signaling of Caþþ cycling and Valvular interstitial cells (VICs) are the primary cell type residing within the surface membrane ion channels, ROS, Nrf-2 antioxidant signaling and auto- heart valves. Under homeostatic conditions, these cells exhibit a quiescent, phagy, Janus-Kinase signaling; NADþ salvage pathways activation; activation fibroblast-like phenotype. However, alterations in the microenvironment, of ER-stress and integrin signaling. Thus, the TGAC8 heart copes with chronic including increased tissue stresses, can promote a transition to an activated, stress increase intrinsic sympathetic stimulation via altered transcription and myofibroblast-like state. While this transition allows heart valves to maintain translation of large number of proteins. unidirectional blood flow during periods of growth and development, it is also a primary pathophysiological mechanism that leads to heart valve disease. 645-Pos Previous models have investigated the tissue and cellular biomechanics of The Therapeutic Implications of Protein Kinase C Inhibition in Endothe- VICs; however, a critical step linking cellular biomechanics to mechanotrans- lial Dysfunction Induced by Cardioplegic-Ischemia/Reperfusion Injury duction is currently lacking. Therefore, a mathematical model of integrin- Justin Kim, Guangbin Shi, Amy Zhao, Frank Sellke, Jun Feng. mediated mechanotransduction was developed to bridge this gap and provide Surgery, Brown (Alpert) Medical School, Providence, RI, USA. key mechanistic insight to existing VIC biomechanics models. Initial steps to- Cardioplegic-ischemia/reperfusion (C-I/R) injury, a condition that arises in car- ward a holistic multiscale model of VICs that incorporates both cellular me- diac surgery patients, triggers endothelial dysfunction, which can cause life- chanics and signal/mechanotransduction will be discussed. threatening cardiac conditions post-surgery. Dramatic increase in intracellular mitochondrial Reactive Oxygen Species (mROS), which prompt endothelial 643-Pos apoptosis, plays a leading role in C-l/R-injury-induced endothelial dysfunction. Characterization of the Contribution of Retinoic Acid Receptor Isoforms Unfortunately, the risk of C-l/R-injury-induced-morbidity and mortality is in the Suppression of Cardiac Hypertrophy elevated for type-2 diabetes mellitus patients. Due to the well-known regulatory Lauren Parker, Ni Yang, Brian O’Rourke, D. Brian Foster. nature of PKC in mROS-producing pathways, the protective effects of PKC in- Johns Hopkins University, Baltimore, MD, USA. hibition against simulated endothelial C-I/R injury were investigated in vitro us- Pathway analyses of proteomic studies in guinea pig and rat hearts subjected to ing a Human Coronary Artery Endothelial Cell (HCAEC) model. Hypoxia was pressure overload-induced hypertrophy and heart failure (HF) suggest altered induced in nondiabetic (ND) and diabetic (DM) HCAEC followed by reoxygena- retinoid signaling may contribute to HF progression. In recent studies, we tion in St. Thomas’ cardioplegia solution with and without 0.5 mM general PKC showed that cardiac levels of the hormone all-trans retinoic acid (ATRA) are inhibitor, chelerythrine, pretreatment. Levels of mROS, apoptosis, and necrosis deficient in human non-ischemic HF patients. This has prompted us to revisit were detected using mitoSOX and Apoptosis/Necrosis fluorescent staining, visu- an old, but poorly understood, observation that ATRA suppresses phenyleph- alized using a Nikon Eclipse TE2000-U Inverted Microscope, and quantified via rine (Phe)-induced hypertrophy in neonatal rat ventricular myocytes (NRVMs). ImageJ. In the presence of chelerythrine, MitoROS and apoptotic levels were Using cross-sectional area (CSA) as an index of hypertrophy, we show, consis- significantly reduced (p < 0.05) for the ND group, suggesting therapeutic poten- tent with prior work, that ATRA suppresses Phe-induced increases in NRVM tial of PKC inhibition to mitigate/prevent C-I/R injury. Interestingly, chelerythr- CSA by average of 70%. We then used the pan-CYP26 inhibitor talarozole ine pretreatment showed no significant improvement in endothelial protection for (Tala) to raise endogenous ATRA levels, and observed that Tala similarly in- the DM group. This phenomenon may be explained by the non-specific nature of hibited Phe-induced increases in NRVM CSA (p<0.001). ATRA exerts its bio- chelerythrine; certain PKC isotypes (e.g. )3 exhibit cardioprotective effects when logical actions through a class of ligand-dependent transcription factors called activated. Thus, future investigations include investigating PKC isotype-specific retinoic acid receptors (RARs), of which there are three forms, a, b and g.We inhibitors and their endothelial protective effects. examined the impact of RARa, RARb, and RARg antagonists on ATRA and Tala mediated hypertrophy suppression. The RARb antagonist LE135 (1 m 646-Pos M) fully blocked the action of ATRA (p<0.0001), while initial studies with Splitting Up: Finding a New Way to Monitor Mitochondrial CaMKII Us- the RARa and RARg antagonists (BMS 195614 (1 m M) and MM 11253 (1 ing SplitGFP m M)) failed to significantly mitigate CSA suppression. Thus, initial pharmaco- Kevin R. Murphy1, Qinchuan Wang1, Jonathan Granger1, Gianna Bortoli2, Jinying Yang1, Xi Zhang1, Elizabeth Luczak1, Rong Li1, Mark E. Anderson1. logic studies indicate ATRA elicits an anti-hypertrophic program, at least in 1 2 part, through activation of RARb. Interestingly, when experiments were Medicine, Johns Hopkins Medicine, Baltimore, MD, USA, Macalester repeated using Tala, hypertrophy inhibition appeared to be mediated by a com- College, St Paul, MN, USA. bination of RARb and RARg. Ongoing experiments are aimed at corroborating Important signaling events are specified by subcellular targeting, but current these observations in NRVMs virally-transduced with siRNA to each of RAR approaches may be inadequate to measure these events in living cells or in isoform. These studies will provide key insights to delineate the mechanisms by vivo. CaMKII (calcium/calmodulin-dependent protein kinase II) is an impor- which ATRA dysregulation contributes to HF pathogenesis. tant mediator of cellular signaling, calcium homeostasis, heart failure, and le- thal cardiac arrhythmias. There are two major CaMKII isoforms in the heart: g 644-Pos and d. Despite the broad functionality CaMKII has in regulating the cell, the Transcriptome and Proteome Alterations of Mice that Overexpress Ad- availability of sensitive and specific tools to dynamically monitor CaMKII in enylate Cyclase Type 8 Underlie a Chronic and Marked Increase in Sino- subcellular compartments is limited. Recently, CaMKII was reported to reside atrial Node (SAN) and Left Ventricle (LV) Performance While Ensuring in mitochondria where it mediates calcium homoeostasis and redox status in the Heart Survival organelle. Current methods of mitochondrial CaMKII detection rely on anti- Kirill Tarasov1, Khalid Chakir1, Yelena Tarasova1, body based detection that cannot reliably distinguish between isoforms, and Yevgeniya Lukyanenko1, Alexey Lyashkov2, Edward G. Lakatta1. in some cases lack specificity. Here we developed a genetic reporter using 1LCS, NIA/NIH, Baltimore, MD, USA, 2TGB, NIA/NIH, Baltimore, MD, SplitGFP to track mitochondrial CaMKII (mitoCaMKII) accumulation. Our USA. strategy ‘splits’ self-complementing GFP, comprised of 11 b-strands, into Cardiac specific overexpression of adenylate cyclase type 8 in the mouse heart two parts. First, GFP strands 1-10 were fused with a mitochondria localization (TGAC8) at 3 m.o. of age, induces a chronic increase in heart rate and contrac- sequence (MTS) and mCherry tag. Second, the GFP strand 11 was fused with tility, that mimics sympathetic stimulation induced by b-adreno receptor CaMKIId. When both components are localized in the mitochondria the GFP (BAR) activation, but emerges in the absence of BAR activation. Chronic sym- self-complements and fluorescence reconstitutes. Using the excitable human pathetic stimulation creates a marked stress on the heart that results in several retinal epithelial cell line (RPE1) we found that our CaMKIId-SplitGFP re- adverse sequelae including chronic cardiac hypertrophy, failure and arrythmias. porter is a sensitive marker of mitoCaMKII. Further, our reporter is capable We hypothesized that a coherence of numerous, diverse molecular signaling net- of detecting dynamic increases of mitoCaMKII in response to b-adrenergic works become activated not only to sustain increase induced by intrinsic Ad- stress. These data suggest that CaMKII-SplitGFP is a useful tool in monitoring þþ enylyl Cyclase (AC)-cAMP-PKA-Ca signaling that drives chronic increase the dynamics and accumulation of mitoCaMKII, expanding the previously TGAC8 heart performance, but also, to ensure cardiac survival during this marked limited repertoire of CaMKII detection methods.

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647-Pos granules in PC12 and A10 cells. PLCb is activated by the Galpha(q) family of G Minimizing the Number of Measurements Required to Predict a Pheno- proteins in response to hormones and neurotransmitters. We find that 30% of typic Landscape in Bacterial Folate Metabolism the proteins associated with cytosolic PLCb1 are markers for stress granules Andrew D. Mathis1, Judith Boldt2, Kimberly A. Reynolds3. including FXR1/2, eIF4, eIF5a, PABPC1 and Ago2. PLCb1 directly binds to 1Green Center for Systems Biology, University of Texas Southwestern eIF5A and also to Ago2, whose role in RNA-induced silencing is halted under Medical Center, Dallas, TX, USA, 2Center for Interdisciplinary Research in stress. PLCb1-Ago2 complexes are disrupted when Gaq is activated allowing Biology, Colle`ge de France, Paris, France, 3Department of Biophysics and Ago2 to move into particles. Lowering the cytosolic level of PLCb1 by siRNA, Green Center for Systems Biology, University of Texas Southwestern Galpha(q) activation or environmental stress (osmotic, cold and heat shock) in- Medical Center, Dallas, TX, USA. creases the size and number of stress granules, and a shift in RNA size to longer A major difficulty when predicting phenotype is that the effect of a mutation lengths. Down-regulating PLCb1 alters the production of a heat shock protein can be dependent on the genetic and environmental background in which it supporting the idea that PLCb1 alters stress responses. Mechanistically, our re- is made. Further, comprehensively quantifying these dependencies or epistases sults fit a simple model in which eIF5a and its associated proteins partition into in all possible backgrounds is simply intractable. So, how can this problem be particles after release from PLCb1. Taken together our studies show a commu- addressed? Some promising solutions are mathematical algorithms that use nication network between environmental signals and stress responses through relatively small sets of incomplete information to approximate large datasets. PLCb1. These strategies include (1) Bliss independence-like models that may be able to predict higher order epistasis from lower order terms, and (2) compressed 650-Pos sensing to find the smallest random sampling of phenotypes that can be used Systems Biology of Control and Regulation of Substrate Selection in Cyto- to accurately approximate the whole dataset. Though these mathematical stra- plasmic and Mitochondrial Catabolic Networks tegies are relatively well established, it has been challenging to collect the data Sonia Cortassa, Miguel A. Aon, Steven J. Sollott. required to test them at the level of genetic and environmental backgrounds. To Lab Cardiovasc Sci, Natl Inst Aging, NIH, Baltimore, MD, USA. collect this data, I developed a high-precision growth assay that combines Substrate selection between fats and glucose is associated with the success CRISPRi gene knockdowns, to manipulate genetic background; continuous of interventions that maintain health (e.g., exercise or caloric restriction), culture, to modulate environmental background; and next-generation or with the severity of diseases (e.g., diabetes or other metabolic disorders). sequencing, to measure thousands of growth phenotypes simultaneously. Using Although the interaction between glucose and fatty-acids (FAs) catabolism this technique, I am currently measuring epistasis between gene pairs, genes has been studied for decades, a quantitative and integrated understanding and environments, and multiple different environments. More specifically, I of the control and regulation of substrate selection through central catabolic am measuring 2nd order epistasis between all possible pairs of 22 enzymes in pathways is lacking. We addressed this gap here using a computational E. coli one-carbon folate metabolism. These measurements are being made model representing cardiomyocyte catabolism encompassing glucose (Glc) in all combinations of 5 critical metabolites produced by the pathway, totaling utilization, pyruvate oxidation in the tricarboxylic acid (TCA) cycle, b to 32 environments. The resulting dataset will contain more than 30,000 pheno- -oxidation of palmitate (Palm) and oxidative phosphorylation in cyto- type measurements. Collection and analysis of this data will provide one bench- plasmic and mitochondrial compartments. The model is described by 82 dif- mark towards determining a minimum number of measurements required to ferential equations and 119 enzymatic and transport reactions accounting for approximate a phenotypic landscape. regulatory mechanisms and key players such as pyruvate dehydrogenase (PDH). We applied metabolic control analysis to the network operating 648-Pos with various ratios of Glc to Palm. The flux and metabolites’ concentration The Role of Pool Size Measurements in Improving Flux Estimations in control were visualized through heat maps providing major insights into Non-Stationary Metabolic Flux Analysis main control and regulatory nodes throughout the catabolic network. Meta- Anna Sher1, Daniel Fridman2, Jamey Young3, Cynthia J. Musante1. bolic pathways located in different compartments reciprocally control each 1Internal Medicine Research Unit, Pfizer Inc., Cambridge, MA, USA, 2Dept. other. For example, glucose uptake and phosphofructokinase (PFK) control of Applied Mathematics, Dept. of Molecular Biochemistry and Biophysics, most processes in catabolism while TCA cycle activities and membrane- Yale University, New Haven, CT, USA, 3Dept. of Chemical and associated energy transduction reactions exert control on all mitochondrial Biomolecular Engineering, Dept. of Molecular Physiology and Biophysics, processes. PFK and PDH, two highly regulated enzymes, exhibit opposite Vanderbilt University, Nashville, TN, USA. behavior from a control standpoint. While PFK activity is a main rate- Over the past several decades, Metabolic Flux Analysis (MFA) has gained controlling step affecting the whole network, PDH plays the role of a major importance in the biomedical and pharmaceutical sciences. MFA is based on regulator showing high sensitivity (elasticity) to substrate availability and a computational procedure used to determine metabolic reaction rates (fluxes) key activators/inhibitors. Overall, these results indicate that control across and in some cases metabolite concentrations (pool sizes) within metabolic path- central catabolism is highly distributed suggesting that fuel selection be- ways where direct observation and measurement of intracellular function is not tween FAs and Glc goes well beyond the mechanisms traditionally postu- feasible. Applying 13C MFA to biological data, such as that in liver meta- lated to explain the glucose-FA cycle. bolism, helps to determine, for example, the fate of glucose in regulatory meta- bolic pathways under physiological and pathophysiological steady state Posters: Optical Microscopy and Superresolution conditions of a cell. However, designing models with reliable flux estimations remains a challenge. A debate that still persists is whether metabolite pool size Imaging I measurements added as constraints in optimization algorithms improve meta- bolic flux predictions. 651-Pos We studied the role of pool sizes using simple model networks with total of 8 Quantifying FRET Efficiency between Fluorescent Proteins using Fluores- and 9 metabolites. To investigate the effect of pool size constraints on flux es- cence Polarization Microscopy timations, we set up a series of scenarios varying inputs into the model for Vishnu Rao. several metabolites (such as information on pool size measurements and noise Physiology, University of Maryland School of Medicine, Baltimore, MD, levels in the mass isotopomer distribution (MID) labeling data). Our findings USA. indicate that in models when a full set of MID labeling data (as oppose to Fo¨rster resonance energy transfer (FRET) between fluorescent proteins can be just M0 labeling data) is available, pool sizes become dispensable in flux esti- measured using fluorescence polarization microscopy. FRET refers to the energy mations. Moreover, in biological networks where a full set of labeling data for transfer that occurs from an excited donor to an acceptor when the two molecules certain metabolites is accessible but the exact model network is unknown, flux are in close proximity. The molecules involved in the energy transfer can be the estimations are more reliable if pool sizes are not included as model constraints. same, homotransfer, or different, heterotransfer. If plane polarized light is used to excite donor fluorescent proteins, the energy transferred to acceptor fluores- 649-Pos cent proteins can be observed via a decrease in the emission polarization. Phospholipase Cbeta Regulates Stress Granule Formation Although changes in polarization can be quantified, conversion to FRET effi- Suzanne F. Scarlata1, Lela Jackson1, Androniqi Qifti2, Osama Garwain1. ciency, the percent of excitation events where energy is transferred, has been 1Dept Chem/Biochem, Worcester Polytech Inst, Worcester, MA, USA, difficult. Both homo- and heterotransfer sensors have been used to measure 2Worcester Polytechnic Institute, Worcester, MA, USA. enzyme activation, protein aggregation, and calcium levels. Here we derive During adverse environmental conditions, mammalian cells regulate protein equations for converting homo- and heterotransfer polarization changes to production by sequestering the translation machinery in membraneless organ- FRET efficiencies following Fo¨rster’s original derivation of the quantitative the- elles (i.e. stress granules) whose formation carefully is regulated. Here, we ory of non-radiative energy transfer. Measurements of heterotransfer FRET ef- show that phospholipase Cbeta (PLCb) prevents premature formation of stress ficiency using our approach match well with spectral and lifetime measurements.

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Efficiencies calculated from polarization changes fall within 10% error of spec- 654-Pos tral and lifetime efficiencies. Further, our solution for homotransfer FRET effi- Automated Single Molecule Clustering of Superresolution Data as an N- ciency agrees with the previous derivations. These formulations will be Body Problem especially useful in applications where conversion to FRET efficiency is advan- Peter K. Relich, Shreyasi Thakur, Melike Lakadamyali. tageous such as determining molecular distances and cluster sizes. Dept Physiology, University of Pennsylvania, Philadelphia, PA, USA. Proteins form molecular complexes in cells that are critical for cellular pro- 652-Pos cesses such as signal transduction, cellular metabolism, and muscle contrac- Unveiling the Inhibitory Synapse Organization Using Superresolution tion. Single molecule based super-resolution microscopy methods such as Microscopy STORM or PALM can resolve the clustered state of these proteins with high 1,2 3 3 Silvia Scalisi , Andrea Barberis , Enrica Maria Petrini , spatial resolution. These methods use photoswitching or on/off binding of flu- 1 1,2 Francesca Cella Zanacchi , Alberto Diaspro . orophores to achieve a sparse image of single molecules in a densely labeled 1Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy, 2Department of 3 sample. The molecules are localized iteratively over time to build a high- Physics, University of Genoa, Genoa, Italy, Neuroscience and Brain resolution image, which is pointllistic in nature. An automated and robust sin- Technologies, Istituto Italiano di Tecnologia, Genoa, Italy. gle molecule clustering algorithm is required to rigorously analyze these single The advent of super-resolution microscopy provided both a substantial improve- molecule data in a quantitative manner. We propose a clustering algorithm ment of the spatial resolution and the possibility to perform quantitative mea- inspired by Symplectic N-Body integrators implemented in modern celestial surements at a nanometric level. In particular, single-molecule localization mechanics simulations. This approach is scalable for big data applications (SML) techniques provide a powerful tool to answer biological questions that and provides a physical definition for segmented clusters. The performance require the observation of subcellular structures. Quantitative single-molecule of this method is compared with a Voronoi based clustering algorithm on simu- analysis allows quantifying the number and observing the distribution of mole- lated and biological data using external and internal validation metrics. cules in several biological systems beyond the diffraction limit [1]. In the last few years, many computational methods employing clustering analysis algorithms 655-Pos [2] have been developed to extract quantitative information from SML data sets. Cusp Artifacts in High Order Superresolution Optical Fluctuation Imag- In neuroscience, SML has been exploited to achieve a detailed characterization ing (SOFI) of density and spatial organization of synaptic proteins [3]. Recently, it has Xiyu Yi, Shimon Weiss. been reported that under plasticity conditions, chemically induced by long- Chemistry and Biochemistry, Univ Calif Los Angeles, Los Angeles, CA, USA. term potentiation (iLTP) of inhibitory synapses, GABAA receptors are immo- Since its introduction, Super-resolution Optical Fluctuation Imaging (SOFI) bilized and confined at synapses in cultured hippocampal neurons. iLTP has attracted interest due to its simplicity and affordability. Many applications, expression relies on the recruitment and accumulation of the scaffold protein methodology enhancements, and derivative methods have since been intro- gephyrin at synaptic areas [4], thereby enhancing the clustering of synaptic duced. One of the most important and attractive features of SOFI is its high or- GABAA receptors and potentiating GABAergic synaptic currents [5]. der cumulant image reconstruction, which could potentially yield unlimited In our work, we use stochastic optical reconstruction microscopy (STORM) com- resolution enhancement. bined with clustering analysis to study the nanoscale distribution of the inhibitory Practical application of high order ( higher than 4) SOFI has been, however, synapse, in particular to count both scaffold postsynaptic proteins (i.e. gephyrin) limited. The reasons for such limitation have never been fully understood and GABAA receptors during iLTP. Furthermore, quantitative super-resolution (nor fully discussed). In this work, careful analysis of the artifacts of high order methods employing DNA origami [6] as a calibration standard allow insights SOFI cumulants is provided. We have identified that there is a portion of highly in proteins copy number and their distribution at the inhibitory synapse. non-intuitive artifacts (dubbed here as ‘cusp’ artifacts) hidden in the high order [1] Deschout H. et al., Nature Methods. (2014) SOFI cumulants, which have been overlooked in previous studies. A series of [2] Nicovich P. R. et al., Nature Protocols. (2017) realistic simulations were performed to study cusp artifacts as a function of [3] Specht C. G. et al., Neuron. (2013) blinking statistics, the spatial distribution of photophysical properties of the [4] Pennacchietti F. et al., Journal of Neuroscience. (2017) sample, the total number of frames processed per dataset, photobleaching, [5] Petrini E. M. et al., Nature Communication. (2014) and noise. Experiments, simulations, and theory all show that high order cumu- [6] Pischedda F. et al., Molecular & Cellular Proteomics (2014) lants and odd-order moments could suffer from cusp artifacts. We demonstrate that cusps-artifacts could be altogether eliminated by utilizing even-order mo- 653-Pos ments constructed from cumulants for image reconstruction. Together with dy- Super-Resolution Microscopy Reveals the Molecular Architecture of namic range compression, these approaches yield improved SOFI images at the Centriole Subdistal Appendages and Its Role in Microtubule/Golgi cost of reduced theoretical resolution gain. Our study provides new insight into Anchoring the nature of high order SOFI cumulants, outlines guidelines for developing and Weng Man Chong, T Tony Yang, Jung-Chi Liao. screening SOFI-optimized fluorescence probes, and suggests improved strate- IAMS, Academia Sinica, Taipei, Taiwan. gies for SOFI data acquisition. The subdistal appendages (SDAs) are centriolar structures important to microtu- bule (MT) nucleation and anchoring. Recent studies suggest its role in linking 656-Pos submerged primary cilia to Golgi. Despite its importance, little is known about Comprehensive Fluorophore Blinking Analysis Platform as a Prerequisite their assembly geometry and structure-function relationship. Here, we use for Palm Data Interpretation dSTORM superresolution microscopy to resolve the organization of multiple Benedikt K. Rossboth1, Rene Platzer2, Florian Baumgart1, SDA proteins and centriole-associated proteins in mammalian cells. dSTORM Hannes Stockinger2, Gerhard J. Schuetz1, Johannes B. Huppa2, imaging reveals that ODF2, a core SDA protein, localizes closed to the centriole Mario Brameshuber1. wall while Cep128 and centriolin form ring-like structure building upon it. 1Institute of Applied Physics, TU Wien, Vienna, Austria, 2Institute for CCDC68, Cep170 and Ninein are two layered structures with one layer in the Hygiene and Applied Immunology, Medical University of Vienna, Vienna, SDA region (the SDA layer) surrounding the core SDA proteins and the other Austria. layer residing close to the centriole proximal end (the proximal layer). Dual- Determining nanoscale protein distribution via Photoactivation Localization color dSTORM shows that these SDA proteins form a triangular structure rooted Microscopy (PALM) mandates precise knowledge of the applied fluorophore’s from the centriole wall, mapping to the SDA architecture resolved with electron photophysics. If not accounted for, blinking of dyes on time-scales of typical microscopy. Interestingly, dSTORM imaging of g-tubulin, the key MT nucle- PALM experiments will invariably cause overcounting artifacts, which become ating element, is found to be enriched between the SDA and the proximal layer even more pronounced in fixed cells with predominantly immobile proteins. of Cep170 at the location where MT fibers are observed. Besides, MT fibers are Here, we developed a lipid bilayer-based imaging platform as a means to deter- also found to attach to various centriole elements including the DAs, SDAs and mine the blinking behavior of individual PS-CFP2 molecules, widely used as a the proximal end of the centriole. Depletion of SDAs upon Cep128 knockout photo-switchable fluorophore in PALM for its reportedly low blinking ten- suppresses MT attachment to SDAs but not DAs. dSTORM imaging also reveals dency. We observed 35% of molecules to appear in a single frame only, while that AKAP450, a scaffold protein connecting the centrosome and Golgi, forms a a considerable fraction of molecules exhibited blinking cycles on time scales of cup like structure surrounding the entire centriole via its interaction with Cep170. up to several seconds. On average, a single PS-CFP2 molecule was detected 3.5 Upon Cep170 depletion, the centrosomal AKAP450 is abolished, causing times during the recording of a super-resolution experiment with considerable detachment of the mother centriole from Golgi. Together, using dSTORM, we implications for cluster quantification. Our strategy is amenable to determining systematically disclosed the structural organization of SDAs and illustrated blinking signatures for any fluorophore of choice to support robust conclusions the structural function of SDAs in MT anchoring and centriole-Golgi associa- related to PALM. Furthermore, the gained blinking statistics can be utilized to tion. discriminate clustered from randomly distributed membrane proteins.

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657-Pos and imaging frame duration. The method exploits the fact that the intensity in a Third Harmonic Generation Imaging using Common Histological Dyes region of interest of the imaging frame during which the intensity change occurred Alexei Kazarine1, Angelica A. Gopal1,2, Paul W. Wiseman1,3. depends on its time during the exposure. It requires simultaneous recording of the 1Department of Chemistry, McGill University, Montreal, QC, Canada, imaging data and a related highly time resolved (HTR) event such as a stimulation 2Department of Physiology, McGill University, Montreal, QC, Canada, pulse, action potential or amperometric spike. When multiple events occurring at 3Department of Physics, McGill University, Montreal, QC, Canada. random times during an exposure are averaged, the time correlation between the Histology has long relied on optical microscopy of tissue stained with colorful HTR events and the fluorescence change can be determined with sub-frame res- absorptive dyes, which enables easy visual differentiation between cell nuclei olution. We present the algorithm of the method, its step response function, and a and various extracellular structures. Histological stains are presently the gold theoretical, computational, and experimental analysis of its precision, providing standard in diagnoses by medical pathologists. While these stains provide an guidelines for camera exposure time settings depending on imaging signal prop- effective way to visualize tissue components, they still require thin sectioning erties and camera parameters for optimal time resolution. ECOM achieves a tem- which results in only two dimensional data that cannot be easily stitched in poral resolution with a standard error s t0=sqrt[3/(2þg)] tframe/SNR,with three dimensions. As tissue inherently exists as a three dimensional structure, tframe=teþtreadout and g=treadout/tframe, limited only by SNR. Supported by ERC it is important to develop histological methods to investigate it in such form grant ADG 322699, the Max-Planck-Society and NIH grant R01GM121787. while maintaining the high spatial resolution of light microscopy. Third harmonic generation (THG) imaging is a nonlinear microscopy technique 660-Pos that is gaining attention in the biomedical community. In this technique, three Method for High Frequency Tracking and Sub-Nm Sample Stabilization in incident photons are combined into one by light-matter interactions with the Single-Molecule Fluorescence Microscopy sample, resulting in high contrast and inherent three dimensional optical Patrick Schmidt1, Benjamin Reichert1, John Lajoie2, Sanjeevi Sivasankar3. 1 2 sectioning. While in practice THG only typically occurs at optical heterogene- Electrical Engineering, Iowa State University, Ames, IA, USA, Physics and 3 ities and interfaces within the biological sample, some materials present Astronomy, Iowa State University, Ames, IA, USA, Biomedical enhanced THG emission due to resonance effects. These materials have also Engineering, University of California, Davis, Davis, CA, USA. been called harmoniphores. Hematoxylin (H), a dye that is often used in com- While fluorescence microscopes and atomic force microscopes are widely bination with eosin (E) as part of the H&E stain, has previously been reported used to visualize, track, and manipulate single biomolecules, the resolution as an effective harmoniphore for distinguishing cell nuclei by 3D THG of these methods is limited by sample drift. To minimize drift, active feedback imaging. methods have recently been used to stabilize single molecule microscopes on In this work, we apply trans THG imaging to skin and liver sections to show the sub-nanometer scale. However, these methods require high intensity lasers that in addition to H&E, other commonly used histological dyes in elastic which limits their application in single molecule fluorescence measurements. and collagen staining can serve as effective THG harmoniphores. We find Furthermore, these feedback methods do not track user-defined regions of the that elastic fibers can be easily visualized by THG microscopy using Verhoeff sample, but rather monitor the relative displacement of an unknown point on a Van Gieson (VVG) staining while collagen is directly highlighted by THG im- fiducial marker, which limits their use in biological force measurements. To aging of picrosirius red staining. By combining THG imaging with common overcome these limitations, we have developed a novel method to image, histological dyes, 3D histological imaging could be achieved. track and stabilize a sample using low laser intensities. We demonstrate the capabilities of our approach by tracking a user-chosen point on a fiducial 658-Pos marker at 8.6 kHz and stabilizing it with sub-nanometer resolution. We further Monitoring Self-Organization Events in the Early Embryogenesis of Cae- showcase the application of our method in single molecule fluorescence norhabditis elegans with Lightsheet Microscopy microscopy by imaging and stabilizing individual fluorescently-tagged Matthias Weiss, Rolf Fickentscher, Philipp Struntz. streptavidin proteins under biologically relevant conditions. We anticipate Experimental Physics I, Univ Bayreuth, Bayreuth, Germany. that our method can be easily used to improve the resolution of a wide range Embryogenesis is a remarkably robust, but still poorly understood self- of single molecule fluorescence microscopy and integrated force-fluorescence organization phenomenon. Due to its simplicity, high reproducibility, optical applications. transparency, and well-characterized genetics, the small nematode Caenorhab- ditis elegans is a superb model organism to study self-organization events dur- 661-Pos ing early development. Using single plane illumination microscopy (SPIM) and Multi-Modal Superresolution Microscopy through Superresolution Radial simulations, we have explored how physical cues determine the cell arrange- Fluctuations (SRRF) ment in the early embryogenesis of C. elegans. As a result, we show that the Jeffrey Oleske. coupling of cellular volumes and cell cycle times in combination with a me- Andor Technology, Shrewsbury, NJ, USA. chanically guided arrangement process is key for a fail-safe embryogenesis Super-resolution radial fluctuations (SRRF) is a synthesis of temporal fluctua- of C. elegans [Biophys. J. 105, 1805 (2013); Phys. Rev. Lett. 117, 188101 tion analysis and localization microscopy. One of the key differences between (2016); Sci. Rep. 7, 9369 (2017)]. Our accompanying simulation model not SRRF and other super-resolution methods is its applicability to live-cell dy- only predicts the quantitative relation of cell volumes and division times until namics because it functions across a very wide range of fluorophore densities the larval state (including fluctuations) but also cell migration paths and posi- and excitation powers. SRRF is applied to data from imaging modes which tions until gastrulation are fully captured. Going beyond mere imaging, we include widefield, TIRF and confocal, where short frame bursts (e.g. 50 frames) have used pixel-wise fluorescence correlation spectroscopy (SPIM-FCS) to can be processed to deliver spatial resolution enhancements similar to or better spatiotemporally quantify the diffusion of proteins in individual cells of the em- than structured illumination microscopy (SIM). On the other hand, with sparse bryo in cytoplasm and on membranes [J. Phys. D 49, 044002 (2016)]. data e.g. STORM, SRRF can deliver resolution similar to Gaussian fitting local- ization methods. Thus SRRF could provide a route to super-resolution without 659-Pos the need for specialized optical hardware, exotic probes or very high power den- Precision of Time Super-Resolution Imaging by Event Correlation Micro- sities. We present a fast GPU-based SRRF algorithm and apply it to imagery scopy from an iXon EMCCD coupled to a multi-modal imaging platform, Dragonfly. Qinghua Fang1, Ying Zhao1, Manfred Lindau1,2. > 1 The new implementation is 300 times faster than the current CPU version Nanoscale Cell Biology, Max-Planck-Institute for Biophysical Chemistry, > 2 running on an Intel Xeon 3.5GHz 4 core processor, and 20 times faster than Go¨ttingen, Germany, Applied & Engineering Physics, Cornell University, the NanoJ GPU implementation, while also being integrated with acquisition Ithaca, NY, USA. for real time use. We explore the image resolution and quality with sCMOS Fluorescence imaging is widely used to monitor dynamic cellular functions un- cameras and various fluorophores including fluorescent proteins and organic der conditions of very low light intensities to avoid photodamage, rapid photo- dyes. bleaching, and excessive loading or overexpression of fluorescent labels. Even with highly sensitive EMCCD or sCMOS cameras exposure times (te) are typi- 662-Pos cally in the 100ms range, although many biological processes such as action Molecular Counting by Photon Statistics in Confocal Fluorescence potentials or synaptic transmission occur on a time scale of 1ms. The low Imaging photon rate and long imaging frame duration therefore impose limits on the Marcelle Koenig, Caroline Berlage, Paja Reisch, Christian Oelsner, time resolution of rapid fluorescence changes. This limitation is analogous to Felix Koberling, Haisen Ta, Rainer Erdmann. the spatial resolution limit set by the objective’s diffraction limited point spread PicoQuant GmbH, Berlin, Germany. function and camera pixel size. Quantifying molecules in subdiffraction-sized structures is crucial for understand- We developed a time super-resolution technique named Event COrrelation Mi- ing various fundamental biological processes. Ideally, a non-destructive measure- croscopy (ECOM) that breaks the time resolution limit given by the photon rate ment would result in an image of a cellular structure along with the number of

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fluorescent molecules in each region of interest. Recently, a considerable amount Rab11, MHC I, and MHC II, suggesting that HA from VLPs underwent of work has gone into developing such methods. Counting by Photon Statistics bi-directional trafficking towards both high-degradative late and low- (CoPS) is a promising new technique based on photon antibunching, which ex- degradative static early and/or recycling endosomal pathways. In contrast, ploits the fact that a single molecule can only generate one photon at a time [1]. soluble HA intensity, cluster density and degree of aggregation decreased at The number of independent fluorescent emitters can be determined by measuring 45 min, suggesting that HA had predominantly entered high-degradative en- photon coincidences on a time-resolved confocal microscope with four single- dosomal pathway. Our study shows that HA delivery to antigen-presenting photon detectors (e.g., MicroTime 200, PicoQuant) [2]. Both a molecular bright- cells in the form of VLPs facilitates antigen uptake and diversifies its endo- ness and the spatial density of fluorophores per image pixel are estimated. By sum- somal processing. ming over all corresponding pixels, the number of molecules in a structure can be calculated. The only calibration required is to determine the instrument’s Point 665-Pos Spread Function (PSF) size. We investigated imaging with CoPS under a wide Flexible Light-Sheet Generation by Field Synthesis 1 2 1 1 range of experimental conditions. The applicability of the method both to artificial Bo-Jui Chang , Mark Kittisopikul , Kevin M. Dean , Reto P. Fiolka . 1Dept Cell Biology, Univ Texas Southwestern Med Ctr, Dallas, TX, samples such as immobilized DNA origami, as well as to biological samples is 2 explored. We also discuss sample preparation requirements and practical guide- USA, Cell and Molecular Biology, Northwestern University, Chicago, IL, lines for performing CoPS measurements. USA. [1] Ta, H., Wolfrum, J., Herten, D.-P., An extended scheme for counting fluo- Light-sheet microscopy has revolutionized biological imaging by affording rescent molecules by photon-antibunching. Laser Phys. 20:119 (2010) rapid and efficient volumetric imaging while minimizing the applied light- [2] Ta, H.et al., Mapping molecules in scanning far-field fluorescence nanosco- dosage and thus phototoxicity. However, due to beam divergence, there is a py.Nat. Commun. 6:7977 (2015) trade-off between the light-sheet thickness and the useful field-of-view for imaging: the confocal parameter, which approximates the propagation length 663-Pos of a light-sheet, shrinks nonlinearly with the size of the beam waist, and Plasma Membrane-Selective Visualization by Multimodal Two-Photon consequently, modest axial resolution is achieved when imaging large speci- Imaging in Living Cells using Second Harmonic Generation mens. To overcome this trade-off, propagation invariant beams have been em- Takaha Mizuguchi, Masato Yasui, Mutsuo Nuriya. ployed in light-sheet generation, such as Bessel or Airy beams. These beams Department of Pharmacology, Keio University School of Medicine, Tokyo, maintain their cross-sectional profile over extended distances and overcome Japan. the beam divergence of Gaussian beams. To generate a light-sheet, a propa- The plasma membrane is the place for intercellular communication and intra- gation invariant beam is scanned laterally to create a time averaged sheet cellular signal transduction. Its selective imaging in living cells is an indispens- of light. Such setups require dedicated optical trains, which increase the able tool for cell biology. Conventionally, fluorescence-based techniques with cost and complexity of a light-sheet microscope. We have recently discovered fluorescent proteins or dyes were used for visualizations of membrane. Howev- and proven the ‘Field Synthesis’ theorem, which describes a universal mech- er, it suffers from a problem with background signals from intracellular mem- anism for light-sheet generation. Field synthesis predicts that any scanned brane structures like the endoplasmic reticulum and endocytosis/exocytosis light-sheet can be synthesized by an incoherent superposition of 1D intensity vesicles. To overcome this, we applied a second harmonic generation (SHG) patterns. We demonstrate that in practice, this results in far less complex op- technique using a non-fluorescent amphiphilic SHG dye Ap3 for plasma tical systems and much higher light-efficiency compared to previous setups membrane-selective imaging. SHG is one of nonlinear optical phenomena using propagation invariant beams. Through careful comparisons, we show that only occurs with the materials lacking centrosymmetry under the phase- that through Field Synthesis we can re-create the time averaged intensity matching condition. The strict requirement for SHG can offer a precise visual- of Bessel and Lattice light-sheets. However, in contrast to scanned Bessel ization of plasma membrane where amphiphilic dyes are adsorbed and aligned beams, we achieve 100% spatial duty cycle, which results in drastically asymmetrically. The non-fluorescent nature of the SHG dye Ap3 can also pro- reduced photobleaching. Compared to lattice light-sheet microscopy, Field vide a multimodal use with two-photon fluorescence-based applications. In this Synthesis is achromatic and can hence perform simultaneous multicolor imag- study, we first characterized that the SHG signals from Ap3 is plasma ing. We present careful comparisons of each illumination mode and demon- membrane-selective by side-by-side comparison with membrane-anchored strate biological imaging applications of light-sheets produced by Field GFP and that it is point-source origin. In addition to the clear identification Synthesis. of plasma membrane, the multimodal imaging with two-photon fluorescence from GFP-fused actin and tubulin fibers revealed the cytoskeletal organization 666-Pos beneath the plasma membrane with a subresolution scale. Taken together, our Line-Scanning Spatial Correlation Spectroscopy for Studying Dynamics in data suggest that the SHG-dye based multimodal imaging enables high- Biomembranes resolution plasma membrane-selective imaging, which is readily applied to Peng Gao, Xiang Gao, Karin Nienhaus, G. Ulrich Nienhaus. cell biology research. Dept Appl Phys, Karlsruhe Inst Tech, Karlsruhe, Germany. Biomembranes that are not attached to a solid support fluctuate incessantly 664-Pos and give rise to sizeable intensity fluctuations in fluorescence correlation Intracellular Tracking of Influenza Hemagglutinin in Human Monocyte- spectroscopy (FCS) experiments, masking intensity fluctuations due to mol- Derived Macrophages Measured by Image Cross Correlation Spectros- ecules diffusing within the membrane. Line-scanning FCS (lsFCS) is an copy elegant approach to avoid these intensity variations by scanning a line Angelica A. Gopal1,2, Alexander I. Makarkov3,4, Nathalie Landry5, many times perpendicularly through the membrane and analyzing temporal Brian J. Ward4, Paul W. Wiseman2,6. intensity pair correlations of the fluorescence from the membrane. Here we 1 Department of Physiology, McGill University, Montreal, QC, Canada, have extended this concept to spatial correlations by displacing each scan 2 Department of Chemistry, McGill University, Montreal, QC, Canada, line from the previous one by a constant amount and analyzing spatial inten- 3 Division of Experimental Medicine, McGill University, Montreal, QC, sity pair correlations between lines, as is done in raster image correlation 4 Canada, Infectious Diseases and Immunity in Global Health Program, spectroscopy (RICS). In line-scanning spatial correlation spectroscopy Research Institute of McGill University Health Centre, Montreal, QC, (lsSCS), the effective observation area is enlarged with respect to lsFCS, re- 5 6 Canada, Medicago Inc., Quebec, QC, Canada, Department of Physics, sulting in better sampling of sparse, slowly moving molecules and averaging McGill University, Montreal, QC, Canada. of the resulting parameters over a larger region. The fraction of photo- The adaptive immune response has been demonstrated to be vital for protec- bleached molecules is also reduced, which is advantageous for precise deter- tion against influenza, viral clearance and long term immunity. Plant-derived mination of area densities of molecules. For cross-correlation analysis, we virus-like particle (VLP) vaccines bearing influenza hemagglutinin (HA) have perform dual-color lsSCS experiments using pulsed interleaved excitation been shown to induce strong humoral and cellular CD4þ T cell-mediated (PIE). immune responses in both pre-clinical and clinical studies. To further our understanding of the VLP vaccine intracellular handling, we used confocal 667-Pos fluorescence microscopy and segmentation paired with image cross correla- Brownian Motion using a Piezo Actuated Microscope Stage tion spectroscopy (ICCS) to track the antigen intracellularly in human Nicholas A. Vickers, Sean B. Andersson. monocyte-derived macrophages (MDMs), where HA was delivered either as Mech Eng, Boston Univ, Boston, MA, USA. VLPs (HA-VLPs) or as soluble HA based on the sequence of A/California/ Single particle tracking (SPT) is an important collection of techniques and al- 07/2009 (H1N1) influenza strain. Pulsing MDMs with HA-VLPs resulted in gorithms for the study of biomolecular transport and understanding the role of a greater amount of HA accumulating intracellularly at 15 min compared to the dynamics of small molecules, proteins, and other organic material in health soluble HA. At 45 min, the HA was strongly co-localized with Rab5, Rab7, and disease on a cellular level. However successful these techniques are, it is

BPJ 9322_9324 136a Sunday, March 3, 2019 difficult to compare them directly, especially as one is most interested in their an average 20% mobile fraction, while we found that there was little to no ex- performance on a specific experimental setup. Here, we demonstrate through change of cadherins or desmosomal plaque proteins in hyperadhesive desmo- Monte-Carlo simulation and experiment, the ability of a piezo actuated micro- somes. This loss of mobility was confirmed using the DP point mutant scope stage to replicate faithfully a discrete-time sampled Brownian motion tra- S2849G, that has been shown to induce hyperadhesion. We conclude that jectory. Simulations consisted of an exact discrete model of the piezo stage changes in protein mobility represent the structural mechanism by which including closed-loop control, the input Brownian motion trajectory, and desmosome hyperadhesion is established. feed forward model inverse control. A range of stage response times, discrete time step durations, and diffusion constants were modeled. The output of the 670-Pos system was compared to the input using mean squared displacement (MSD), Photon-Free Calibration of CMOS Cameras for Precise Single Molecule Kullback-Leibler divergence comparing the single time step displacement dis- Localization Microscopy Reconstruction tributions, and stage tracking error. We found that MSD was preserved over a Robin Diekmann, Jonas Ries. large range of stage response times, discrete time step durations, and diffusion CBB Unit, EMBL Heidelberg, Heidelberg, Germany. constant values. Physical experiments consisted of an epifluorescent micro- Scientific-grade CMOS (sCMOS) cameras have evolved into a preferred detec- scope taking widefield images of quantum dots fixed to a microscope slide tor type for single molecule localization microscopy (SMLM). Compared to moved using the piezo actuated microscope stage following a Brownian motion EM-CCDs, sCMOS cameras do not only feature potentially increased localiza- trajectory control input. We envision this work will allow for the characteriza- tion precisions and framerates, but also come at typically lower cost. Transi- tion and evaluation of SPT microscopes and localization algorithms by moving tioning to industry-grade CMOS cameras can decrease the cost much further, fixed fluorophores through a known Brownian motion trajectory to provide a while in principle maintaining the advantages of sCMOS detectors. Conse- known ground truth in an experimental setting. Future work includes using quently, such highly affordable cameras are recently gaining popularity in other biologically relevant motion models such as confined diffusion and the community. However, considerable pixel-to-pixel variations in terms of elastic tethering as the motion model, and a comparison of SPT microscope noise, offset and gain which are well-known for sCMOS cameras can be configurations and estimation algorithms through simulated motion with a even pronounced for CMOS cameras and impair the localization uncertainties. piezo stage. Explicit consideration during the reconstruction procedure is able to largely circumvent this effect if the detector is characterized accordingly. 668-Pos To this end, we present a novel approach to characterize CMOS cameras. It re- Spatiofunctional Enzyme Droplets in Cellular Metabolism lies on thermally generated electrons only, but allows for similar data process- Erin L. Kennedy, Miji Jeon, Patricia S. Boyd, Farhan Augustine, Songon An, ing as in the traditional approach of using photoelectrons. Going without the Minjoung Kyoung. need of external stimuli, this makes in-situ camera characterization possible. University of Maryland Baltimore County, Baltimore, MD, USA. We show that industry-grade CMOS cameras which have been characterized Rate-limiting enzymes in the glucose metabolism have been observed to form by this approach can efficiently applied to common SMLM schemes of spatially distinct enzyme assemblies throughout the cytoplasm. While these as- PALM, dSTORM and DNA-PAINT. semblies are observed to regulate metabolic flux, the mechanism of function 671-Pos and formation is not well known. We provide support of the glucose meta- Single Molecule Measurements Based on Information Theory bolism assembly formation through liquid phase separation using lattice light Sheng Liu, Fang Huang. sheet microscopy. The spatially dynamic enzyme assemblies exhibit behaviors Purdue Univ, West Lafayette, IN, USA. of liquid phase droplets. Our multi-color spatial analysis also indicates that a Single molecule studies aim at extracting desirable measurements, such as lo- significant number of the enzyme assemblies are spatially associated with the cations and orientations, from single-molecule emission patterns. Numerous mitochondria. By inhibiting the mitochondria, we observed spatiofunctional algorithms and microscopy techniques have been developed over the last de- behaviors between the assemblies and the mitochondria. The formation and or- cades towards this aim in the aspect of either increasing the measurement di- ganization of enzyme assemblies in relation to other organelles suggests an mensions or the estimation precisions. However, without considering the orchestrated regulation of metabolic functions. This work provides a founda- detailed designs of a data analyzing algorithm or a microscope system, the tion for mapping the spatially dynamic metabolic networks of the glucose amount of information contained in a given single-molecule emission pattern metabolism within live cells. can be quantified using the Fisher information matrix which provides a lower bound of the measurement uncertainty, named Crame-Rao lower bound 669-Pos (CRLB). Here we examined the CRLB of estimating various information car- Calcium Independence of Epithelial Junctions is Regulated by Protein ried by single-molecule emission patterns, such as molecule locations and ori- Mobility entations, and wavefront distortions, from several types of existing point 1 1 1 Emily I. Bartle , Tara M. Urner , Tejeshwar C. Rao , spread functions, such as astigmatism PSF, double-helix PSF, dipole PSF, Andrew P. Kowalczyk2, Alexa L. Mattheyses1. 1 2 interferometric 4Pi PSF and PSFs that were distorted by systematic or sample CDIB, UAB, Birmingham, AL, USA, Cell Biology, Emory University, induced aberrations. Furthermore, from the perspective of information theory, Atlanta, GA, USA. we also evaluated the change of information content by various image pro- Desmosomes are macromolecular cell adhesion complexes that provide me- cessing techniques such as noise reduction and super-resolution microscopy chanical integrity to epithelial tissues. Unlike other cell junctions, desmosomes reconstruction. can adopt a unique calcium-independent adhesive state where adhesion is maintained in the absence of calcium, also known as hyperadhesion. Regula- 672-Pos tion of adhesion between the calcium-dependent and calcium-independent Human Stem Cell Structures Measured with Concentration-Calibrated states is critical for balancing desmosome strength with plasticity in processes Super-Resolution Microscopy including epithelial tissue homeostasis, embryogenesis, and wound healing. Derek Thirstrup, Winfried Wiegraebe, Allen Institute for Cell Science Though changes in signaling coordinated with desmosome adhesion are well Team. understood, adhesion is ultimately determined by binding of cadherins at the Microscopy, Allen Institute for Cell Science, Seattle, WA, USA. desmosome core, and the structural differences between states remain un- The mission of the Allen Institute for Cell Science is to understand and predict known. Here we investigate the mechanism of hyperadhesion by exploring cell behaviors. The current project aims to create a stem cell state space by con- the physical properties of desmosomes, including protein order, desmosome ar- joining genomic, imaging, and phenotypic data from cells. chitecture, and protein mobility. We used a human keratinocyte cell line (Ha- We tagged most major molecular machineries (organelles, membranes and CaT) and confirmed treatment with the PKCa inhibitor Go¨6976 induced cytoskeleton) of human induced pluripotent stem cells (hiPSCs) with fluores- hyperadhesion. We then used fluorescence polarization microscopy to measure cent proteins, and acquired large, reproducible datasets of each cell line for cadherin order. Previously we showed that the cadherin Desmoglein 3 (Dsg3) model input using an automated spinning-disk microscopy pipeline (www. was ordered in calcium-dependent desmosomes and became disordered after allencell.org). We used the CRISPR/Cas9 system to fuse the fluorescent pro- calcium depletion. Here we show when calcium was depleted from hyperadhe- teins directly to the protein of interest under the endogenous promoter. There- sive desmosomes, Dsg3 became disordered following the same kinetics, even fore, the measured intensity of fluorescence emission is proportional to the though desmosomes remained adhesive. This demonstrates that cadherin order protein distribution, localization, and dynamics of individual cells. is not what drives hyperadhesion. Next, we investigated desmosome plaque ar- To improve the accuracy of the integrated cell models we are building, we chitecture by super-resolution STORM and found no changes between collected data with higher spatial resolution and measured absolute protein con- calcium-dependent or -independent states. Finally, we investigated protein centrations. We increased the resolution by about 1.7x using the Zeiss LSM 880 mobility using FRAP. In calcium-dependent desmosomes cadherins exhibited confocal microscope equipped with an Airy fast detector based on pixel

BPJ 9322_9324 Sunday, March 3, 2019 137a reassignment. We created intensity/concentration calibration standards using lecular bridges formed by the transluminal physical interaction of the outer and cytosolic eGFP, cell-lysate, as well as purified eGFP in solution. We calibrated inner nuclear membrane KASH and the SUN proteins, respectively. Mammals the concentration standards with FCS (Fluorescence Correlation Spectros- encode six KASH proteins and five SUN proteins. In vitro, KASH proteins bind copy). The concentration standards allow quantitative comparison of data to the domain interfaces of trimeric SUN2 proteins resulting in the formation of collected on different systems. a SUN2/KASH hetero-hexamer. Recently, we extended the application of We validated these approaches with quantitative Western blots, ELISA, and single-color fluorescence fluctuation spectroscopy (FFS) to quantify protein UV/VIS spectroscopy. We will discuss the advantages and limitations of the oligomerization within the NE of living cells by developing mean segmented different standards, as well as the limitations, corrections, and controls neces- Q-factor (MSQ) and time-shifted MSQ (tsMSQ) to address experimental chal- sary because of mono-allelic expression of eGFP-tagged proteins, inactive lenges unique to the environment of the NE. Using FFS, we demonstrated that eGFP, and environmental changes of eGFP brightness. SUN2 trimerizes in vivo, whereas the luminal SUN-binding KASH peptide of These results will add additional, quantitative constraints to our integrated the KASH protein nesprin-2 (KASH2) remains monomeric. To be able to iden- models based on concentrations as well as finer structural detail due to the tify the oligomerization of SUN2/KASH within the NE of living cells, we improved resolution. describe here the development of dual-color (DC) tsMSQ. We first validated DC tsMSQ using nuclear envelope proteins tagged with EGFP and mCherry 673-Pos as model systems. Finally, we applied DC tsMSQ to detect the formation of Nuclear Pores as Universal Reference Standards for Quantitative Micro- hetero-complexes of EGFP-tagged SUN2 and mCherry-tagged KASH2. The scopy development of DC tsMSQ will enable future efforts aimed at the mechanistic Jervis V. Thevathasan, Ulf Matti, Maurice Kahnwald, dissection of LINC complex assembly and its regulation within the NE as well Sudheer Kumar Peneti, Bianca Nijmeijer, Moritz Kueblbeck, Jan Ellenberg, as investigations of binary protein systems within this important sub-cellular Jonas Ries. compartment. This work has been supported by a grant from the NIH (R01 Cell Biology and Biophysics, EMBL, Heidelberg, Germany. GM64589). Recent advances in superresolution microscopy now allow us to address struc- tural questions in cell biology with optical methods. However, a quantitative 676-Pos interpretation is often limited by sub-optimal performance and calibration of Automating Localization Microscopy the microscope, undetermined performance of the fluorescence label and imag- Joran Deschamps, Yiming Li, Markus Mund, Jonas Ries. ing conditions, unknown labeling efficiencies and systematic errors in counting Cell Biology & Biophysics, European Molecular Biology Laboratory, protein numbers. Here we show that the use of reference standards can over- Heidelberg, Germany. come these limitations and greatly improve quantitative microscopy. To this Single-molecule localization microscopy (SMLM) has become a popular tool end we exploit the precise 3D arrangement and stoichiometry of proteins in in cell biology as it allows imaging cellular structures with a precision of the nuclear pore complex. We present a set of genome edited cell lines in which tens of nanometers. However, acquiring superresolved images with SMLM is we endogenously labeled the nucleoporin Nup96 with eGFP, SNAP- or HALO- inherently slow, requiring up to hours for a single image and careful user inter- Tag or the photoconvertible fluorescent protein mMaple. We demonstrate their vention in between experiments. This effectively limits the throughput of use as a) simple and robust resolution standards for calibration and quality con- SMLM to a handful of images of the structure of interest per study. We present trol, b) accurate assays to quantify absolute labeling efficiencies in superreso- tools to overcome this limitation by automating a wide-field microscope to lution microscopy and c) precise counting reference standards for absolute perform SMLM experiments. This microscope is capable of SMLM imaging stoichiometry measurements. As a resource shared with the community, these over the course of days thanks to advanced electronics and an open-source plu- cell lines will enable many groups to assess the quality of their microscopes and gin for Micro-manager. In the meantime, SMLM experiments are analysed dur- labels and to perform quantitative, absolute measurements. ing acquisition using a freely accessible software in Matlab. Finally, the 674-Pos microscope features a flat illumination system, providing homogeneous statis- Fluorescence Lifetime Imaging Microscopy using Compressed Phasors tics across large fields of view. Altogether these developments in optics, hard- Ryan A. Colyer, Sarah Grant, Sarah Eplett. ware and software can be transferred to any single-molecule wide-field Cabrini Univ, Garnet Valley, PA, USA. microscope, thus enabling automated localization microscopy. Fluorescence Lifetime Imaging Microscopy (FLIM) has high value for spatial 677-Pos determination of chemical conditions or binding events, but in biological sam- ples typical setups require long acquisition times or potentially phototoxic Quantitative and Motion-Corrected Super-Resolution Imaging of Endo- light intensities. Attempts at widefield acquisition systems with cameras or some Dynamics in Living Cells custom detectors have suffered from collection inefficiencies or high unit Elias M. Puchner, Santosh Adhikari. costs. The method presented here is a novel approach designed to provide Dept Phys & Astron, Univ Minnesota, Minneapolis, MN, USA. low intensity widefield acquisition with a single point detector and a digital Quantitative Super-Resolution Microscopy is evolving into a powerful tech- nique to study biological processes below the optical diffraction limit. Intracel- micromirror device (DMD). The method exploits recent advancements in lular calibration approaches for quantitative Photoactivated Localization compressive sensing, and combines these with phasor analysis to produce im- ages constructed from structurally compressed phasors. This approach permits Microscopy (qPALM) have provided a major development in the field, allow- lifetime acquisition across half of the image at a time, while photons are able ing researchers to determine the absolute number of biomolecules on individual to be collected by a single point detector. This allows the combination of organelles and to simultaneously resolve their size with about 20nm resolution. lower intensity illumination FLIM with a flexible choice of detector However, a general limitation of PALM has been the need to fix cells in order to avoid blurring caused by motion. Here, we present our new live-cell PALM properties. approach, in which we use dual-color imaging to track nano-scale organelles 675-Pos to correct for their motion during data acquisition. We employed our motion- Illuminating SUN2/KASH Hetero-Complex Formation Within the Nuclear corrected PALM technique to simultaneously characterize the size of individ- Envelope of Living Cells with Dual-Color Time-Shifted MSQ ual endocytic vesicles and endosomes in live yeast cells and quantified the Kwang-Ho Hur1,2, Jared Hennen1, John Kohler1, Siddarth Reddy Karuka1, number of accessible PI3P binding sites they contain. The analysis of numerous G.W. Gant Luxton2,3, Joachim D. Mueller1,3. cells revealed a characteristic vesicle maturation trajectory (composition and 1School of Physics and Astronomy, University of Minnesota, Minneapolis, size) as well as mechanistic information indicating that PI3P production pre- MN, USA, 2Department of Genetics, Cell Biology, and Development, cedes fusion into larger endosomes. These results are consistent with our pre- University of Minnesota, Minneapolis, MN, USA, 3Department of vious study in fixed cells and yield additional insights into the dynamics of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA. endosome maturation and fusion. By analyzing various diffusing endocytic The nuclear envelope (NE) is a cellular compartment consisting of an inner and vesicles and simultaneously resolving their size, we observe in real time how outer nuclear membrane separated by the perinuclear space. Research on their diffusion coefficient depends on their size and environment and how cellular mechanotransduction identified the nucleus as a mechanosensor, which they mature to endosomes by fusion. Our dual-color live-cell PALM technique responds to the mechanical properties of the extracellular environment. Force not only gives novel information about the mode of transport of these specific transmission across the NE into the nucleoplasm is facilitated by linker of organelles, but also provides a new avenue for quantitative super-resolution im- nucleoskeleton-and-cytoskeleton (LINC) complexes, which are conserved mo- aging in living cells.

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Posters: Single-Molecule Spectroscopy I somal DNA becomes rigidified, as evidenced by the loss of normal jiggling motion of DNA loci. The diffusive motion of a subset of ribosomes is also frozen, 678-Pos and the mean diffusion coefficients of the DNA binding protein HU and the non- Time-Tagged Single Photon Counting Examination of Rotation of endogenous protein Kaede decrease twofold. Additionally, LL-37 causes DNA Receptor-Bound Quantum Dots decompaction and alleviates the segregation between DNA and ribosome. We 1 1 2 1 attribute it to the alleviation of crowding effect and segregative phase separation Dongmei Zhang , Jason Pace , Deborah A. Roess , B. George Barisas . 8 1Department of Chemistry, Colorado State University, Fort Collins, CO, between negatively charged ribosome and DNA. Roughly 10 LL-37 copies flood USA, 2Department of Biomedical Sciences, Colorado State University, Fort the cell. Most LL-37 remains bound within the cell after extensive rinsing with Collins, CO, USA. fresh growth medium, and growth never recovers. The results suggest that the Rotation of single membrane receptors can be observed by examination of high concentration of polycationic peptide forms a dense network of noncovalent polarized optical signals from receptor-bound asymmetric nanoparticles such ‘‘pseudo-crosslinks’’ within the DNA meshwork and among 70S-polysomes, as fluorescent quantum dots (QD) or nanogold. For example, we have examined thus rigidifying the entire cytoplasm. The bacterial cytoplasm comprises a the slow, hindered rotation of the Type I Fce receptor (FceRI) on 2H3 RBL concentrated collection of biopolymers that are predominantly polyanionic. In cells using polarized fluorescence imaging of receptor-bound Qdot655. With normal cells, this provides a kind of ‘‘electrostatic lubrication’’ enabling reason- imaging methods, only receptor rotational correlation times (RCT) slower ably facile diffusion in spite of the high biopolymer volume fraction. However, than the camera frame time can be examined and suitable low-light cameras this same polyanionic nature renders the cytoplasm highly susceptible to massive typically require 1ms or longer per frame. However, time-resolved phosphores- adsorption of polycationic agents after membrane permeabilization. If this phe- cence anisotropy shows the hydrodynamic RCT of FcεRI to be about 40 msat nomenon proves widespread across cationic agents and bacterial species, it will 25C. To examine such rapid reorientation, an alternate approach is to illumi- help explain why bacterial resistance to AMPs develops only very slowly. nate individual QD on the cell surface with a focused laser beam, collect fluo- 681-Pos rescence using a confocal detector and direct signals polarized parallel and A Divisive Segmentation and Clustering Scheme for Accelerated and perpendicular to the laser polarization into separate APD detectors. A time- Improved Single-Molecule Time Series Idealization (DiSC) tagged single photon counter records the channel of each detected photon David S. White1,2, Marcel P. Goldschen-Ohm3, Randall H. Goldsmith2, and its arrival time with a precision of 165 ps. The auto- and cross- Baron Chanda1,4. correlations of the two signals are calculated directly from arrival times without 1Dept Neuroscience, Univ Wisconsin Madison, Madison, WI, USA, 2Dept binning, combined using adjustable constants such as the g-factor, and these Chemistry, Univ Wisconsin Madison, Madison, WI, USA, 3Dept constants optimized to obtain the maximum statistical independence between Neuroscience, Univ Texas Austin, Austin, TX, USA, 4Biomolecular the anisotropy and intensity time-autocorrelation functions (TAC). While Chemistry, Univ Wisconsin Madison, Madison, WI, USA. RCTs below 1 ns are theoretically accessible, useful rotational information ex- Single-molecule methods offer unprecedented view into the heterogenous dy- R ists only when RCT*photon count rate 1. Thus, given photon count rates, our namicsunderlying chemical and biological interactions. As their popularity grows, R m data potentially provide information on RCTs 20 s. However, intensity sotoo does the demand for efficient and accurate methods toreliablyextract kinetic changes due to QD blinking may feed through to some extent into calculated parameters from large, often noisy, time series data sets. Conventional methods anisotropies and thus slightly distort apparent anisotropy TAC decay shapes. include change-point analysis and hidden Markov modeling; the former being Efforts aimed at removing this possible complication are underway. computationally efficient and parameter free at the cost of lower accuracy, and the latter being the opposite. Here, we introduce DiSC, a new idealization scheme 679-Pos that combines the strengths of both these methods for parameter free acceleration Stable Off-Path Structures in the Folding Dynamics of Two Consecutive ofhidden Markov modeling. DiSC identifies segments and clusters simultaneously Telomeric DNA G-Quadruplexes in a divisive manner, wherein the growth of each node is controlled by a specified Emil L. Kristoffersen, Victoria Birkedal. information criterion. The identified clusters and change-points are then used as iNANO/Dept Chem, Aarhus University, Aarhus C, Denmark. priors for hidden Markov modeling to improve the overall fit at a fraction of the G-quadruplexes are non-canonical DNA structures able to form in guanine-rich standard computational cost. We validate the performance of DiSC using simu- DNA sequences. Hundred thousands of potential and confirmed G-quadruplex lated data and determine DiSC is both faster and more accurate for state and forming regions have been identified throughout the human genomic DNA (Hup- change-point identification than the standard cutting-edge methods. pert and Balasubramanian NAR 2005; Chambers et al. Nat. Biotechnol. 2015; Be- drat, Lacroix, and Mergny NAR 2016). Amongst those sequences, many are able 682-Pos to support formation of multiple consecutive G-quadruplexes. One such region is Three-Color Single-Molecule FRET and Fluorescence Lifetime Analysis of the 300 nucleotide long repeating (TTAGGG) overhang of the mammalian telo- Fast Protein Folding mere. Formation of G-quadruplexes has been shown to block important DNA Janghyun Yoo, John M. Louis, Irina V. Gopich, Hoi Sung Chung. modifying enzymes and the G-quadruplex interphase is a potential drug target. Laboratory of Chemical Physics, National Institute of Diabetes and Digestive Using single molecule FRET TIRF microscopy with alternating laser excita- and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA. tion, we investigated folding of multiple G-quadruplex structures and identified We describe the theory, experiment, and analysis of three-color Fo¨rster reso- distinct conformational stages taken up by the constructs in the presence of nance energy transfer spectroscopy for probing conformational dynamics of various monovalent cations. Our results show a large conformation diversity a fast-folding protein, a3D. To site-specifically label fluorophores, a cysteine in the presence of Naþ and Kþ. In experiments with Naþ, where antiparallel residue and an unnatural amino acid were labeled first, and then we appended G-quadruplex formation was favored, we found that time-traces could be sorted a cysteine residue to the C-terminus of the protein by the sortase-mediated liga- into two distinct non-interconverting population stages. Only <3.5% time- tion for the third dye. All three FRET efficiencies were determined using alter- traces was observed in which both population stages were visited indicative nating excitation of the donor and acceptor 1. Due to fast folding on a of a high interconversion energy barrier. The observed populations included millisecond time scale, we used a maximum likelihood method that analyzes several conformations of the fully folded structures and structures with only photon trajectories without binning the data. The extracted kinetic parameters one antiparallel G-quadruplex. Some of these very stable conformations block agree well with the previously measured parameters with two-color FRET, sug- full folding and thus are off the folding pathway. Taken together, our observa- gesting the third fluorophore does not affect the folding dynamics of the pro- tions suggest a model where G-quadruplexes form randomly along the telo- tein. The FRET efficiencies for all three dye pairs were calculated after meric overhang in a way that sometimes block full condensation. various corrections. They were compared with the FRET efficiencies obtained from the two-color segments collected in the same experiment. The FRET ef- 680-Pos ficiencies of the folded state agree with those from the two-color segments, Rigidification of the E. coli Cytoplasm by the Human Antimicrobial Pep- whereas the unfolded state FRET efficiencies are different. This happens tide Ll-37 Revealed by Superresolution Fluorescence Microscopy because fluctuations of all three inter-dye distances contribute to the three- Yanyu Zhu, Sonisilpa Mohapatra, James C. Weisshaar. color FRET efficiency. This difference can be accounted for by using the Dept Chemistry, Univ Wisconsin Madison, Madison, WI, USA. Gaussian chain model with the parameters obtained from the two-color seg- In an era faced with increasing antibiotic resistance, the design and development ments. Therefore, three-color FRET provides additional information on the of new anti-microbial agents is in desperate need. Evolutionarily conserved anti- flexibility of molecules that cannot be obtained from a combination of two- microbial peptides (AMPs) are useful prototypes and promising drugs in this color FRET experiments. Using the delay times of photons from the laser pulse, context. We apply super-resolution microscopy and single-particle tracking to fluorescence lifetimes were determined using the maximum likelihood anal- reveal novel effects of the cationic human cathelicidin LL-37 on the E. coli cyto- ysis. The correlation between FRET efficiencies and lifetimes of the donor, plasm. Seconds after LL-37 penetrates the cytoplasmic membrane, the chromo- acceptor 1, and acceptor 2 was visualized in two-dimensional FRET

BPJ 9325_9327 Sunday, March 3, 2019 139a efficiency-lifetime histograms, which can be used to demonstrate the presence 686-Pos of conformational dynamics in a protein. Extending Fluorescence Longevity with a Laguerre-Gaussian Trapping Laser for the Combination of Optical Trapping and Single Molecule Fluo- 683-Pos rescence Investigation of Single Particle Tracking Performance by Different Parti- Zheng Zhang1,2, Joshua N. Milstein1,2. cle Filter and Smoother Algorithms 1Department of Physics, University of Toronto, Toronto, ON, Canada, Ye Lin1, Sean B. Andersson1,2. 2Department of Chemical and Physical Sciences, University of Toronto 1Division of Systems Engineering, Boston University, Boston, MA, USA, Mississauga, Mississauga, ON, Canada. 2Department of Mechanical Engineering, Boston University, Boston, MA, Optical trapping and single-molecule fluorescence are two powerful techniques USA. for studying biophysical phenomena. These techniques often provide comple- Single particle tracking (SPT) is a powerful class of techniques for exploring mentary information on conformational changes and interactions of the dynamics of single molecules moving inside living cells. In order to extract a biological molecule. However, combining these two techniques poses a great information about the biophysical processes under study, one desires both the challenge since the photobleaching lifetime of many fluorophores are strongly trajectories of the tracked particles as well as the parameters of their motions, decreased when exposed to the high intensity of the optical trapping laser. In such as diffusion coefficients, confinement lengths, and similar values. Typi- this work, we show that when a Laguerre-Gaussian laser beam, in contrast to cally this information is determined in two steps, first through trajectory estima- a Gaussian beam, is used for axial optical trapping, the photobleaching lifetime tion from the image data and then parameter estimation from the trajectories In of the fluorophore is significantly extended when placed within the dark region prior work, we have introduced a general algorithm known as Sequential Monte of the wavefront. This provides a foundation for combining axial optical twee- Carlo-Expectation Maximization (SMC-EM) that leverages nonlinear system zers with single molecule fluorescence microscopy. identification tools using particle filters and particle smoothers to handle nonlinear models of motion as well as nonlinear models of observation, allow- 687-Pos ing us to incorporate camera models, varying point spread functions, and other Toward Single Molecule FRET Studies of DNA Mismatch Repair in Live experimental realities. These scheme does not separate the estimation of trajec- Bacteria tory and motion parameters. SMC-EM is in fact a family of algorithms Pengning Xu, Andrew Hensley, Edward Chan, Keith R. Weninger. described by the choice of filter and smoother. In this work, we undertake a sys- Physics, North Carolina State University, Raleigh, NC, USA. tematic study of the effect the choice of these different sub-algorithms, DNA Mismatch Repair (MMR) is an important correction system that contrib- focusing on diffusion models as well as the simplest setting that is biologically utes to genomic fidelity during DNA replication. The protein MutS initiates relevant. The algorithms are compared with respect to a variety of measures, DNA MMR by recognition of base mismatch or insertion/deletion loop and including speed of convergence, computational complexity, trajectory estima- activation of downstream repair processes. Single molecule FRET has previ- tion accuracy, parameter estimation accuracy, and robustness to noise. ously been used in purified, in vitro studies to characterize conformational tran- sitions in MutS as it interacts with a DNA mismatch and converts to a sliding 684-Pos clamp. The relationship of these conformational transitions to the setting inside Bayesian Approach to Fluorescence Correlation Spectroscopy Data Anal- live cells remains to be demonstrated. Here we report on efforts to combine ysis - The Danger of Least-Square Fitting in vivo single molecule tracking with single molecule FRET to study the Taq Helmut H. Strey. MutS behavior inside live E. coli. Dept Biomed Eng, Stony Brook University, Stony Brook, NY, USA. 688-Pos We present a Bayesian approach to fluorescence correlation spectroscopy Single-Molecular Pull-Down for Quantifying Epigenetic Modifications in (FCS) parameter estimation. Traditionally, FCS data are analyzed by least- Cell-Free DNA square fitting the autocorrelation function of the fluorescent intensity signal. Yang Du1, Yongyao Wang1, Jiyan Liu2, Jiajie Diao1. We show by simulation that a least-square analysis of FCS autocorrelation 1Cancer Biology, College of Medicine University of Cincinnati, Cincinnati, functions is problematic both in the sense that the analysis results in an OH, USA, 2Medical Oncology, Cancer Center, State Key Laboratory of order-of-magnitude overestimation of confidence in the fitting parameters, Biotherapy, West China Hospital, West China Medical School, Chengdu, but more importantly in systematic shifts of parameters away from the true China. value. This effect is more pronounced the shorter the data set is. Motivated DNA epigenetic modifications contribute critical regulatory functions to the by this result, we developed a Bayesian framework for the analysis of FCS underlining genetic sequence. 5-Methylcytosine (5mC), the fifth base and data that takes single photon arrival times as input. So far, we have solved 5-Hydroxymethylcytosine (5hmC), the sixth base are the two major DNA the time-independent probability distribution of photon arrival times for a 3- epigenetic modifications in mammalian genome and synergistically play d Gaussian beam shape and we are in the process of comparing our method crucial roles in development and pathogenesis. They display tissue and cell to traditional photon counting histograms in FCS. In the future, we will incor- type specific distribution in our genomes and aberrant 5mC/5hmC changes porate the dynamics of fluorescent molecule diffusion into the model. are well-accepted hallmarks of many diseases including cancers. Detection and quantification of 5mC and 5hmC in clinical samples provide important in- 685-Pos formation for diagnosis and prognosis purposes. The current methods to quan- Experimental Dissection of Excluded Volume Effects from Quinary Inter- tify DNA epigenetic modifications such as HPLC-MS, antibody dot blot/ actions in Macromolecular Crowding immunostaining and HPLC-MS either are semi-quantitative or require large Evan J. Burdsall, Vincent J. Altimari, Brandon F. Jarmusik, amount of DNA, thus limiting their use in clinical setting. Here, we report a Everett D. Spencer, Zachary A. Norris, Michael M.J. Lim, sensitive low-input method to quantifying epigenetic modifications in cfDNA. Jeffrey D. Hettinger, Nathaniel V. Nucci. Department of Physics & Astronomy, Rowan University, Glassboro, NJ, USA. 689-Pos Current models of diffusion are, at best, approximations of what happens High-Precision FRET Reveals Sequence Dependent Structures of RNA in vivo. Fick’s Law and Stokes-Einstein approximate dilute solution behavior, Three-Way Junctions but they do not account for in vivo environmental factors that modulate particle Olga Doroshenko1, Hayk Vardanyan1, Aiswaria Prakash1, Sascha Froebel1, motion such as electrostatic forces, hydrophobic interactions, and excluded vol- Stanislav Kalinin1, Simon Sindbert1, Oleg Opanasyuk1, Christian A. Hanke1, ume. Such factors represent the contributions from macromolecular crowding Sabine Mueller2, Holger Gohlke3, Claus A.M. Seidel1. that lead to diffusional anomalies seen in living cells. In the past, separation 1Molecular Physical Chemistry, Heinrich Heine University, Dusseldorf, of excluded volume effects from the quinary interactions created by weak elec- Germany, 2Ernst-Moritz-Arndt-Univ Greifswald, Greifswald, Germany, trostatic and hydrophobic forces has been challenging. We are using a combi- 3Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine nation of fluorescence correlation spectroscopy (FCS) and microfluidic devices University, Duesseldorf, Germany. to tease apart these contributions such that they may be differentially quanti- Fo¨rster-Resonance-Energy-Transfer (FRET) restrained high-precision struc- fied. FCS allows precise measurement of single molecule fluorescence thereby tural modeling is the powerful tool for detection conformational changes in permitting assessment of local mobility in the presence and absence of crow- the biomolecular structure. In this study we combine single-molecule multi- ders. By modulating the crowder surface area under conditions of constant parameter fluorescence detection (MFD) and ensemble Time-Correlated Single excluded volume, quinary forces may be assessed and compared to the diffu- Photon Counting measurements (eTCSPC) to investigate structure and dy- sional impact of excluded volume alone. Our long-term goal is to derive a namics of RNA three-way-junctions (3WJs) which are derived from the hairpin more precise mathematical model for how a particle will diffuse in an intracel- ribozyme. Bulge and sequence variations were considered as dominant factors lular environment. *EJB and VJA contributed equally to this work. influencing junction conformations of RNAs. To investigate the influence of

BPJ 9325_9327 140a Sunday, March 3, 2019 these factors on RNA 3WJ conformations a set of six different molecules with Posters: Molecular Dynamics I different sequences were generated, two of which have two and five unpaired nu- cleotides in the junction region. Overall 258 FRET pairs were measured and 692-Pos investigated with the analysis framework [1] that includes probability distribu- Fast Simulation Methods for Chemistry and Biology Based on Quantum tion analysis (PDA) [2] for FRET distance determination and FRET position and Mechanics screening (FPS) toolkit for structural model generation. We also compare Pedro E.M. Lopes. numerous measurements from different constructs in order to calibrate error esti- Fastcompchem.com, Baltimore, MD, USA. mation accurately. The results demonstrate that each RNA 3WJ construct has The computational studies of large biomolecular systems are typically based on only one predominant conformer. Furthermore bulges in the junction region molecular mechanics, where systems are described at the atomic level using determine orientation and rotation of helices, inducing coaxial stacking. The empirical force fields. Nonetheless, methods based on force fields have severe stacked helices are different for the 3WJs with different bulges whereas struc- problems that are well known. For example, these methods have problems tures without bulges do not exhibit coaxial stacking. Noteworthy Mg concentra- describing the rich coordination chemistry of transition metals (ex. metallopro- tion in solution modulates structural stabilization, preventing conformational teins) or physical phenomena such as charge transfer. The requirement of spe- changes. Our results show that small changes in the sequence make dramatic cific parameterization also limits their applicability. changes in RNA 3WJ tertiary structures which impacts the biological function. There is a need to develop new computational methods based on Quantum Me- [1] Kalinin, S. et al, Nature Methods, 9, 1218-1225 (2012) chanics (QM) to study large and heterogeneous systems. Quantum methods are [2] Antonik, M. et al, J.Phys.Chem, 110,6970-6978 (2006) typically limited by the calculation of Electron Repulsion Integrals (ERIs) and 690-Pos diagonalization of large matrices. Initial work focused on the development of Investigating How Chirality of a Threading Binuclear Ruthenium Com- fast techniques for the calculation of ERIs and the fastest algorithm ever devel- plex Affects the DNA Threading Intercalation using Optical Tweezers oped has been published [1]. Adam A. Jabak1, Nicholas Bryden1, Fredrik Westerlund2, Per Lincoln3, Subsequently, work has begun on fast techniques for diagonalization/density Micah J. McCauley4, Ioulia F. Rouzina5, Mark C. Williams4, matrix optimization. The goal is to use the new algorithms for diagonalization Thayaparan Paramanathan1. and ERIs computation in a new generation of semi-empirical methods for bio- 1Dept of Physics, Bridgewater State University, Bridgewater, MA, USA, logic simulations, materials science and drug-design. The new computational 2Dept of Biology and Biological Engineering, Chalmers University of method is based on a highly parameterized tight-binding Hamiltonian that in- Technology, Gothenburg, Sweden, 3Dept of Chemistry and Chemical corporates terms for electron-electron repulsion, electron-nucleus attractions Engineering, Chalmers University of Technology, Gothenburg, Sweden, and dispersion. The algorithm is extremely fast and takes advantage of the lat- 4Dept of Physics, Northeastern University, Boston, MA, USA, 5Department est hardware developments. For the first time, MD simulations of large bio- of Chemistry and Biochemistry, Ohio State University, Columbus, OH, USA. molecules will be possible based on QM. Using optical tweezers, we have been able to study the interactions of small mol- Results from the simulation of small proteins will be presented. The test systems will ecules and prospective cancer drugs with DNA. One type of these molecules, be small solvated proteins (ex. Crambin). Special emphasis will be placed on the known as threading intercalators, has a flat planar moiety in between the mole- calculation of important properties that cannot be captured by classical force fields. cule’s bulky side chains. In order to bind with DNA they have to thread their The parameterization strategies of the semi-empirical method will be discussed. bulky side chains in between the base pairs. Due to this requirement for binding, 1. P.E.M. Lopes, Fast calculation of two-electron-repulsion integrals: a numer- these molecules tend to have high binding affinities and slow kinetics. We have ical approach, Theor Chem Acc 136 (2017) 112 been investigating the binding properties of the ruthenium-based threading in- 4þ 693-Pos tercalator ^^-[m-bidppz(phen)4Ru2] ,or^^-P for short. This complex has the exact chemical components but an opposite chirality to the previously stud- Error Analysis for Small-Sample, High-Variance Data: Cautions for Boot- ied DD-P complex. Preliminary data show that the change in chirality affects the strapping and Bayesian Bootstrapping DNA binding kinetics, the association and dissociation rates, to at least a three- Barmak Mostofian, Daniel M. Zuckerman. fold in favor of the left-handed ^^-P complex. The data also confirm that both Oregon Hlth and Sci Univ, Portland, OR, USA. the left-handed and right-handed molecule have a similar binding affinity as a Recent advances in molecular simulations allow the direct evaluation of kinetic result of their common intercalating moiety. This comparison will give us a bet- parameters such as rate constants for protein folding or unfolding. However, ter understanding of how chirality affects the binding to DNA and will contribute these calculations are usually computationally expensive and even significant towards improved designs of potential cancer treatment drugs. computing resources may result in a small number of independent rate estimates spread over many orders of magnitude. Such small-sample, high-variance data 691-Pos are not readily amenable to analysis using the standard uncertainty (i.e., ‘‘stan- Three Color Single-Molecule FRET of Fast IDP Binding and Folding dard error of the mean’’) because unphysical negative limits of confidence inter- Jae-Yeol Kim, Janghyun Yoo, Hoi Sung Chung. vals result. Bootstrapping, a natural alternative guaranteed to yield a confidence LCP, NIDDK NIH, Bethesda, MD, USA. interval within the minimum and maximum values, also exhibits a striking sys- Molecular interactions of intrinsically disordered proteins (IDPs) are different tematic bias of the lower confidence limit. As we show, bootstrapping artifactu- from those between folded molecules because IDP binding involves large convor- ally assigns high probability to improbably low mean values. A second mational changes. To understand the mechanism of IDP binding, three-color Fo¨r- alternative, the Bayesian bootstrap strategy, does not suffer from the same deficit ster resonance energy transfer (FRET) will be very useful because both the and is more logically consistent with the type of confidence interval desired, but interaction between two molecules and conformational changes can be probed must be used with caution nevertheless. Neither standard nor Bayesian boot- simultaneously. In this work, we investigated binding of a disordered protein, strapping can overcome the intrinsic challenge of under-estimating the mean the transactivation domain (TAD) of p53 and one of its binding partners, nuclear from small-size, high-variance samples. Our conclusions are based on extensive coactivator binding domain (NCBD) of CBP. TAD labeled with Alexa488 analysis of model distributions and re-analysis of multiple independent atom- (donor) and Alexa594 (acceptor 1, A1) site-specifically was immobilized on a istic simulations. Although we only analyze rate constants, similar consider- glass surface and incubated with CF680R (acceptor 2, A2)-labeled NCBD in so- ations will apply to other types of high-variance calculations, potentially lution. To determine all three FRET efficiencies, alternating excitation of the including highly non-linear averages like the Jarzynski relation. donor and A1 is typically used. However, inbinding experiments, direct excitation of A2 attached to NCBD in solution by A1 excitation laser produces a high back- 694-Pos ground signal. Therefore, we devised a method using a single donor excitation that MDFF Error Analysis: A Tool for Determining Stereochemical and Ther- determines all three FRET efficiencies by utilizing incomplete labeling of A2. modynamic Correct Structures When TAD binds to unlabled NCBD, the FRET efficiency between the donor Daipayan Sarkar1, John Vant2, Mrinal Shekhar3, Jane S. Richardson4, and A1 (E1) can be determined. Using this information, it is possible to determine Robert Skeel5, Abhishek Singharoy2. the other two FRET efficiencies in principle. However, due to the fast binding and 1Mechanical Engineering, The University of Texas at Dallas, Richardson, dissociation kinetics, the bound and unbound states are not well separated in TX, USA, 2School of Molecular Sciences, Arizona State University, Tempe, binned FRET efficiency trajectories (1 ms bin time), which prevents pre- AZ, USA, 3Biophysics and Quantitative Biology, University of Illinois determination of E1. We employed the maximum likelihood method developed Urbana-Champaign, Urbana, IL, USA, 4Biochemistry, Duke University, by Gopich and Szabo that analyzes photon trajectories directly without binning Durham, NC, USA, 5School of Mathematical and Statistical Sciences, the data. By globally analyzing the data using a kinetic model that includes bind- Arizona State University, Tempe, AZ, USA. ing to both labeled and unlabled NCBD, rate coefficients of the sub-millisecond Popular methods such as molecular Dynamics Flexible Fitting (MDFF), xMDFF kinetics and all three FRET efficiencies were determined accurately. for low resolution X-ray Crystallography and very recently developed resolution

BPJ 9325_9327 Sunday, March 3, 2019 141a exchange, ReMDFF, provide a precise tool to fit atomic structures of a protein field, partitioning of a small peptide, hydrogen-bond dynamics, and membrane inside an electron density map to improve the resolution of the protein structure. mixing also show very little dependence on HMR and the time step. We also Typically, MDFF incorporates the electron microscopy (EM) data as an external tested a shorter, 9-A˚ cutoff (vs. the standard CHARMM cutoff of 12 A˚ ), finding potential (UEM) added to the standard molecular dynamics force field, such that significant deviations in almost all properties tested. We conclude that HMR is the high-density areas in the map correspond to energy minima, where the atoms a valid approach for membrane systems but a 9-A cutoff is often not. are subject to forces proportional to the gradient of the EM map. However, the 697-Pos error involved in biasing the standard potential with UEM has not been explored Optimal Temperature and Pressure Evaluations in Molecular Dynamics yet. Re-casting UEM in an integro-differential form, reveals a hidden error en- ergy term reminiscent of long-range interactions between stationary masses. Simulations with a Large Time Step This additional contribution to the force filed unnecessarily contorts the molec- Jaewoon Jung, Chigusa Kobayashi, Yuji Sugita. ular geometries during an MDFF refinement. We perform, a quantitative assess- Riken, Kobe, Japan. ment of the effect of this hidden error term in MDFF’s grid force field using In molecular dynamics (MD) simulations, an accurate evaluation of tempera- recently published structures using EM to Protein Data Bank, of varying resi- ture and pressure is essential for controlling temperature as well as pressure dues, 200 - 20,000 with 3-8 A˚ resolution and their corresponding electron den- in the isothermal-isobaric conditions. In MD, conventional approaches of tem- sity maps. The initial systems are prepared using default MDFF settings, for perature and pressure evaluations from kinetic energy do not include Hessian different grid force scaling parameter. The protein structure files are modified terms properly, and are not appropriate with a large time step. To enable an by replacing the charge with atoms mass multiplied by a factor proportional MD simulation with a large time step, we suggest more optimal temperature to the error term. The mass-based coulomb like potential is then calculated using and pressure evaluations. Temperature is evaluated by combining two types NAMD Energy where the structures corresponding to low and high error term of kinetic energies: kinetic energies defined at half- and full-time steps. Pres- values are used and run through MolProbity for comparison of their relative sta- sure is evaluated only from the kinetic energy at half-time step. These provide bilities. Our results indicate, re-weighting an ensemble of structures based on the optimal instantaneous temperature and pressure up to the third order of the time error-energy term, provides improvement in determining both stereochemical step while conventional ones are accurate only up to the first order of the time and thermodynamic correct structures of proteins from electron density maps. step. The method is tested for a one-dimensional harmonic oscillator, pure wa- ter molecules, a Bovine pancreatic trypsin inhibitor (BPTI) protein in water 695-Pos molecules, and a hydrated 1,2-dispalmitoyl-sn-phosphatidylcholine (DPPC) Loos: A Tool for Making Tools to Analyze Molecular Dynamics Simula- lipid bilayer in water molecules. In all tests, our temperature/pressure evalua- tions tions well reproduce the usual physical properties for time steps up to 5 fs. Alan Grossfield, Tod D. Romo. Dept Biochem/Biophys, Univ Rochester Med Ctr, Rochester, NY, USA. 698-Pos The enormous improvements in computational power in recent years means that Determining Free Energy Differences Through Non-Linear Morphing we can routinely runs simulations of biologically interesting systems out to the Martin Reinhardt, Helmut Grubmueller. microsecond scale or longer. However, in many cases their scientific impact is Department of Theoretical and Computational Biophysics, Max Planck limited by an inability to extract useful information from this wealth of data. Institute for Biophysical Chemistry, Go¨ttingen, Germany. We created LOOS to unlock this previously unrealized potential. Molecular dy- The Bennett Acceptance Ratio (BAR) method is widely used to estimate the free namics is unique among biophysical techniques in that the raw data can be inter- energy difference between two given states of a many-body system, such as a rogated in a multitude of ways; getting the most out of the data usually requires macromolecule, where Molecular Dynamics or Monte Carlo simulations are typi- exploration and iterative refinement. LOOS is designed to make this process easy cally used for sampling. Because insufficient phase space overlap of the two states and efficient, to minimize the friction between a user and her data. can lead to large biases in the results, the calculation is commonly split up into LOOS can be viewed in two ways: 1) It is a suite of well-validated, easy-to-use several smaller steps using intermediate states defined along a so-called morphing tools for analyzing molecular dynamics simulations. These tools include common path — typically a linear interpolation between the start and the end states’ Ham- tasks, such as trajectory manipulation, as well as more advanced techniques such iltonians. Here, using BAR, we develop an optimization scheme to find the path as analysis of simulation convergence, 3D histograms, and Voronoi decomposi- that yields the most accurate free energy values from the much more general class tion, with a particular focus on membranes and membrane-bound proteins. 2) It is of paths that includes also non-linear interpolations. For a broad range of one- a tool for making tools. At its heart, LOOS is a Cþþ library covered with a Python dimensional test systems, we show that the free energy sampling error can be interface, for the optimal mix of high performance and ease of development. decreased by 25% to 35% compared to linear interpolations, with a theoretical up- LOOS is designed for rapid application development, so that most common tasks per limit of 50%. Further, correlations arising from using the same sample in one (reading files, selecting atoms, aligning structures, computing distances, etc) are state to evaluate differences to more than one other state canbe taken into account. one-line operations. LOOS is licensed under the GPLv3 license and available via Finally, we demonstrate that the optimization is still valid for a large number of GitHub (https://github.com/GrossfieldLab/loos). intermediate states with very sparse sampling for each, enabling further optimi- zation by trading off sampling versus number of intermediate states. 696-Pos On the Validity of Hydrogen Mass Repartitioning for CHARMM36 Mem- 699-Pos brane Systems in NAMD Exploring Optimal Resource Allocation for Weighted Ensemble Resam- James C. Gumbart1, Curtis Balusek1, Hyea Hwang2, Chun Hon Lau3, pling of Rare Events Karl Lundquist1, Anthony Hazel1, Anna Pavlova1, Diane Lynch4, Jeremy T. Copperman1, David Aristoff2, Daniel M. Zuckerman1. Patricia Reggio4, Yi Wang3. 1Department of Biomedical Engineering, Oregon Health and Science 1Dept Physics, Georgia Tech, Atlanta, GA, USA, 2Materials Science and University, Portland, OR, USA, 2Department of Mathematics, Colorado State Engineering, Georgia Tech, Atlanta, GA, USA, 3Dept Physics, Chinese University, Fort Collins, CO, USA. University Hong Kong, Hong Kong, Hong Kong, 4Chemistry, UNC A key goal of molecular dynamics simulations in biology is the accurate and pre- Greensboro, Greensboro, NC, USA. cise measurement of the rates and mechanisms of rare events, for example ligand The time step of atomistic molecular dynamics (MD) simulations is determined binding/unbinding, or protein folding. A fundamental problem in the field lies in by the fastest motions in the system. Typically, these are bond vibrations be- determining the best way to use finite computing resources to obtain this informa- tween hydrogen atoms and their parent heavy atoms, which require a 1-fs tion. The framework of the weighted ensemble (WE) resampling procedure pro- time step to ensure stability of the simulation. Imposing constraints on vides the ability to sample thousands of unbiased reactive trajectories spanning hydrogen-parent-atom distances permits a 2 to 3-fs time step, but angle vibra- initial and final states of interest in simulation times on the scale of the event dura- tions prevent any further increase. An increasingly popular solution is to in- tion, which is typically exponentially shorter than the first-passage time. The WE crease the mass of the hydrogen atoms to 3 amu and decrease the mass of resampling approach, although unbiased, comes with the cost that the variance the parent atom by an equivalent amount. This approach, known as hydrogen can be large, limiting the value of computed quantities. Building on recent ideas mass repartitioning (HMR), permits time steps up to 4 fs with reasonable simu- regarding the sources of variance in a resampled trajectory ensemble [https:// lation stability. While HMR has been applied in many published studies to date, arxiv.org/abs/1806.00860], we implement an adaptive variance-minimization it has not been extensively tested for membrane systems. Here, we compare the procedure that dynamically optimizes allocation of computing resources across results of simulations of a variety of membranes and membrane-protein sys- the space. This procedure is based on leveraging a coarse-grained Markov model tems run using a 2-fs time step and a 4-fs time step with HMR. For pure mem- of the system which can be known a priori or created on the fly. The variance is brane systems, we find practically no difference in structural properties, such as (approximately) minimized at each intermediate step by estimating the optimal area-per-lipid and order parameters, and very little difference in kinetic prop- resource allocation defined by the coarse-grained model. We present results erties such as the diffusion constant. Conductance through a porin in an applied regarding the application of these methods to the folding of small proteins.

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700-Pos eters for a wide range of functional groups and heterocycles. This approach for the New Tools for Conformational and Binding Free Energy Simulations optimization of all aspects of DGenFF parameters is built on the atom typing Giacomo Fiorin1, Grace Brannigan2, Jeroˆme Henin3. methodology of CGenFF. Development of electrostatic parameters is being per- 1ICMS, Temple University, Philadelphia, PA, USA, 2Rutgers University formed through generation of a database of partial atomic charges and atomic po- Camden, Camden, NJ, USA, 3LBT - IBPC, CNRS, Paris, France. larizabilities based on quantum mechanical wave function partitioning and fitting We offer free and open source tools for simpler, faster, and more robust free en- to electrostatic potentials. Optimization of intramolecular parameters targets ergy simulations in NAMD, assisted by interactive collective variable design in quantum mechanical data on geometries, vibrational frequencies and potential en- VMD. We introduce the Collective Variables Dashboard, a graphical interface ergy surfaces supplemented with experimental data when available. Lennard- within VMD that enables an interactive workflow to analyze existing structures Jones parameters are generally transferred directly from the biomolecular Drude and simulation trajectories in terms of collective variables. The Dashboard lets force field supplemented with QM data on interactions with water and rare gases. scientists explore simulations simultaneously as molecular representations and Validation of the DGenFF parameters is performed against a range of condensed in collective variable space, and navigate between those representations in a phase experimental data including pure solvent properties, crystals and aqueous fluid way. It offers a sandbox to experiment with and fine-tune collective vari- solution properties. Efforts to automate the approach for DGenFF will facilitate ables, whose values are updated on-the-fly. Once relevant variables have been its extension to a vast number of drug-like molecules allowing comprehensive obtained, the Dashboard outputs a configuration file that can be read directly application of a fully polarizable empirical force field for computer-aided drug by the Collective Variables Module in NAMD or LAMMPS for enhanced sam- design and the study of physical phenomena in a wide range of chemical systems. pling simulations or in situ analysis. For binding free energy, we have designed, and implemented in NAMD, the Interleaved Double-Wide Sampling (IDWS) 703-Pos method, which samples both directions of an alchemical transformation at no Comparison of Functional Group Affinity Patterns from the Additive additional cost, allowing for statistically optimal MBAR reconstruction of Versus Drude Polarizable Force Fields from the Site-Identification by free energy differences. IDWS is complemented by a new type of restraint Ligand Competitive Saturation (SILCS) Approach 1 2 1 3 scheme for absolute binding free energy calculations. Distance-to-bound config- Himanshu Goel , Delin Sun Sun , Wenbo Yu , Alexander D. MacKerell . 1School of Pharmacy, University of Maryland, Baltimore, MD, USA, uration restraints, implemented using the Collective Variables Module, allow 2 3 for efficient confinement of a ligand around a binding pose with a single degree Lawrence Livermore National Laboratory, Pleasanton, CA, USA, Dept of freedom. As a result, our framework for calculating an absolute binding free Pharm Sci, Univ Maryland, Baltimore, MD, USA. energy requires only two simulations in condensed phase and one in vacuum. The Site Identification by Ligand Competitive Saturation (SILCS) methodology is of utility for characterization of functional group affinities for the different re- 701-Pos gion of protein or macromolecular surfaces. This approach includes sampling of Optimized Parameters for the Drude Polarizable Force Field for Small the conformational space for multiple solutes (representing various functional Organic Molecules groups) and the protein target present in an aqueous solution. Until now, this tech- Chetan Rupakheti1, Alexander D. MacKerell2, Benoit Roux1. nique was performed for the system where pairwise or additive force field param- 1Biochemistry and Molecular Biology, University of Chicago, Chicago, IL, eters were employed. In the additive force field, the partial atomic charges are USA, 2School of Pharmacy, University of Maryland, Baltimore, MD, USA. kept fixed which results in limiting its ability to accurate model different interac- Meaningful efforts in computer-aided drug dsign (CADD) need accurate molec- tions in a diverse set of biological environments. Consequently, a viable alterna- ular mechanical force fields to quantitatively characterize protein-ligand interac- tive to overcome this issue is to include explicit electronic polarization which tions. The most widely used strategy to ascribe force field parameters for new includes alternation of the molecular dipoles as per the environment. The Drude compounds apply pre-defined tabulated values following knowledge-based polarizable force field is the direct extension of the additive force field. The aim of rules. Two widely used approaches following this strategy are the General Amber this work is to analyze, compare and discuss the results obtained from the additive Force Field (GAFF) and the CHARMM General Force Field (CGenFF). While versus Drude polarizable force field in the context of the SILCS molecular these approaches are extremely useful, an important limitation of using pre- dynamics simulation methodology. For this purpose, we considered two proteins, tabulated parameter values is that they may be inadequate in the context of a spe- P38 kinase and Factor Xa, an aqueous solution along with the multiple solutes. cific molecule. To resolve this issue, we previously introduced the General Auto- We present the free energy maps (FragMaps) representing the affinity patterns mated Atomic Model Parameterization (GAAMP) to automatically generate the of the functional groups represented by the solutes against the protein surface parameters of atomic models of small molecules using the results from ab initio for additive and Drude simulations. Moreover, we calculated the ligand grid quantum mechanical (QM) calculations as target data [DOI:10.1021/ct4003477]. free energy (LGFE) and compared with the experimental binding affinities. The GAAMP protocol uses QM data to optimize the bond, valence angle, and dihedral angle internal parameters, and atomic partial charges. However, the 704-Pos Lennard-Jones (LJ) 6-12 parameters cannot easily be refined from QM data Gromaps: A Gromacs-Based Toolset to Analyse Density Maps Derived and are unchanged from the initial atom types assignments severely limiting from Molecular Dynamics Simulations 1 2 3 4 the achievable accuracy by these models. Here, we systematically optimize the Rodolfo Briones , Christian Blau , Carsten Kutzner , Bert L. de Groot , 5,6 LJ parameters of the Drude force field to reproduce experimental neat liquid den- Camilo Aponte-Santamarı´a . 1Forschungszentrum Julich,€ Julich,€ Germany, 2Theoretical Computational sities and enthalpies of vaporization for a large set of compounds, covering a wide 3 range of chemical functionalities. The optimization is carried out by constructing Biophysics, Stockholm University, Solna, Sweden, Theoretical and an objective function and exploiting the analytical gradient information within an Computational Biophysics, Max Planck Institute for Biophysical Chemistry, Go¨ttingen, Germany, 4Computational Biomolecular Dynamics Group, Max optimization framework. The new set of LJ parameters will help to improve the 5 accuracy of the computed physical properties of diverse small organic molecules. Planck Institute for Biophysical Chemistry, Go¨ttingen, Germany, Max Planck Tandem Group in Computational Biophysics, University of Los These developments will provide the necessary tools to widely apply drude polar- 6 izable force field model for drug discovery endeavors. Andes, Bogota´, Colombia, Interdisciplinary Center for Scientific Computing (IWR), University of Heidelberg, Heidelberg, Germany. 702-Pos We introduce a computational toolset, named GROmars, to obtain and compare Polarizable General Force Field for Drug-Like Molecules: Drude General time-averaged density maps derived from molecular dynamics (MD) simula- Force Field (DGenFF) tions. GROmars efficiently computes density maps, by fast multi-Gaussian Payal Chatterjee1, Esther Heid2, Christian Schro¨der2, spreading of atomic densities onto a 3-dimensional grid. It complements existing Alexander D. MacKerell1. map-based tools by enabling spacial inspection of atomic average localization 1Dept Pharm Sci, Univ Maryland, Baltimore, MD, USA, 2Institute of during the simulations. Most importantly, it allows the comparison between Computational Biological Chemistry, University of Vienna, Vienna, Austria. computed and reference maps (e.g. experimental), through calculation of differ- The Classical Drude oscillator polarizable force field offers an empirical approach ence maps, and local and time-resolved global correlation. These comparison op- to explicitly represent induced electronic polarization that is currently not erations proved useful to quantitatively contrast perturbed and control included in fixed charge representation of additive force fields. This includes simulation-data sets and to examine how much biomolecular systems resembles atomic polarizability on all non-hydrogen atoms, lone pairs and anisotropic polar- both synthetic and experimental density maps. This was especially advantageous ization in hydrogen bond acceptors and atom-based Thole scaling factors, while for multi-molecular systems, in which standard comparisons, like RMSDs, are the rest of the functional form is similar to that of CHARMM additive force field. difficult to compute. In addition, GROmars incorporates absolute and relative The Drude force field currently extends to proteins, nucleic acids, lipids, carbohy- spatial free energy estimates to provide an energetic picture of atomistic locali- drates and a limited set of drug-like (organic) molecules. To expand the coverage zation. This is a GROMACS-based toolset, thus allowing for static or dynamic of the Drude General Force Field (DGenFF) beyond functional groups common to selection of atoms, or even coarse-grained beads, for the density calculation. biological molecules we have undertaken the systematic optimization of param- Furthermore, masking of regions was implemented, to speed up calculations

BPJ 9325_9327 Sunday, March 3, 2019 143a and to facilitate the comparison with experimental maps. Beyond map compar- N-methylacetamide molecules as the model system, we then assess the cooperativ- ison, GROmars provides an straightforward method to detect solvent cavities ity of protein backbone hydrogen bonds using quantum chemistry calculations at the and average charge-distribution in biomolecular systems. We employed all these MP2/aug-cc-pVTZ level. MM calculations have also been carried out and functionalities to inspect lipid and water localization in aquaporin channels, the compared with the QM results. While additive force fields all lead to null coopera- binding of cholesterol to the G Protein Coupled Chemokine Receptor Type 4, tivity, polarizable force fields - including the Drude and the AMOEBA protein force and the identification of permeation pathways through the dermicidin antimicro- fields - have been found to reproduce the trend and the magnitude of QM results. bial channel. Based on these examples, we anticipate a high applicability of We further study the contribution of hydrogen bonding cooperativity in protein GROmars for the analysis of MD simulations and their comparison with exper- dynamics using long timescale explicit solvent MD simulations with the Drude- imentally determined densities. GROmars is open-source and it is available at 2013 polarizable force fields. Based on the simulation results of several peptide https://mptg-cbp.github.io/gromaps.html [submitted to Biophysical Journal]. systems, we show that the cooperativity of NH-OC backbone hydrogen bonding has significant contribution in the thermodynamics and kinetics of secondary 705-Pos structure formation and plays a major role in the aggregation of intrinsically disor- Exploring Hydrogen Bond Geometry in RNA with F-SAPT dered proteins (IDPs). Our work demonstrates the importance of including explicit Louis G. Smith, Chapin E. Cavender, Alan Grossfield, David H. Mathews. polarization in modeling the cooperativity of protein folding and aggregation. Dept Biochem & Biophys, Univ Rochester, Rochester, NY, USA. Molecular dynamics (MD) simulations with all-atom models provide important 708-Pos information for developing hypotheses and interpreting experimental data. MD Waterfall Sampling: An Online Sequential Monte Carlo Strategy for estimates the potential energy as a function of molecular conformation using a Conformational Sampling of Biomolecular Systems set of classical equations and parameters called a force field. The accuracy of Mir Ishruna Muniyat, Justin L. MacCallum. this force field is thus necessary but not sufficient for accurate simulations. Chemistry, University of Calgary, Calgary, AB, Canada. Generally, to sample conformation energy landscapes sufficiently given current Conformational sampling of biomolecular systems remains challenging because computing power, MD simulations use force fields that assume a fixed point their energy landscapes are often rugged with large energetic and entropic barriers. charge model. The central importance of the structure-function relationship in These challenges can be partially overcome using Replica Exchange Molecular RNA biology has made accurate MD simulations of RNA and RNA-protein Dynamics (REMD) simulations. Hamiltonian replica exchange (H-REMD) is a complexes an important goal of the field. For RNA, currently available force particularly promising technique, as it allows for the introduction of biasing poten- fields with fixed point charges do not adequately model the potential energy; tials along one or more reaction coordinates that can guide sampling, potentially simulations of small RNA do not reflect the known solution conformations. overcoming both entropic and enthalpic barriers. However, we have observed One hypothesis that may partially explain these issues is that this functional that the introduction of such biasing potentials can lead to poor kinetic behavior, form does not adequately model the angular dependence of hydrogen bonds. where some replicas become trapped in a limited region of the reaction coordinate. Because the hydrogen bonds are represented as a partial charge interaction, Due to the coupling between replicas due to swapping, this creates a barrier for they are not as able to model interactions where the natural geometry of the other non-trapped replicas to traverse this region of the reaction coordinate. Under bond along the donor, hydrogen, and acceptor is not co-axial, such as in the these circumstances, Annealed Importance Sampling (AIS), a form of Sequential case of hydrogen bonds with carbonyl lone pairs as acceptors, which should Monte Carlo (SMC), may provide for improved sampling. Here, we propose a not favor a linear hydrogen bond along the axis of the double bond. In a fixed novel sampling strategy called ‘‘waterfall sampling’’ which can potentially point charge model hydrogen bond they do. To test this, we are using Func- perform better on systemswithkinetic and entropic barriers ata reasonable compu- tional Group Symmetry Adapted Perturbation Theory (F-SAPT) to estimate tational cost. Waterfall sampling combines ideas from reservoir-REMD and AIS. the energies of hydrogen bonds as a function of their relative orientation. We An immediate advantage of waterfall sampling is that the movement of replicas in compare these energies to those estimated by current force fields, and will replica space is directed, eliminating poor round-trip times for large systems. use the results to guide the design of more sophisticated functional forms. Furthermore, there is no coupling between replicas, so waterfall sampling is less 706-Pos susceptibletothekinetictrapping describedabove.We test our methodona variety Markov Modeling of Protein Diffusion on Telomeric DNA of simple systems, including systems with both entropic and enthalpic barriers, and Milosz Wieczor, Antoni Marciniak, Jacek Czub. we further explore its applicability on a more complex protein folding problem. Dept Phys Chem, Gdansk Univ Tech, Gdansk, Poland. Telomeres, the termini of linear chromosomes, solve two major issues of genome 709-Pos maintenance in Eukaryota: hide the loose ends of DNA from the DNA repair ma- Charting the Hydrophobic Effect: Computing Spatially Resolved Absolute chinery, and fix the end-replication problem by allowing for repeated shortening Hydration Shell Entropies and extension of the tandem repeating fragments. The protective nucleoprotein Leonard P. Heinz, Helmut Grubmueller. complex is not static, though, with e.g. cell-cycle dependent opening and closing Department for Theoretical & Computational Biophysics, Max Planck of the telomeric loop necessary for DNA replication. Due to the repetitive nature Institute for Biophysical Chemistry, Go¨ttingen, Germany. of the telomeric sequence, all telomeric DNA-binding proteins also constantly Biophysical systems are governed by their free energy and thus often depend on a slide along the DNA, moving between adjacent binding sites. In particular, the fine-tuned interplay between enthalpy and entropy. For protein folding, e.g., the sliding of POT1 along single-stranded DNA prevents the unwanted formation solvation contributions are crucial, which also gives rise to the hydrophobic ef- of G-quadruplexes on the 30-terminal overhang, while TRF1 and TRF2 slide fect. A quantitative understanding of the thermodynamics of solvation, and in along the double-stranded portion to assemble the shelterin. particular the associated entropies, is therefore essential. While a variety of Here, we use molecular simulations augmented with Markov models and free methods allow assessing the solute entropy, solvation shell entropies are notori- energy methods to model the diffusion of TRF1 and POT1 along both telomeric ously difficult to obtain from computer simulations, because the shallow energy and polyA DNA in atomistic resolution. Instead of a simple geometrical, center- landscape requires sampling of an extremely large configuration space. Here we of-mass position based approach, we use intermolecular contacts as a more solve the sampling problem by exploiting the permutation symmetry of the sol- informative descriptor of diffusive processes, particularly when subdiffusion vent particles which reduces the configuration space that has to be sampled by the comes into play. Comparison with experimental data allows to probe the sensi- Gibbs factor 1/N!, leaving the physics of the system unchanged. We perform a tivity of the model to its parameters. With this approach, we outline a general mutual information expansion to obtain both translational and rotational solvent methodology for the atomistic modelling of sliding-like diffusion along biopoly- entropy values from the permutationally reduced trajectory. The expansion mers, a crucial stage in the dynamic formation of sequence-specific complexes. yields entropy contributions of individual solvent particles as well as of groups of particles, such as different solvation shells, thereby providing a spatial reso- 707-Pos lution. We applied our method to compute entropies of solvation in multiple sys- Capturing the Cooperativity of Backbone Hydrogen Bonding with Polariz- tems, such as n-alkanes and small peptides and obtained results that are in able Force Fields agreement with other methods and experimental values. Jing Huang. School of Life Sciences, Westlake University, Hangzhou, China. 710-Pos The hydrogen bonds (H-bonds) between backbone NH and CO groups are funda- Hamiltonian Replica Exchange for Enhanced Sampling of the Conforma- mental for the stability, structure and dynamics of proteins. A key feature of such tional Landscape for Intrinsically Disordered Proteins hydrogen bonding interactions is that multiple H-bonds can enhance each other Justin S.H. Kim1,2, Sarah Rauscher1,2. when aligned, as such in the a-helix or b-sheet secondary structures. To better 1Physics, University of Toronto, Toronto, ON, Canada, 2Chemical & understand this cooperativity effect, we first propose a new physical quantity to Physical Sciences, University of Toronto Mississauga, Mississauga, ON, evaluate the cooperativity of intermolecular interactions. Using H-bond aligned Canada.

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Intrinsically disordered proteins (IDPs) are a class of proteins that lack a initial and the final stage is large, biasing collective variables (cv) that are unique native three-dimensional structure. Molecular dynamics (MD) is an some combination of atomic coordinates, allows sampling of all representative invaluable method to study the dynamics of folded proteins. However, there configurations and accurate calculation of free energy profiles. In this work, us- have been challenges in its application to simulating IDPs. One of these chal- ing all atom molecular dynamics simulation with biasing schemes like well- lenges is the need to sample a vast number of structural conformations. To tempered metadynamics, umbrella sampling and replica exchange umbrella help overcome this challenge, one of the methods that was introduced is sampling, we calculate the free energy profile for translocation of small mole- the temperature replica exchange (TRE) algorithm. This method uses a cules through a model 1,2-dioleoyl-sn-glycero-3-phosphocholinebilayer as a random walk in temperature space to help overcome energy barriers. Howev- function of distance from the bilayer center. We find that only for neutral sol- er, this method can require vast computing resources and the efficiency of the utes, the free energy calculation is straightforward. Using the committor anal- TRE algorithm is not well characterized. We use a variant of Hamiltonian ysis, we find that non-converged profiles result due to insufficient sampling of replica exchange (HRE), which instead of temperature uses the scaling of the transition state. We also demonstrate the usage of acceptance probabilities the Lennard-Jones protein-water interactions. In conducting the algorithm in in a replica exchange calculation as a way to identify sampling problems. Based this manner, the computational requirements are reduced compared to TRE. on our results, we provide useful guidance to detect and overcome convergence Here, we used MD simulations on an IDP system to compare the sampling failure to infer useful physical behavior of a system. efficiency between these sampling algorithms. We tested this algorithm on the RS1 region of the serine/arginine-rich splicing factor 1; an IDP with 714-Pos many consecutive repeating RS residues. In on-going work, we are comparing Enhanced Sampling, Kinetics Calculation and Structural Database Anal- the computational efficiency of the HRE algorithm to TRE, conventional MD ysis Aiming at Computational Drug Design simulation, as well as other HRE algorithms. Kei Moritsugu1, Tohru Terada2, Yoshihiko Nishino1, Akinori Kidera1. 1Yokohama City Univ, Yokohama, Japan, 2Grad Sch Agr Life Sci, Univ 711-Pos Tokyo, Bunkyo-ku, Japan. Protein-Water and Solvent-Mediated Interactions in Multi-Scale Simula- Integrative study using molecular dynamics (MD) simulations and structural tions bioinformatics will be essential for computational drug design. Here, we pre- Matthias Heyden. sent application studies for pharmacological targets using our newly devel- School of Molecular Sciences, Arizona State University, Tempe, AZ, USA. oped multiscale enhanced sampling (MSES), weighted ensemble and Water is the ubiquitous solvent in biomolecular systems. Water contributes to Motion Tree. In MSES, the structural sampling of large proteins at atomic res- the stability of natively folded proteins, it mediates intermolecular interactions olution is enhanced by coupling with accelerated dynamics of the associated via the hydrophobic effect and through hydrogen bonds, it provides the dielec- coarse-grained model. The weighted ensemble developed by Zuckerman and tric medium that helps to separate charges, and its dynamic properties are coworkers allows the rate constants of protein structural changes to be effi- coupled to structural fluctuations of solvated molecules. ciently calculated by use of many short-time MDs. The Motion Tree, calcu- The work presented here focuses on the extraction of thermodynamic signa- lated by a hierarchical clustering of the residue-residue distance fluctuation, tures from atomistic molecular dynamics simulations of biomolecular sys- illustrates significant dynamic domains inherent in the structural ensemble. tems. We developed a spatially resolved analysis of solvation free energy Firstly, we applied MSES to an important drug discovery target, glucokinase contributions for biomolecules. As a result, we can distinguish hydrophobic (GCK). MSES enabled to compare two sets of the free energy landscapes for and hydrophilic hydration sites on a biomolecular surface based on local ther- the glucose-bound and glucose-unbound forms, demonstrating the drastic modynamic parameters in an atomistic, explicit-solvent simulation. This al- change of the free energy surface depending on the glucose concentration. lows us to compare to simpler classification schemes of solvent accessible The energy landscapes, together with the weighted ensemble simulations, al- surfaces that are frequently used in implicit solvation models. We study in lowed the rate constant of the GCK structural change in milli-second time- detail protein-water interactions of intrinsically disordered proteins, which scale to be estimated. Secondly, a comprehensive analysis of protein kinase were recently identified as a critical component for realistic computational structures was carried out using Motion Tree. Motion Tree calculated from models. 1107 crystal structures of human tyrosine kinases demonstrated a significance We then utilize this microscopic information to improve the description of sol- of the domain motion between kinase N-lobe and C-lobe, as well as the local vent effects in computationally efficient simulation models, which rely on im- motions of both activation loop and C-helix. Structural classifications by the plicit representations of the solvent. Such multi-scale simulations models allow three motions and by assigning the information on kinase activities (DFG-in/ for simulation studies of biomolecular recognition, self-assembly or molecular C-helix-in), drug interactions, etc., yielded a general picture of how protein crowding in realistic biomolecular environments at moderate computational kinases are inactivated due to what kinds of drug molecules on which binding costs. pockets. Furthermore, MSES was performed to illustrate the importance of the 712-Pos domain motion for the protein kinase activation, seen in such as EGFR Connectivity, Dynamics and Biomolecular Energy Transport dimerization. Justin Elenewski1, Kirill Velizhanin2, Michael Zwolak1. 1National Institute of Standards and Techology, Gaithersburg, MD, USA, 715-Pos 2Los Alamos National Laboratory, Los Alamos, NM, USA. When Additive Molecular Dynamics Fails: Quantum Effects in Calcium- Anomalous heat transport abounds in bimolecular systems, where complex en- Dependent Lectin/Carbohydrate Complex Martin Lepsik1, Mickael G. Lelimousin1, Emanuele Paci2, Anne Imberty1. ergy landscapes and pronounced nonlinearities can collude to dramatically 1 2 impact behavior. The underlying - and often overlooked - pathways are none- CERMAV CNRS, Grenoble, France, Faculty of Biological Sciences, Univ theless essential, acting to dissipate waste heat from enzyme catalysis and facil- Leeds, Leeds, United Kingdom. itating the development of mechanical motion. While spectroscopic techniques The developments of additive carbohydrate force fields increased the reliability of molecular dynamics simulations (MD) of protein-carbohydrate complexes. can directly probe these dynamics, their broad use will require a clear theoret- 2þ ical relation between macromolecular structure and routes for heat propagation. The presence of bridging Ca ions can, however, pose problems for structural and energetic description due to quantum effects, such as charge transfer. To We approach this problem by levering non-equilibrium simulations and simpli- 2þ fied models to devise general principles for heat transport in model proteins. In overcome this limitation, we have developed Ca parameters with effective doing so, we demonstrate that heat transport is highly sensitive to topological electronic polarisation for use with additive force fields and applied them to characteristics of the conformational ensemble. We further delineate a cross- a calcium-dependent lectin/carbohydrate complex. Such a treatment improved 2þ 2þ over between energy localization and free propagation, lending insight into pro- the structural description of the binding site (Ca Ca distance matching tein function and suggesting metrologies that probe bimolecular dynamics that of the crystal structure) but failed to reproduce the pattern of protein/car- through their response to non-equilibrium perturbations. bohydrate interactions and the location of specific bridging water molecules. This system thus necessitated the use of polarizable force fields. Coupled 713-Pos with quantum mechanical/molecular mechanical (QM/MM) calculations, we þ Using Committor and its Distribution to Assess the Convergence of Free were able to describe the polarization phenomena of the Ca2 ions, protein Energy Calculations carboxylate side chains, carbohydrate hydroxyls and specific water molecules Nihit Pokhrel, Lutz Maibaum. in the active site which together define the structure and energetics of the com- þ Dept Chemistry, Univ Washington, Seattle, WA, USA. plex. In summary, we utilized new Ca2 parameters and advanced computa- Molecular simulation has been extensively used to calculate free energy asso- tional techniques (polarizable MD, QM/MM calculations) on the ciated with any molecular phenomenon in order to understand the thermody- crystallographic structure to gain atomistic and energetic understanding of þ namics and kinetics of the process. When the energetic barrier between the the Ca2 -dependent lectin/carbohydrate complex.

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716-Pos namics, the Force Matching (FM) [1] and, further, the Multi-Scale Coarse- Overcoming the Embeddability Problem: A More Robust Calculation of Graining (MSCG) approaches to biomolecular systems have been developed Kinetic Information from Sparsely Sampled Molecular Dynamics Simula- by the Voth group [2], in which the forces computed at the all-atom level are tions averaged over the degrees of freedom omitted from a coarse-grained representa- Curtis Goolsby, Mahmoud Moradi. tion. However, because isotropic site-site interactions have been assumed, Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. aggressive coarse graining is not possible and the resulting force fields are not Markovian models provide a convenient way for estimating kinetic properties of transferable. We propose a new approach to FM, which overcomes both prob- biomolecular systems from molecular dynamics (MD) trajectories. These for- lems. Anisotropic forces have been introduced by least-squares fitting the forces malisms present attractive characteristics which potentially can overcome sam- computed over the entire coarse-grained sites to the all-atom forces. To achieve pling problems by constructing long-term kinetic and thermodynamic transferability, the factor-expansion approach developed in our earlier work [3] information from short trajectories. Markov State Models (MSM) traditionally has been implemented, in which the PMF of a system is expanded into Kubo use the time-dependent transition probability matrix (TPM) to estimate different cluster-cumulant functions [4], termed factors, that can be identified with the properties of the system as opposed to the time-independent transition rate matrix respective energy terms; the mean forces are partitioned likewise. The new (TRM) or the generator matrix, which is formally the matrix logarithm of TPM FM variant has been applied to our highly reduced physics-based UNRES model divided by the lag time, used to construct the TPM. This is partly done because of of polypeptide chains [5], in which only united peptide groups and united side the possibility of the matrix logarithm of sparse TPMs not being a valid TRM, e.g. chains are the interaction sites. Comparison of the all-atom dynamics and non-physical/complex rate predictions. This problem, known as the embeddabil- coarse-grained dynamics will be presented. ity problem is what our work addresses. We present a comparative study of MSM [1] S. Izvekov & G.A. Voth, J. Phys. Chem. B, 109, 2469-2473 (2005) results using the standard TPM approach to results using an embeddability- [2] A. Das et al., J. Chem. Phys., 136, 194115 (2012) corrected TRM in situations of increasingly sparse sampling. Our work attempts [3] A.K. Sieradzan et al., J. Chem. Phys., 146, 124106 (2017) to overcome this problem and devise a more robust method for kinetic predictions [4] R. Kubo, J. Phys. Soc. Japan, 17, 1100-1120 (1962) from MD trajectories. As a means to testing our work, we characterize the ther- [5] A. Liwo, et al., J. Molec. Model., 20, 2306 (2014) modynamics and kinetics of three systems, a simple bistable potential, a more complex multi-state toy model, and finally, as a means of presenting the general 720-Pos applicability of our methods, a realistic membrane protein in explicit membrane Comparison of Forcefields in the Prediction of Intrinsic Residue-Specific simulated using all-atom MD. We use this study to compare the effectiveness of 8 Backbone Dihedral Angle Distributions of Blocked Amino Acids Jared M. Lalmansingh1, Jeong-Mo Choi2, Rohit V. Pappu2. algorithms, ranging from simple deterministic methods to stochastic Monte 1 Carlo methods, in situations of progressively insufficient sampling. Department of Physics, Washington University in St. Louis, St. Louis, MO, USA, 2Department of Biomedical Engineering, Washington University in St. 717-Pos Louis, St. Louis, MO, USA. Heterogeneous Solvation in Distinctive Protein-Protein Interfaces Re- The intrinsic conformational preferences of amino acids in water are governed by vealed by Molecular Dynamics Simulations the distributions of backbone dihedral angles, f and c. These intrinsic preferences Clarisse Gravina Ricci1, James A. McCammon2. can be gleaned from simulations of or measurements on blocked amino acids of 0 1Dept Pharmacology, Univ Calif San Diego, San Diego, CA, USA, the form N-acetyl-Xaa-N -methylamide, where Xaa denotes the amino acid resi- 2Chemistry & Bioche, Univ Calif San Diego, La Jolla, CA, USA. due of interest. In these constructs, the backbone dihedralangle preferences are not Water, despite being a driving force in biochemical processes, has an elusively influenced by local and non-local interactions that prevail in longer peptide and complex microscopic behavior. While water can increase its local density near protein sequences. Accordingly, comparative assessments of conformational dis- amphiphilic protein surfaces, water is also thought to evaporate from hydrophobic tributions extracted fromsimulations of blocked amino acids provide a direct route surfaces and cavities, an effect known as ‘‘dewetting’’. The existence and extent for comparing molecular mechanics forcefields. Recently Choi and Pappu devel- of dewetting effects remains elusive due to the difficulty in observing clear ‘‘dry- oped a refined description of blocked amino acid conformational distributions that ing’’ transitions in experiments or simulations. Here, we use explicit solvent mo- are based on a combination of coil library statistics and spectroscopic data. The lecular dynamics (MD) simulations to study the molecular solvation at the refined library renders conformational distributions in terms of conformational ba- binding interfaces of two distinctive molecular complexes: the highly hydrophilic sins that are defined by basin centers, basin envelopes, and basin weights. barnase-barstar and the highly hydrophobic MDM2-p53. Our simulations, in Here, we describe the approach used to extract basin structures given a set of conjunction with simple volumetric analyses, reveal a strikingly different water conformations in terms of f and c values. The backbone dihedral angle distri- behavior at the binding interfaces of these two molecular complexes. In both com- butions are partitioned into discrete basins based on a steepest descent method. plexes, we observe significant changes in the water local density as the two pro- We also provide quantitative, comparative assessments of commonly used mo- teins approach, supporting the existence of a clear dewetting transition in the case lecular mechanics forcefields by comparing the conformational distributions of MDM2-p53, with an onset distance of 5.6-7.6A˚ . Furthermore, the solvation obtained using these forcefields to the refined coil library for blocked amino analysis reported herein is a valuable tool to capture and quantify persistent or acids. Our results show significant, residue- and forcefield-specific deviations transient dewetting events in future explicit solvent MD simulations. of conformational distributions from the newly established, experimentally derived touchstones for these distributions. We show how the deviations can 718-Pos be used to improve forcefield parameters using the ABSINTH model as an Comparing Binding Affinity Results from Pyrosetta and Coarse-Grained example. A similar procedure might be adaptable for other molecular me- Simulations chanics forcefields, although a balanced approach is required to account for in- Kyle P. Martin, F. Marty Ytreberg. teractions that are unrepresented in blocked amino acids. Department of Physics, Univ Idaho, Moscow, ID, USA. The binding affinity is a direct measure of the strength of interaction between pro- 721-Pos teins and their calculation remains a challenge. It is known that statistical- Development of a New Calcium Ion Model for Simulating Biomolecules mechanics-based all-atom simulations can generate accurate affinity estimates, Aihua Zhang, Hua Yu, Chunhong Liu, Chen Song. but these calculations are cost-prohibitive. We are interested in developing and us- Center for Quantitative Biology, Peking University, Beijing, China. ing methods that have a balance between accuracy and simulation efficiency. In Calcium ions play crucial roles in many important biological processes, such as this study, we compare binding affinity estimates from between two such methods. muscle contraction, signal transduction in nervous system, cell signaling regula- We used PyRosetta to estimate the binding affinity changes due to single amino tion and cell fate determination. In the last few years, a couple of novel structures of Ca2þ permeable ion channels have been determined, which makes the compu- acid mutations in three protein-protein systems. Results were then compared to 2þ previous work done with coarse-grained simulations, and to experimental results. tational studies of the Ca -related bio-molecules highly desirable. However, the existing Ca2þ models in the classical non-polarizable force field are inaccu- 719-Pos rate in calculating the interaction energies between calcium ions and protein Extension of the Force Matching Method to Anisotropic Coarse-Grained molecules, which has hindered the relevant computational studies. We are devel- þ Transferable Force Fields: Application to the Unres Model of Proteins oping a new Ca2 model in the framework of the classical non-polarizable force Jozef A. Liwo1, Cezary R. Czaplewski2. field. The aim is to not only reproduce the energetical and dynamical properties 1Fac Chem, Univ Gdansk, Gdansk, Poland, 2Chemistry, University of of calcium ions in solution, but also better describe the interaction energies be- Gdansk, Gdansk, Poland. tween calcium ions and proteins. Although still under development, the new Coarse-grained models of biological macromolecules are nowadays extensively Ca2þ model has shown very promising results in terms of permeability through used in simulations because of tremendous extension of the time- and size-scale ion channels in our simulations. It will probably allow many bio-molecules in- that they offer. To provide the link between coarse-grained and all-atom dy- teracting with Ca2þ to be simulated in a more quantitative way.

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Posters: Biosensors I were constructed and co-transfected into HEK-293 cells. Results from the inter- molecular FRET supports the dimerization hypothesis as large voltage- 722-Pos dependent FRET signals were observed. However, when one FP of the FRET How to Make Excitable Cells pair was replaced with a farnesylated version the voltage-dependent FRET Merrilee A. Thomas. signal was much smaller than when both FPs are fused to independent VSDs, Neuroscience and Cell Biology, Montana State University, Bozeman, MT, USA. suggesting that the VSD is involved in the dimerization of GEVIs. Heterotri- Genetically encoded, fluorescent voltage sensors could be powerful tools in meric FRET studies where GEVIs fusing CFP, YFP and RFP were transfected neuroscience. There are now several proof-of-principle versions that show prom- into HEK-293 cells in equal concentrations showed a large stoke shift, where ise. Improving on these, however, has been challenging because testing them in- voltage-dependent RFP emission signal was observed with CFP excitation (exci- volves low throughput, labor intensive processes. Our goal is to develop a simple, tation: 420 nm, emission: 645 nm long pass), further suggesting that the VSD reproducible, optogenetic process for testing prototypes. We chose to use Bacu- must be responsible for the association of the FPs. This paradigm enables the lovirus to introduce the optogenetic components because this gives us control development of new FRET-based probes, especially probes for 2-photon imag- over expression levels of each component and is remarkably reproducible. To hy- ing. Research reported in this publication was supported by the National Institute perpolarize HEK 293 cells, we express the Inwardly-rectifying potassium chan- Of Neurological Disorders And Stroke of the National Institutes of Health under nel. For the actuator, we chose bpac, a blue light activated adenylyl cyclase from Award Number U01NS099691. the soil bacterium Beggiatoia, (Stierl et al. 2011). 480 nM light activates this enzyme, but 560 nM light used to image mApple fluorescent proteins does not. 725-Pos In turn, the cAMP gates a mutant cyclic nucleotide-gated ion channel, (Rich et Surface-Enhanced Raman Spectroscopy (SERS)-Active Nanopipette for al. 2000). Then we optimized the incorporation of a voltage-gated sodium chan- Single Cell Intracellular pH Sensing nel, (Ren et al. 2001) . We then transiently transfected the voltage sensor Flicr1 Jing Guo, Jin He. and fluorescence changes marked voltage membrane changes indicating the Physics Department, Florida International University, Miami, FL, USA. HEK cells are excitable and can be controlled using 488nM light (Abdelfattah Single cell intra-cellular pH is very important in cells as it influence cell behav- et al. 2016). With these components working, we are characterizing the optimal iors including proliferation and migration. Functionalized nanoparticles-based expression levels of each component, and the response properties of the system. Surface-Enhanced Raman and Fluorescence spectroscopy have been used for This approach can be easily deployed in automated screening systems. A limita- intra-cellular pH sensing. The main obstacle for above mentioned methods tion is that the incorporation of a blue-light actuator means it will be limited to are labeling and nanoparticle uncontrollable localization inside a cell. To over- testing red fluorescent, or IR, sensors. I am using a Cairn splitter for dual imaging. come those drawbacks, we developed a SERS-active glass nanopipette sensor My hope is that as I see the near IR calcium sensor excite on one side of the Cairn that included a nanostructured gold layer functionalized with a pH reporter optosplitter I can examine if any of the voltage sensors that I am mutagenizing molecule 4-Mercaptobenzoic acid whose SERS spectrum changes as a function work on the left side of the screen in the same cell. of pH. This SERS-active nanopipette probe allows for label-free, localized pH probing of single cell. Moreover, the apex of SERS-active nanopipette still 723-Pos open after gold deposition, this open channel could be used in drug delivery Role of Electric Field Changes in Fluorescence Response of Red Fluores- by electrophoretic nanoinjection. It is possible dynamic monitoring of cell cent Genetically-Encoded Ca2D Indicators response to delivered drug through SERS changes. Rosana S. Molina, Thomas E. Hughes, Mikhail Drobizhev. Cell Biology & Neuroscience, Montana State University, Bozeman, MT, USA. 726-Pos Red fluorescent genetically encoded indicators (GECIs) are engineered proteins Label-Free Enzyme Activity Measurements with Quantum-Limited Bio- that consist of a circularly permuted red fluorescent protein fused to a Ca2þ- sensors sensing domain. A Ca2þ-induced conformational change in the Ca2þ-sensing Arvind Balijepalli1, Son T. Le1,2, Nicholas B. Guros1,3, Antonio Cardone1,3, domain translates into a change in fluorescence, making them useful to track Niranjana D. Amin4, Jeffery B. Klauda3, Harish C. Pant4, Curt A. Richter1. 1 2 Ca2þ activities within cells and tissue. Within a GECI, amino acid residues NIST, Gaithersburg, MD, USA, Theiss Research, LaJolla, CA, USA, 3 4 create a local electric field surrounding the fluorescent protein chromophore UMD, College Park, MD, USA, NIH, Bethesda, MD, USA. that modulates its photophysical properties. By measuring the ratio of the two- Accurate and rapid measurements of enzyme activity are critical to the devel- photon cross section obtained with circularly and linearly polarized light, we opment of therapeutics that target neurological disorders. An effective label- can determine the angle between the transition dipole moment and the change free measurement of enzyme activity can be achieved through the detection in the permanent dipole moments of the ground and excited states of the chromo- of small changes in the solution pH during enzyme-mediated phosphorylation phore. Combining this information for a select group of red GECIs with informa- of substrate proteins. Here, we report the measurements of solution pH by using tion about their one-photon and two-photon absorption properties, we calculated ionic-liquid gated 2D field-effect transistors (FETs) with 200x higher sensi- the two orthogonal components Ex and Ey of the electric field in the chromophore tivity than previously reported. The FETs are fabricated with single- plane along its long and short axes, respectively. We found that for these red GE- monolayer 2D transition metal dichalcogenide semiconducting films and are 2þ CIs, the Ex component changes upon binding Ca . The changes in the electric top-gated with an ionic-liquid. The high ionic-liquid polarizability allows field correlate with changes both in the peak two-photon cross section and the strong coupling between the top gate (ionic liquid) and a back-gate dielectric quantum yield. We quantitatively explain the change in quantum yield by (substrate oxide) which enables amplification of a voltage applied to the considering the twisted intramolecular charge transfer process as a doorway ionic-liquid gate. This amplification is limited by the intrinsic quantum capac- for nonradiative relaxation and its dependence on the Ex component of the elec- itance of the single-monolayer MoS2 semiconducting channel; thus, the de- tric field. Our model may inform the rational design of more efficient red GECIs vices operate near their theoretical limits. We leveraged the high for two-photon microscopy. performance of the devices to enable pH measurements with sensitivities that greatly exceed the Nernst value of 59 mV/pH (at room temperature). The 724-Pos dramatically improved sensitivity allows the detection of pH changes as small ^ A Voltage Dependent Heterotrimeric FRET Signal Suggests Multimeric as 80x10 -6. The method allowed the rapid and quantitative estimation of the Association for the Voltage Sensing Domain of the Voltage Sensing Phos- kinetics of enzymes that are implicated in Alzheimer’s disease. We have phatase demonstrated the ability of ionic-liquid-gated FETs to detect small changes Lee Min Leong1,2, Bok Eum Kang1,2, Bradley J. Baker1,2. in the solution pH during phosphorylation of the substrate protein, histone 1Center for Functional Connectomics, Korea Institute of Science and H1. We compared these FET measurements against enzyme activity measure- Technology, Seoul, Republic of Korea, 2Division of Bio-Medical Science & ments obtained from a radioactively labeled ATP assay (which involves Technology, KIST School, Korea University of Science and Technology, handling radioactive materials and is slow to perform). The FET-based mea- Seoul, Republic of Korea. surements are in excellent quantitative agreement with the ATP assay, while Changes in membrane potential can be measured optically using genetically enabling results in minutes and can lead to rapid therapeutic screening. voltage indicators (GEVIs). One of the most popular GEVIs, ArcLight, is con- structed by the fusion of the voltage sensitive domain (VSD) to the fluorescent 727-Pos protein (FP), Super Ecliptic pHluorin A227D. Primary studies propose that the Luminescent Molecular Sensors for the Selective Detection of Neurodegen- mechanism coupling the change in membrane potential to fluorescence changes erative Disease Protein Pathology in CSF is via the dimerization of the cytoplasmic FP. Intermolecular Fo¨rster Resonance Florencia Monge, Adeline Fanni, Shanya Jiang, David G. Whitten, Energy (FRET) was used to further study the GEVI dimerization mechanism. Kiran Bhaskar, Eva Y. Chi. Novel constructs fusing the VSD to either FRET donors or FRET acceptors Univ New Mexico, Albuquerque, NM, USA.

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Alzheimer’s disease (AD) is a leading cause of death worldwide for which we chambers, the applied electric field will drive these molecules to the other cham- have no clinically useful diagnostic tools, nor do we have a treatment. The dis- ber through the nanopore, as a result of the increased resistivity by the passing ease is characterized by the misfolding and aggregation of the amyloid beta (Ab) molecules, the ionic current drops. Tau proteins are characterized by measuring and tau proteins that cause neuronal damage. Neuronal loss occurs decades prior the parameters of current drop events produced by their translocation through a to the onset of symptoms, making the pre-symptomatic stage an ideal target win- solid-state nanopore: the time duration, the current drop amplitudes and the inte- dow for detection and treatment. Research has shown that levels of proteins and grated area which are directly proportional to the excluded volume of tau protein. small molecules in the cerebrospinal fluid (CSF) can be used as biomarkers for The nanopore experiments were performed under applied voltages from 60-210 detecting and monitoring certain neurodegeneration processes, such as cognitive mV at pH 4.0 and 12.0 with 0.1 M, 0.5 M, 1 M, 1.5 M, and 2 M KCl. Our data show decline. Changes in the levels of Ab and tau in the CSF are also being investi- that at pH 7.4 and 12.0 the current drop and time duration amplitudes both in- gated as AD biomarkers. Herein we have characterized two small phenylene crease with increased applied voltages. The current amplitude is found to be lin- ethynylene (PE)-based conjugated polyelectrolytes, anionic OPE1, and cationic early increased with salt concentration at both pHs. The time duration increases at OPE2, as novel sensors for Ab and tau aggregates in the CSF. We have previ- pH 7.4 and decreases at 12.0 with increased salt concentrations. Furthermore, our ously demonstrated the efficiency of these OPEs for the selective in vitro and results show that tau protein is aggregated to multimers when tau with heparin ex vivo detection of amyloid protein aggregates. In this work, we tested the se- (tau : heparin = 1:4) are incubated at 50 oC in 1 M KCl at pH 12.0 for 12 days. lective sensing of OPEs for amyloid protein aggregates in the CSF. Aggregates 730-Pos of model proteins, hen egg white lysozyme, bovine insulin, and disease-relevant a proteins, Ab40, PHF6 (VQIVY), and human-derived tau oligomers, were added -Synuclein Interaction with and Translocation by the MspA Porin 1 2 3 to CSF pooled from healthy human subjects and sensor selectively, limits of Philip A. Gurnev , David P. Hoogerheide , Jens H. Gundlach , Andrew H. Laszlo3, Sergey M. Bezrukov1. detection and sensitivity were quantified using a combination of techniques, 1 including fluorescence measurements, reverse phase-HPLC, and transmission Section on Molecular Transport, NICHD, NIH, Bethesda, MD, USA, 2Center for Neutron Research, National Institute of Standards and electron microscopy. Our results indicate that the larger, cationic OPE2 molecule 3 has high selectivity for the amyloids in CSF over its anionic analog. The selective Technology, Gaithersburg, MD, USA, Department of Physics, University of sensing of OPEs was validated for its use to detect pathological Ab and tau bio- Washington, Seattle, WA, USA. markers in CSF, which could lead to early detection and diagnosis of AD. We have recently reported (Hoogerheide et al., Biophysical Journal 2018, 114, 772-776) the use of ‘‘selectivity tags’’ to track the translocation of a non- 728-Pos uniformly charged polypeptide through the mitochondrial VDAC nanopore on Novel Sensors for Detecting Alzheimer’s Disease Related Tau Protein Ag- the single molecule level. We now demonstrate the generality of the selectivity gregates tag technique with another widely used, robust b-barrel nanopore, the mycobacte- Salomon L. Alires1, Florencia A. Monge2, David G. Whitten3, Eva Y. Chi3. rial porin MspA. First, we show that a-Synuclein (a-Syn), a naturally occurring, 1Department of Biology, University of New Mexico, Albuquerque, NM, intrinsically disordered polypeptide associated with Parkinson’s disease pathology USA, 2Biomedical Engineering Graduate Program, Center for Biomedical and mitochondrial bioenergetics, can interact with the bilayer-reconstituted Engineering, University of New Mexico, Albuquerque, NM, USA, 3Center D90N/D91N/D93N/D118R/E139K/D134R MspA pore (‘‘MspA’’). a-Syn causes for Biomedical Engineering, Department of Chemical and Biological a discrete 80% blockage of the MspA current, with the blockage on-rate rising Engineering, University of New Mexico, Albuquerque, NM, USA. exponentially with the applied field. The residence time, on the other hand, dis- The pathological hallmarks of Alzheimer’s disease (AD) are the accumulation of plays a crossover behavior, indicating that at voltages >50 mV the entire a-Syn extracellular amyloid-beta (Ab) plaques and intracellular tau neurofibrillary tan- polymer passes through the pore. Second, threading a-Syn, which has a ‘‘diblock gles. Currently, there is no reliable method to simultaneously detect both plaques copolymer’’ structure with regions of opposite uniform charges at its ends, through and tangles in vivo for early disease diagnosis. We have recently shown that a new MspA strongly modulates the electrostatics and ionic selectivity of MspA’s class of synthetic sensors based on an oligo phenylene-ethynylene (OPE) scaffold constriction, with an increased sensitivity compared to the original findings re- selectively detected the fibrillar conformation of model amyloid proteins and AD ported for the VDAC nanopore (Hoogerheide et al., 2018). Under conditions of related Ab peptides. Upon binding to amyloid fibrils, restriction to intramolecular 0.2/1 M KCl gradient across the membrane, the a-Syn interactions with MspA pro- rotation of OPEs lead to backbone planarization and fluorescence ‘‘turn on’’. In duce readily detectable current sub-levels, which correspond to differently this study, we tested the sensing capability of OPEs on a synthetically derived six charged regions of a-Syn polypeptide as it interacts and threads through the amino acid tau peptide VQIVYK also called paired helical filament six (PHF6). pore and allow for direct single-molecule detection of a-Syn translocation success. This sequence is found in the third repeat microtubule-binding domain of the tau Third, we employ the selectivity tag technique to observe the effect of various co- protein and its hyperphosphorylation is noted to be critical in the formation of solvents (e.g. salt species) on the process of a-Syn-MspA translocation. neurofibrillary tangles. Based on previous work, we hypothesize that OPEs can selectively detect the fibrillar conformer of PHF6. Under various incubation con- 731-Pos ditions, two distinct types of PHF6 fibrils were made and characterized through Nanopore Spectroscopy: A Single Molecule Approach to Analyze Protein transmission electron microscopy and circular dichroism. The selective binding Structural Dynamics potential of OPE was compared with thioflavin-T in detecting PHF6 fibrils and Min Chen, Xin Li. analyzed with fluorescence and ultraviolet-visible spectroscopy. Our results Dept Chemistry, Univ Massachusetts Amherst, Amherst, MA, USA. show that negatively charged OPE1- displayed higher affinity and selectivity It has been well accepted that proteins are dynamic systems that undergo confor- (over monomers) for both types of fibrils compared to positively charged mational transitions among different states during ligand binding and recogni- OPE2þ. Importantly, thioflavin-T only detected one type of PHF6 fibrils. tion, catalysis and regulation. Because the structural dynamics of protein are Thus, OPE1- exhibits superior sensing capability for tau PHF6 fibrils compared central to their function, it is highly desirable to design experiments that allow to the more commonly used thioflavin-T. As such, OPE1- has shown to be an the quantitative analysis of the structural dynamics of proteins in order to under- effective sensor for Ab fibrils as well, hence it has the potential to simultaneously stand their function and regulation. While most structural biological approaches detect both plaques and tangles for better disease diagnosis. are useful to study the states that are highly populated, homogeneous and static, intermediate states that are short-lived and comprise only a small fraction of the 729-Pos overall population are invisible to conventional structural methods. Single mole- Electric Field and Ionic Strength Dependent Translocation of Tau Protein cule measurements are well suited to explore the heterogeneity of conforma- Through Solid-State Nanopore tional states. However, current frequently used single-molecule approaches, Mitu C. Acharjee1, Haopeng Li2, Jiali Li3. e.g., FRET, AFM, optical tweezers and single-molecule fluorescence spectros- 1Dept Microelectronics-Photonics, Univ Arkansas, Fayetteville, AR, USA, copy have a temporal resolution of 10 ms or lower, which is unsuitable for 2Cell and Molecular Biology, Univ Arkansas, Fayetteville, AR, USA, 3Dept following fast motions and probing the short-lived intermediate states. Physics, Univ Arkansas, Fayetteville, AR, USA. Nanopore-based measurements, evolved from patch clamp techniques, can Aggregation of tau protein is one of the leading causes of neurodegenerative dis- resolve events within 10 ms providing an excellent tool to trace fast motions eases like Alzheimer’s, Parkinson’s, Pick’s etc. In this report, nanopores are beyond the temporal resolution of fluorescence-based methods. By trapping a fabricated in free-standing silicon nitride membrane on silicon substrate by ion single maltose-binding protein (MBP) within a ClyA nanopore, we have re- beam sculpting method to the desired sizes, 10-25 nm diameters. The nanopore corded the conformational changes of MBP upon ligand binding. Our studies chip is placed in between two chambers made of polydimethylsiloxane. The have revealed multiple ligand-bound states that were not observed by other ap- chambers are filled with potassium chloride (KCl) solution. A stable ionic current proaches. Thus our results have demonstrated the performance of nanopore anal- is established by applying a biased voltage on a pair of silver chloride electrodes ysis for quantitative measurement of protein ensembles and their inter-state across the nanopore membrane. When charged tau proteins are put into one of the transitions.

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732-Pos whichgreatly enhances the membrane stability while allowing silicon chipintegra- Nanozyme Modified Electrochemical Biosensors as Rapid Screening Tools tion. We demonstrate the insertion of alpha-HL in lipid and polymer bilayers, for Biomolecules which allows increased stability and higher applied voltages to be applied. Monica Florescu, Melinda David, Adrian Serban. Medicine, Transilvania University of Brasov, Brasov, Romania. 735-Pos Label-free nanobiosensors offer big advantages for real-time analysis due to Label-Free Detection of Solo Oligonucleotide Lesion Based on Site-Direct their high sensitivity, specificity and quick response. Several nanoparticles, Mutagenized Aerolysin Nanopore such as gold nanoparticles, are able to catalyse reactions, similar as enzymes, Jiajun Wang1, Meng-yin Li1, Jie Yang1, Xue-yuan Wu1, Jin Huang2, and they can be used for label-free nanozyme modified electrochemical sensors Yi-lun Ying1, Yi-tao Long1. as rapid screening tools for biomolecules. 1Key Laboratory for Advanced Materials School of Chemistry & Molecular Biomarkers concentration, like thyroxin hormone, is linked to a large variety of Engineering, East China University of Science and Technology, Shanghai, medical conditions, like hypo/hyperthyroidism. The best therapy in deficit sit- China, 2School of Pharmacy, East China University of Science and uations is the biomolecules replacement, which consists in administration of Technology, Shanghai, China. medication (levo-thyroxine synthetic molecule) to adjust their concentration Nanopore sensing is one of the prominent single molecule detection tools due in blood to a physiological level. There are studies showing that hypothyroid- to its ultimate spatial and time resolution. Such tool offers wide application in ism is a state of increased oxidative stress. Therefore, some treatments advise studying peptide and protein-protein interaction, DNA translocation monitorin- taking antioxidant supplements together with the medication. There are many gand even amino acid directional movement control. Aerolysin expressed by A. food supplements (hydro-soluble plant extracts) that are supposed to contain hydrophila can form watered filled lumen composed by 7-fold repeats, which a variety of antioxidant compounds to inhibit the oxidation of biomolecules un- provides 10-14 A˚ confined nano-space. When reconstituted into artificial der oxidative stress. Herewith, the detection of these biomolecules still lacks a bilayer, it self-assembled as nanopore. WT Aerolysin has fulfilled the discrim- simple, reliable tool, such as label-free nanosensors. ination of oligonucleotides at different length, single base discrimination (XA3, m In this work, nanozyme modified electrochemical sensors for antioxidant com- X=A, G, T, C), single methylcytosine detection (CA3 and CA3) etc. pounds, but also levo-thyroxine rapid screening tools were evaluated and optimized. Previously, by carefully examining the Aerolysin nanopore structure, R220 is Electrochemical measurements such as cyclic voltammetry, differential pulse vol- credited with the bone fide sensing responsibility whilst K238 determines the sub- tammetry and amperometric detection were used and detection limits in nanomolar strate sojourn. We hereby present a rationally designed Aerolysin nanopore (de- range were obtained. The results were correlated with those obtained with classical noted as AL) by optimizing the amino acid at 238 position. Primarily check the and sensitive method of spectrophotometry and good results were obtained. AL in the case of discriminating (dX)4 (X=A, G, T, C), it has one order of magnitude This work was supported by a grant of the Romanian Ministry of Research and longer than the WT Aerolysin. The AL has been optimized so that been capable to Innovation, CCCDI - UEFISCDI, project number PN-III-P1-1.2-PCCDI-2017- read the slight electrical difference among random chains (XGTA) that is not acces- 0062, contract no. 58, within PNCDI III. We hereby acknowledge the structural sible from any reported biological nanopores. As expected, single damaged nucle- founds project PRO-DD (POS-CCE, O.2.2.1., ID 123, SMIS 2637, No 11/2009) otide base could be identified from any position of a 4-base chain, moreover, lesion for providing the infrastructure used in this work. occurs at duel positions (ACGCGA, AmCGmCGA, AmCGCGA) could also be sensed. Further application would be performed utilizing such label-free tool to 733-Pos sense DNA lesion from whole blood or even in situ diagnose in a living cell. Temperature Studies Reveal the Roles of Entropy and Enthalpy in Polymer-Porin Interactions 736-Pos Joseph W. Robertson1, Joseph Reiner2, Christopher Angevine2, Kinetic Analysis of Single Molecule Electrodiffusion in a Biological Nano- Nuwan Kothalawala3, Amala Dass3. pore with Two Binding Sites 1Physical Measurement Laboratory, Natl Inst Standards Tech, Gaithersburg, MD, Norbert Ankri1, Mordjane Boukhet2, Gerhard Baaken2, USA, 2Physical Measurement Laboratory, Virginia Commonwealth University, Murugappan Muthukumar3, Jan C. Behrends4. Richmond, VA, USA, 3University of Mississippi, Oxford, MS, USA. 1UNIS, INSERM U-1072, Universite Aix Marseille, Marseille, France, Transport of proteins and small molecules across membranes often relies on 2Ionera Technologies GmbH, Freiburg, Germany, 3Polymer Science, transmembrane porins. Attempts to quantify this interaction have been based University of Massachusetts Amherst, Amherst, MA, USA, 4Dept Physiol, on modeling the nanopore as a free energy barrier from which the polymer es- Univ Freiburg, Freiburg, Germany. capes. The analysis is performed at room temperature and the kinetics have When single short adenine oligonucleotides (d.p. 3-5) block the pore formed by been used to extrapolate the free energy barrier formed by the polymer-pore the bacterial toxin aerolysin, the resultant resistive pulses are complex with two interaction. This approach is limited because the free energy is explicitly depen- levels (short and deep vs. long and shallow) in a variety of configurations, the dent on the solution temperature. Furthermore, attempts to separate the entropic probability of which depends on voltage. We account for this by a four-state ki- and enthalpic components of the free energy barrier require temperature depen- netic mechanism with two undistinguishable open states i,o and two blocked dent measurements of the polymer-pore kinetics. Here we report on the applica- states m1 (short-deep) and m2 (long-shallow) linked as o<->m1<->m2->i. tion of laser-based nanopore heating to study the interaction of near-neutral M1 is directly accessible from o which designates the presence of the nucleotide polymers (PEG, peptides) with an alpha hemolysin nanopore sensor. In addition, 0 at the pore s cis-side mouth, while m2 is inaccessible from state i, as nucleotides we report on the thermodynamic properties of alpha-hemolysin pores that are unable to enter from the trans-side irrespective of voltage. In this framework, contain thiolate-capped gold clusters (Au (SG) ) and results show the gold 25 18 several experimentally accessible statistical quantities such as the frequency or clusters significantly increase the enthalpy of interaction between the polymers probability of returns to m1 from m2, the fraction of restive pulses ending in m1 and pore. Voltage studies show a linear dependence with the enthalpy and en- and the mean dwell times in m2 as well as the mean total duration of resistive tropy for most of the polymers studied as expected. Finally, we find a significant pulses acquire mechanistic significance and allow direct kinetic predictions us- dependence between polymer residence time and the entropic component of the ing a Q-matrix approach. For A3, the data are consistent with a charged particle free energy barrier for the polymers studied herein. This suggests that to increase moving through a one-dimensional energy landscape with two minimain an elec- the interaction strength between the polymer and pore, and thus the sensitivity of trically biased random walk. For longer nucleotides (e.g. A5) the success rate for the pore for polymer analysis, one should focus on modifying system entropy. translocation is higher than predicted by the rate constants determined from the 734-Pos other observables. It appears likely that this is due to an ability of the longer olig- Improved Bilayer Membrane Stability for Nanopore Sensing Applications omers to simultaneously link with the two binding sites, producing an excess of Xinqi Kang1, Mohammad Amin Alibakhshi2, Meni Wanunu2. returns from m2 to m1, which, however, does not entail a propensity to result in 1Bioengineering, Northeastern University, Boston, MA, USA, 2Physics, translocation failures. Such double tethering of DNA might promote transloca- Northeastern University, Boston, MA, USA. tion of longer chains by producing an extended conformation and may also Over the past decade, biological nanopores have emerged as biosensors for versa- contribute to the strong rectification of transport observed for aerolysin. tile applications, including sensing large molecules such as biomarkers, PEG, DNA, RNA, and small molecules such as peptides and drugs. Traditionally, bio- 737-Pos logical poresare incorporated into planar lipid membranes, or polymer membranes Optimizing Nucleic Acid Biomarker Detection in a Solid-State Nanopore which is painted on circular Teflon apertures. A limitation of the Teflon aperture is Through Probabilistic Modeling the stability of resulting membranes. Herein, we design an aperture for supporting Samuel Bearden1, Osama K. Zahid2, Adam R. Hall3. 1 the lipid bilayers and polymer bilayers with high stability. We employed a gear Biomedical Engineering, Wake Forest Health Sciences, Winston Salem, NC, 2 3 shaped 100 mm aperture produced in SU-8 films that sit on top of silicon windows. USA, SBES, Wake Forest Univ, Winston-Salem, NC, USA, Biomedical Compared with circular apertures, the gear shape apertures store more solvent, Engineering, Wake Forest Health Sciences, Winston-Salem, NC, USA.

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We have established a solid-state (SS-) nanopore assay with high specificity for small size of metal ions, biological nanopore blockage by a single ion results nucleic acid targets, enabling applications like epigenetic screening, micro- in shallow resistive pulses. here, we found that metal ions can embedded in bio- RNA analysis, and pathogen detection . Despite its proven specificity and sensi- logical nanopore and further amplified ion blockage. in addition, metal ions tivity, translational biomarker detection at clinically relevant concentrations is embedded biological nanopores are capable of translocating ssdna with hindered by very low event rates and long acquisition times. It has been noted enlarged blockage amplitude and enhanced capture rate due to a channel cavity that most small-molecule translocations are not detectable due to low signal to which has been finely tuned by ion embedding. these phenomena suggest novel noise ratio or fast timescales. Here, we describe the probability that a given ion sensing concepts and potential applications with metal ion-embedded bio- translocation will produce a detectable event by considering tunable system pa- logical nanopores. rameters, and use it to show that these key experimental variables can influence the measured event rate in a predictable fashion. This model elucidates the de- 741-Pos pendencies of nanopore/analyte interactions and suggests methods by which High-Throughput Optical Sensing from Immobilized Biological Nano- measurement sensitivity can be optimized. pores in a Micro-Bilayer Array 1 2 Event probability can be determined experimentally by comparing expected Yuqin Wang , Shuo Huang . 1Nanjing Univ, Nanjing, China, 2Chemistry and Chemical Eng, Nanjing (Rexp) to observed (Robs) event rates for dsDNA constructs of various lengths Univ, Nanjing, China. bound to monovalent streptavidin. Rexp can be derived analytically from the recent developments in nanopore sequencing have created a sensation in the Nernst-Planck equation and Knudsen diffusion coefficient, and Robs measured as a function of fundamental experimental parameters, including the driving sequencing industry but the measurement throughput is still limited for practical potential, target concentration, nanopore radius, and data acquisition rate. Us- uses. in contrast to electrophysiology readout, optical single channel recording (oscr) permits simultaneous recording from numerous nanopores. however, ther- ing these data to generate probability distributions, we show that Robs can be predicted for arbitrary experimental conditions from first principles. With mal drifts of biological nanopores set obstacles for signal acquisition during imag- this approach, we are able to better understand and even influence the sensi- ing. by finely adjusting the osmosis-oriented interactions between the lipid/ tivity of our SS-nanopore assay. The explicit use of the probability distribution substrate interfaces, we have proposed a simple but universal approach to immo- provides insight into the mechanisms that govern event generation, and our bilize nanopores for high-quality oscrs. a finger-tip measurement device and a approach may be expanded to take into account any fundamental system multiplex bilayer array have also been developed, which will be applicable to parameter. Tuning system parameters according to the underlying probability portable and micro detections that are inaccessible to a patch-clamp probe. such distribution will enable optimized detection of low-abundance or challenging developments significantly enhance the utility of nanopore sensing by expanding nucleic acid biomarker targets. its uses in diverse areas, such as high throughput dna sequencing, drug screening and biophysical studies. 738-Pos Real-Time Nanopore Couting of Amplicons for Ultrasensitive and Lable- 742-Pos Free Sequence-Specific DNA Detection Microscopic Imaging of Restriction Engineered Biological Nanopores for Zifan Tang, Weihua Guan. Highly Specific Spotting of Epigenetic Markers Electrical Engineering, The Pennsylvania State University, State College, Shuo Huang. PA, USA. Chemistry and Chemical Eng, Nanjing Univ, Nanjing, China. Ultrasensitive, rapid, and cost-effective sequence-specific detection of nucleic Biological nanopores, which originate from ion channel proteins, are capable of sequencing genomic DNAs but with a limited throughput even in a commer- acids (DNAs and RNAs) is of great significance for various applications. There 2þ is an increasing interest in developing label-free nucleic acid detection methods cialized form. By digitally encoding fluorescence from Ca fluxes through in- > ^ due to their potential for minimization and integration. Significant efforts have dividual nanopores, an array of single molecule sensors ( 10 4) could be been made towards nanopore-based label-free sensor that allows nucleic acids visually monitored in parallel within a finger-tip sized device. Nanopore sen- to be analyzed electronically. The detection specificity is usually achieved by sors have long been restricted to either natural ion channel proteins or fabri- functionalizing the nanopore area or probe molecules. However, the sensitivity cated solid state apertures, which have limited spatial resolution for of nanopore to detect nucleic acids is usually limited due to the diffusion identifying a single nucleotide without any interference from its neighbour limited transport process. In this work, we explored the feasibility of using bases. Our recent investigation unveils that precise engineering within the the nanopore as a real-time single molecule counter during the amplification pore restriction could be performed with an enhanced sensing performance process. This novel approach combines the label-free nanopore sensor with by signal amplification. The demonstrated progress in the measurement the exquisite sensitivity offered by amplification for sequence-specific nucleic throughput and the spatial resolution for a nanopore sensor could benefit funda- acid detection. A glass nanopore provides an electronic eye to sample the num- mental biophysical studies in single molecule and practical precision medicine ber of amplicons in real-time thus remove the need for optical sensors. As the applications such as epigenetic marker spotting, which is to be demonstrated in amplification process goes along, the numbers of molecules being detected per our talk. unit time grow exponentially. This approach would allow ultrasensitive and 742.1-Pos label-free sequence-specific DNA detection and paves the way for integrated Development of Photoactivatable Optical Bio-Sensors of Physiological point-of-care nucleic acid testing. Activities 1 2 1 739-Pos Sungmoo Lee , Yoon-Kyu Song , Bradley J. Baker . 1Center for Functional Connectomics, Korea Institute of Science and DNA Based Nanopore Sensing 2 Haichen Wu. Technology, Seoul, Republic of Korea, Graduate School of Convergence Institute of Chemistry Chinese Academy of Sciences, Beijing, China. Science and Technology, Seoul National University, Seoul, Republic of Nanopore sensing is an attractive, label-free approach that can measure single Korea. molecules. Earlier work in nanopore sensing mainly focused on building Photoactivatable fluorescent proteins enable specific localization of a region of sensing elements inside the nanopore through protein mutation and chemical interest by activating a small population of fluorophores. Genetically encoded modification. However, in recent years, DNA molecules have been used as fluorescent sensors of physiological activities often suffer from the high back- sensing probes for the detection and quantification of a wide variety of analytes. ground fluorescence from a densely labeled tissue. Therefore, the development In this talk, several examples are given to illustrate how the DNA probes are of photoactivatable variants of optical bio-sensors would facilitate accurate and used to detect large size biomacromolecules including DNA/RNA, antigens, targetable study by highlighting only a certain area or single cells reducing the antibodies, enzymes, etc. in nanopores. unwanted baseline fluorescence. In this presentation, we will introduce our rational approach to develop photoactivatable versions of existing bio-sensors 740-Pos such as a voltage indicator or a pH indicator. We have so far constructed a Embedding Single Metal Ions Within a Biological Nanopore for Amplified circularly permuted FP, a FRET (Fluorescence Resonance Energy Transfer) Ion and ssDNA Sensing pair and a cytoplasmic FP types. This includes a photoactivatable variant of Jiao Cao1, Shuo Huang2. our recently published genetically encoded voltage indicator, Bongwoori-R3. 1Nanjing University, Nanjing, China, 2Chemistry and Chemical Eng, Nanjing This variant was successfully photoactivated and its voltage sensitivity was Univ, Nanjing, China. demonstrated in mammalian cells. This research was supported by the National besides sensing biomacromolecules, biological nanopores are capable of Institute of Neurological Disorders and Stroke of the National Institutes of resolving inorganic ions such as zn2þ, CO2þ OR CD2þ. however, as the Health under the award number U01NS099691.

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