Sunday, February 12, 2017 15a

Symposium: Proteins in Vivo: From the external amyloid aggregates rapidly surround the cells and act as a recruitment point for soluble protein, triggering amyloid fibril elongation. A fraction of Ribosome Through the Chaperone to the Native aggregates surrounding the cardiomyocytes is internalized via micropinocyto- State sis (same mechanism followed by soluble proteins). AL amyloid fibrils are shown to cause cell growth arrest at low concentration. Soluble proteins induce 80-Symp apoptosis, demonstrating different cytotoxic mechanisms between soluble pro- Channeling Nascent Proteins Towards the Native State: Role of the tein and amyloid aggregates. Ribosome and Molecular Chaperones In vitro amyloid formation experiments show that heterologous recruitment of Silvia Cavagnero, Rayna M. Addabbo, Matthew D. Dalphin, Yue Liu, light chains is kinetically determined by the conformation of the amyloidogenic Miranda F. Mecha. conformational precursor and modulated by the differential ability of each pro- Chemistry, University of Wisconsin-Madison, Madison, WI, USA. tein to either nucleate or elongate fibrils. Despite much progress over the last six decades, we are still far from understand- Studies on the early events of fibril formation followed by dynamic light scat- ing the mechanism of protein folding and aggregation in the cell. This lack of in- tering, chromatography and electron microscopy have shown differences in the formation poses tremendous challenges to progress in many areas of life sciences, species formed by amyloidogenic proteins (stable small oligomers) compared and it severely impedes key efforts in biomedical research. Learning more about to control proteins (large oligomers observed). the interplay between protein folding and aggregation in bacterial cells has a direct Overall, our studies emphasize the complex interactions between light chain impact on the development of strategies to treat microbial infection and on the and cells that result in fibril internalization, protein recruitment, and cytotox- optimization of protein-based drug production in the pharmaceutical industry. icity that may occur in AL amyloidosis. In bacteria, the majority of soluble cellular proteins fold or aggregate co- Symposium: Biophysics of the Cytoskeletal- translationally and immediately post-translationally. The ribosome and molecular chaperones play a key role in this process. This presentation will report progress, Membrane Interface failures and surprises on our molecular-level understanding of how the ribosome, ribosomal proteins and cotranslationally active molecular chaperones modulate 83-Symp the balance between protein folding and aggregation in the cell. By probing the Modeling Membrane Tubules with Lipid Droplets and Migrasomes structure and dynamics of nascent proteins by fluorescence depolarization in the Michael M. Kozlov. frequency domain, multidimensional NMR, biochemical tools and kinetic simu- Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv lations, we will attempt to recapitulate some fundamental concepts of general sig- University, Tel Aviv, Israel. nificance, and suggest how these concepts can be specifically exploited to Membrane tubules of few tens of nanometer cross-sectional diameters and understand and reprogram the bacterial translation machinery to maximize the micron-scale lengths represent a basic structural component of intra-cellular yield and timely production of correctly folded proteins. organelles, such as endoplasmic reticulum and Golgi Complex, and emerge from plasma membranes in the course of cell crawling on extra-cellular 81-Symp matrices and substrates. Besides barrier functions, the tubular membranes Ribosomes in Motion: The Dynamics of Nature’s Protein Synthesis serve as platforms for formation of peculiar cell organelles, Lipid Droplets Machinery and Migrasomes, whose properties are to be understood in terms of simple Ruben L. Gonzalez Jr. physics. Lipid Droplet can be regarded as lenses of hydrophobic substance Department of Chemistry, Columbia University, New York, NY, USA. (triacylglycerol, sterol esters) growing up between the two membrane leaflets The ribosome can be regarded as a molecular machine that converts chemical and into micron-large buds, which, possibly, detach from the membrane to form thermal energy into productive mechanical work. This chemo- and thermome- emulsion-like droplets. Migrasomes are micron-size spherical bodies discov- chanical view of ribosome function continues to fuel efforts to identify the mobile ered, recently, to form on membrane tethers pulled out of the cell body in components of the ribosomal machine, characterize the structural dynamics of the course of cell migration. We address the micromechanics of the two organ- these components, and develop an understanding of how these dynamics are regu- elles to gain understanding of physics behind their formation and evolution. lated in order to direct mechanical processes during protein synthesis. It is within We analyze the shape and energy of a membrane tubule containing a lipid this context that our research group has developed a fully reconstituted, fluores- droplet in dependence of the droplet size and the elastic properties of the cently labeled in vitro translation system and used it for single-molecule fluores- tubular membrane and the lipid monolayers covering the droplet surface. cence imaging studies of protein synthesis. Together with ensemble biochemical We determine the conditions of the droplet detachment from the tubule. We investigations of protein synthesis by the ribosome and structural studies of func- suggest a physical model of Migrasome as a rigid membrane domain, which tional ribosomal complexes, single-molecule fluorescence imaging of protein syn- swells within a membrane tubule into a sphere-like body as a result of interplay thesis continues to provide unique and powerful mechanistic insights into this between the membrane bending rigidity and lateral tension. By computing the fundamental biological process. In this talk, I will present recent and ongoing domain shapes for varying membrane elastic properties and the domain sizes work from our research group aimed at developing an ever-deeper understanding and comparing the results with the available images, we suggest the criteria of the function of the ribosome during protein synthesis. In addition, I will discuss of Migrasome formation. what we envision lies ahead as single-molecule fluorescence imaging approaches continue to evolve and expand to address increasingly complex mechanistic and 84-Symp regulatory aspects of this fundamental biological process. Multiscale Simulation of Proteins at the Membrane-Cytoskeleton Interface Gregory A. Voth. 82-Symp University of Chicago, Chicago, IL, USA. From Native to Amyloid in the Test Tube and in Cells: A Journey Protein-protein interactions at the cell membrane play a crucial role in dictating of Misbehaving Antibodies cell morphology, adhesion, motility, and cytokinesis. Examples include the Marina Ramirez-Alvarado1, Marta Marin-Argany1, Christopher J. Dick1, coupling of transmembrane integrins with the cytoskeleton as well as Luis M. Blancas-Mejia1, Pinaki Misra1, Yi Lin2, Angela Williams3, formin-driven polymerization of actin filaments in fission yeast ring assembly. Jonathan S. Wall3. The intrinsic mechanosensitivity of these proteins is an important determinant 1Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA, of emergent large-scale effects from collections of membrane-associated com- 2Medicine-Division of Hematology, Mayo Clinic, Rochester, MN, USA, plexes such as focal adhesions and cytokinetic nodes. Whether integrin and 3Medicine and Radiology, The University of Tennessee Graduate School of formin-nodes clustering respond to mechanical cues remains controversial in Medicine, Knoxville, TN, USA. the field, due to inherent limitations of experimental approaches in isolating Light chain (AL) amyloidosis is an incurable disease characterized by the specific protein contributions. Coarse-grained (CG) simulations of such sys- misfolding, aggregation, and systemic deposition of amyloid composed of tems can be indispensable tools for distinguishing between such competing sce- immunoglobulin light chains. We have conducted thermodynamic and fibril narios. In this talk, I will discuss how we apply CG simulations to recapitulate formation studies of both variable domain and full length immunoglobulin pro- the complex behavior observed by our experimental collaborators and place teins involved in AL amyloidosis. We have found that there is thermodynamic them into a rigorous framework to learn more about the microscopic behavior range -under physiological conditions- in which these proteins populate at play. First, using a new model of single-point, two state integrin particles on a partially folded states that favor amyloid formation. quasi-2D membrane, we demonstrate how cell spreading on soft substrates is We have studied the internalization of these same AL proteins in soluble form regulated by integrin activation and how multiple integrin subtypes differen- and as amyloid fibrils into human cardiomyocytes. Our results show how tially transmit traction stress. By further including explicit coupling to a

BPJ 7693_7704 16a Sunday, February 12, 2017

fluctuating cytoskeleton, we have studied the effect of single actin filament and yiiP. CotP appears to have a role in Co2þ homeostasis as the deletion polymerization on integrin clustering and ligand binding. Second, I will discuss mutant strain DcotP is more sensitive and accumulate this ion. In inhibition recent work on the mechanosensitivity of formin proteins in yeast cytokinetic halo experiments, DczcD and DyiiP strains showed low Zn2þ sensitivity. How- nodes. I will show results from our computational CG model explaining how ever, in iron-rich media and in the presence of Zn2þ the strains secreted higher modulating formin activity by myosin generated forces on actin filaments levels of the iron chelator pyoverdine. The non-pyoverdine producer triple mu- can increase the efficiency of cell division. tants, DzntA-DpvdA-DczcD and DzntA-DpvdA-DyiiP, displayed increased Zn2þ sensitivity and increased intracellular Zn2þ accumulation compared to the 85-Symp double mutant DzntA-DpvdA. Most importantly, the strains DczcD, DyiiP, How Actin Cytoskeleton Dynamics Induce Membrane Tubulations DpvdA-DczcD and DpvdA-DyiiP showed a 4- to 8-fold increased sensitivity Ce´cile Sykes1,2. 1 2 to imipenem and ciprofloxacin and a 2-fold increased sensitivity to chloram- Curie Institute, Paris Cedex 05, France, CNRS, Paris, France. phenicol and gentamycin. Complemented strains attained similar levels of anti- E-mail: [email protected] biotic resistance as the PAO1 parental strain. These observations correlated Web: http://umr168.curie.fr/en/Sykes-group with increased sensitivity to EDTA-lysozyme treatment and overexpression Cell deformations are part of many vital processes such as division, motility or of OprN and OprD porins in DczcD and DyiiP vs. WTand DcotP. We hypoth- intracellular transport. They result from the continuous re-organization of the esize that while CotP is a Co2þ efflux transporter CzcD and YiiP export Zn2þ to actin cytoskeleton underneath the membrane. In order to unveil how biochem- the periplasm and that their function is required for regulation of OprD synthe- ical assembly can change membrane shape, we develop a minimal system sis, outer membrane stability and therefore antibiotic tolerance. based on purified components to control all the relevant parameters. We reconstruct a finely tuned actin network on cell-sized liposomes to 88-Plat address the respective role of membrane and actin network in membrane tu- Functional Characterization of the Urea Transporter UreI bulation. The actin network is reconstituted at the outer surface of fluores- from Helicobactor Pylori cently labeled liposomes with purified proteins that allow to follow Andreas Horner1, Christine Siligan1, Johannes Preiner2, simultaneously membrane and actin dynamics. The system is tuned to Sergey A. Akimov3, Peter Pohl1. generate a branched cortical network next to the membrane. We demonstrate 1Institute of Biophysics, Johannes Kepler University Linz, Linz, Austria, that the actin cytoskeleton induces the formation of membrane tubes with 2Center for Advanced Bioanalysis GmbH, Linz, Austria, 3Laboratory of different morphologies that are reminiscent of endocytosis and finger-like Bioelectrochemistry, A.N. Frumkin Institute of Physical Chemistry and protrusions in cells: tubes are spontaneously pulled outside the liposomes Electrochemistry, Russian Academy of Sciences, Moscow, Russian and spikes grow towards liposome centers. By decreasing membrane tension Federation. via osmotic shock, we show that spike formation is promoted without More than 50% of the world’s population is infected with Helicobactor pylori,a affecting tubes pulling. These results suggest that these structures are two pathogenic bacterium responsible for numerous gastroduodenal disorders such as independent deformations relying on different mechanisms, both based on chronic gastritis, peptic ulcer disease, and gastric cancer. A small proton-gated membrane deformation induced by cytoskeletal dynamics. This investiga- inner membrane channel HpUreI represents H.pylori’s life insurance. It ensures tion highlights how membrane tubulation depends on a mechanical balance survival in the acidic gastric juice, by means of urea transport from the periplasm between the membrane and the actin network. to the cytoplasm, where urea is hydrolyzed by urease. In turn, the hydrolysis products ammonia and carbon dioxide buffer the periplasm to pH 6.1. MD sim- 86-Symp ulations suggested that water is transported along with urea through the channel. Redefining the Role of the Arp2/3 Complex: Regulation of Morphology at We tested that hypothesis by estimating the unitary transport rates for both the Leading Edge molecules. To this end we overexpressed HpUreI in E.coli, purified the trans- Karen L. Anderson1, Christopher Page1, Mark F. Swift1, Praveen Suraneni2, porter and reconstituted the protein into large unilamellar vesicles (LUVs). Mandy EW Janssen1, Thomas D. Pollard3, Rong Li2, Niels Volkmann1, We observed water flux by osmotically deflating the vesicles or inducing urea Dorit Hanein1. transport under isosmotic conditions at neutral and acidic conditions. We 1Sanford Burnham, La Jolla, CA, USA, 2Department of Cell Biology, Johns extracted the unitary transport rates from the intensity of scattered light by (i) ex- Hopkins School of Medicine, Baltimore, MD, USA, 3Molecular Cellular & tending our recent treatment of the Rayleigh-Gans-Debye equation for solvent Developmental Biology and of Cell Biology and of Molecular Biophysics & transport (1) across vesicular membranes to solute transport and (ii) determining Bi, Yale University, New Haven, CT, USA. the number of reconstituted transporters (2) and their oligomeric state (1) Arp2/3 complex is thought to be the primary protrusive force generator in cell with fluorescence correlation spectroscopy before and after detergent-mediated migration by controlling the assembly and turnover of the branched filament vesicle solubilization. In addition we confirmed the hexameric arrangement by network that pushes the leading edge of moving cells forward. However, acquiring high-speed AFM images of the proteoliposomes spread on mica. mouse fibroblasts without functional Arp2/3 complex migrate at rates similar (1) Horner A, et al. (2015) The mobility of single-file water molecules is gov- to wild-type cells. Correlative fluorescence and large-scale cellular cryo- erned by the number of H-bonds they may form with channel-lining residues. tomography studies combined with automated actin-network analysis allow Science Advances 1(2):e1400083. (2) Hoomann T, Jahnke N, Horner A, Keller examining the effect of the absence of functional Arp2/3 complex on the ar- S, & Pohl P (2013) Filter gate closure inhibits ion but not water transport chitecture of actin networks in fibroblast cells at the single-filament scale. through potassium channels. Proc. Natl. Acad. Sci. U. S. A. 110(26):10842- Because Arp2/3 complex can only fulfill its function in the cell when all 10847. seven subunits are assembled, genetic elimination of the ARPC3 subunit en- -/- sures that functional Arp2/3 complex is absent in the ARPC3 cells versus 89-Plat the wild type. Quantitative analysis at the single filament scale of these Understanding the Conformational Diversity of Proton-Coupled isogenic cell lines suggests that the role of Arp2/3 complex in cell motility Oligopeptide Transporter (POT) Family contains, in addition to generating dendritic actin networks at the leading Balaji Selvam, Diwakar Shukla. edge and filopodia regulation, an element that is responsible for the fine- Department of Chemical and Biomolecular Engineering, University of tuning of actin-bundle morphology at the cell periphery as well as in Illinois, Urbana, IL, USA. protrusions. Proton-coupled oligopeptide transporters (POTs) are members of the major facilitator superfamily (MFS) proteins involved in uptake of nutrients across Platform: Membrane Pumps, Transporters, and of the cell membrane. These proteins undergo intrinsic conformational changes Exchangers I between outward-facing (OF), occluded (OC) and inward-facing (IF) states to 1.1 - transport substrate molecules. AtNRT is a plant nitrate (NO3 ) transporter 87-Plat identified in Arabidopsis thaliana, belonging to the POT family. It shares Pseudomonas Aeruginosa CDF Transporters CzcD and YiiP are Involved close structural and functional homology to the bacterial di/tri-peptide trans- 2D in Zn Efflux, Outer Membrane Permeability and Antibiotic Resistance porter PepTSo. In our study, we investigated the functional mechanism of 1.1 Agostina Salusso, Daniel Raimunda. AtNRT and PepTSo. Both transporter protein crystal structures are obtained INIMEC-CONICET-UNC, Co´rdoba, Argentina. in the IF state and dynamics between functionally important intermediate states Transporters of the cation diffusion facilitator (CDF) family form dimers remains elusive. We performed all atom molecular dynamics simulations over that export transition metals from the cytosol. The opportunistic pathogen microsecond timescales to explore the conformational landscape of AtNRT1.1 Pseudomonas aeruginosa presents three homologous CDF genes, czcD, cotP and PepTSo. Since the conformational changes of these proteins occur at large

BPJ 7693_7704 Sunday, February 12, 2017 17a timescales, an adaptive sampling approach has been employed to efficiently 92-Plat sample the free energy landscapes. MD results reveal key gating residues Determination of Ion Channel and Pump Fluxes for Chinese Hamster that act as transition switches for intracellular and extracellular opening of Ovary Cells using Rubidium Uptake Measurement the transporter. In AtNRT1.1, the residues Lys164-Tyr480 of helices 4 and 10 Azita Fazelkhah. stabilize the OC state and helices 1 and 7 move ~15A˚ to reach the OF state. University of Manitoba, Winnipeg, MB, Canada. In PepTSo, Ser131-Tyr431 of helices 4 and 10 act as gating residues to stabilize Cells expend a significant portion of their energy and have considerable appa- the OC state while the OF state is obtained by breaking the interaction between ratus to control the flow ions across the cell membrane. Ions concentrations Arg32-Asp316 of helices 1 and 7. Our finding sheds light on the conformational have also been demonstrated to be important to processes such as apoptosis 1.1 dynamics and functional mechanisms of AtNRT and PepTSo leading to and implicated in monitoring cells metabolism for biopharmaceutical produc- better understanding of the role of hidden intermediate states in membrane tion. Accurate quantitative modelling of ion transport is challenging since the transporters. densities of ion channels and pumps are known for relatively few cells. In this work the ion channels and pumps fluxes for CHO cells were estimated using 90-Plat Rubidium uptake measurements combined with Ouabain inhibition of pumps. Insights into the Cation Selectivity and Cooperativity with Sugar Attached CHO cells were incubated in a buffer containing 5.4 mM Rb and in in Salmonella Typhimurium Melibiose Permease some cases Ouabain was also added to the buffer in different concentrations Hariharan Parameswaran, Lan Guan. over a period of two hours to inhibit the Naþ-Kþ ATPase pump to study passive Cell Physiology & Molecular Biophysics, Texas Tech University Health and active ion fluxes across the membrane. Sciences Center, LUBBOCK, TX, USA. Using Inductively Coupled Plasma Optical Emission Spectrometry, the Rb The melibiose permease of Salmonella typhimurium (MelBSt), a member of content of the lysed cells after two hours of incubation was 10.6 mg/L, Major Facilitator Superfamily, utilizes the electrochemical gradient of Naþ, þ þ which is a result of Rb transfer through the channels and pumps. From this, Li or H for accumulation of galactosides. Based on crystal structures and the Rb flux through the active and passive pathways is estimated to be functional studies, it has been proposed that MelBSt has a single galactoside- 8.510 12 mol.cm 2.s 1. binding site and all three cations compete for a single cation site. Using purified The Rb entry through the diffusion pathway was estimated using Ouabain to þ þ þ wild-type or mutants MelBSt, direct measurement of Na or melibiose binding inhibit the Na -K ATPase activity. With 5 mM concentration of Ouabain were performed with isothermal titration calorimetry. The results show that the þ the inhibition was maximized and the Rb uptake is reduced by 80% compared binding affinity for Na or melibiose is mutually increased by about 6-8 fold, to the control. From this result the Rb flux through the channels is estimated to exhibiting a positive cooperativity. To reveal the cation selectivity between 12 2 1 þ þ þ be 2.8 10 mol.cm .s . Na and H , the dissociation constant (Kd)ofNa binding was measured as þ þ The obtained results were used in temporal ion concentration simulations using a function of pH. The absolute KD value for Na of 600mM and for H of þ methodology of Hernandez 2000. These simulations are in agreement with the 0.4 mM (i.e. pKa = 6.4) are determined, showing that MelBSt selects for H þ work where ion pumps were inhibited via ATP production inhibition in Mito- over Na . With a pKa value slightly lower than the neutral pH, MelB is able chondria using Oligomycin (B. Saboktakin Rizi, Biomicrofluidics, 2014). to use either Hþ or Naþ as the coupling cation. Furthermore, Hþ release during Naþ binding was analyzed by measuring the overall heat changes in three re- 93-Plat þ action buffers with different ionization enthalpies. While Kd for Na is similar, Functional Effect of Human FXYD2, FXYD4, FXYD6, and FXYD7 the enthalpy changes obtained in the three buffers fit to a linear function with a on Human Alpha 1 Beta 1 Sodium-Potassium ATPase slope of 0.3 and 0.03 at pH 7.45 or 8.2, respectively. It is likely that about Sharan Bijlani, Natalia Armas Capote, Grace Shim, Dylan Meyer, þ þ 30% MelBSt proteins engage H release during Na binding at pH 7.45 and Pablo Artigas. nearly all MelBSt is deprotonated at pH 8.2. The results also support the notion Cell Physiology, Texas Tech University Health Science Center, Lubbock, that Naþ and Hþ exclusively bind to a single site. This study provides insights TX, USA. þ þ þ þ into the molecular mechanisms operated by MelB to utilize either Naþ or Hþ. The Na ,K -ATPase builds and maintains the Na and K gradient across the plasmalemma. Functional Naþ,Kþ-ATPase requires association of a-andb-sub- 91-Plat units. A third single transmembrane protein of the FXYD family (FXYD1- Modulation of Energy Conversion Through Manipulation of the FXYD7) modulate Naþ,Kþ-ATPase function in a tissue specific manner. Here, Retinal Thermal Equilibrium by an Aromatic Residue in the Seven- we used electrophysiology on oocytes expressing human a1b1Naþ,Kþ-ATPase Transmembrane Receptor Bacteriorhodopsin to evaluate the functional effects of association with human FXYD2, 4, 6 & 7. Un- Xiaoyan Ding1,2, Yujiao Gao1, Chao Sun1, Haolin Cui1, Juan Wang1, þ der two-electrode voltage clamp, in the presence of 125 mM Na o, application of Yanan Yang1, Dinu Iuga3, Fang Tian2, Anthony Watts4, Xin Zhao1. þ K o activated outward currents (due to 3Naþ/2Kþ exchange) with voltage- 1Department of Physics, East China Normal University, Shanghai, China, dependent apparent dissociation constant. For a1b1, K0.5,0 =1.150.1 mM and 2Department of Biochemistry and Molecular Biology, Penn State College of K0.5100=1.550.3 mM (at 0, n=6, and 100 mV, n=6, respectively). The Medicine, Hershey, PA, USA, 3Department of Physics, University of 4 K0.5,0 for all FXYDs was 1-1.2 mM; the K0.5100 was ~1 mM for FXYD2 and Warwick, Coventry, United Kingdom, Department of Biochemistry, FXYD6 and ~2 mM for FXYD 4 and FXYD7. Thus, these FXYDs induced University of Oxford, Oxford, United Kingdom. þ changes in K o affinity could be relevant at hyperpolarized potentials. We Light-driven proton pumps, a number of the microbial rhodopsin family seven- þ measured the transient charge movement observed in the absence of K o,which transmembrane receptors, distribute widely among Archaea, Eubacteria, and describes the voltage-dependent equilibrium between Naþ-bound and externally Eukaryota for harvesting and converting light energy in a wider spectral range. open Naþ-free states. The steady-state charge-voltage curves were fitted with a The common feature of those proteins is that the retinal chromophore maintains þ Boltzmann distribution. At constant [Na ]o a shift in the distribution’s center a cis-trans thermal equilibrium in the dark-adapted state. Absorption of a þ (V1/2) to positive voltages signals an increase in Na o affinity while a shift photon causes photo isomerization of the chromophore from the all-trans to to negative voltages indicates a reduction, with a 25 mV per two fold change the 13-cis, 15-anti configuration and triggers a series of structural rearrange- þ in Na o affinity. The V1/2 were (in mV) 41 50.5 (a1b1, n=6), 16 52 ments in the protein that initiates the vectorial translocation of a proton out (FXYD4, n=4), 26 5 0.3 (FXYD7, n=2), 57 52 (FXYD2, n=7), and of the cell. However, how the cis-trans thermal equilibrium impacts on the pro- þ 60 53 (FXYD6, n=7). These results suggest similar increase in Na o affinity ton translocation photocycle, and further impacts on the ATP formation is still þ by FXYD4 or FXYD7 and identical reduction in Na o affinity by FXYD2 and poorly understood. Here, as a novel study to address this challenge, Y185F FXYD6. Measurement of K0.5 for NaþKþ-ATPase activation by intracellular mutant, an aromatic residue at the retinal binding pocket of bacteriorhodopsin, Naþ in inside-out patches is underway. Supported by R15-NS081570-01A1. is identified to shift the cis-trans isomerization thermal equilibrium to a cis dominated state and causes a weakening of the M state, loss of the O state 94-Plat and elongation of the proton pumping cycle, and further decreases the ATP for- The Energy-Transduction Domain of the SERCA Calcium Pump is a mation rate. Different interactions of Tyrosine 185 with the retinal chromo- Prime Therapeutic Target in Heart Failure and Obesity phore are observed in the dark-adapted state and the M state, indicating that Joseph M. Autry1, Michel Espinoza-Fonseca1, Bengt Svensson1, Tyrosine 185 may serve as a rotamer switch to maintain the proton transloca- Christine B. Karim1, Stephanie J. Valberg2, John K. Lee1, David D. Thomas1. tion kinetics and further the energy conversion in bacteriorhodopsin. 1Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Reference: Ding, X., et al. 2016. Mediation mechanism of tyrosine 185 on Minneapolis, MN, USA, 2Large Animal Clinical Sciences, Michigan State the retinal isomerization equilibrium and the proton release channel in the University, East Lansing, MI, USA. seven-transmembrane receptor bacteriorhodopsin. Biochim. Biophys. Acta We have used a comprehensive array of techniques to characterize the 1857:1786-1795. energy-transduction domain of the sarco/endoplasmic reticulum calcium

BPJ 7693_7704 18a Sunday, February 12, 2017 transport ATPase (SERCA), including x-ray crystallography, solution bio- incomplete. Our previous investigations have demonstrated the dominance of chemistry, fluorescence spectroscopy, molecular dynamics simulation, and tetradotoxin-resistant (TTX-R) NaV1.8 channels in muscle afferent somata, small-molecule activation. The energy-transduction domain of SERCA me- but the TTX-sensitive (TTX-S) channel types have not yet been investigated. diates long-range allosteric coupling of ATP hydrolysis in the cytosolic Using immunohistochemistry, we have recently demonstrated expression of 2þ headpiece to Ca transport in the transmembrane domain. In heart failure, TTX-S NaV1.6 and NaV1.7 channels in the somata and axons of group IV neu- decreased SERCA activity correlates with disease progression in patients; rons, but the contribution of these channels to NaV current is unknown. We increasing SERCA activity by drug or gene therapy reverses heart failure have tested three NaV1.7 channel-selective blockers on group IV neurons in animal models. Here we solved the x-ray crystal structure of SERCA in (diameter < 33 mm) to determine the contribution of NaV1.7 current. The complex with a novel small-molecule activator, thus identifying a unique blockers were ProToxin-II (100 nM; IC50 = 0.3 nM), Ssm6a (100 nM; IC50 = binding site on the energy-transduction domain. Conformation-specific pro- 25 nM) and ICA-121431 (ICA, 1 mM, IC50 = 19 nM). To our surprise, teolytic cleavage and intramolecular glutaraldehyde cross-linking was used ProTx-II and Ssm6a failed to block NaV current in group IV neurons. The to determine activator effect on SERCA headpiece structure. Fluorescence average affect on the TTX-S current was 1.2 5 5.0% for ProTx-II and spectroscopy was used to identify kinetic transitions accelerated by the acti- þ17.2 5 38% for Ssm6a (both NS). For ICA, we found a 60 5 23% block vator. We propose a structural and biochemical mechanism for small- of the TTX-S current (p < 0.05). Based on the published IC50s we expected molecule activation of SERCA. Interestingly, the activator site is located all three blockers to produce similar effects, and do not yet understand reason vicinal to the binding site of sarcolipin (SLN), a muscle phosphoprotein for the absence of block by ProTx-II and Ssm6a. It is interesting that human and that regulates SERCA energy expenditure and muscle metabolism to provide rat NaV channels can respond differently to blockers. For human channels, ICA resistance against diet-induced obesity and extreme cold through non- potently blocks hNaV1.3, but not hNaV1.7, whereas the block is opposite for rat 2þ shivering thermogenesis. SLN uncoupling of ATP hydrolysis from Ca channels, rNaV1.3 is insensitive and hNaV1.7 is potently blocked (McCormack transport by SERCA was examined using microsecond MD simulation, et al. (2013) PNAS 110: E2724). Based on the ICA result, we tentatively finding that the cytosolic domain of SLN induces a salt bridge-mediated conclude that ~60% of the TTX-S current in group IV neurons is generated structural rearrangement of the energy-transduction domain, which allosteri- by NaV1.7 channels. cally uncouples SERCA by disrupting Ca2þ occlusion at residue E309 in transport site II, and thereby facilitating futile Ca2þ backflux in muscle. 97-Plat Our results demonstrate that the energy-transduction domain is a key control Multiple Mechanisms of Propofol Inhibition of the Voltage-Gated Sodium 19 element that provides a structural motif for activation of Ca2þ transport by Channel Nachbac: A F NMR Investigation cardiac SERCA2a in heart failure and for uncoupling ATP hydrolysis by Yali Wang1, Marta M. Wells1, William Dailey2, Roderic Eckenhoff2, muscle SERCA1a in obesity. Pei Tang1, Yan Xu1. 1University of Pittsburgh, Pittsburgh, PA, USA, 2University of Pennsylvania, Platform: Voltage-gated Na Channels Philidelphia, PA, USA. Voltage-gated sodium channels are important molecular targets for anes- 95-Plat thetics. The intravenous general anesthetic propofol was found to inhibit Tetrodotoxin Resistance: Natural Experiments to Understand Voltage- NaChBac, a prokaryotic sodium channel from Bacillus halodurans, but little Gated Sodium Channel Structure and Function is known about where propofol binds and how propofol binding inhibits the Shana L. Geffeney1, Gabriela Toledo2, Charles T. Hanifin1. function of this channel. Here, we used 19F-NMR saturation transfer differ- 1Biology, Utah State University-Uintah Basin, Vernal, UT, USA, 2Biology, ence (STD) spectroscopy to quantify propofol interactions with NaChBac University of Virginia, Richmond, VA, USA. and to understand the inhibitory mechanisms by propofol. 19F probes were Here we report data from our long-term studies of naturally occurring tetro- introduced, one at a time, by tagging 1,1,1-trifluoro-3-bromo acetone to dotoxin (TTX) resistant voltage-gated sodium channels (VGSCs) in TTX- site-directed single cysteine mutations that had been computationally pre- resistant metazoans. These results identify critical biophysical properties dicted to be involved in propofol binding. Quantitative 19F-NMR STD be- in individual members of the VGSC gene family as well as differences be- tween the 19F probes and 4-fluoropropofol, a fluorinated propofol analog tween members. Members of the VGSC gene family are expressed in show that propofol binds with different affinities to the sites in different re- different excitable tissues and encode proteins with different biophysical gions of NaChBac. The order of fluoropropofol binding strength, reflected properties. Characterizing isoform-specific properties is critical for under- in the cross relaxation rate constant (s), from the strongest to the weakest, standing functional differences between excitable cells because, in nerve is as follows: the channel activation gate at the cytoplasmic end of the and muscle cells, the compliment of VGSCs expressed controls cell excit- pore, the voltage sensing domain near the gating charge-carrying residues, ability. TTX is a neurotoxin used by diverse taxa including octopuses, newts, the ion selectivity filter at the end of the P1 loop, the S4-S5 linker, and the and fishes. The toxin causes paralysis and death by binding to and blocking central cavity of the pore. No STD was detectable in the extracellular inter- the pore of VGSCs. Amino acid substitutions in the channel pore alter TTX face of channel. The finding of multiple binding sites is consistent with the binding as well as change biophysical properties such as voltage-dependence previous observations with the volatile anesthetics isoflurane and sevoflurane. of activation and slow inactivation. Multiple species have evolved resistance Our results suggest that propofol inhibits NaChBac through multiple sites with to TTX through pore substitutions in their VGSCs. We have used these distinct mechanisms. As a channel blocker, propofol interferes with the acti- natural experiments to identify shared substitutions in TTX-resistant vation gate and selectivity filter and obstructs conductance by binding to the VGSC orthologs (e.g. NaV1.4) and paralogs (e.g. NaV1.5 and NaV1.7) of inner pore. As an inhibitory modulator, propofol binding interrupts charge multiple TTX-resistant animals. By characterizing the properties of channels movement in the voltage-sensing domain and restricts the pivot motion of encoded by synthesized snake NaV1.4 genes, our work identifies interactions the S4-S5 linker. The study provides a molecular basis for the understanding between pore residues and demonstrates that multiple convergent amino acid of propofol inhibitory action on voltage-gated sodium channels. This research substitutions are compensatory. Specific amino acid changes rescue channel was supported by grants from the NIH. function when they occur in combination with substitutions that dramatically increase TTX resistance. Additionally, our work confirms that TTX resis- 98-Plat tance evolved prior to ecological exposure in snake and salamander lineages. The Complete Crystal Structure of an Open Activated Sodium Channel These patterns suggest that selective pressures on channel function rather Jennifer Booker, Altin Sula, Claire E. Naylor, B.A. Wallace. than a requirement for TTX resistance supported alterations in channel struc- Insitute of Structural and Molecular Biology, Birkbeck College, University of ture in our study taxa. By comparing convergent and divergent substitutions, London, London, United Kingdom. we identify critical biophysical differences between members of the VGSC Voltage-gated sodium channels (Navs) play essential roles in excitable tis- gene family. sues, with the activation and opening of these channels resulting in the initial phase of the action potential. A new high resolution (2.45 A) crystal struc- 96-Plat ture of the NavMs prokaryotic sodium channel, has provided the first view of Using Specific Blockers to Identify TTX-S NaV Channels Subtypes in Rat a complete sodium channel structure, enabling visualisation of the interac- Muscle Afferent Neurons tions of the voltage sensor (VS), S4-S5 linker, pore, and C-terminal domains. Renuka Ramachandra, Keith S. Elmslie. The structure has a canonical activated conformation for its VS S4 helix, Pharmacology, AT Still Univ/Kirksville College of Osteopathic Medicine, with a novel S4-S5 linker conformation that leads to an open selectivity filter Kirksville, MO, USA. whichinturnleadstoanopenactivationgateattheintracellularmembrane Muscle pain is transmitted by group III and IV afferents, but our knowledge of surface. This ends in a C-terminal domain (CTD) that acts as a ‘‘glue’’ be- the NaV channel types that generate the action potentials in these neurons is tween the monomers that comprise the tetrameric channel. The activated VS

BPJ 7693_7704 Sunday, February 12, 2017 19a

conformation produces a heretofore unseen extensively-hydrogen bonded to reports on heterologously expressed Nav1.5, mutation of the calmodulin and salt-bridged interaction motif involving a sodium-channel specific S3 binding site did not perturb electrophysiologic properties of Nav1.5 in Trp, the S4-S5 linker, the end of the S6 transmembrane region and the cardiomyocytes. top of the CTD. The interaction motif involves a number of residues which 101-Plat are conserved across eukaryotic and prokaryotic sodium channels, but 2D not other ion channels. This structure demonstrates for the first time the Investigating Ca -Dependent Regulation of Sodium Channels via physical linkage between channel activation and opening, enabling a com- Thermodynamic and Structural Analysis of Nav1.4 and Nav1.5 Carboxy plete model for the mechanism of sodium channel activation/opening and Tail Interactions with Calmodulin Jesse B. Yoder1, Sandra B. Gabelli2, L. Mario Amzel1. closing. (*= these authors contributed equally; supported by grants from 1 the U.K. BBSRC) Biophysics & Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USA, 2Medicine, Biophysics & Biophysical 99-Plat Chemistry, Johns Hopkins University School of Medicine, Baltimore, The Cardiac Channel NaV1.5 Inactivation is Modulated by its C-Terminal MD, USA. EF-hand Domain Voltage-gated sodium channels (Nav) are essential for cardiac and skeletal Bernd R. Gardill, Ching-Chieh Tung, Ricardo E. Rivera-Acevedo, Filip Van muscle function. Channelopathic mutations in cardiac sodium channel Petegem. (Nav1.5) and skeletal muscle sodium channel (Nav1.4), particularly in their Biochemistry and Molecular Biology, University of British Columbia, cytoplasmic carboxy tails (CTNav), give rise to numerous arrhythmias and Vancouver, BC, Canada. myotonias. In Nav regulation, CTNav partners with the Ca2þ-sensing protein The cardiac isoform of voltage-gated sodium channels Nav1.5 is responsible calmodulin (CaM). We aim to understand how CaM regulates channel func- for the rapid upstroke of the action potential after excitation. The underlying tion. Ca2þ-free CaM (apoCaM) is a regulatory modulator of Nav, and gene of the alpha-subunit SCN5A is mostly expressed in the myocardium apoCaM bound to the CTNav increases the channel’s open probability. although some expression in other tissue has been reported. Auxiliary pro- CaM also participates in the Ca2þ-dependent inactivation (CDI) of Nav1.4. teins like calmodulin have been identified to modify channel gating. Muta- In contrast, Nav1.5 does not show CDI and no role of Ca2þ-CaM is known tions in SCN5A and calmodulin have been linked to several arrhythmias, in Nav1.5. To understand the Ca2þ-control of CaM regulation of these sodium including Brugada syndrome and Long-QT syndrome type 3. The channel channels we have collected binding data of CTNavs with CaM, in the pres- architecture is organized in four homologous transmembrane repeats and a ence and absence of Ca2þ. Binding data of CTNavs with CaM mutants 2þ C-terminal EF-hand domain. After channel opening Nav1.5 undergoes inac- with Ca -binding knocked out in either of CaM’s functional domains (lobes) tivation within milliseconds which leads to a non-conducting state of the have also been collected to understand the distinct roles of CaM’s lobes. channel. The cytoplasmic DIII-IV linker is known as inactivation gate. It Collectively, these binding data have allowed us to predict CTNav and contains the hydrophobic IFM motif which has been shown to stabilize CaM populations as a function of Ca2þ concentration. To gain information the inactivated state. A previous study suggested a direct interaction of the on the structural changes induced by Ca2þ on the CTNav-CaM complexes, C-terminal part of the DIII-IV linker and the C-terminal EF-hand domain we conducted small angle scattering (SAXS) experiments on CTNav1.4- of the channel stabilizing the inactivation gate in the inactivated state. CaM and CTNav1.5-CaM in the presence and absence of Ca2þ. The distance Here, we present an alternative model where the EF-hand destabilizes the in- distribution function, P(r), shows changes in both complexes upon addition of activated state through binding to the DIII-IV linker. Isothermal titration Ca2þ. Computational modeling of flexible forms of the complexes reveals calorimetry was utilized to characterize binding of WT and mutant inactiva- conformations of CTNav-CaM, in the presence and absence of Ca2þ, that tion gate peptides to the EF-hand domain. NMR spectroscopy enabled us are compatible with our SAXS data. The molecular envelope of the to map the exact interaction site on the EF-hand domain. Two-electrode CTNav1.5-apoCaM matches well with the apoCaM-CTNav1.5 crystal struc- voltage clamp measurements in Xenopus laevis oocytes provided data on ture previously determined by our lab. inactivation gate mutants and EF-hand disease mutants in the context of full length channel. Mutations that abolished EF-hand binding lead to a pro- 102-Plat found left-shift of steady-state inactivation, suggesting a role of the EF-hand Targeting the Cardiac Sodium Channel to Increase Excitability domain in destabilizing the inactivated state. of Stem-Cell Derived Cardiomyocytes Valentin Sottas, Cristian Mihnea Trache, Nina D. Ullrich. 100-Plat Heidelberg University, Heidelberg, Germany. Transgenic Approach to Exploration of Calmodulin’s Role on Sodium The cardiogenic potential of stem-cell derived cardiomyocytes (iPSC-CM) and Channel Function within Cardiomyocytes their prospective use for cardiac cell therapy crucially depends on their excit- Jeffrey Abrams, Alexander Katchman, Lin Yang, Steven Marx. ability and functional integration in myocardial tissue. Indeed, previous studies Columbia University Medical Center, New York, NY, USA. from our group have shown that cell excitability and intercellular coupling are Many disease-causing mutations within Nav1.5 are localized to its C-terminal strongly reduced in iPSC-CMs compared to primary cardiomyocytes. For clin- domain. This region interacts with numerous auxiliary proteins. Amongst ical aspects, impaired excitability and electrical signal propagation may lead to the most controversial of these interactions is that with calmodulin. To clarify conduction slowing and the development of arrhythmia. In this project, we calmodulin’s role on sodium channels expressed within cardiomyocytes, focus on the idea that cardiomyocyte excitability and conduction properties we employed a transgenic approach.We generated transgenic mice with are interrelated and depend on the expression of the cardiac sodium channel doxycycline-inducible, cardiac-specific expression of either FLAG-epitope- Nav1.5 and the major gap junction forming protein connexin-43 (Cx43). We tagged C374Y-Nav1.5 (WT), or C374Y / IQ1908-1909AA-Nav1.5 (IQ/AA). tested the hypothesis that molecular remodeling of both proteins enhances The C374Y mutation increases tetrodotoxin sensitivity, enabling electrophys- cell excitability as reflected in Nav1.5 activity and action potential (AP) prop- iologic distinction between endogenous and transgenic Nav1.5. The IQ/AA erties with the aim to approach native cardiomyocyte function. Using a combi- mutation is known to decrease binding affinity with calmodulin. Limb-lead nation of molecular modulation and electrophysiological evaluation in voltage ECG’s were measured under anesthesia. Biophysical properties of Nav1.5 and current clamp modes, our data demonstrate that enhanced Nav1.5 expres- were measured in isolated cardiomycoytes. Quantification of late current sion in iPSC-CMs significantly increased sodium current (INa in pA/pF: control was determined by normalizing persistent current detected with 100 mM 40.5510.5, Nav1.5 118.3527.2) and upstroke velocity (dv/dtmax, in V/s: [Na]o/3mM [Na]i, to peak current measured with 3 mM [Na]o/3mM [Na]i. 156518 vs. 276529, respectively) of the AP, a critical determinant of cell Transgenic sodium channels trafficked to the sarcolemma and were detected excitability. Typically, a fraction of iPSC-CMs also exhibited spontaneous using FLAG-specific antibodies. Electrophysiologic characteristics of WT APs with low dv/dtmax (<50 V/s) driven without Nav1.5, a hallmark of imma- Nav1.5 were comparable to Nav1.5 in non-transgenic mice. The IQ/AA trans- turity. However, after Nav1.5 overexpression, all recorded APs showed fast genic channel also trafficked to the membrane and demonstrated comparable dv/dtmax. Furthermore, INa was also increased in Cx43-overexpressing iPSC- electrophysiologic properties. The amount of late current determined by the CMs (INa 66.2519.8 pA/pF) suggesting that Cx43 may influence Nav1.5 ratio between persistent current and peak sodium current was not different expression and thereby cell excitability. This notion was further confirmed in in non-transgenic, transgenic WT, and transgenic IQ/AA mice. QT interval immunostainings of Cx43-overexpressing iPSC-CMs demonstrating increased was not prolonged in IQ/AA mice recorded using limb-lead ECG under iso- Nav1.5 expression at the plasma membrane, which suggests a common regula- flurane anesthesia. In contrast, mice expressing transgenic FLAG-epitope- tion pathway between both proteins. In conclusion, modulation of Nav1.5 and tagged F1759A-Nav1.5, a lidocaine resistant sodium channel that also has Cx43 expression greatly enhances the excitability of iPSC-CMs and may increased persistent current, showed prolonged QT intervals. These data represent a powerful new target for improving the functional maturation of demonstrate a novel approach to study mutant human Nav1.5. In contrast iPSC-CMs.

BPJ 7693_7704 20a Sunday, February 12, 2017

Platform: Optical Microscopy and Super- that tracks the mobility of different oligomeric species within live cell nuclear architecture.1 pCOMB amplifies the signal from the brightest species present resolution Imaging: Novel Approaches and and filters the dynamics of the extracted oligomeric population based on arrival time between two locations. Here we use this method to demonstrate Analysis I a dependence of signal transducer and activator of transcription 3 (STAT3) 103-Plat mobility on oligomeric state. We find that upon entering the nucleus Super-Resolution Imaging of Unlabeled Proteins on DNA STAT3 dimers must first bind DNA to form STAT3 tetramers, which are Anna EC Meijering, Andreas S. Biebricher, Erwin JG Peterman, also DNA-bound but exhibit a different mobility signature. Examining the dimer-to-tetramer transition by a cross pair correlation analysis (cpCOMB) Gijs JL Wuite, Iddo Heller. reveals chromatin accessibility to modulate STAT3 tetramer formation. Physics and Astronomy, Vrije Universiteit Amsterdam, Amsterdam, Thus the pCOMB approach is suitable for mapping the impact oligomerisa- Netherlands. Direct visualization of DNA-protein interactions at the single-molecule level is tion has on transcription factor dynamics. of ever increasing importance to unravel the salient details of a wide range of 1. Hinde, E.*, E. Pandzic, Z. Yang, I. H. W. Ng, D. A. Jans, M. A. Bogoye- DNA-associated processes. Recent emergence of super-resolution imaging has vitch, E. Gratton and K. Gaus*. 2016. Quantifying the dynamics of the the potential to further boost the impact of such investigations. However, cur- oligomeric transcription factor STAT3 by pair correlation of molecular brightness. Nature Communications. 7(11047). rent imaging methods heavily rely on (fluorescent) labeling strategies, which can be challenging and potentially even interfere with the molecular interac- 106-Plat tions under scrutiny. Here, we introduce a new label-free method to image Reconstructing Spatial Features of Nucleocytoplasmic Transport using the presence and location of DNA-bound proteins with super-resolution on Projected Cargo Localizations optically manipulated DNA. The method is based on localization microscopy Li-Chun Tu1, Maximiliaan Huisman1, Yu-Chieh Chung2, Carlas Smith1, of DNA-intercalating dyes that locally bind to bare DNA but not to protein- David Grunwald1. bound DNA sections, yielding an inverted image that reveals the ‘shadows’ 1RNA Therapeutics Institute, University of Massachusetts Medical School, of the proteins on the DNA: inverse binding-activated localization microscopy Worcester, MA, USA, 2No affiliation, Worcester, MA, USA. (iBALM). We present the proof of principle of iBALM as well as of functional Time-resolved single molecule experiments have provided tremendous infor- variations to this method and provide experimental and theoretical data that mation pertaining to molecular dynamics and localization over the last decade. describe the spatial and temporal resolution that can be obtained. iBALM Biological processes take place in a light sensitive environment, on time scales has the potential to become a valuable addition to the single-molecule toolkit from sub-millisecond (ms) to hours and length scales from nanometer to milli- and enable direct visualization of processes that were previously not possible meter, presenting a number of experimental challenges. One such challenge is due to limitations posed by labeling. the need for speed in image acquisition to correctly follow single molecule 104-Plat movements during translocation through the nuclear pore complex (NPC). Varying Label Density to Probe Membrane Protein Nanoclusters in Translocation through the central channel has repeatedly been reported to be STORM/PALM faster than 20 ms. Florian Baumgart1, Andreas Arnold1, Konrad Leskovar1, Kaj Staszek1, Biochemical and structural data of the components that make up the NPC have Martin Foelser1, Julian Weghuber2, Hannes Stockinger3, led to the question if specific spatial transport routes exist within the NPC in Gerhard J. Schuetz1. vivo. Millisecond time resolutions and three-dimensional spatial resolution in 1Institute of Applied Physics, TU Wien, Vienna, Austria, 2University of the range of only a few nm are needed in order to resolve the path traveled Applied Sciences Upper Austria, Wels, Austria, 3Medical University of by transport receptors and cargos by single molecule real-time microscopy. Vienna, Vienna, Austria. These imaging requirements are challenging and are not met by current tech- Superresolution microscopy has facilitated the investigation of cellular struc- nology; however, it was suggested by Ma & Yang that highly time-resolved tures at length scales far below the optical diffraction limit. When applied to 2D tracking data can be interpreted as projected cargo densities and subse- the plasma membrane, the presence of a variety of protein nanoclusters was quently transformed into a 3D cargo distribution. Such distributions would pro- revealed, which lead to speculations whether nanoclustering was a general vide valuable insights into the function of NPC mediate transport in cells. Here feature of plasma membrane proteins. Particularly in T lymphocytes, clustering we present a thorough analysis of the conditions needed for this method to work of signaling proteins has been proposed to represent a fundamental mechanism for the nuclear pore complex and the limits to which the results may be for cell activation. Recently, however, doubts were raised whether imaging interpreted. artifacts inherent to PALM/STORM might have influenced or even caused the observation of some of those protein clusters. To approach these con- 107-Plat cerns, we developed a method to robustly discriminate clustered from Following a Giant’s Footsteps: Single-Particle and Super-Resolution random distributions of molecules detected with single molecule localization Approaches to Decipher the Nuclear Transport of Hepatitis B Virus Capsids microscopy-based techniques like PALM and STORM.1 The approach is based Giulia Paci, Niccolo` Banterle, Christine Koehler, Edward A. Lemke. on deliberate variations of the labeling density of the samples and quantitative EMBL (European Molecular Biology Laboratory), Heidelberg, Germany. cluster analysis. Our method circumvents the problem of clustering artifacts Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs), generated by the blinking statistics of the fluorophores used. It can be readily and is a tightly regulated process. The Hepatitis B Virus (HBV) capsid is applied to PALM and STORM experiments where either overexpressed pro- one of the largest cargoes ever shown to be imported intact to the nuclear teins are present over a broad range of expression levels or antibody concentra- side of the NPC. The import of such large cargoes - close in dimension to tions are titrated to achieve different degrees of labeling. the expected maximum size of the NPC central channel - is largely unknown, 1 Baumgart et al., Nat. Meth. 13: 661-664 (2016) as well as to what extent plasticity of the NPC transport conduit is required to accommodate the transport. To decipher the molecular details of this host- 105-Plat pathogen interaction, we developed a dual strategy that allows us to visualize Tracking Oligomeric Transcription Factor Dynamics by Pair Correlation with high spatiotemporal resolution the viral capsid, as well as the elusive of Molecular Brightness (pCOMB) permeability barrier of the NPC. We follow nuclear import of fluorescently Elizabeth Hinde1, Elvis Pandzic1, Zhengmin Yang1, Ivan Ng2, labelled HBV capsids in mammalian cells using single-particle tracking on a Marie Bogoyevitch3, David Jans4, Enrico Gratton5, Katharina Gaus1. purpose optimized self-designed HILO microscope. We observe an average 1EMBL AUSTRALIA NODE IN SINGLE MOLECULE SCIENCE, transport time in the hundreds of milliseconds range, orders of magnitude faster University of New South Wales, Sydney, Australia, 2Program in Emerging than what expected for such a large cargo. In addition, slower interactions occur Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore, at the cytoplasmic filaments and nuclear basket, pointing to docking and un- 3Department of Biochemistry and Molecular Biology, University of docking interactions being the rate limiting factor. Complementarily, to deci- Melbourne, Melbourne, Australia, 4Department of Biochemistry and pher the changes occurring in the NPC during viral transport, we developed Molecular Biology, Monash University, Melbourne, Australia, 5Laboratory a method to image site-specifically labelled nucleoporins in the cell using for Fluorescence Dynamics, Biomedical Engineering, University of super-resolution microscopy (SRM). We combine site-specific tagging of California Irvine, Irvine, CA, USA. proteins, through an enhanced Amber suppression system, with DNA-PAINT Oligomerisation of transcription factors controls their translocation into the microscopy, enabling the visualization of even low abundance proteins such nucleus and DNA binding activity. Here we present a fluorescence micro- as nucleoporins. This synergistic approach gives us the potential to resolve scopy method termed pCOMB (pair correlation of molecular brightness) the plasticity of the permeability barrier of NPCs, allowing us a dual view on

BPJ 7693_7704 Sunday, February 12, 2017 21a the viral transport from both the cargo and the ‘‘gate’’ (NPC) level in an unprec- improvements in photostability in living cells. These fluorophores enable edented manner. robust multi-color, wash-free imaging with a large photon budget. We are investigating their phototoxicity and mechanism in order to maximize the pho- 108-Plat tostability, to generalize this approach to different fluorophores, and to apply Development of a Simultaneous Six-Color Fluorescence Microscope with these fluorophores to diverse biological settings, including living cells, tissues, Single-Molecule Sensitivity and animals. We freely share our fluorophores with the academic community. Jingyu Wang, Jamie Barnett, Luke Springall, Neil M. Kad. This work is supported by HHMI and NIH (U01 EB 021236). School of Biosciences, University of Kent, Canterbury, United Kingdom. Prokaryotic Nucleotide excision repair (NER) is a complex mechanism involving six proteins: UvrA, UvrB, UvrC, UvrD, DNA polymerase 1 and Platform: Membrane Protein Structures I DNA ligase. To holistically study the kinetics of NER, multiple labelling and 111-Plat imaging channels are required to differentiate the parties involved in the dam- Structure Inhibition and Regulation of a Two-Pore Channel TPC1 age search and repair activities. Using Quantum dot (Qdot)-conjugated UvrA, Alexander F. Kintzer, Robert M. Stroud. UvrB, UvrC, UvrD, DNA polymerase 1 and a Qdot labelled lesion we aim to Biochemistry and Biophysics, University of California at San Francisco, San detect and determine the significance of all the complexes that contribute to Francisco, CA, USA. repair. For this purpose, we have adapted an existing fluorescence microscope Membrane transport serves vital functions in the cell, providing nutrients for into a multi-color simultaneous fluorescence microscope by spectrally sepa- growth, relaying electrical signals, evading pathogens, and maintaining rating 6 emission channels based on available Qdots: 525 nm, 565 nm, homeostasis. Voltage- and ligand-gated ion channels propagate cellular elec- 585 nm, 625 nm, 655 nm and 705 nm. These six channels were created using trical signals by coupling changes in membrane potential and the binding of parallel optical splitting devices distributed into two paths and imaged on two molecules to channel opening and selective passage of ions through the identical state-of-the-art Hamamatsu, ORCA-Flash4.0 V2 scientific CMOS membrane barrier. Two-pore channels (TPCs) are intracellular ion channels cameras. Each low noise, high QE (>80%) camera sensor has 2048 x 2048 that integrate changes in membrane potential, second messengers, and phos- pixels, therefore every spectral channel has 682 x 2048 pixels; sufficiently large phorylation to control endolysosomal trafficking, autophagy, cellular ion and to image elongated DNA tightropes. Although the Qdots can be illuminated by amino acid homeostasis, and ultra-long action potential-like signals. They a single 488 nm source, the fluorescence microscope also uses four excitation broadly impact human diseases related to trafficking including filoviral in- lasers: 405 nm, 488 nm, 561 nm and 637 nm combined into a single path. This fections, Parkinson’s disease, obesity, fatty liver disease, and Alzheimer’s allows for numerous combinations of channels to be excited and separately disease. The response of TPCs to multiple cellular inputs suggests a multi- modulated using an Arduino-based control element linking the cameras to state gating mechanism. Nevertheless, the mechanisms that govern cycles the lasers. Using this system, we present data obtained on the heterogeneity of activation and deactivation or ‘gating’ in the channel remain poorly un- of the complexes formed during NER. derstood. To understand the bases for ion permeation, channel activation, 109-Plat the location of voltage-sensing domains and regulatory ion-binding sites, Intracellular Delivery of Membrane Impermeable Photostable Fluorescent and phosphoregulation, we determined the crystal structure of TPC1 from ˚ Probes into Living Cells for Super-Resolution Microscopy Arabidopsis thaliana to 2.87A resolution. This reveals for the first time Yuji Ishitsuka1, Kai Wen Teng1, Pin Ren1, Yeoan Youn1, Xiang Deng2, how TPC channels assemble as ‘quasi-tetramers’ from two non-equivalent Pinghua Ge1, Andrew Belmont2, Paul R. Selvin1. tandem pore-forming subunits. We determined sites of phosphorylation in 1Department of Physics, University of Illinois Urbana-Champaign, Urbana, the N-terminal and C-terminal domains that are positioned to allosterically IL, USA, 2Department of Cell and Developmental Biology, University of modulate channel activation by cytoplasmic calcium. One of the two voltage Illinois Urbana-Champaign, Urbana, IL, USA. sensing domains (VSD2) encodes voltage sensitivity and inhibition by Specific labeling of the cellular target with fluorophore is one of the funda- luminal calcium locks VSD2 in a ‘resting’ conformation, distinct from the mental requirement in all fluorescence imaging techniques including single activated VSDs observed in structures of other voltage-gated ion channels. molecule and super-resolution microscopy techniques. While extracellular tar- The structure shows how potent pharmacophore trans-Ned-19 allosterically gets may be tagged with virtually any kind of probes, intracellular labeling of acts to inhibit channel opening. In animals, trans-Ned-19 prevents infection living cells is limited to the use of fluorescent proteins and limited selection of by Ebola virus and Filoviruses by blocking fusion of the viral and endolyso- membrane permeable dyes. Here we show that pore forming proteins can be somal membranes, thereby preventing delivery of their RNA into the host used to temporarily permeabilize the cells and allow delivery of various fluo- cytoplasm. The structure of TPC1 paves the way for understanding the com- rescent probes, ranging from organic dyes (<1 kDa) to fluorescent immuno- plex function of these channels and may aid the development of antiviral globulin antibody (~150 kDa), for specific labeling of intracellular targets for compounds. live fluorescence cell imaging. We demonstrate that permeabilized cells can efficiently be recovered to carry on normal cellular processes shown by nuclear 112-Plat translocation of nanobody-labeled p65 proteins in response to chemical stimu- Novel Mechanism of Gating in the TrkH-TrkA Complex 1 1,2 1,3 1 lation. One of the most photostable but membrane impermeable organic fluo- Hanzhi Zhang , Zhao Wang , Mingqiang Rong , Yaping Pan , 1,2 1 rophore, Atto 647N, has been delivered into cells to label actin fibers and Wah Chiu , Ming Zhou . 1Baylor College of Medicine, Houston, TX, USA, 2National Center for kinesin dimers and to perform super-resolution fluorescence microscopy imag- 3 ing dSTORM and single molecule tracking, respectively. This technique opens Macromolecular Imaging, Houston, TX, USA, Kunming Institute of up a large number of stable, but otherwise membrane impermeable fluores- Zoology, China Academic of Science, Kunming, China. The superfamily of Kþ transporters (SKT) is ubiquitous in bacteria, fungi and cence labeling probes that are available for investigating transfected and endog- þ enous intracellular targets. plants. SKT proteins are required for survival of bacteria in low K conditions and are involved in salt regulation in fungi and plants. Bacterial SKTs have two 110-Plat components, a membrane embedded protein that resembles an ion channel and Bright and Photostable Fluorophores for Advanced Fluorescence a cytosolic protein that regulates channel gating [1]. Crystal structures of two Microscopy bacterial SKT systems were reported recently [2,3]. In both structures, the Qinsi Zheng1, Jonathan B. Grimm1, Anand K. Muthusamy1, membrane embedded component forms a homodimer onto which a homotetra- Robert H. Singer1,2, Luke D. Lavis1. meric ring of the cytosolic protein docks. Single-channel activities of one of the 1Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, complexes, the TrkH (membrane embedded) -TrkA (cytosolic) complex, were USA, 2Albert Einstein College of Medicine, New York, NY, USA. recorded and analyzed: ATP or ATP analogs such as AMPPNP activates the Advanced fluorescence microscopy, including single-molecule and super- channel while ADP closes it [2]. The structure of the TrkH-TrkA complex is resolution imaging, demands bright and photostable fluorophores. We have likely in a closed conformation because it was crystallized in the presence of recently reported a general approach to improve fluorophores brightness in NADH, a ligand that does not activates the channel. In order to understand living cells by substituting the N,N-dimethylamino groups found in classic how ATP or its analogs induces channel opening, we solved the structure of dyes with four-membered azetidine rings (Nature Methods 12, 244250 the TrkH-TrkA complex in the presence of AMPPNP to 3.29 A˚ by x-ray crys- (2015)). In an unpublished work we have synthesized new derivatives contain- tallography. When compared to the previous structures, the new structure ing substituents on the azetidine ring. Using this approach we were able to fine shows that each TrkA protomer binds to two AMPPNP molecules, and that tune the wavelength and fluorogenecity of the fluorophore without affecting the TrkA tetramer assumes an elongated conformation that likely induces a brightness. Here, we report that several of these novel substituted-azetidine change in the TrkH. Conformational changes in the TrkH involve significant fluorophores, as well as the substituted-xanthene ones, exhibit substantial changes in the dimer interface and are different from any other channels of

BPJ 7693_7704 22a Sunday, February 12, 2017 known structure. These new observations and hypotheses will be validated and transporter-mediated sterol transport and to analyze the disruptive effects of mu- tested by mutational and functional analyses. tations causing sitosterolemia. The structure will serve as a structural template [1] Levin EJ, Zhou M. Recent progress on the structure and function of the for homology modelling to a wide range of transport system that is regulated TrkH/KtrB ion channel. Curr Opin Struct Biol. 2014;27:95-101. by ABCG transporters and by ABC2 superfamily. [2] Cao Y, Pan Y, Huang H, et al. Gating of the TrkH ion channel by its asso- ciated RCK protein TrkA. Nature. 2013;496(7445):317-22. 116-Plat [3] Vieira-pires RS, Szollosi A, Morais-cabral JH. The structure of the KtrAB Structural and Mechanistic Basis of Proton-Coupled Metal Ion Transport potassium transporter. Nature. 2013;496(7445):323-8. in the SLC11/NRAMP Family Cristina Manatschal, Ines A. Ehrnstorfer, Raimund Dutzler. 113-Plat Department of Biochemistry, University of Zurich, Zurich, Switzerland. Structural and Functional Characterization of a Calcium-Activated Divalent metal ion transporters (DMTs) of the SLC11/NRAMP family trans- Cation Channel From Tsukamurella Paurometabola port iron and manganese across cellular membranes. These proteins are highly Bala Dhakshnamoorthy, Ahmed Rohaim, Huan Rui, Lydia Blachowicz, conserved across all kingdoms of life and thus likely share a common transport Benoit Roux. mechanism. Our previous crystal structure of Staphylococcus capitis DMT University of Chicago, Chicago, IL, USA. (ScaDMT) has established the structural relationship of the SLC11 family The selectivity filter is an essential functional element of Kþ channels that is with the amino acid transporter LeuT and it revealed the location of a conserved highly conserved both in terms of its primary sequence and its three- transition-metal ion binding site in the center of the transporter. In this struc- dimensional structure. Here, we investigate the properties of an ion channel ture, ScaDMT adopts an inward-facing conformation. Recently, we have deter- from the Gram-positive bacterium Tsukamurella paurometabola with a selec- mined the crystal structure of Eremococcus coleocola DMT (EcoDMT) in an tivity filter formed by an uncommon proline-rich sequence. Electrophysiolog- outward-facing conformation. Together these structures define the endpoints ical recordings show that it is a non-selective cation channel and that its activity of the transport cycle. Functional assays with proteoliposomes established depends on Ca2þ concentration. In the crystal structure, the selectivity filter EcoDMT as secondary active transporter that couples the symport of Mn2þ 2þ adopts a novel conformation with Ca ions bound within the filter near the and protons with a KM in the low micromolar range. Mutants of residues of pore helix where they are coordinated by backbone oxygen atoms, a recurrent the metal ion binding site severely affected both, Mn2þ and proton transport motif found in multiple proteins. The binding of Ca2þ ion in the selectivity filter thus suggesting that the transport of protons requires conformational changes controls the widening of the pore as shown in crystal structures and in molec- of the transporter. Inspection of both structures revealed two protonatable res- ular dynamics simulations. The structural, functional and computational data idues close to the metal ion binding site that have changed their accessibility to provide a preliminary characterization of this calcium-gated cation channel. either side of the membrane as potential candidates for proton acceptors. Mutation of one of these residues, a conserved histidine on a-helix 6b, resulted 114-Plat in metal ion transport that appears to be no longer coupled to protons, which Crystal Structure of a Low CO2-Inducible Protein, implies that this residue likely plays a central role in proton transport. Taken LCI1 in Chlamydomonas Reinhardtii together, our studies have revealed the conformational changes underlying Tsung-Han Chou. transition-metal ion transport in the SLC11 family by the alternate access Physics and Astronomy, Iowa State University, Ames, IA, USA. mechanism and they provide important insights into the determinants of its The assimilation of atmospheric carbon dioxide (CO2) by microalgae and coupling to protons. plants for photosynthesis has not been fully understood. Gaining insight into the intricate structural details of the CO2 scavenging mechanism by the photo- 117-Plat synthetic green algae Chlamydomonas reinhardtii can pave the way for utiliz- Structural Basis of Concentrative Nucleoside Transport ing abundant CO2 as a valuable alternative fuel resource. Here, we present a Marsha M. Hirschi, Zachary L. Johnson, Seok-Yong Lee. crystal structure of the Chlamydomonas reinhardtii LCI1 channel, which is Duke University, Durham, NC, USA. involved in the CO2-concentrating mechanism (CCM) to assimilate inorganic Nucleosides are essential molecules for the living cell. As precursors to nucle- carbon resources. Combined with X-ray crystallography, mass spectrometry otides they serve to fuel the salvage pathway of DNA and RNA synthesis. and computational simulation, our data indicate that the LCI1 membrane pro- Certain tissues, such as the brain and bone marrow, lack the capacity for de tein forms a trimeric assembly, in which each protomer conducts uncharged novo synthesis and therefore rely completely on the influx of nucleosides. CO2 and shuttles this inorganic carbon species across the cell membrane. Concentrative nucleoside transporters (CNTs) utilize sodium or proton gradi- ents to transport nucleosides across the cell membrane. These secondary active 115-Plat transporters also play an essential role in the termination of adenosine Structural Role of ABCG5/ABCG8 in Sterol Transport signaling, which controls important cellular processes such as neuromodulation Jyh-Yeuan (Eric) Lee, Daniel Rosenbaum, Helen Hobbs. and cardiovascular function. In addition to natural substrates CNTs are also the UT Southwestern Medical Center at Dallas, Dallas, TX, USA. conduit for many anti-cancer and anti-viral drugs, making them of special in- ATP binding cassette (ABC) transporters play critical roles in maintaining sterol terest from a pharmacological point of view. Notably, CNTs are the main trans- homeostasis in eukaryotic organisms, including yeast, plants and mammals. In port route for a popular pancreatic cancer drug, gemcitabine. We previously humans, the heterodimeric ABCG5/ABCG8 (G5G8) mediates the excretion of reported on the structure of CNT from Vibrio cholerae in complex with various cholesterol and dietary plant sterols into bile and into the gut lumen. Mutations substrates and nucleoside-like drugs. Each of these structures captured the inactivating either ABCG5 or ABCG8 cause sitosterolemia, a rare autosomal transporter in the inward-facing occluded conformation. In order to describe recessive genetic disorder characterized by plant sterol accumulation, hyper- the transport mechanism in further detail we performed crystallization studies cholesterolemia, and premature coronary atherosclerosis. ABCG5 and ABCG8 with CNT from Neisseria wadsworthii (CNTNW). CNTNW is highly homolo- are half ABC transporters;each subunit consists of an N-terminal nucleotide- gous to human CNT3, sharing 38% sequence identity and nearly identical sub- binding domain (NBD) and a C-terminal transmembrane domain (TMD). The strate binding sites. Here we present crystal structures of CNTNW captured in NBDs dimerize to form two catalytically asymmetric nucleotide-binding sites alternative conformations. We confirmed the physiological relevance of the (NBS), one that is catalytically active (NBS2) and the other inactive (NBS1). conformations by crosslinking experiments. Our structural analyses and func- To understand the structural basis for G5G8-mediated sterol transport we devel- tional studies provide new insights into the mechanism of CNTs and cellular oped a large-scale purification of human G5G8 by exploiting Pichia patoris nucleoside uptake in molecular detail. yeast. We crystallized the transporter in lipid bilayers, solved its structure in a nucleotide-free state at 3.9 A˚ resolution, and generated the first atomic model 118-Plat of an ABC sterol transporter. G5G8 presents a new structural configuration for Investigating the Structure of the Drug Transporter EmrE the TMD of ABC transporters, which is present in a large and functionally Maureen Leninger, James R. Banigan, Geliana Abramov, diverse ABC2 superfamily. We discover that the TMD and the NBS are coupled Nathaniel J. Traaseth. through networks of interactions that differ between NBS1 and NBS2, reflecting Chemistry, New York University, New York, NY, USA. the catalytic asymmetry of the transporter. A series of conserved polar residues in Multidrug resistance in bacteria is a critical challenge in public health and drug the TMD form polar networks that we proposed play a role in transmitting sig- discovery. One of the primary mechanisms of resistance is efflux pumps, which nals from the ATPase catalysis in the NBS to sterol transport on the TMD. couple an energetically favorable process with the export of a drug molecule Molecular dynamic simulation and long-range coevolution analysis revealed against its concentration gradient.1 Efflux pumps from the small multidrug an inward-upward TMD movement that predicts a significant conformational resistance protein family are ubiquitous among bacteria. These secondary change between the TMD subunits. Thus, the G5G8 structure provides a active transporters couple the efflux of a wide variety of toxic compounds molecular framework that allows us to propose a mechanistic model for ABC with the proton gradient of the inner membrane.2 To gain insight into this

BPJ 7693_7704 Sunday, February 12, 2017 23a transport process we carried out a series of biophysical experiments, including intensity time curve that increases in a sigmoidal fashion to a steady state. solution and solid-state NMR spectroscopy using the multidrug transporter This regular pattern is reproducible by melittin, LL37 and alamethicin, but EmrE. EmrE is an asymmetric and antiparallel homodimer, which couples not by CCCP or daptomycin. Remarkably, a similar regular pattern was repro- the efflux of cationic drugs with the import of protons. Previously, we revealed duced in GUVs. Indeed the steady-state membrane permeabilization induced a large change in structure and dynamics due to the acid/base chemistry at a by AMPs is quantitatively the same in spheroplasts and GUVs. There are how- conserved glutamic acid residue.3 In this work we focus on describing the ever interesting dissimilarities in details that reveal differences between bacte- site-specific structural changes within the substrate binding pocket that drive rial and lipid membranes. Spheroplast studies reveal a steady state membrane the large conformational change in EmrE. Specifically, our oriented solid- permeability independent of AMP concentration from 0.05 to 5.0 mM, that is state NMR and solution NMR experiments reveal backbone and side chain difficult to reproduce by lipid vesicles due of the fragility of pure lipid bilayers. structural perturbations upon binding protons or drugs that reveal a specific Tension in GUVs also influences the action of AMPs, whereas the spheroplast mechanism involving aromatic residues in the binding pocket. Finally, we built membranes are tensionless. Despite these differences, our results provide a on our previous findings 3 to show how conformationally biased mutants allow strong support for using model lipid bilayers to study the molecular interactions for the unambiguous determination of monomer-specific restraints for struc- of AMPs with bacterial membranes. tural elucidation of EmrE in lipid bilayers. References 121-Plat 1. Pu, Y.; Zhao, Z.; Li, Y.; Zou, J.; Ma, Q.; Zhao, Y.; Ke, Y.; Zhu, Y.; Chen, H.; Characterization of Anti-Biofilm Peptide Activity: A Biophysical Approach Baker, Matthew A. B.; Ge, H.; Sun, Y.; Xie, Xiaoliang S.; Bai, F., Enhanced Li-av T. Segev-Zarko1, Ron Saar-Dover1, Vlad Brumfeld2, Maria Luisa Efflux Activity Facilitates Drug Tolerance in Dormant Bacterial Cells. Molec- Mangoni3, Yechiel Shai1. ular Cell 2016, 62 (2), 284-294. 1Biomolecular sciences, Weizmann institute of science, Rehovot, Israel, 2. Nikaido, H.; Pages, J. M., Broad-specificity efflux pumps and their role in 2Chemical Research Support, Weizmann institute of science, Rehovot, Israel, multidrug resistance of Gram-negative bacteria. FEMS Microbiol Rev 2012, 3Biochemical Sciences, La Sapienza University, Rome, Italy. 36 (2), 340-63. The increasing number of multidrug resistant bacteria to available antibiotics 3. Gayen, A.; Leninger, M.; Traaseth, N. J., Protonation of a glutamate residue is a growing problem worldwide. One major strategy for resistance and an modulates the dynamics of the drug transporter EmrE. Nat Chem Biol 2016, 12 important reason for failure of therapy in the clinic is biofilm formation. To (3), 141-145. cope with unfavorable surroundings many bacteria live as biofilms, sessile micro-colonies adherent to surfaces that secrete an extracellular polymeric Platform: Membrane Active Peptides substance. An attractive alternative to conventional antibiotics are antimicro- and Toxins I bial peptides (AMPs), innate immune system molecules that target the bacte- rial cytoplasmic membrane, causing membrane disruption and cell death. 119-Plat AMPs biophysical properties are extensively studied regarding planktonic An In-Cell Solid-State NMR Portrayal of the Action Mechanism bacteria but much less so regarding biofilm formation. By designing and syn- of Antimicrobial Peptides with Intact Bacteria thesizing a series of model peptides that share the same amino acids compo- Marwa Laadhari1, Alexandre A. Arnold1, Andre´e E. Gravel1, sition but differ in their biophysical properties, we investigated how different Frances Separovic2, Isabelle Marcotte1. steps of biofilm formation are affected by the AMP’s features. We modified 1Chemistry, Universite du Quebec a Montreal, Montre´al, QC, Canada, the peptides characteristics using different approaches of charge segregation 2Chemistry, University of Melbourne, Melbourne, Australia. and amino acids D enantiomers. We used Pseudomonas aeruginosa, an oppor- Antimicrobial peptides (AMPs) are promising candidates to act against drug- tunistic Gram-negative bacteria, which is a leading cause of severe pulmonary resistant bacteria since they can disrupt the bacterial lipid barrier, leading to infections in cystic fibrosis patients and medical device contamination. Our cell death. 2H solid-state NMR is a valuable tool to study at a molecular level work demonstrates that the peptides combat biofilm at different stages of its the action of AMPs on the lipid organization and dynamics within the mem- formation and maintenance: (1) killing bacteria at their planktonic stage (2) brane bilayer. While these studies are usually performed with model phospho- preventing bacterial adhesion to biomaterials and (3) degrading pre-formed lipid membranes, the complexity of the bacterial cell wall prompts the biofilm. We show that substitution of L-to-D amino acids alters the peptides development of in-cell NMR techniques to take into account interactions of biophysical properties and improves their activity against each stage in the all constituents. In particular, Gram() bacteria are protected by lipopolysac- biofilm life cycle. By investigating which biophysical properties are essential charides in their outer membrane while a thick peptidoglycan covers the lipid for anti-biofilm activity we also discovered new mechanisms of peptides bilayer of Gram(þ) bacteria. We have thus developed novel 2H solid-state activity. NMR tools to study in vivo the bactericidal action mechanism of aurein 1.2 and caerin 1.1, AMPs excreted by frog skin. Using deuterated palmitic acid, 122-Plat we have established a protocol to 2H-label phospholipids in Gram(þ) Bacillus Effects of Lipid Composition, Peptide Charge, and Molecularity on the subtilis, and optimized the deuteration protocol of Gram() Escherichia coli. Structure of Antimicrobial Peptide Transmembrane Pores Via a combination of static and magic-angle spinning (MAS) experiments, Almudena Pino Angeles, Themis Lazaridis. our results support the interaction of the AMPs with membrane lipids leading Chemistry, City College of New York, New York, NY, USA. to decrease in lipid chain order. This effect is, however, observed at higher pep- The structure of transmembrane pores formed by antimicrobial and cell- tide concentration with B. subtilis and most likely attributable to an interaction penetrating peptides depends on the properties of the membrane and the pep- with negatively-charged components in its cell walls, such as teichoic acids. tide. To examine the effect of lipid composition, we simulated magainin 2 Our study shows that the nature of the cell wall plays a role upon the actual con- (MAG2) antiparallel tetramers in DMPC, DMPG and DMPC/PG 1:1 bilayers. centration of AMPs on the bacterial membrane and highlights the importance of Together with the results from our previous work (1), we observe that the studying membrane interactions with intact bacteria. MAG2 pore size initially decreases as the anionic content of the membrane in- creases. However, in pure DMPG the observed pore is similar in size to that in 120-Plat DMPC, but with considerably more unfolded peptides. We also simulated a Action of Antimicrobial Peptides on Bacterial and Lipid Membranes: A PGLa parallel tetramer in POPE/PG 3:1 and observed that 1-2 monomers either Direct Comparison adopt or tend to adopt a surface orientation during the 5 ms of the simulation, in Joseph E. Faust, Pei-Yin Yang, Huey W. Huang. partial agreement with NMR data (2). Physics & Astronomy, Rice University, Houston, TX, USA. We explored the effect of molecularity by simulations of hexamers and octamers Are lipid bilayers a good model for studying bacterial membranes? We study of melittin and octamers of MAG2 and comparison with previous tetramer re- this problem by comparing the action of antimicrobial peptides (AMPs) on sults (1,3). The results show a stable wide pore for the MAG2 octamer, with E. coli spheroplasts and on giant unilamellar vesicles (GUVs) made from all the monomers involved in the formation and support of the water channel. model lipid bilayers. We observed AMPs inducing membrane permeability On the contrary, in both melittin oligomers the number of peptides in contact in spheroplasts or GUVs under well defined concentrations of AMPs and dye with the pore is reduced during the simulation. Finally, we explored the effect molecules. The permeability allowed the dye molecules to diffuse in and out of peptide charge by comparing simulations of hexamers of melittin (charge of the cells or vesicles, from which we were able to measure the instantaneous þ5) and its gain-of-function variant MELP5 (charge þ2) (4). Whereas the me- membrane permeability by fluorescence recovery after photobleaching littin hexamer broke up into two trimers, one surrounding a pore and one dry, the (FRAP). We also compared the effect of AMPs with that of metabolic poison MELP5 hexamer simulation resulted in a stable wide pore. CCCP and daptomycin that kills bacteria by leaking potassium ions. The action (1) Pino-Angeles A, Leveritt JM 3rd and Lazaridis T. PloS Comput Biol (2016) of AMPs on spheroplasts is unique in producing an intracellular fluorescence 12(1):e1004570

BPJ 7693_7704 24a Sunday, February 12, 2017

(2) Stranberg E, Zerweck J et al. Biophys J (2013) 104(6):L9-11 biologically active than P3 for reasons that are not understood. Here, we (3) Leveritt JM 3rd, Pino-Angeles A and Lazaridis T. Biophys J (2015) compare the disruptive effects of P1 and P3 on lipid bilayers and DNA, and 108(10):2424-6 investigate the role of their amino terminal Cu and Ni (ATCUN) binding motif (4) Wiedman G, Fuselier T et al. J Am Chem Soc (2014) 136(12):4724-31 in their mechanism of action. We rely on a combination of complementary methods, including CD- and NMR-monitored titrations, structural solid-state 123-Plat NMR, neutron diffraction with deuterium labeling, surface plasmon resonance, Spontaneous Formation of an Ensemble of Structurally Diverse Membrane microscopy, biological activity assays, and voltammetry. We find by solid-state Channel Architectures from a Single Antimicrobial Peptide Maculatin NMR that both peptides use an a-helical structure to interact with bacterial cell 1 1 2 2 Yukun Wang , Charles Chen , Dan Hu , Jakob Ulmschneider , membrane mimics and isolated DNA. Interestingly, while P1 is more mem- Martin Ulmschneider1. 1 2 brane active than P3, the latter is more disruptive to DNA. Through neutron Johns Hopkins University, Baltimore, MD, USA, Shanghai Jiaotong diffraction studies, we demonstrate that P1 inserts more deeply in lipid mem- University, Shanghai, China. branes and induces a major conformational rearrangement of the lipid head- Antimicrobial peptides (AMPs) are an ancient, powerful, and ubiquitous groups, which leads to bilayer defects and significant water penetration into component of the innate immune defence in all domains of life and play a the interstices of the membrane. Remarkably, both peptides bound to Cu2þ key role in controlling the human microbiome. Many AMPs are known to act as nucleases that damage isolated DNA within minutes and P3 is signifi- selectively target and form pores in microbial membranes, killing a wide vari- cantly more effective than P1. In the context of bacterial cells, we show that ety of pathogens at low micromolar concentrations. Fundamental questions copper plays an essential role in planktonic, biofilm, and persister cell eradica- remain, however, regarding the molecular mechanisms of membrane targeting, tion. Furthermore, we find that the more membrane-active P1 kills bacteria pore formation and function, and the extent to which poration contributes rapidly while the more DNA-damaging P3 results in slower cell death. Taken towards antimicrobial activity. Here we report an experimentally guided and together, these results identify host defense metallopeptides with complemen- validated unbiased long-timescale simulation methodology that yields the com- tary and multi-pronged mechanisms of action as valuable templates to consider plete mechanism of spontaneous pore assembly in the membrane for the amphi- for the design of novel strategies against drug resistant bacteria. philic pore-forming AMP maculatin at atomic resolution. Rather than a single well defined pore, maculatin was found to form an ensemble of well defined 126-Plat temporarily functional channels that continuously form and dissociate in the 20D Years and no End in Sight: Histidine-Rich Designer Peptides membrane. All channels are formed from highly symmetric low-oligomeric as- with pH-Dependent Membrane Topology and with Multifacet Biomedical semblies of up to 8 membrane-spanning peptides that mimic integral membrane Potential protein channels in structure. Each channel has a different architecture, func- Christopher Aisenbrey1, Philippe Bertani1, David Fenard2, Anne Galy3, tional lifetime, and ion conductivity and overall membrane permeabilization Elise Glattard1, Martin Gotthardt4, Antoine Kichler5, Nan Liu4, is dominated by higher order oligomers, which form ion channels that also Arnaud Marquette1, Regine Suss€ 4, Louic Vermeer1, Dennis Wilkins-Juhl1, conduct water at high rates. The highest order oligomers were also found to Justine Wolf1, Burkhard Bechinger1. efficiently conduct small dyes. Channel architectures as well as their relative 1Chemistry, University of Strasbourg/CNRS, Strasbourg, France, 2Innovation weighting in the ensemble are sensitive to minor mutations as well as variation Technologique Lentivirus, Ge´ne´thon, Evry, France, 3Ge´ne´thon/Inserm of lipid tail length. All pores are formed spontaneously by the consecutive addi- UMR951, Evry, France, 4Pharmazeutische Technologie und Biopharmazie, tion of individual helices to a transmembrane helix or helix bundle, in contrast University of Freiburg, Freiburg, Germany, 5Laboratoire ‘‘Vecteurs: to current poration models postulated for AMPs. The diversity of channel archi- Synthe`se et Applications The´rapeutiques’’ (V-SAT), CNRS, Strasbourg, tectures formed by a single sequence is remarkable and could explain why no France. sequence-function relationships in AMP sequences have been discovered to The synthetic LAH4 peptides were designed to investigate the interactions that date. Structural ensembles formed by AMP may be the key to preventing bac- determine the membrane topology of helical peptides (1). Their core consists of terial resistance. alanines, leucine and four histidines arranged to form an amphipathic helix, as 124-Plat well as two lysines at each terminus. Through protonation of its histidines (pKs between 5.4 and 6.0) the alignment of the helices is transmembrane at neutral An Exact Model of Daptomycin Binding to Lipid Bilayers < Antje Pokorny, Tala O. Khatib. pH and in-plane at pH 5.5 (1). The LAH4 peptides exhibit membrane pore- Chemistry and Biochemistry, Univ. North Carolina Wilmington, formation and antimicrobial action at both neutral and at acidic pH including Wilmington, NC, USA. against clinical isolates where the low pH configuration is more active (2). The Daptomycin is a cyclic lipopeptide of clinical importance in the treatment of LAH4 peptides have been found to also exhibit potent DNA and siRNA transfec- multi-drug resistant infections, including those caused by methicillin- tion activities (3). Therefore they can act as a non-viral vector and has indeed been resistant S. aureus (MRSA) strains. Similar to other antimicrobial peptides, used for the delivery of quantum dots or protein-based vaccines. Furthermore, daptomycin binds with preference to the anionic cytoplasmic membranes typi- transduction by adeno-associated viruses or lentiviruses is enhanced by LAH4 cally found in prokaryotes. However, in contrast to most linear, alpha-helical (4) or non-peptidic mimetics of this family of peptides (5). Recent and ongoing peptides, daptomycin binds to lipid bilayers only in the presence of calcium biophysical, structural and cell biological investigations will be reported which ions and its activity is absolutely calcium-dependent. We measured the interac- aim to understand these activities at atomic resolution (3, 6-8). tion of daptomycin with anionic lipid membranes using kinetic binding exper- (1) B. Bechinger, J.Mol.Biol. 263, 768 (1996). iments and equilibrium titrations. The data were analyzed using an exact model (2) A. J. Mason, et al., J. Biol. Chem. 284, 119 (2009). describing the interactions of daptomycin with the lipid bilayer that includes (3) B. Bechinger, et al. J Pept Sci (2017, in prep.). solution and membrane-bound states, and the influence of calcium ions on (4) S. Majdoul, Seye, A.K., Kichler, A., Holic, N., Galy, A., Bechinger, B., daptomycin-lipid interactions. Fenard, D., J. Biol. Chem., 291, 2161 (2016) (5) C. Douat, C. et al. Angew. Chem Int. Ed 54, 11133 (2015) 125-Plat (6) C. Aisenbrey, B. Bechinger, Langmuir 30, 10374 (2014). The Metallopeptides Piscidin 1 and Piscidin 3 Employ Membrane and (7) R. Macha´n, P. Jurkiewicz, T. Steinberger, B. Bechinger, M. Hof, Langmuir Nuclease Activity to Eradicate Planktonic, Biofilm, and Persister Cells 30, 6171 (2014). Myriam L. Cotten1, M. Daben J. Libardo2, Ali Adem Bahar3, Riqiang Fu4, (8) A. Farrotti, G. Bocchinfuso, A. Palleschi, N. Rosato, E. S. Salnikov, N. Vitalii I. Silin5, Dacheng Ren3, Mihaela Mihailescu5, Alfredo Angeles-Boza2. Voievoda, B. Bechinger, L. Stella, BBA 1848, 581 (2015). 1Applied Science, College of William and Mary, Williamsburg, VA, USA, 2Chemistry, University of Connecticut, Storrs, CT, USA, 3Biomedical and Platform: Micro- and Nanotechnology Chemical Engineering, Syracuse University, Syracuse, NY, USA, 4National High Magnetic Field Laboratory, Tallahassee, FL, USA, 5Institute for 127-Plat Bioscience and Biotechnology Research, University of Maryland, Rockville, Improving the Temporal Resolution of Nanopore Recordings MD, USA. Siddharth Shekar1, Chen-Chi Chien2, David Niedzwiecki2, Marija Drndic2, Piscidin 1 (P1) and piscidin 3 (P3) are highly potent host-defense peptides Kenneth Shepard1. (HDPs) active against drug resistant bacteria, and therefore important for the 1Electrical Engineering, Columbia University, New York, NY, USA, design of novel anti-infective therapeutics. Both peptides are cationic and 2Physics and Astronomy, University of Pennsylvania, Philadelphia, interact with the anionic lipid membranes of bacteria. They also translocate PA, USA. across these membranes and colocalize with intracellular DNA. While Solid-state nanopores are being pursued for a number of applications both peptides have similar three-dimensional structures, P1 is generally more including, most notably, DNA sequencing. One of the challenges that

BPJ 7693_7704 Sunday, February 12, 2017 25a nanopores present is the fast rate at which molecules translocate. Significant 130-Plat improvements in the measurement bandwidth can be obtained through Salt Rejection using Conically Shaped Pores with Patterned Surface the optimization of detection electronics and reduction in nanopore membrane Charges capacitance. We present a low-noise, custom-designed complementary metal- Crystal Yang1, James Boyd2, Yinghua Qiu2, Zuzanna S. Siwy2. oxide-semiconductor (CMOS) amplifier chip capable of recording transloca- 1Dept. of Chemistry, University of California, Irvine, Irvine, CA, USA, tion dynamics in nanopores at bandwidths up to 10 MHz. We integrate 2Dept. of Physics and Astronomy, University of California, Irvine, Irvine, state-of-the-art silicon nitride nanopores with this amplifier to achieve signal CA, USA. to noise ratios (SNRs) of better than 10 at 5 MHz bandwidth in ssDNA trans- In light of global potable water scarcity, whether it be due to increasing agri- location experiments. We observe transient features with durations as short as cultural demands, higher standards of living, or overdrawing from under- 200 ns in some translocation events, features that would have been hidden at ground sources, the need for efficient desalination techniques is of high lower recording bandwidths. We also use our platform to record ssDNA trans- importance. Reverse osmosis membranes offer excellent rejection of salt location through glass-passivated silicon-nitride nanopores with membrane but the throughput of the technology is very low due to the sub-1nm effec- capacitances of less than 1 pF, further extending the achievable recording tive size of the membrane voids. We propose a different approach to achieve bandwidth. At these speeds, the potential exists to realize free-running DNA desalination using membranes containing pores with opening diameter of sequencing using solid-state nanopores. few nm, and walls with high density of surface charges and surface charge patterns. We found that even 5 nm in diameter nanopores and containing a 128-Plat junction between a zone with positive surface charges and a zone with nega- Identification of Single Nucleotides in SiN Nanopore tive surface charges could offer rejection of salt up to 80% from 100 mM Haojie Yang, Zhongwu Li, Yunfei Chen, Wei Si. KCl. Increasing the pore diameter can increase the flux of water even two Jiangsu Key Laboratory for Design and Manufacture of Micro-Nano orders of magnitude compared to fluxes achievable with reverse osmosis Biomedical Instruments, Southeast university, Nanjing, China. membranes. Experiments were performed with track-etch membranes with A nanopore with 1.8 nm in diameter is fabricated on a reduced SiN membrane densities of pores up 109 per cm2. Membranes in polyethylene terephthalate by focused ion beam and high energy electron beam. Through measuring the and polyimide were tested. Experimental observations were supported by the blockade ionic current for single nucleotides passing through the solid-state numerical modeling performed by solving Poisson-Nernst-Planck and nanopore, it is found that the current blockade can be used to statistically Navier-Stokes equations. Dependence of salt rejection on salt concentration, detect and identify all four types of nucleotides. The current blockade fitted applied pressure and surface charge pattern is investigated in detail. Discus- by Gauss function share the same trend as the volume of four type of nucle- sion on the range of electrostatic interactions in aqueous solutions as a func- > > > otide (dGTP dATP dTTP dCTP). We find the dwell time distributions tion of surface charge density and surface charge arrangement will also be are asymmetric in shape. They are characterized by sharply increasing peaks provided. at shorter times followed by broader decays at longer times. The distribution shapes were fitted to first-passage time distributions obtained from the 1D 131-Plat Fokker-Planck equation. The velocity and diffusion constant can be obtained Genetically Encoded DNA-Protein Hybrid Origami from the fitting function. It is found that larger single nucleotide mass has a Florian Praetorius, Hendrik Dietz. smaller diffusion coefficient, the same as the Einstein relation. The larger sin- Technische Universit€at Munchen,€ Garching near Munich, Germany. gle nucleotide volume has a faster velocity. In order to explain the experi- Here we describe an approach to bottom-up fabrication with nanometer- mental phenomena, molecular dynamics (MD) simulations were conducted precision that allows integrating the functional diversity of proteins in using a SiN pore with 1 M KCl. Results show that anions dominate the ionic designed three-dimensional structural frameworks. We reimagined the suc- migration in the nanopore, namely the concentration and velocity of anions in cessful DNA origami design principle using a set of custom staple proteins the nanopore are much higher than that of cations. The electroosmotic flow to fold a double-stranded DNA template into a user-defined shape. Each sta- which generated by anions has the same moving direction as single nucleo- ple protein recognizes two distinct double-helical DNA sequences and can tides. We consider singe nucleotides can be driven by both electroosmotic carry additional functionalities. The staple proteins we present here are flow and electric field through the nanopore. Because the four types of nucle- based on the transcription activator-like (TAL) effector proteins. Due to their otides have the same surface charge, the single nucleotide with larger volume repetitive structure these proteins offer a unique programmability that en- has a higher velocity provided by the electroomotic flow.In our experiment ables us to construct numerous staple proteins targeting any desired DNA result shows single nucleotide can be statistically identified by the SiN nano- sequence. Our approach is general, meaning that many different objects pore, and the MD simulation result shows the single nucleotide transmission may be created using the same set of rules, and it is modular, because com- mechanism in the 1.8 nm SiN nanopore. ponents can be modified or exchanged individually. We present rules for constructing megadalton-scale DNA-protein hybrid nanostructures; intro- 129-Plat duce important structural motifs, such as curvature, corners, and vertices; Formation of Synthetic Nanopores with Diameters from 20-50 nm by describe principles for creating multi-layer DNA-protein objects with Laser-Assisted Dielectric Breakdown enhanced rigidity; and demonstrate the possibility to combine our DNA- Cuifeng Ying, Jared Houghtaling, Bodo Wilts, Michael Mayer. protein hybrid origami with conventional DNA nanotechnology. Since all Adolphe Merkle Institute, Fribourg, Switzerland. components can be encoded genetically, our structures should be amenable Fabrication of nanopores in synthetic substrated by controlled breakdown to biotechnological mass-production. Moreover, since the target objects can (CBD)[1] is a robust and well characterized method that allows sub-2 nm nano- self-assemble at room temperature in near-physiological buffer, our hybrid pore fabrication, which is a challenge for traditional fabrication methods origami may also provide an attractive method to realize positioning and such as transmission electron microscopy (TEM). Due to its stochastic pore for- scaffolding tasks in vivo. We expect our method to find application both mation process, however, attempts to widen a nanopore using CBD can result in in scaffolding protein functionalities and in manipulating the spatial arrange- random formation of multiple nanopores instead of increasing the size of a ment of genomic DNA. single pore. Since CBD is a broadly-accessible, tuneable, and cost-effective method, it would be useful to fabricate nanopores with diameters from 20 to 132-Plat 50 nm for applications beyond DNA sequencing. This work focuses on fabri- DNA Nanoparticles Programmed from the Top Down with Variable cating such large nanopores by optically localizing the formation and enlarge- Design Motifs ment of defects in the SiN membrane[2]. We show that the breakdown time Sakul Ratanalert, Remi Veneziano, Mark Bathe. of SiN membranes (i.e. time for dielectric breakdown at constant applied poten- MIT, Cambridge, MA, USA. tial difference across the membranes) can be lowered significantly by focusing A hallmark of naturally evolved RNA assemblies, such as the ribosome, a laser beam on the membrane. To avoid forming multiple nanopores during ribonuclease-P, and tRNAs, is their ability to fold from a single nucleic acid the enlargement process, low voltage pulses were applied. In this study, strand into complex, functional 3D structures with diverse biological functions differences of parameters between nanopores fabricated by standard CBD in the cell. A long-standing aim of synthetic structural biology has been to mimic and laser-assisted CBD are discussed. The size, number and shape of the result- these properties of self-folding and functionality using a single strand of DNA, as ing nanopores are assessed by various techniques, including protein transloca- a stepping stone to RNA, because of its fewer secondary structure conformations tion experiments. and increased preference to hybridize in solution. Deriving the rules for such [1] Kwok, H.; Briggs, K.; Tabard-Cossa, V. PLoS One 2014, 9, (3), e92880. [2] self-assembly also allows for the ability to extend the design space to structures Pud, S.; Verschueren, D.; Vukovic, N.; Plesa, C.; Jonsson, M. P.; Dekker, C. beyond the set of naturally evolved molecules. Toward this end, we have devel- Nano letters 2015, 15, (10), 7112-7. oped a top-down sequence design approach based on the principle of scaffolded

BPJ 7693_7704 26a Sunday, February 12, 2017

DNA origami. First, we present a fully autonomous algorithm to produce a into specific three-dimensional structures to promote self-cleavage of specific single-stranded DNA scaffold and complementary staple strands, which fold phosphodiester bonds. Metal cations stabilize the folded RNA, which usually into nearly arbitrary target 3D objects, from Platonic solids to non-spherical to- correlates with biochemical activity. We used ensemble and single molecule pologies, with near quantitative synthetic yield, demonstrated for a dozen struc- FRET to compare the folding dynamics of a 195 nt bacterial group I ribozyme tures experimentally (Veneziano, Ratanalert, et al., Science, 2016). Second, we and a 54 nt Twister ribozyme from Oryza sativa. Whereas the group I ribo- present a powerful approach that eliminates staple strands entirely, offering the zyme remains stably folded in 2 mM MgCl2 that is sufficient for its activity, ability to program a single DNA molecule to fold into these arbitrary 3D shapes Twister ribozyme folds and unfolds on the second timescale even in 100 mM on its own, similar to natural RNA assemblies. We examine the folding pathways MgCl2. Nevertheless, Twister still self-cleaves in 50 mM MgCl2, although of each of these design modalities using quantitative PCR and present a thermo- visits to the folded state are transient and infrequent. Surprisingly, transition dynamic model to optimize sequence design, folding temperature, and yield metals activated Twister ribozyme even more efficiently than Mg2þ.Ongoing (Ratanalert et al., in prep, 2016). Together, these algorithms solve a long- efforts to understand how folding dynamics tune RNA activity will be standing challenge of synthetic structural biology to program nearly arbitrary discussed. 3D geometries using synthetic nucleic acids. 136-Symp 133-Plat Adventures with RNA Graphs pH-Responsive Reversible Regulation of Enzyme Activity by DNA-Based Tamar Schlick. Nanostructure Courant Inst, New York Univ, HHMI, New York, NY, USA. Seong Ho Kim1,2, So Yeon Kim1,2. RNA’s modular, hierarchical and versatile structure makes possible diverse, 1University of Science and Technology, Seoul, Korea, Republic of, 2Korea essential regulatory and catalytic roles in the cell. It also invites systematic Institute of Science and technology, Seoul, Korea, Republic of. modeling and simulation approaches. Among the diverse computational and Here, we present a novel method for reversibly regulating enzyme activity by theoretical approaches to model RNA structures, graph theory has been applied caging an enzyme into a pH-responsive DNA-based tetrahedron nanostructure. in various contexts to study RNA structure and function. I will present an over- pH-dependent opening/closing of the DNA cage was verified by measuring view of recent graph theoretical approaches for predicting and designing RNA fluorescence resonance energy transfer between two vertex corners of the topologies using graphical representations of RNA secondary structure, data- tetrahedron. The position of the covalent enzyme attachment in DNA was care- mining tools for junction topology prediction, and hierarchical sampling of fully chosen such that the attached enzyme faced inward the DNA cage. Both graphs based on statistical potentials. As evident from the work of many groups proteinase K protection assay and single-molecule based pull-down assay in the mathematical and biological sciences, graph theoretical approaches offer showed that the encapsulated enzyme were exposed to either proteinase K a fruitful avenue for designing novel RNA topologies and predicting tertiary or target antibody by pH-dependent opening of DNA cage. Remarkably, we structures from given secondary structures. found that the caging/uncaging process were reversible, implying that enzyme Of possible interest activity toward relatively larger substrates than DNA cage can be reversibly - H.H. Gan, S. Pasquali and T. Schlick, Nucl. Acids Res. 31:2926 (2003) regulated. Considering that the DNA cage is widely used as a delivery carrier, - N. Kim et al., J. Mol. Biol. 341:1129 (2004) our method can be further extended to reversibly regulate cell function by - G. Quarta and K. Sin and T. Schlick, PLoS Comput. Biol. 8: e1002368 (2012). pH-dependent activity control of delivered enzyme. - C. Laing, S. Jung, N. Kim, S. Elmetwaly, M. Zharan, and T. Schlick, PLOS One 8(8): e71947 (2013). 134-Plat - N. Kim, C. Laing, S. Elmetwaly, S. Jung, J. Curuksu, and T. Schlick, Proc. Eco-Friendly Processing for Engineering Bio-Safe Quantum Dots and Natl. Acad. Sci. USA 111: 4079 (2014). their Interaction with Biological Systems - M. Zharan, C. S. Bayrak, S. Elmetwaly, and T. Schlick, Nuc. Acids Res. 43: Marta d’Amora1, Marina Rodio1, Alberto Diaspro1,2, Romuald Intartaglia1. 1 2 9474 (2015). Istituto Italiano di Tecnologia, genova, Italy, Department of Physics, - N. Baba, S. Elmetwaly, N. Kim, and T. Schlick, J. Mol. Biol. 428: 811 (2016). University of Genoa, Genova, Italy. - L. Hua, Y. Song, N. Kim, C. Laing, J. T. L. Wang, and T. Schlick, PlOS One Inorganic nanomaterials have gained attention for delivery vehicles, gene 11: e0147097 (2016). detection systems, labeling and therapeutic applications. Many efforts have been reported in the synthesis of heavy metal quantum dots (QDs), for long- 137-Symp term, real-time cell labeling applications. Exposure to these QDs in living tis- The Structural and Mechanistic Origins of Catalysis in Nucleolytic sue endanger several issues due to their chemical composition, artificial ligand Ribozymes and/or the employed solution routes. In particular, surface coating/stabilization David M. Lilley, Timothy J. Wilson, Yijin Liu. of nanomaterials by chemical organic molecules, such as citrate have shown to Life Sciences, University of Dundee, Dundee, United Kingdom. trigger different interaction at cellular level [1]. However, it is still difficult to The nucleolytic ribozymes are a structurally diverse and widespread group of draw a definite conclusion. Therefore, other alternatives, taking into account catalytic RNA species. They accelerate transesterification reactions around a the nanoparticles fabrication strategy and the minimum toxicity of the carrier million fold, resulting in the site-specific cleavage or ligation of RNA. The itself, are crucial for potential success of nanomaterials in the clinical setting. potential entities that can participate are the nucleobases, 20-hydroxyl groups [2,3] Owing to its biocompatibility and biodegrability, silicon based nanoma- and hydrated metal ions. Probable catalytic strategies are the facilitation of terials are ideal candidates for in vivo applications [4]. Here, we will report a in-line attack, stabilization of the phosphorane transition state, deprotonation safe engineering approach based on liquid phase pulsed laser ablation tech- of the nucleophile and protonation of the oxyanion leaving group (these last nique enabling the generation of photoemissive, highly pure (i.e. free of chem- two being general base-acid catalysis). ical ligand at the surface) silicon quantum dots. Furthermore, we will present While much of the above was studied in ribozymes such as hammerhead, the physicochemical interactions of these non-toxic nanotools having unique hairpin and VS, these principles are well illustrated by newer ribozymes. We surface chemistry with biological systems. have solved the crystal structure of the twister ribozyme, which adopts a double (1) Mu et al., Chem Rev., 114, 7740. (2014) pseudoknot fold with a central active site. This well illustrates the four strate- (2) Intartaglia et al., Nanoscale, 4, 1271, (2012) gies summarized above. We have recently solved the structure of a new ribo- (3) Rodio et al., J. Colloid Interface Sci. 465, 242 (2016) zyme, that appears mechanistically very different, with a key role for a (4) Park et al, Nature Materials, 8, 331 (2009) bound metal ion where an inner-sphere water molecule acts as a general base. Symposium: RNA Structures and Dynamics Platform: Membrane Receptors and Signal 135-Symp Transduction I Cleaving Fast and Slow: Strategies for Self-Assembly of Catalytic RNA Sarah A. Woodson1, Subrata Panja1, Boyang Hua2, Krishnarjun Sarkar1, 138-Plat Taekjip Ha3. Oligomerization of the Epidermal Growth Factor Receptor Organizes 1Dept Biophysics, Johns Hopkins Univ, Baltimore, MD, USA, 2Dept Kinase-Active Dimers into Competent Signaling Platforms Biophysics & Biochemistry, Johns Hopkins Medical School, Baltimore, MD, Sarah R. Needham1, Laura C. Zanetti-Domigues1, Anton Arkhipov2, USA, 3Dept Biophysics & Biochemistry, HHMI & Johns Hopkins Medical Venkatesh P. Mysore3, Dimitrios Korovesis1, Selene K. Roberts1, School, Baltimore, MD, USA. Christopher J. Tynan1, Daniel J. Rolfe1, Michael Hirsch1, Ribozyme RNA motifs are widespread in nature and their adaptation to Alireza Lajevardipour4, Andrew H.A Clayton4, Peter J. Parker5,6, varied growth conditions has been sparsely investigated. Ribozymes fold Yibing Shan3, David E. Shaw3,7, Marisa L. Martin-Fernandez1.

BPJ 7693_7704 Sunday, February 12, 2017 27a

1Central Laser Facility, Science and Technology Facilities Council, signaling geometry at cell-cell contact. These are designed and produced by Oxfordshire, United Kingdom, 2Allen Institute for Brain Science, Seattle, employing sheet-like ligand decorated DNA nanostructures anchored on artifi- WA, USA, 3D. E. Shaw Research, New York, NY, USA, 4Faculty of Science, cial supported lipid bilayers. Such structures are rendered from polyhedral flat Engineering and Technology, Swinburne University of Technology, Victoria, sheet meshes that allow high modularity in design of shapes and protein spatial Australia, 5Division of Cancer Studies, King’s College London, London, distributions. The DNA flat sheets are functionalized with monomeric ephrin- United Kingdom, 6Francis Crick Institute, London, United Kingdom, A5 conjugates with spatial distributions that allow or not dimerization of the 7Department of Biochemistry and Molecular Biophysics, Columbia receptor. These substrates will allow an unparalleled control of the dimerization University, New York, NY, USA. of the receptor and will elucidate the role of cytoskeleton during the formation Epidermal growth factor receptor (EGFR, or HER1/ErbB1) is a cell-surface of the signaling assembly. receptor tyrosine kinase that plays a fundamental role in regulation of Together with biochemical techniques, high-resolution microscopies, and cellular metabolism, growth and differentiation. Dysregulation of EGFR or computational tools, this approach will give insights into molecular mecha- other members of the human epidermal growth factor receptor (HER) nisms of spatial organization of ligands and receptors during clustering and family (HER2/ErbB2/Neu, HER3/ErbB3, and HER4/ErbB4) can lead to the their effects on receptor-mediated signaling. development of various cancers. EGFR signaling is activated by ligand- induced receptor dimerization. Ligand binding also induces EGFR oligomeri- 141-Plat zation, but the structures and functions of the oligomers are poorly understood. SAM Domain Inhibits Oligomerization and Auto-Activation Although the majority of EGFR research has largely focused on a of EphA2 Kinase 1 1 2 3 dimerization-dependent activation mechanism, recent analyses suggest that Xiaojun Shi , Deanna M. Bowman , Vera M. Hapiak , Ryan W. Lingerak , 2 4 2 1 oligomerization also plays a crucial role in EGFR signaling. Here, we use flu- Ji Zheng , Matthias Buck , Bingcheng Wang , Adam W. Smith . 1Department of Chemistry, University of Akron, Akron, OH, USA, orophore localization imaging with photobleaching (FLImP) to probe the 2 structure of EGFR oligomers. We find that at physiological EGF concentra- Rammelkamp Center for Research, MetroHealth Medical Center, Cleveland, OH, USA, 3Department of Biology, University of Akron, Akron, tions, EGFR assembles into oligomers, as indicated by pairwise distances of 4 receptor-bound fluorophore-conjugated EGF ligands. The pairwise ligand OH, USA, Department of Physiology & Biophysics, Case Western Reserve distances correspond well with predictions from our structural model of the University, Clevenland, OH, USA. oligomers constructed from molecular dynamics simulations. The model sug- Eph receptors are the largest family of receptor tyrosine kinases (RTKs) and gests that oligomerization is mediated extracellularly by unoccupied ligand- bind to membrane-tethered ligands called ephrins. Eph-Ephrin interactions binding sites and that oligomerization organizes kinase-active dimers in are involved in various biological processes, such as neural development, tis- ways optimal for auto-phosphorylation in trans between neighboring dimers. sue patterning and vascular growth. Several structural studies have shown We argue that ligand-induced oligomerization is essential to the regulation that the extracellular domain (ECD) of EphA2 binds ephrin and forms an of EGFR signaling. oligomer that promotes kinase activation. However, the role of the intracel- lular domain (ICD) in this process remains largely unexplored. Here, we 139-Plat report on the unique role of the sterile a motif (SAM) domain in regulating Kinetics of G Protein-Coupled Receptor Dimerization from Markov State EphA2 kinase activation. First, western blots and cell function assays indi- Model Analysis of Coarse-Grained Simulations cated that deletion of the SAM domain leads to constitutive activation of Diego Prada-Gracia, Kristen Marino, Davide Provasi, Marta Filizola. the kinase. We then carried out fluorescence correlation spectroscopy Department of Pharmacological Sciences, Icahn School of Medicine at (FCS) measurements to investigate the dynamics and lateral organization Mount Sinai, New York, NY, USA. of EphA2 receptors in live cancer cell membranes. The FCS results showed Experimental and computational studies published over the past decade have that deletion of the SAM domain led to receptor oligomerization. This sug- mostly focused on the structural and thermodynamic properties of putative gested that constitutive activation of the SAM-deletion construct observed in G Protein-Coupled Receptor(GPCR) dimers. Although equally crucial for the cell functional assays were a result of receptor oligomerization. Ligand addressing the role of dimerization in GPCR function, a thorough description activation with ephrinA1 (EA1) induces oligomerization of the receptors, but of the timescales required to form different dimeric interfaces has yet to be pro- deletion of the SAM domain led to larger oligomers than those with the full vided. In this study, we approach this question by applying Markov State Model length receptor. FCS measurements were performed on cancer cells treated analysis to coarse grained simulations of a prototypic GPCR, the mu-opioid with two kinds of soluble EA1 ligand, dimeric EA1-Fc and monomeric EA1 receptor (MOR), carried out in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- (mEA1), were used. While inconsistencies exist in the literature, our FCS re- choline/cholesterol membrane model using an adaptive sampling strategy. sults demonstrated that both dimeric and monomeric EA1 activation led to We identify several dimerization interfaces of MOR grouped in four macro- oligomerization and activation of EphA2 through induction of the distinct states with significantly different dimerization rates, and characterize the role clusters. Together, our work clarifies the unique function of SAM domain of lipid dynamics, protein-lipid and specific protein-protein interactions in in EphA2 signaling. modulating the kinetic properties of receptor dimerization. Furthermore, we discuss the interplay between receptor activation and dimer formation by 142-Plat comparing the results obtained for the activated and inactive receptor con- Single-Molecule Analysis of the Supramolecular Organization of the M2 a formations of MOR, and observe substantial differences in the dimerization Muscarinic Receptor and the G i1 Protein 1 1 2 kinetics for the two conformations. Claudiu Gradinaru , Dennis D. Fernandes , Rabindra Shivnaraine , James Wells3. 140-Plat 1Physics, University of Toronto, Mississauga, ON, Canada, 2Molecular DNA Nanotechnology for Understanding Ephrin Receptor Clustering and Cellular Physiology, Stanford University, Palo Alto, CA, USA, Alessandro Bosco, Erik Benson, Bjo¨rn Ho¨gberg, Ana Teixeira. 3Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada. Medical Biochemistry and Biophysics (MBB), Karolinska Institutet, G Protein-coupled receptors constitute the largest family of transmembrane Stockholm, Sweden. signaling proteins and the largest pool of drug targets, yet their mechanism Membrane protein biophysical context has been proved to be fundamental in of action remains obscure. That uncertainty relates to unresolved questions the modulation of the cell-cell contact signalling generated by ligand receptor regarding the supramolecular nature of the signaling complex formed by re- recognition, however tools and techniques that are able to control membrane ceptor and G protein. We therefore have characterized the oligomeric status microenvironment at the nanoscale and then interpret the molecular mecha- of eGFP-tagged M2 muscarinic receptor (M2R) and Gi1 by single-particle pho- nisms are still scarce. DNA nanotechnology offers a powerful tool to target re- tobleaching of immobilized complexes (Shivnaraine et al, J. Am. Chem. Soc., ceptor macromolecular assembly at the nanoscale. 2016, 138, pp 11583-11598). The method was calibrated with multiplexed In recent years it has been shown that in breast cancer cell lines, the spatial or- controls comprising 1-4 copies of fused eGFP proteins. The photobleaching ganization of ephrin controls the formation of cluster of Eph receptors and this patterns of eGFP-M2R were indicative of a tetramer and unaffected by musca- phenomenon has been linked to tumor aggressiveness. In our lab, we recently rinic ligands; those of eGFP-Gi1 were indicative of a hexamer and unaffected designed and charachterized rod-like DNA structures decorated with ephrins by GTPg S. A complex of M2R and Gi1 was tetrameric in both, and activation with nanometric precision that can be used to target Eph receptors. In partic- by a full agonist plus GTPg S reduced the oligomeric size of Gi1 without ular, we showed that the nanoscale spacing of pre-dimerized Fc-fused affecting that of the receptor. A similar reduction was observed upon activa- ephrin-A5 directs the levels of EphA2 receptor phosphorilation and activation tion of eGFP-Gai1 by the receptor-mimic mastoparan plus GTPgS; a consti- in human breast cancer cells and modulates their invasive properties. More- tutively active eGFP-Gai1 mutant was predominantly dimeric. The oligomeric over, we are currently developing substrates that recreate the intramembrane nature of Gi1 in live CHO cells was demonstrated by means of Fo¨rster

BPJ 7693_7704 28a Sunday, February 12, 2017 resonance energy transfer and dual-color fluorescence correlation spectros- 145-Plat copy in studies with eGFP- and mCherry-labeled Gai1; stochastic FRET Plasma Membrane Diffusion Modes of FcεRI Receptor for Immunoglobin was ruled out by means of non-interacting pairs. Our results suggest that E Measured with Imaging Fluorescence Correlation Spectroscopy the complex between M2R and holo-Gi1 is an octamer comprising four copies Nirmalya Bag, David Holowka, Barbara Baird. of each, and that activation is accompanied by a decrease in the oligomeric Department of Chemistry and Chemical Biology, Cornell University, Ithaca, size of Gi1. The structural feasibility of such a complex was demonstrated NY, USA. in molecular dynamics simulations. Spatio-temporal organization of the plasma membrane plays a significant role in cell signaling. A prevailing view is that the plasma membrane spatially seg- 143-Plat regates into ordered lipid (Lo-like) nanodomains co-existing with disordered Live Cell Super-Resolution Microscopy Measures Membrane-Driven lipid (Ld-like) regions. Our laboratory, using super-resolution fluorescence im- Sorting of B Cell Receptor Signaling Partners aging, previously showed that immunoglobin E bound to its receptor, FcεRI, Sarah A. Shelby, Sarah L. Veatch, Matthew B. Stone. (IgE-FcεRI complex) undergoes time-dependent redistribution on the mem- University of Michigan, Ann Arbor, MI, USA. brane surface after multivalent ligand stimulation, upon which the receptor is B cells are acutely sensitive to stimulation through antigen-induced clus- phosphorylated by membrane-anchored Lyn kinase. A large body of evidence tering of the B cell receptor (BCR). Transduction of the activation signal, shows that this interaction of IgE-FcεRI and Lyn occurs in the ordered regions which is key for the adaptive immune response, involves rearrangement of the plasma membrane enriched in cholesterol. We are examining the nature of membrane-resident signaling molecules relative to clustered BCR. The and dynamics of plasma membrane organization as experienced by IgE-FcεRI details and dynamics of this re-organization have been difficult to directly in resting state and after antigen stimulation. We employ imaging total internal observe due to the small dimensions of signaling complexes and the poten- reflection fluorescence correlation spectroscopy (ITIR-FCS), which maps tially weak or transient interactions that drive their formation. To overcome lateral diffusion with pixel resolution, to investigate the diffusion distribution these obstacles, we have utilized live-cell super-resolution localization mi- of IgE-FcεRI complexes on the ventral surface of live RBL cells in native croscopy to simultaneously image BCR and other membrane species during and experimentally modulated (e.g., cholesterol depleted) conditions. We antigen-induced cell activation. The sensitivity of this quantitative tech- also conduct spot variation FCS (svFCS), which analyzes space-dependence nique has allowed us to measure the effects of subtle forces, such as those of diffusion coefficients from the same set of ITIR-FCS data, to reveal the ex- originating from lipid compositional heterogeneity, that influence the inter- istence of nanoscopic obstacles (such as nanodomains) that affect the diffusion actions of BCR with its signaling partners. We found that minimal of IgE-FcεRI complexes. These observations are compared with the diffusion membrane-anchored probes that partition into ordered domains of model behavior of protein markers that are known to prefer ordered or disordered re- membranes co-localize with BCR clusters, while anchors that partition gions of plasma membrane. These results, in conjunction with super-resolution into disordered domains are excluded from clusters, suggesting that BCR imaging, which determines the size and density of these complexes, will pro- clusters can nucleate local enrichment of a specific lipid composition. We vide a new level of insight into plasma membrane organization and its dynamic are exploring the effects of membrane-driven sorting on recruitment of remodeling upon ligand-receptor stimulation. BCR signaling proteins to receptor clusters and how it contributes to their specific role in signal transduction. In particular, we have found that the Platform: Cell Mechanics, Mechanosensing, palmitoylated transmembrane adapter proteins LAT2, LIME, and PAG, and Motility I which serve distinct adapter functions in BCR signaling, are diferentially recruited to BCR clusters. This differential recruitment is due to a combi- 146-Plat nation of interactions between the transmembrane domains of these proteins Integrin Catch Bond Kinetics Mediate Mechanosensing during Cell with the membrane and to specific protein-protein interactions. Our results Spreading suggest that the membrane influences local sorting of downstream signaling Tamara C. Bidone1, Patrick W. Oakes2, Yvonne Beckham3, proteins around BCR clusters and provide insight on the forces driving Margaret L. Gardel3, Gregory A. Voth1. regulation of BCR signaling. 1Chemistry, University of Chicago, Chicago, IL, USA, 2Rochester University, Rochester, NY, USA, 3University of Chicago, Chicago, IL, USA. 144-Plat Cell spreading and polarization are morphogenetic responses to extracellular Mechanism of CD36 Signal Transduction by F-actin and Lipid matrix adhesion. Cultured fibroblasts polarize when plated on rigid, but Nanodomains not compliant substrates. In this study, we demonstrate that fibroblasts have Swai Mon Khaing, Nicolas Touret. increased spread area on compliant substrates when integrins are directly acti- Biochemistry, University of Alberta, Edmonton, AB, Canada. þ vated via manganese (Mn2 ), even upon inhibition of myosin motor activity. CD36, a multi-ligand plasma membrane receptor, has been implicated in im- Integrin activation is primarily regulated by changes in tertiary and quaternary munity, metabolism and angiogenesis. We have recently demonstrated that þ structure and Mn2 treatments enhance the lifetime of integrin/ligand bonds. CD36 nanoclustering at the plasma membrane is key to the initiation of The decrease of integrin/ligand unbinding rate under tension suggests a CD36 signaling. In endothelial cells (ECs), the binding of thrombospondin-1 mechanosensing role of activated integrins in regulating adhesion formation. (TSP-1, an endogenous extracellular matrix anti-angiogenic factor) to CD36 Using a combination of atomistic molecular dynamics and coarse grain nanoclusters activates an associated Src family kinase, Fyn, leading to ECs Brownian dynamics simulations, we identify integrin intermediates along the apoptosis, hence, inhibiting angiogenesis. We are interested in elucidating activation pathway and show that modulation of integrin catch bond kinetics the mechanisms of CD36-Fyn enrichment and the role of lipid nanodomains þ upon Mn2 -activation promotes integrin binding on compliant substrates. and actin cytoskeleton during TSP-1 induced signaling in ECs. We hypothesize Combining experiments with simulations, our results support the idea that that lipid nanodomains play a role in bringing together CD36-Fyn to F-actin different regimes of ligand binding and cell spreading result from variations regions through adaptor molecules which forms a signaling platform. Using in integrin catch bond kinetics. This suggests an alternative mechanosensing microscopy methods on HeLa cells co-transfected with Fyn and various fluo- pathway based on integrin activity and independent of myosin activity. rescent lipid biosensors and stained for F-actin (Phalloidin-AF647), we deter- mined that Fyn is enriched on F-actin area at sites of phosphatidylinositol 147-Plat 4,5-bisphosphate enrichment (PIP2). During TSP-1 stimulation on Human Molecular Tension Sensors Reveal a Minimally Tensioned Integrin State Microvascular Endothelial Cells (HMEC), the CD36-Fyn-F-actin enrichment in Living Cells shift to domains containing PI(3,4,5)P3, suggesting a role for the phosphoino- Steven J. Tan, Chang C. Alice, Armen H. Mekhdjian, Alexander R. Dunn. sitide 3-kinase in signaling. The role of this kinase is further investigated using Stanford University, Stanford, CA, USA. inhibitor targeting PI3-Kinase subunits for signal transduction. Additionally, Integrins mediate cell adhesion to the extracellular matrix and enable the con- we will be using rapamycin dimerization system to understand the role of struction of complex, multicellular organisms. Despite this biological pro- these lipid nanodomains in CD36-Fyn signaling. Furthermore, to characterize minence, fundamental aspects of integrin-based adhesion remain poorly the adaptor molecules involved in connecting F-actin to lipid nanodomains understood. Notably, the magnitude of the mechanical load experienced by in- and/or CD36 nanoclusters, we are conducting APEX2 proximity labelling dividual integrins within living cells is unclear, due principally to limitations and fractionation approaches followed by mass spectrometry (MS) analysis. inherent to existing techniques. We used Fo¨rster resonance energy transfer The MS screen will further narrow down proteins that are biotinylated, adjacent (FRET)-based molecular tension sensors (MTSs) to measure the distribution to CD36 and enriched in F-actin. Altogether, our investigation will provide in- of forces exerted by individual integrin heterodimers in living cells. Taking sights into understanding the activity of plasma membrane receptor nanoclus- advantage of the sensors’ modular nature, we engineered MTS variants that tering and signaling. display a minimal RGD sequence derived from fibronectin (MTSRGD) or the

BPJ 7693_7704 Sunday, February 12, 2017 29a

th th 9 and 10 type III domains of fibronectin (MTSFN), and are sensitive to low spatial resolution of 0.2 mm, ten times higher than conventional methods. Using (0-7 pN) or intermediate (8-11 pN) loads. Using these sensors, we found that a TGT, we determined that platelet adhesion and spreading requires integrin ten- large fraction of integrins in living cells exert forces <3 pN, while a minority sion lower than 12 pN, a force much less than the ~40 pN integrin tension subpopulation experiencing substantially higher loads was enriched in large required by regular mammalian cell adhesion. This surprising result suggests adhesion complexes. Treatment with the filamentous (F)-actin disruptor cyto- that platelets have extraordinary adhesion and spreading ability which might chalasin D revealed that only the high-load integrin population required an be beneficial for the rapid response of platelets to injuries. We also monitored intact actin cytoskeleton, and that the low-load (<3 pN) population was suffi- calcium oscillation in platelets to confirm that platelets are activated at this cient to mediate cell adhesion on short, ~15 minute timescales. Integrin engage- low integrin tension level. Next, using TGT as a tension sensor, we provided ment with the fibronectin synergy site, a secondary binding site specifically for the high-resolution (0.2 mm) integrin tension map for platelet adhesion for the a5b1 integrin, led to increased recruitment of a5b1 integrin to adhesions, but not first time. Real-time tension mapping shows that integrin tensions at a high level to an increase in overall cellular traction generation. Consistent with previous (>54 pN) are initially concentrated in one or two micron-sized punctate regions reports, the presence of the synergy site did, however, increase the resistance of upon platelet adhesion. After full platelet spreading, these integrin tensions cells to detachment via externally applied load. Based on these data, we suggest spread out to the entire platelet-substrate interface and the tension level decreases that a large pool of engaged, but minimally tensioned integrins may provide a into the range of 12~54 pN. These high integrin tensions are not required for synergy site-dependent adhesive reserve that imparts cells and tissues with me- platelet adhesion and activation, but may be required for platelet contraction, chanical integrity in the presence of widely varying mechanical loads. In as these tensions were abolished by myosin II inhibition without compromising ongoing work, we take advantage of the unique capabilities of MTSs to deter- platelet adhesion and activation. Overall, our research has initiated the biome- mine how subpopulations of load-bearing integrins are altered in response to chanical study of platelets at the molecular tension level, and revealed a rich external mechanical perturbations, and how (and whether) distinct integrin sub- dynamics of integrin tensions in platelets. types bear differing levels of mechanical load. 150-Plat 148-Plat Receptor Nucleation and Clustering in Cellular Adhesion and Mechanical Nascent Adhesoins that Form on all Substrates by Recruiting Unliganded Signal Transduction Integrins and are Important for Mechanotransduction Kabir H. Biswas1, Kevin L. Hartman1, Ronen Zaidel-Bar1,2, Rishita Changede1, Haogang Cai2, Michael P. Sheetz1. Jay T. Groves1,3. 1National University of Singapore, Mechanobiology Institute, Singapore, 1Mechanobiology Institute, National University of Singapore, Singapore, Singapore, 2Engineering, Columbia University, New York, NY, USA. Singapore, 2Department of Biomedical Engineering, National University of Integrin adhesions assemble and mature in response to ligand binding and me- Singapore, Singapore, Singapore, 3Department of Chemistry, University of chanical factors, but the molecular-level organization is not known. We report California, Berkeley, Berkeley, CA, USA. that ~100-nm clusters of ~50 b3-activated integrins form very early adhesions E-cadherin-based cell-cell adhesions are key to development and maintenance of under a wide variety of conditions on RGD surfaces. These adhesions form the epithelial tissue, and a loss of these adhesions may contribute to cancer devel- similarly on fluid and rigid substrates, but most adhesions are transient on rigid opment. These are mechanosensitive structures in that they are strengthened un- substrates. Without talin or actin polymerization, few early adhesions form, but der tension. Mechanotransduction in these adhesions has been postulated to be expression of either the talin head or rod domain in talin-depleted cells restores mediated, in part, by a force-dependent conformational activation of a-catenin, early adhesion formation. Mutation of the integrin binding site in the talin rod which allows it to interact with vinculin, in addition to F-actin, resulting in decreases cluster size. We suggest that the integrin clusters constitute universal strengthening of junctions. Here, using E-cadherin adhesions reconstituted on early adhesions and that they are the modular units of cell matrix adhesions. synthetic, nanopatterned membranes, we show that activation of a-catenin is They require the association of activated integrins with cytoplasmic proteins, dependent on E-cadherin clustering, and is sustained in the absence of mechan- in particular talin and actin, and cytoskeletal contraction on them causes adhe- ical force or association with F-actin or vinculin. Adhesions are formed by sion maturation for cell motility and growth. Using gold nano-patterning we filopodia-mediated nucleation and micron-scale assembly of E-cadherin clusters, observe that these clusters grow using unliganded but activated integrins. which could be distinguished as either peripheral or central depending on their This indicates a mechanism to assemble the clusters rapidly around an activated relative location at the cell-bilayer adhesion. While F-actin, vinculin and phos- and liganded integrin. Integrins are not enzymes hence they would need the aid phorylated myosin light chain associate only with the peripheral assemblies, acti- of other enzymes for mechanotransduction. Nascent adhesions have distinct vated a-catenin is present in both peripheral and central assemblies, and persisted functions on compliant and rigid substrates wherein, the epidermal growth fac- in the central assemblies in the absence of actomyosin tension. Impeding tor receptor is recruited to these clusters only on the rigid substrates, in absence filopodia-mediated nucleation and micron-scale assembly of E-cadherin adhe- of EGF. This receptor acts as a mechanoenzyme that is required for mechano- sion complexes, by confining bilayer bound E-cadherin extracellular domain transduction within fibroblasts. Taken together our studies show that nascent movement on nanopatterned substrates, reduced levels of activated a-catenin. adhesions form on substrates of vastly varying rigidities. These modular adhe- Taken together, although the initial activation of a-catenin requires micron- sions are formed by a cohort of unliganded integrins recruited to adhesions sites scale clustering that may allow development of mechanical forces, sustained by cytoplasmic factors and they are central to bring about different mechano- force is not required for maintaining a-catenin in the active state. transduction on different substrates. 151-Plat 149-Plat Tau Like Proteins Reduce Torque Generation in Microtubule Bundles High-Resolution Integrin Molecular Tension Dynamics during Platelet Michael Krieg1, Jan Stuehmer2, Juan G. Cueva1, Richard Fetter1, Adhesion and Activation Kerri Spilker1, Daniel Cremers2, Kang Shen1, Alex R. Dunn3, Xuefeng Wang1, Yongliang Wang1, Dana N. LeVine2. Miriam B. Goodman1. 1Physics and Astronomy, Iowa State University, Ames, IA, USA, 1Stanford University, Stanford, CA, USA, 2TU Munich, Munich, Germany, 2Department of Veterinary Clinical Sciences, Iowa State University, Ames, 3Stanford, Stanford, CA, USA. IA, USA. All animals and plants, even protozoa, have evolved specialized molecular sen- Platelets are small disc-shaped cell fragments circulating in the bloodstream. sors that convert mechanical stress into behavioral responses. The touch recep- Upon injury, platelets adhere and aggregate on injured subendothelium to tor neurons (TRNs) in Caenorhabditis elegans respond to gentle body touch and form blood clots to stop bleeding. Irregular platelet adhesion and aggregation are especially arguably better characterized on a physiological and ultrastruc- may cause acute coronary syndrome or other cardiovascular diseases. Membrane tural level than somatosensory neurons in other animals. C. elegans is a unique protein integrins mediate platelet adhesion and transmit tensions to activate model organism in which to study the mechanics of neurons due to their simple platelets, therefore playing important roles in platelet functions. Nevertheless, shapes, the known wiring diagram, transparent body, and a rich repertoire of cellular force study in platelets is very limited and integrin molecular tensions simple behaviors. As in other animals, neuron morphology is critical for func- were never calibrated in platelets, presumably due to the 2~3 mm size of platelets tion in C. elegans. We have previously shown that a functional, pre-stressed which is at the resolution limit of conventional cell traction force microscopy. spectrin network is critical for mechanosensation and neuron stability under Here we applied a novel molecular tension sensor and modulator named TGT body-evoked forces (Krieg, Nat Cell Bio, 2014). How the constituent mole- (tension gauge tether) to study integrin tensions in platelets. TGT is a rupturable cules of these different neurons establish a functional organization and how molecular linker with a programmable tension tolerance (Ttol). As a tension nanometer sized molecules can determine cell shape in the millimeter scale modulator, TGT globally restricts integrin tensions under the designed level of and enable axons to resist external forces is still not understood. We addressed Ttol, enabling the study of integrin-tension dependency for a certain cellular func- this question using light, electron and STED microscopy and found that TRNs tion. As a tension sensor, TGT maps integrin tensions by fluorescence with a defects in the organization of the axonal spectrin lattice and microtubule

BPJ 7693_7704 30a Sunday, February 12, 2017 bundles undergo deformations highly similar to a twisted rod under compres- cellular proteins to form the pre-integration complex (PIC). Viral integrase (IN) sion. Our data suggests that tau-like proteins minimize microtubule lattice in- is a key component of the PIC and is involved in several steps of replication teractions and the prevent torque generation that leads to extreme neuron notably in reverse transcription, nuclear import, chromatin targeting and inte- deformations. These experimental results, together with mechanical modeling gration. Viral components such as IN cannot perform these functions on their of the neuron, suggest that spectrin tension and microtubule bundle mechanics own and need to recruit host cell proteins to efficiently carry out the different are crucial for stabilizing chiral cytoskeletal networks and produce a special- processes. IN is a flexible protein, property allowing its interaction with multi- ized cell shape that we propose is critical for neuronal function. ple partners and enabling its multiple functions in viral replication. The molec- ular mechanisms and dynamics of these processes remain largely unknown. 152-Plat Purification of proteins that participate in these large transient complexes is Torque Generation in the Bacterial Flagellar Motor impeded by low amounts, heterogeneity, instability and poor solubility. To Jasmine A. Nirody1, Richard M. Berry2, George Oster1. 1 2 circumvent these difficulties we develop methodologies that enable the produc- University of California, Berkeley, Berkeley, CA, USA, University of tion of stable complexes for structural and functional studies [1] as well as sys- Oxford, Oxford, United Kingdom. tem for the production of multi-protein complexes from mammalian cells The bacterial flagellar motor (BFM) drives swimming in a wide variety of enabling assembly of entire complexes within cells. Using these strategies bacterial species. Its fundamental role in a variety of biological processes, we reconstruct in vitro stable and soluble complexes around IN. We use including chemotaxis and biofilm formation, has made understanding its dy- cryo-EM combined with X-ray crystallography to solve structures of the IN/ namics an important question in biophysics. We put forward a mechanically- LEDGF/DNA [2] and IN/LEDGF/INI1/DNA [3] complexes. Other IN com- specific model for motor rotation, pinpointing critical residues and structures plexes involved in the PIC nuclear translocation and integration as well as for motor function. We implicate a steric interaction between the rotor and IN post-translational modifications (phosphorylation and acetylation) have the torque-generating complexes (stators). Two surprising predictions of our been characterized and are under study. model are: (1) the duty ratio of the motor is not close to unity as previously [1] Levy et al. (2016) Nature comm. 7: 10932 believed; and (2) motor rotation is loosely coupled to ion flux. We show that [2] Michel et al. (2009) EMBO J., 28, 980-991 these predictions, while contrary to previous reports, are consistent with current [3] Maillot et al. (2013) PLoS ONE 8(4): e60734 experimental evidence. We also put forward several further experiments and measurements designed to directly test the validity of this model and its 155-Plat implications. Mechanisms of Sequence Dependent Translational Stalling 153-Plat Lars V. Bock1, Paul PH Huter2, Stefan Arenz2, Michael Graf2, Fission Yeast Contractile Ring Tension Increases ~2-Fold Helmut Grubmueller3, Daniel Wilson2, Andrea C. Vaiana3. 1 Throughout Constriction and Regulates Septum Closure Theoretical and Computational Biophysics, Lars Bock, Go¨ttingen, 2 3 but does not Set the Constriction Rate Germany, Gene Center, Munich, Germany, Theoretical and Computational Sathish Thiyagarajan1, Harvey Chin2, Erdem Karatekin3, Biophysics, Max Planck Institute for Biophysical Chemistry, Go¨ttingen, Thomas D. Pollard4, Ben O’Shaughnessy2. Germany. 1Physics, Columbia University, New York, NY, USA, 2Chemical Ribosomal stalling during protein synthesis in bacteria occurs in different ways Engineering, Columbia University, New York, NY, USA, 3Cellular and and under different conditions. Stalling of specific peptide sequences can be a Molecular Physiology, Yale University, New Haven, CT, USA, 4Molecular, pre-programmed means of detecting the presence of potentially lethal antibi- Cellular, and Developmental Biology, Yale University, New Haven, CT, otics and constitute the initial step of a complex resistance pathway. An USA. example of this is the stalling of ErmBL peptide synthesis in the presence of What is the role of the cytokinetic contractile ring? The tensile actomyosin ring the antibiotic erythromycin. In other cases, stalling seems to be an effect of un- is central to cytokinesis, and widely thought to drive cell cleavage as it con- usually slow, sequence dependent, rates of amino acid incorporation. This is the stricts. Here we directly addressed this question by measurements of cytoki- case for translation of proteins containing poly-proline stretches. Poly-proline netic ring tension and mathematical modeling. In the model organism fission sequences are known to stall ribosomes, normal translation rates are achieved yeast, as in other fungi, ring constriction is accompanied by septation, the in- only by recruiting a special elongation factor (EF-P in bacteria). Here, we ward growth of cell wall in the wake of the constricting ring that seals daughter investigate the stalling mechanisms in the two scenarios described above by cells in new cell wall. explicit-solvent, all-atom molecular dynamics simulations of the ribosome. We measured ring tensions in live fission yeast protoplasts using a novel The simulations are started from high-resolution cryo-EM structures and per- method based on measuring membrane tension and the furrow geometry. As formed under stalling and non-stalling conditions. We find networks of allo- constricted progressed, ring tension increased from ~ 400 pN to ~ 800 pN. steric interactions between the nascent peptide chain and the ribosome that To our knowledge these are the first measurements of the evolving ring tension differently affect the positioning and the dynamics of the peptidyl tRNA rela- throughout constriction. tive to the A-site tRNA in such a way as to hinder peptide bond formation de- We used these tension values in a mathematical model of septum growth, medi- pending on the presence of the antibiotic (in the first scenario) or the absence of ated by beta-glucan synthases (Bgs) at the septum edge, hypothesized mecha- the elongation factor (second scenario). The simulation results not only explain nosensitive (Thiyagarajan et al., 2015). The stochastic septum growth produced the stalling mechanism, but can also predict the effect of mutations on stalling. faceting, defects and edge roughness. In simulations, ring tension modulated In the case of erythromycin induced stalling, these predictions have been exper- Bgs growth rates in a curvature-dependent fashion, suppressing defects and imentally confirmed by a toe-printing assay. Our results illustrate the fine de- roughness so septum edges were nearly circular. Simulated edges had low tails of how the efficiency of peptide bond formation can be modulated by roughnesses (~5%) and a roughness exponent ~0.5, consistent with septum external factors in a way that depends on the specific sequence being translated. edges we measured in live cells. Our model revealed a mechanosensitivity ~15% per pN per Bgs complex. 156-Plat Thus, ring tension regulates septum growth to ensure the septum closes as a Quantitative Analysis and Modeling of Translation using Ribosome shrinking circle (not a slit) and daughter cells are properly sealed by new Profiling Data: How Biophysical Properties of the Ribosome Exit Tunnel cell wall. However, the model showed constriction rates are set by the septum and the Nascent Polypeptide Modulate the Elongation Rate growth machinery, while ring tension had little effect on the mean rate, explain- Khanh Dao Duc1, Zain H. Saleem2, Yun S. Song1. 1 ing why experimentally the rate is constant in time. Mathematics & Biology, University of Pennsylvania, Philadelphia, PA, USA, 2Physics, University of Pennsylvania, Philadelphia, PA, USA. Platform: Protein Structure and Conformation I Ribosome profiling provides a detailed view into the complex dynamics of translation. Although the precise relation between the observed ribosome foot- 154-Plat print densities and the actual translation elongation rates remains elusive, the The HIV-1 Pre-Integration Complexes: Structures, Functions data clearly suggest that elongation speed is quite heterogeneous along the tran- and Dynamics script. Previous studies have shown that elongation is locally regulated by mul- Nicolas Levy, Karine Pradeau-aubreton, Sylvia Eiler, Julien Batisse, tiple factors, but the observed heterogeneity remains only partially explained. Oyindamola Oladosu, Benoit Maillot, Marc Ruff. To dissect quantitatively the different determinants of translation speed, we IGBMC, Illkirch, France. here use a probabilistic model of the translation dynamics to estimate After retroviral infection of a target cell, during the early phase of replication, transcript-specific initiation and local elongation rates from ribosome profiling the HIV-1 genomic viral RNA is reverse transcribed by the viral reverse tran- data. Using this model-based approach, we infer the extent of interference be- scriptase to generate the double-stranded viral DNA that interact with viral and tween ribosomes on the same transcript (which cannot be observed directly

BPJ 7693_7704 Sunday, February 12, 2017 31a from current ribosome profiling data), and show that it varies substantially We generated 1000 random networks for number of nodes from 8 to 16. The across different genes and different positions. However, we show that neither probability is widely distributed from network to network, and close to the ribosomal interference nor the distribution of slow codons is sufficient to log-normal distribution . This fact means that there are a small number of net- explain the observed variation in the mean elongation rate along the transcript. works that are robust against the mutation. The funnel structure becomes rarer Surprisingly, by optimizing the fit of statistical linear models, we find that the exponentially as the protein becomes longer. hydropathy of the nascent polypeptide segment within the ribosome plays a ma- jor role in governing the variation of the mean elongation rate. In addition, we 159-Plat find that positively and negatively charged amino acid residues near the begin- Mechanistic Insights into NSF Mediated SNARE Complex Disassembly ning and end of the ribosomal exit tunnel, respectively, are important determi- Minglei Zhao, Ucheor Choi, Axel Brunger. nants of translation speed. This result is consistent with the electrostatic Stanford University, Stanford, CA, USA. properties of the ribosomal exit tunnel, which we study by exploring the geom- Evolutionarily conserved SNARE (soluble N-ethylmaleimide sensitive factor etry of the tunnel and solving the Poisson Boltzmann equation. attachment protein receptors) proteins form a complex that drives membrane fusion in eukaryotes. The ATPase NSF (N-ethylmaleimide sensitive factor), 157-Plat together with the adaptor protein SNAP (soluble NSF attachment protein), dis- Is Protein Single Molecule Dynamics under Force Described by Two or assembles the SNARE complex, making individual SNAREs available for sub- More States? sequent rounds of fusion. We determined structures of ATP- and ADP-bound Jagannath Mondal1, Ronen Berkovich2, Bruce Berne3. NSF, and the NSF/SNAP/SNARE supercomplex (known as 20S particle) using 1TIFR Center for Interdisciplinary Sciences, Hyderabad, India, 2Ben Gurion single-particle cryo-electron microscopy (cryo-EM). We further dissected the University, Beer Sheeva, Israel, 3chemistry, Columbia University, New York, mechanism of NSF mediated SNARE complex disassembly using single- NY, USA. molecule fluorescence microscopy based on the cryo-EM structures. Single molecule force spectroscopy is a useful technique for investigating me- chanically induced protein unfolding and refolding under reduced forces by 160-Plat monitoring the end-to-end distance of the protein. The data is often interpreted Cell-Free Synthesis of Site-Specifically Double-Labeled Proteins for More via a ‘‘two-state’’ model based on the assumption that the end-to-end distance Accurate Single-Molecule FRET Studies 1 1 1 alone is a good reaction coordinate and the thermodynamic behavior is then Mayuri Sadoine , Michele Cerminara , Noemie Kempf , 1 1,2 ascribed to the free energy as a function of this one reaction coordinate. In Alexandros Katranidis ,Jo¨rg Fitter . 1Institute of Complex Systems ICS-5, Forschungszentrum Juelich, Juelich, this paper, we determined the free energy surface (PMF) of GB1 protein from 2 atomistic simulations in explicit solvent under different applied forces as a func- Germany, 1. Physikalisches Institut (IA), RWTH Aachen, Aachen, tion of two collective variables (the end-to-end-distance, and the fraction of Germany. native contacts r). The calculated 2-d free energy surfaces exhibited several Single molecule FRET (smFRET) is a powerful tool for looking at protein distinct states, or basins, mostly visible along the r coordinate. Brownian dy- folding and conformational dynamics (1). The application of smFRET to inter- namics (BD) simulations on the smoothed free energy surface show that the pro- esting proteins involved in pathologies or drug targeting is limited when the tein visits a metastable molten globule state and is thus a three state folder, not protein of interest is difficult to express in vivo due to toxicity and where the two state folder inferred using the end-to-end distance as the sole reaction co- cell-free expression is required. Moreover, site-specific labeling is an important ordinate. These BD simulations reproduce the unfolding and collapse-refolding issue for smFRET (2), but there is still a lack in simple and robust methods for patterns observed in the force-clamp experiments. This study lends support to site-specific labeling of in vitro synthesized proteins for smFRET studies. The recent experiments that suggest that GB1 is not a two-state folder. conventional method is to double label the protein via cysteine residues (3), but as a consequence of a single chemistry, labeling specificity is limited and would 158-Plat lead to an inaccurate picture of the studied population (4). Incorporation of un- Finding Protein Folding Funnels in Random Networks natural amino acids carrying an orthogonal chemistry can increase labeling Macoto Kikuchi. specificity as it was reported (4). However, described methods were so far Cybermedia center, Osaka University, Toyonaka, Japan. mainly considered for smFRET studies performed on in vivo synthesized Many attempts have been made for understanding protein folding dynamics us- proteins. ing network representations of folding pathway. For example, the Markov state By using human calmodulin (hCaM) as a model protein we have developed an model has been widely used in analyzing the transitions between protein con- alternative method for site-specific labeling of cell-free synthesized proteins. formations of trajectories obtained by molecular dynamics simulations. Site-specificity of our system allowed us to obtain sharper FRET histograms In the present work, I also focus on the network structures of protein folding compared to the conventional labeling method. In addition, by demonstrating dynamics, but from a somewhat different perspective. Currently most widely functionality of the labeled hCaM through binding experiments performed in accepted theoretical framework of the protein folding is so-called funnel pic- presence of ligands and partners, we have shown the biological relevance of ture, which states that the number of conformations decreases as the energy our method. lowers from the denatured state towards the native state. In other words, the en- In summary, we have developed a robust and simple method that enables the ergy landscapes of proteins have been designed through Darwinian evolution so accurate study of cell-free synthesized proteins in smFRET. This method can that proteins readily fold to their native states. A question I would like to ask be used for studying proteins that cannot be expressed in vivo, as it would be here is ‘‘How rare are such folding-friendly funnel structures?’’. for toxic or membrane proteins. As a first attempt to answer this question, I introduce a random network model 1. Tan, Y. W., J. A. Hanson, J. W. Chu, and H. Yang. 2014. Confocal single- with each nodes assigned a random energy. Nodes represent the metastable molecule FRET for protein conformational dynamics. Methods Mol. Biol. conformation ensembles. One of the nodes is considered as denatured state 1084:51-62. and another the folded state. Other nodes represent the intermediate conforma- 2. Joo, C., and T. Ha. 2012. Labeling proteins for single-molecule FRET. Cold tion ensembles between them. Edge connecting the nodes are possible transitions Spring Harb. Protoc. 2012:1009-1012. between the nodes. The network structure is assumed to be determined by the 3. Kim, Y., S. O. Ho, N. R. Gassman, Y. Korlann, E. V. Landorf, F. R. Collart, native state conformation, and the energy assignment is considered to reflects and S. Weiss. 2008. Efficient site-specific labeling of proteins via cysteines. the amino-acid sequence. We then define the ideal funnel structure for this model Bioconjug. Chem. 19:786-791. as follows: Starting from the denatured state, if all the paths connecting the nodes 4. Seo, M. H., T. S. Lee, E. Kim, Y. L. Cho, H. S. Park, T. Y. Yoon, and H. S. in energy-lowering direction reach the folded state, then such a network is in a Kim. 2011. Efficient single-molecule fluorescence resonance energy transfer ideal funnel structure. The task now is to compute the probability that the ideal analysis by site-specific dual-labeling of protein using an unnatural amino funnel is realized for a given network by changing the assignment of energy. acid. Anal. Chem. 83:8849-8854.

BPJ 7693_7704 32a Sunday, February 12, 2017

Platform: Protein Dynamics and Allostery I 163-Plat Can we Automatically Detect Biologically Relevant Order Parameters 161-Plat in Molecular Simulation? Comparing Long Timescale Simulations Molecular Simulations to Unravel the Allosteric Interplay between the of Multiple Kinases SH2 Domain and A-loop Plasticity in Protein Kinases Sonya M. Hanson, Joshua H. Fass, John D. Chodera. Giuseppina La Sala1, Laura Riccardi1, Roberto Gaspari2, Andrea Cavalli2,3, Computational Biology Program, Memorial Sloan Kettering Cancer Center, Oliver Hantschel4, Matteo Dal Peraro5,6, Marco De Vivo1,7. New York, NY, USA. 1Laboratory of Molecular Modeling & Drug Discovery, Istituto Italiano di Kinases are a ubiquitous component of important signaling pathways, including Tecnologia, Genova, Italy, 2CompuNet, Istituto Italiano di Tecnologia, those frequently involved in cancer. Understanding the conformational hetero- Genova, Italy, 3Department of Pharmacy and Biotechnology, University of geneity of kinases has been shown to be important for understanding their func- Bologna, Bologna, Italy, 4Swiss Institute for Experimental Cancer Research tion and their role as therapeutic targets. While thousands of structures are (ISREC), EPFL, Lausanne, Switzerland, 5Institute of Bioengineering, EPFL, available via the PDB of kinases in various states, understanding how these Lausanne, Switzerland, 6Swiss Institute of Bioinformatics (SIB), Lausanne, states are kinetically and energetically related is key to making sense of these Switzerland, 7IAS-S/INM-9 Computational Biomedicine, structures. Molecular dynamics has the power to give us this insight, however Forschungszentrum Julich,€ Julich,€ Germany. in simulating kinases structures from hundreds of states found in the PDB or The deregulation of protein kinases is often related with the development of from a single starting structure, we have found that defining order parameters several malignancies such as cancer. Therefore, inhibition of protein kinases to understand these dynamics can be far from straightforward. Analyzing our is an established and often effective pharmacological strategy. However, simulations, we optimized our order parameters to reflect the slowest processes, point mutations in kinases are frequently the cause of drug resistance. To the starting structure of the simulation, or known kinase order parameters from overcome this issue, many efforts are directed towards the design of allosteric the literature. To better facilitate this comparison, we have built a Python drugs, with the goal to inhibit the mutated forms of kinases. To understand library (kinalysis) that can map any kinase structure or set of structures onto the molecular basis of the allosteric control of protein kinases is essential previously established kinase states (defined by DFG flip, C-helix switch, acti- for the design of novel drugs. In this work, we focus on the prototype Abl vation loop conformation, etc.). This can then be used to understand how well kinase. Experimental studies have demonstrated that the binding of the our generalizable methods for defining metastable states captures biologically SH2 domain on the kinase domain enhances the A-loop trans- relevant conformational changes. There is a careful balance between biasing an autophosphorylation, a required step for full kinase activity. Hence, the analysis with what you expect to see and allowing a simulation to tell you SH2 domain acts as an allosteric modulator. However, it is yet poorly under- something you didn’t know before. In the long term, we hope this study informs stood how the SH2 domain affects the A-loop accessibility. Here, we coupled how generalized methods of defining simulation-driven coordinates rather MD simulations with free energy calculations to investigate possible mecha- than coordinates hand-picked for a specific system add to our understanding nisms for such allosteric control in Abl. We found that the A-loop plasticity is of protein conformational dynamics. affected by a complex network of interactions that involve several Abl struc- 164-Plat tural features, such as the DFG motif, the HRD motif and the SH2 domain. Evolution of Regulatory Diversity in the Kinase Superfamily Hence, we hypothesize that such elements might constitute additional layers Jai Pandey1, Orna Resnekov2, David Pincus1, Kimberly A. Reynolds3. that control the Abl allosteric machinery. These results provide important 1Whitehead Institute, MIT, Boston, MA, USA, 2Molecular Medicine hints for deciphering the complex signaling network for Abl activation. Research Institute, Sunnyvale, CA, USA, 3Green Center for Systems Moreover, these findings may help the rational design of Abl binders capable Biology, UT Southwestern Medical Center, Dallas, TX, USA. of interfering with such activation mechanism. Finally, since the SH2 domain Allosteric regulation evolves readily in proteins and homologs often display is found in several other protein kinases, we propose that such a mechanism structurally and biochemically distinct allosteric mechanisms. This is exempli- may be extended to several kinases which are currently under investigations fied by the eukaryotic protein kinase superfamily, in which individual kinase in our labs. family members are regulated by various combinations of protein interaction, dimerization, and/or post-translational modification. Given that allostery re- quires the coordinated activity of multiple, spatially distributed amino acids, 162-Plat it is not obvious how such an apparently complex molecular feature evolves Subunit Exchange and Activation of Human CaMKII Variants once, let alone at a multiplicity of sites. We show that allosteric regulation Ana P. Torres OCampo, Brendan Page, Margaret Stratton. taken across a diversity of protein kinases preferentially occurs at specific, UMass Amherst, Amherst, MA, USA. 2þ conserved surface sites that are distinguished by their pattern of amino acid Ca -calmodulin dependent protein kinase II (CaMKII) assembles into an sequence co-evolution. Further, we show that these sites can be used to engi- oligomeric ring in which the kinase domains are organized around a central neer new allosteric control by introducing synthetic phosphorylation sites hub. Notably, the stimulated activity of CaMKII persists even after the with- 2þ into yeast Kss1 that result in activation of the pheromone response pathway drawal of a calcium stimulus. CaMKII acquires this Ca -independent activ- through Ras-dependent Protein kinase A signaling. These findings strongly ity at a threshold frequency and this property is implicated in long-term support a model for the evolution of new allosteric regulation in which a potentiation (LTP). Indeed, transgenic mice expressing mutant versions of conserved cooperative scaffold facilitates the emergence of regulation at spe- CaMKII have limited LTP and defects in learning and memory. We have pre- cific sites distributed across the protein surface. Thus, the natural architecture viously shown that CaMKII has a remarkable property, which is that activa- of proteins provides a facile route to rewire cell signaling cascades by evolution tion of CaMKII triggers the exchange of subunits between holoenzymes, or engineering. including inactive ones, enabling the Ca2þ-independent activation of neigh- boring subunits. Our results have implications for an earlier idea that subunit 165-Plat exchange in CaMKII may have relevance for long-term memory formation. The G41S Variant of Human Cytochrome C Enhances Apoptosis via These studies were done using primarily human CaMKIIa, isoform 2. There Increased Dynamics are four human CaMKII genes, CaMKIIa and b are found in the brain, CaM- Andreas Ioannis Karsisiotis1, Oliver M. Deacon1, Michael T. Wilson1, KIId is in the heart, and CaMKIIg is found throughout the body. Each of these Colin Macdonald2, Tharin M.A. Blumenschein2, Geoffrey R. Moore2, genes has several splice variants encoding ~20 different isoforms. The pri- Jonathan A.R. Worrall1. mary difference between these isoforms is in the composition and length of 1School of Biological Sciences, University of Essex, Colchester, United the variable linker domain that connects the kinase to the hub. Previous Kingdom, 2School of Chemistry, University of East Anglia, Norwich, United studies have shown that the length of this linker determines the threshold fre- Kingdom. quency for activation. A comprehensive biochemical study of existing human In addition to its well-known role as a component of the electron transport chain, CaMKII isoforms has not been completed. We have expanded our study of mitochondrial cytochrome c can also act as a peroxidase in the early stages of the frequency activation and subunit exchange to the remaining isoforms of intrinsic apoptosis pathway. Ferricytochrome c contains a hexacoordinated CaMKII in order to investigate whether these properties are ubiquitous and heme iron, for which H18 and M80 are the axial ligands. Residual peroxidase why specific isoforms are selectively expressed in different cell types. Our activity under native conditions arises from a minor populated pentacoordinated new data show that as the variable linker domain is lengthened, less CaM form, in which M80 is not coordinated to the heme iron. At the onset of apoptosis, is needed for activation. However, above a certain linker length, there is no binding to cardiolipin increases the proportion of the pentacoordinated form, added effect. increasing peroxidase activity which eventually leads to apoptosis. Mutations

BPJ 7705_7715 Sunday, February 12, 2017 33a in the human gene for cytochrome c, which result in enhanced mitochondrial light on the thermodynamic contributions that enable fM affinities. Supported apoptotic activity, cause thrombocytopenia 4, an inherited autosomal dominant by grants from the NIH, The Mathers Foundation and NSF. thrombocytopenia, characterised by a deficiency in the number of platelets in the blood and leading to abnormal bleeding. The first such mutation to be reported 168-Plat was G41S. Here we use stopped-flow kinetic studies of azide binding to human Pressure Effects on Dissociation of CheY-FliM Complex Studied ferricytochrome c, backbone amide H/D exchange and 15N-relaxation dynamics by Molecular Dynamics Simulations 1 1 2 measured by NMR spectroscopy to compare the wild type and G41S forms of Hiroaki Hata , Yasutaka Nishihara , Masayoshi Nishiyama , Ikuro Kawagishi3, Akio Kitao1. human cytochrome c. We show that alternative conformations are kinetically 1 and thermodynamically more readily accessible for the G41S variant than for Institute of Molecular and Cellular Biosciences, The University of Tokyo, U Tokyo, Japan, 2The Hakubi Center for Advanced Research, Kyoto the wild-type protein. Residue 41 is located in the 40-57 -loop, and the 3 increased loop dynamics in the G41S variant promote the dissociation from University, Kyoto, Japan, Department of Frontier Bioscience, Hosei the heme iron of the M80 ligand, revealing a direct conformational link between University, Tokyo, Japan. the loop and the axial ligand to the heme iron. Increased dissociation of M80 in- The rotational switching of the bacteria flagella motor is controlled by binding of creases the population of a peroxidase active state, which is a key non-native the signaling molecule CheY onto FliM which is a part of motor basal body. The conformational state in apoptosis. rotational switching plays a central role in the bacterial chemotaxis. Recently, it was reported that high hydrostatic pressures of >120 MPa can induce the rota- 166-Plat tional switching even in the absence of CheY [1]. It was also suggested that hy- Global Disordering in Stereo-Specific Protein Association dration of the switch complex at high pressure induces structural changes similar Arun Gupta1, Ines Reinartz2, Alessandro Spilotros3, Venkateswara R. Jonna1, to those caused by the binding of CheY. To gain further insights into the high Anders Hofer1, Dmitri I. Svergun3, Alexander Schug2, Magnus Wolf-Watz1. pressure effect on the motor switching, we investigated differences in conforma- 1University of Umea˚, Umea˚, Sweden, 2Karlsruhe Institute of Technology, tion of monomeric CheY and also CheY-FliM complex at different pressure con- Karlsruhe, Germany, 3European Molecular Biological Laboratory, Hamburg ditions using molecular dynamics (MD) simulations. Then, pressure effects on Outstation, Hamburg, Germany. the binding stability of the CheY-FliM complex was studied by dissociating Protein-protein recognition is of fundamental importance for a myriad of the complex. The dissociation of the protein complex was observed using an biological processes and is ultimately a prerequisite for life as we know it. efficient sampling method, PaCS-MD (Parallel Cascade Selection Molecular There exist several established mechanisms that promote formation of Dynamics) [2]. In PaCS-MD, the cycle of short MD simulations and selection stereo-specific protein complexes. Many of these mechanism involve confor- of the structures close to the product structure for the next cycle are repeated, mational changes of one or both proteins in dimeric assemblies as observed in which enhances the conformational transitions without any additional external ‘‘conformational selection’’ and ‘‘coupled folding and binding’’ scenarios. In biases. From the obtained MD trajectories, the dissociation behavior was char- ‘‘coupled folding and binding’’ events, at least one of the proteins undergoes a acterized using coordinates such as the center of mass (COM) distance and the global ordering event. By using an integrated computational and experimental number of native contacts between CheY and FliM. Moreover, potentials of approach we have discovered that also global disordering can be a productive mean force along the COM distance were calculated from probability distribu- route for formation of a stereo-specific protein complex. This mechanism was tions in steady state obtained by Markov state models. Those potentials of observed for the chaperone binding domain of the Yersinia effector protein mean force provided binding free energies of the protein complex. Based on YopH upon binding to its specific chaperone SycH. These two proteins are the results, we will also discuss mechanisms underlying influences of high hy- crucial for type III secretion system mediated infectivity by Yersinia and drostatic pressures on the binding. Such insights would provide a further under- several other gram negative pathogens. NMR relaxation dispersion experi- standing towards an accurate regulation of protein-protein interactions. ments demonstrated that the otherwise well folded YopH protein dynamically [1] Nishiyama, M. et al. 2013. J. Bacteriol. 195:1809-1814. doi: 10.1128/ samples an expanded high-energy state that corresponds to the SycH binding JB.02139-12. competent conformation. A structure of the protein complex determined [2] Harada, R. and Kitao, A. 2013. J. Chem. Phys. 139:035103. doi: 10.1063/ from a hybrid SAXS and computational approach revealed that YopH wraps 1.4813023. around SycH in a horse shoe like conformation. The binding model was vali- dated by site specific YopH mutations that promoted the disordering event and Platform: General Protein-Lipid Interactions I at the same time displayed improved binding affinity towards SycH. Taken together the data illustrates a tight coupling between a proteins unfolding 169-Plat and functional free energy landscapes and add valuable mechanistic insight Predicting Cholesterol Interaction Sites on GPCRs by Molecular into protein-protein recognition. Simulation Edward R. Lyman1, Clement Arnarez2, Eric Rouviere2. 167-Plat 1Department of Chemistry and Biochemistry, University of Delaware, Role of Conformational Entropy in Extremely High Affinity Protein Newark, DE, USA, 2Department of Physics and Astronomy, University of Interactions Delaware, Newark, DE, USA. Jose A. Caro. G-protein coupled receptor function depends on the lipid environment, in Biochemistry & Biophysics, University of Pennsylvania, Perelman School of particular on cholesterol. Given that brute force mutagenesis of the entire Medicine, Philadelphia, PA, USA. membrane-facing surface is not practical, an approach is presented to identify Interactions of extreme affinity (Kd ~ fM) underlie many biochemical processes putative cholesterol interaction sites on the surface of GPCRs. In unbiased sim- necessary to life. The physical determinants of such large binding energies are ulations in the presence of cholesterol, specific residues are identified as loci of D not well understood. Specific interactions at the interface ( Hbinding) and the cholesterol interaction, identified on the basis of long-lived, reproducible D release of solvating water (T Ssolvation) are usually assumed to dominate the cholesterol binding. Results will be presented for several GPCRs, with a special D binding energetics. The role of conformational entropy (T Sconf) in deter- focus on the A2A adenosine receptor. mining binding affinity has remained elusive, in part due to the difficulties in measuring such changes in entropy experimentally. Recent developments in 170-Plat the Wand laboratory have bridged this gap by using solution NMR measure- Membrane Cholesterol and the Adenosine A2a Receptor ments of dynamics to empirically calibrate a ‘‘conformational entropy meter.’’ Claire McGraw, Anne S. Robinson. It has enabled quantitative measurements of the change in conformational en- Tulane University, New Orleans, LA, USA. tropy in protein-ligand binding. The toxin-antitoxin system studied here, G-protein coupled receptors (GPCRs) represent the largest family of receptor barnase-barstar, forms a complex with fM affinity (DGbinding ~ 19 kcal/mol) proteins in the living world, having approximately 800 human genes predicted; without undergoing any major structural changes and retaining a hydrated however the high-resolution crystal structures of only 26 GPCRs have been re- interface. To explore the role of conformational entropy, the fast (ps-ns time- ported (Ghosh et al., 2015). The first human GPCR to be crystallized was the scale) motions of backbone and side chains of the two proteins were measured b2-adrenergic receptor (b2AR) in 2007 (Cherezov et al., 2007; Rasmussen et in both the free (unbound) and the complexed (bound) states using NMR spec- al., 2007; Rosenbaum et al., 2007). Shortly thereafter an alternate crystal troscopy. Furthermore, hydration dynamics were measured in water and in the form of the b2AR revealed a specific cholesterol binding site between helices confined space of a reverse micelle. The dynamic response observed leads to an I, II, III and IV. From this work a cholesterol consensus motif (CCM) was es- unfavorable change in TDSconf, with a more rigid, still hydrated interface. This tablished, which defined specific interactions between cholesterol and the re- comprehensive study of both protein and ‘‘ligand’’ (in this case, another pro- ceptor. Utilization of this CCM predicted that as many as 25% of all class A tein) and the measured site-specific changes in dynamics and hydration sheds GPCRs could have a specific interaction with cholesterol (Hanson et al., 2008).

BPJ 7705_7715 34a Sunday, February 12, 2017

Our focus is the effects of cholesterol on the activity of the Adenosine A2a re- (ld) domains, and the selective localization and clustering of membrane pro- ceptor (A2aR), a class A GPCR. Membrane cholesterol concentrations were teins. Notch substrate is uniformly distributed in the ld phase while APP sub- varied in Human embryonic kidney (HEK-293) cells by the use of methyl- strate clusters prefer the boundary between ld and lo domains in agreement b-cyclodextrin (MbCD), which is capable of capturing cholesterol in its inner with fluorescence microscopy measurements carried out on proteolipobeads, cavity (Pucadyil et al., 2006). We tested the role that bulk cholesterol depletion suggesting that proteolysis and specificity of these substrates are modulated played in expression, ligand binding and downstream synthesis of cyclic AMP by membrane microenvironement. In addition, AFM time-course measure- (cAMP) in HEK-293 cells. ments suggests that g-secretase recruits specific membrane components and Continuing work from our lab will study the specific interaction between thus creates a favorable lipidic environment for its assembly and activity. cholesterol and A2aR by making point mutations in the residues of the CCM. 173-Plat In A2aR those residues include Tyr43(2.41), Ser47(2.45), Lys122(4.43), Ile125(4.46) and Trp129(4.50) (Lee et al., 2013). Our lab will investigate Lipid/Polydiacetylene Vesicle Composition Alters Mutant Beta-Amyloid whether these point mutations effect the interaction between cholesterol and Peptide Interaction Elizabeth A. Yates1, Michael P. Dorsey1, Brice M. Nguelifack2. purified receptor, as well as the receptors activity in both yeast and mammalian 1 cell model systems. Chemistry, United States Naval Academy, Annapolis, MD, USA, 2Mathematics, United States Naval Academy, Annapolis, MD, USA. 171-Plat Alzheimer’s disease (AD) is commonly identified by the formation of proteina- Probing Cholesterol-Dependence of Integrin-Urokinase Receptor ceous fibrillar aggregate deposits known as amyloids, and the presence of Complex Formation using Confocal Dual-Color Fluorescence Intensity neuritic amyloid plaques containing aggregated b-amyloid (Ab). Point muta- Analysis tions grouped in the hydrophobic core of Ab (positions 21–23) are linked to fa- Yifan Ge1, Jiayun Gao2, Rainer Jordan3, Christoph A. Naumann1. milial forms of AD. Understanding Ab peptide interactions with lipid surfaces 1Chemistry and Chemical Bology, Indiana University-Purdue University will provide detailed information into how surface interactions can drive protein Indianapolis, Indianapolis, IN, USA, 2Chemistry and Chemical Biology, aggregation. We hypothesized changes in the lipid composition of a vesicle will Indiana University-Purdue University Indianapolis, Indianapolis, IN, USA, result in varied mutant Ab peptide aggregation morphology and kinetics, further 3Macromolecular Chemistry, Technical University Dresden, Dresden, modulating peptide-lipid interactions. To test our hypothesis we utilized a color- Germany. imetric, lipid vesicle-binding assay exposed to specific Ab peptides. The assay The modern view of a plasma membrane is that of a complex supramolecular integrates a lipid within a polydiacetylene (PDA) matrix forming a blue vesicle assembly of notable lipid/protein heterogeneity, in which the local lipid envi- when polymerized by UV irradiation. Lipid/PDA vesicles are able to detect ronment may have a profound impact on membrane protein distribution and peptide-lipid interaction by quantitatively measuring a blue-to-red conversion function. Yet, the underlying mechanisms of lipid-mediated regulation of mem- as a percent colorimetric response (%CR). We investigated the interaction of brane protein functionality remain elusive, in part due to the limitations of Wild Type (Ab1-40) and five mutant Ab peptides (E22G Arctic, E22Q Dutch, existing methodologies and the often small size and transient nature of lipid/ A21G Flemish, D23N Iowa, and E22K Italian) with lipid/PDA vesicle systems protein heterogeneities in cellular membranes. Exemplary, we still have rather comprised of total brain lipid extract (TBLE), dimyristoylphosphatideylcholine limited knowledge about the significance of lipid composition in hetero-protein (DMPC), and dimyristoylphosphatidylglycerol (DMPG). Time-resolved absor- complex formation in such membrane systems. To address this fascinating, bance measurements of the ‘‘blue’’ (630 nm) and ‘‘red’’ (490 nm) wavelengths but poorly understood topic, here we introduce a single molecule-sensitive were obtained. Statistical analysis (ANOVA and t-tests) was performed to test confocal experimental strategy, comprised of dual-color fluorescence peak for significant differences between peptides among the lipid/PDA compositions, analysis and photon counting histogram (PCH) method, which not only allows as well as compare each mutant Ab peptide to Wild Type within the context of the detection of hetero-protein complexes in well-defined lipid environments, each lipid. The most peptide-lipid interaction was observed on the negatively but also provides accurate insight into the composition of such complexes in charged DMPG/PDA vesicle system, and the least on the neutral DMPC/PDA the membrane. By applying the described methodology, here we demonstrate system. Dutch and Flemish induced the smallest, while Arctic induced the largest the formation of complexes of avb3 integrins and GPI-anchored urokinase %CR on all lipid/PDA vesicles, respectively. These studies illustrate the role of plasminogen activator receptors (uPAR) in cholesterol-containing model lipid lipid composition and Ab interactions in the onset of familial AD. mixtures. PCH analysis of fluorescence intensity signals of complexes confirms 174-Plat furthermore that avb3-uPAR complexes are comprised of one integrin and two uPAR proteins. Remarkably, formation of comparable hetero-protein com- Synaptotagmin Interactions with Membranes: Measuring the Force of plexes is largely suppressed in a cholesterol-free lipid composition, high- Calcium Triggering of Neurotransmission Clemence Gruget1, Jeff Coleman2, Shyam Krishankumar3, lighting the importance of cholesterol in avb3-uPAR complex formation. Our James E. Rothman2, Fre´de´ric Pincet1, Stephen Donaldson1. results are intriguing in light of the already established significance of choles- 1 terol in integrin function in cellular membranes. Laboratoire de Physique Statistique, Ecole Normale Supe´rieure, Paris, France, 2School of medicine, Yale University, New Haven, CT, USA, 172-Plat 3University College London, London, United Kingdom. Partitioning of g-Secretase and its Substrates in Lipid Microdomains A critical step of neurotransmission is the rapid and synchronized fusion of syn- Marilia Barros1, William Houlihan2, Lane Gilchrist2, Yueming Li1. aptic vesicles with the pre-synaptic plasma membrane of the neuron upon cal- 1Memorial Sloan-Kettering Cancer Center, New York, NY, USA, 2The City cium entry, allowing the release of neurotransmitters immediately upon arrival College of the City University of New York, New York, NY, USA. of an action potential. A calcium binding protein anchored in the synaptic g-Secretase is a multiprotein complex that catalyzes intramembranous cleav- vesicle, the Synaptotagmin-1 (Syt1), has been identified as the calcium sensor age of mutiple transmembrane I proteins. The sequential proteolysis of the of this process, being able to accelerate fusion by more than four orders of amyloid precursor protein (APP) by g-Secretase generates amyloid beta pep- magnitude in presence of calcium. Despite this crucial role, the molecular tides that form plaques, one of the main pathological hallmarks of Alzheimer’s mechanism involved remains unclear. disease. g-Secretase also cleaves Notch the abnormal signaling of which can The cytosolic domain of Syt1 consists of tandem Ca2þ- binding C2 domains lead to cancer. The lipid environment regulates the location and activity of (C2A and C2B) attached to the membrane via a juxtamembrane linker domain. many transmembrane proteins and defines membrane micro or nanodomains, Different sites of the protein are responsible for its specific roles. First, the which serve as spatio-temporal platforms for proteins to function properly, interaction of the polybasic region of the C2B domain with the anionic lipid and therefore have a modulatory impact on the intramembrane proteolysis. PIP2 is needed for the initial docking of the synaptic vesicle at the plasma mem- Mechanistically, the lipids may affect substrate recognition by binding to brane. Second, upon calcium binding, the aliphatic loops on each C2 domains g-secretase and substrates and by excluding or enriching both partners within partially insert into the membrane, enabling the SNARE proteins to complete a microdomain. Despite substantial advances in elucidating how this enzyme membrane fusion. complex functions, the effect of the local membrane lipid microenvironment While the mechanistic details described above are mostly well-accepted, on g-secretase cleavage of substrates is still poorly understood. Here, we char- importantly, the binding energies of Syt1 with membranes have never been re- acterize the partitioning of APP and Notch1 substrate and g-secretase using ported. Therefore, the aim of our work is to measure the energetics of the major solid supported membranes to determine whether the function and activity of membrane binding sites of Syt1, i.e. the polybasic motif and the calcium-loop g-Secretase and its substrates r is influenced by the membrane lateral organiza- insertions of C2B and C2A. For this we use a Surface Force Apparatus (SFA), a tion. As a membrane model we chose a canonical raft mixture containing phos- device that provides a direct measurement of the interaction force between two phatidylcholine, sphingomyelin and cholesterol. Atomic force microscopy surfaces as a function of their separation distance, with nanoscale resolution. (AFM) data revealed membrane heterogeneities, ordered (lo) and disordered This system has successfully been used to measure the energy of the SNARE

BPJ 7705_7715 Sunday, February 12, 2017 35a proteins assembly, but the previous experiments did not include Syt1. In our set lattice thus reveals molecular interactions that generate the hexagonal coat. up, both surfaces are coated with a lipid bilayer. One of them mimics the syn- To determine the role of the hexagonal NEC lattice in budding, we mutated aptic vesicle membrane, on which we bind Syt1. The opposing bilayer mimics residues at the oligomeric interfaces observed in the crystals with mutagen- the inner leaflet of the plasma membrane and contains PIP2 and PS lipids. We esis. Perturbation of the oligomeric interfaces through mutagenesis blocked will present results obtained with various lipid compositions and relevant mu- NEC-mediated budding in vitro confirming that formation of the hexagonal tations of the protein and how these interactions are impacted by the presence NEC lattice drives budding. The NEC structure provide a three-dimensional of calcium. Ultimately, we plan to provide a complete mapping of the ener- blueprint for further dissection of its unique budding mechanism. Moreover, getics of the critical membrane interaction sites of Syt1. the structure represents the first atomic-level view of an oligomeric array formed by a membrane-deforming protein. 175-Plat Study of Insertion of Dengue E into Lipid Bilayers by Neutron Reflectivity Platform: Cardiac, Smooth, and Skeletal Muscle and Molecular Dynamics Simulations Juan M. Vanegas1, Frank Heinrich2, David M. Rogers3, Bryan D. Carson4, Electrophysiology Sadie La Bauve4, Brianna C. Vernon5, Bulent Akgun6,7, Sushil Satija8, 177-Plat Aihua Zheng9, Margaret Kielian10, Susan B. Rempe5, Michael S. Kent5. Action Potential Heterogeneity in Murine Sinoatrial Node Myocytes 1Dept. of Physics, University of Vermont, Burlington, VT, USA, 2Physics, Christian Rickert, Catherine Proenza. Carnegie Mellon University, Pittsburgh, PA, USA, 3Chemistry, University of Physiology & Biophysics, University of Colorado, Denver, CO, USA. South Florida, Tampa, FL, USA, 4Bioenergy and Defense Technology, Sinoatrial node myocytes (SAMs) act as cardiac pacemakers by generating spon- Sandia National Labs, Albuquerque, NM, USA, 5Nanobiology, Sandia taneous action potentials (APs). SAMs are characterized by the expression of the National Labs, Albuquerque, NM, USA, 6Bogazici University, Istanbul, ‘‘funny current’’ (I ) that contributes to the spontaneous diastolic depolarization Turkey, 7National Institute of Stsndards and Technology, Gaithersburg, MD, f phase of the sinoatrial AP. SAMs exhibit a large heterogeneity of morphological USA, 8National Institute of Standards and Technology, Gaithersburg, MD, and electrophysiological properties. However, limited information is available USA, 9Institute of Zoology, Chinese Academy of Sciences, Beijing, China, about the intrinsic variability in AP waveforms in SAMs. In this study, we co- 10Dept of Cell Biology, Albert Einstein College of Medicine, Bronx, recorded APs and I from acutely isolated SAMs from mice using voltage and NY, USA. f current clamp recordings in the same cells. We refined the definitions of The envelope (E) protein of Dengue virus rearranges to a trimeric hairpin to commonly used AP waveform parameters to document the heterogeneity of mediate fusion of the viral and host membranes. Insertion of E into host mem- APs in both perforated patch (PP) and whole-cell (WC) recordings. These defi- branes is essential to the process, serving to anchor E into the target membrane nitions were then implemented in an open-source data analysis program and possibly also to disrupt local order within the membrane. Both aspects are (‘‘paramAP’’) written in Python 3 using NumPy, SciPy, and Matplotlib. Para- likely to be affected by the depth of insertion, the orientation of the trimer with mAP was utilized to characterize APs from more than 100 SAMs. Correlation respect to the membrane normal, and the interactions that form between the analysis was used to evaluate relationships among AP waveform parameters trimer and the membrane. In the present work, we resolved the depth of inser- and to identify parameters that are strongly correlated with I and AP firing tion, the tilt angle, and the fundamental interactions for the soluble portion of f rate. Furthermore, we document time-dependent changes in AP waveform Dengue E trimers (sE) associated with planar lipid bilayer membranes of parameters during both PP and WC recordings. In summary, paramAP is a various combinations of POPC with POPG, POPE, and cholesterol by neutron powerful tool to help standardize AP waveform analysis and parameter defini- reflectivity (NR) and by molecular dynamics (MD) simulations. The tip of E tions in murine SAMs. Our data suggest that commonly-held assumptions about containing the fusion loop (FL) is located at the interface of the headgroups sinoatrial node APs should be extended to accommodate a wider range of AP and acyl chains of the outer leaflet of the lipid bilayers, in good agreement waveforms. In addition, the time-dependent changes in AP waveform parame- with prior predictions. The NR measurements and the MD simulations both ters should be considered when comparing data from different studies. indicate that E tilts with respect to the membrane normal upon insertion, pro- moted by either the anionic lipid POPG or cholesterol. The simulations show 178-Plat that tilting of the protein correlates with hydrogen bond formation between ly- SK4 Ca2D-Activated KD Channels Regulate Sinoatrial Node Firing Rate sines located on the sides of the trimer close to the tip (K246 and K247) and and Cardiac Pacing In Vivo nearby lipid headgroups. These hydrogen bonds provide the majority of the Bernard Attali1, David Weisbrod1, Hanna Bueno1, Joachim Behar2, interaction energy whereas interactions involving the FL are a minor contribu- Shiraz Haron-Khun1, Dor Yadin3, Asher Peretz1, Michael Arad3, tion. POPG promotes formation of these hydrogen bonds through direct inter- Yael Yaniv2. actions with K246, K247, and other polar residues whereas cholesterol 1Physiology, Sackler Medical School, Tel Aviv University, TelAviv, Israel, indirectly facilitates formation of these hydrogen bonds as a result of a greater 2Biomedical Engineering Faculty, Technion-IIT, Haifa, Israel, 3Leviev Heart hydrated volume in the headgroups. Simulations in which the protein was held Center, Sheba Medical Center, Tel Hashomer, TelAviv, Israel. in a vertical orientation with respect to the membrane show that these strong The sinoatrial node (SAN) controls the heart rhythm under physiological con- hydrogen bonding interactions of K246 and K247 with lipid headgroups causes ditions. We recently showed that SK4 calcium-activated potassium channels significant local membrane deformation of a 70:30 POPC:POPG bilayer as the (SK4) are important for automaticity of cardiomyocytes derived from human lipids wrap around the periphery of the E trimer. We propose that these inter- embryonic stem cells. Here we tested whether SK4 are expressed in adult actions play the dominant role in membrane anchoring and may also play a role mouse SAN and play a role in pacemaker function. TRAM-34, a selective in initiating mixing of the outer leaflets during the fusion process. blocker of SK4, significantly reduced the firing rate and depolarized the maximal diastolic potential in SAN cells. Western blots revealed the presence 176-Plat of an SK4 protein in mouse SAN. In vivo ECG recording in mice showed that The Great Nuclear Escape: Structure-Based Mechanism of Membrane intraperitoneal injection of SK4 blockers produced bradycardia and prolonged Budding during Nuclear Egress of Herpesviruses the PR interval. Mathematical modeling predicted that addition of SK4 to the 1 2 Ekaterina E. Heldwein , Janna M. Bigalke . model increases SAN firing rate, while its removal decreases pacemaker fre- 1Department of Molecular Biology and Microbiology, Tufts University 2 quency. This work shows that SK4 play a role in SAN pacemaker function School of Medicine, Boston, MA, USA, Tufts University School of by acting at late repolarization and that they are potential therapeutic targets Medicine, Boston, MA, USA. for treating cardiac arrhythmias. Herpesviruses are unusual among enveloped viruses because they bud twice yet acquire a single envelope. They are also the only known mammalian vi- 179-Plat ruses that bud into the nuclear envelope. Recently, we discovered that the Characteristics of Ivabradine-Sensitive Currents in Mouse Sinoatrial Node herpesvirus nuclear egress complex (NEC) could bud membranes without Myocytes the help of other proteins and that it formed a coat-like hexagonal scaffold in- Emily J. Sharpe1, Stephanie C. Gantz2, Pin Liu2, Bruce P. Bean2, side the budding membrane. This discovery established the NEC as the first Catherine Proenza1. virally encoded budding machine that operates at the nuclear, as opposed to 1University of Colorado - Denver, Aurora, CO, USA, 2Harvard Medical cytoplasmic, membrane but left unknown the structure of the NEC coat and School, Boston, MA, USA. its role in the budding process. To bridge this gap in our knowledge, we deter- Cardiac pacemaking is driven by spontaneous action potentials (APs) in sino- ˚ mined the 2.8-A crystal structure of the NEC from Herpes Simplex virus atrial node myocytes (SAMs). The funny current (If) is thought play a role in (HSV). In crystals, NEC packs into a hexagonal lattice that mimics the hex- the generation of pacemaker activity in SAMs, but the degree to which it agonal NEC coats within budded vesicles. The crystal structure of the NEC contributes is incompletely understood. In this study, we used AP clamp

BPJ 7705_7715 36a Sunday, February 12, 2017

experiments and the If blocker, ivabradine, to investigate the role of If in icantly improved our understandings of electrophysiological functions of pacemaking. Spontaneous APs were recorded from acutely-isolated SAMs electrically excitable organs (heart, brain). These dyes allow to measure simul- from mice, and ~10 s of the AP recording was incorporated into a voltage taneously membrane potential changes from multiple sites of biological clamp command. This AP clamp protocol elicited both inward and outward preparations in a non-invasive way. However, conventional dyes have excita- ivabradine-sensitive currents in SAMs. If was defined as the ivabradine- tion spectra in the blue/green range, which limits the depth of penetration sensitive current remaining in the presence of a pharmacological cocktail of due to high absorbance and photon scattering properties. Therefore, the devel- sodium, calcium and potassium channel blockers. Measurable inward opment of longer wavelength VSDs could have many advantages namely: ac- ivabradine-sensitive current was evident during diastole in 57% of all cells tion potentials recording from tissue’s deeper layers, enhanced voltage and was generally larger in cells with the most negative diastolic voltages. sensitivity, and improved spectral properties such that simultaneous monitoring Although we found that 30 mM ivabradine also blocked both L- and T-type cal- of multiple parameters (i.e. ion transient, pH). cium currents in SAMs, the majority of the ivabradine-sensitive inward current The goal of our study is to develop and characterize novel VSDs with large in the AP clamp experiments appeared to be If because control experiments Stoke shifts and near-infrared spectral properties. Three families of VSDs showed that the drug cocktail blocked ~85% of the calcium currents. Ivabra- were thus synthesized from two heterocyclic fluorophores and their properties dine also blocked a substantial outward current during the AP upstroke and (spectral properties, membrane staining, and dye phototoxicity) assessed in iso- repolarization phases. This outward ivabradine-sensitive current likely includes lated ventricular cells and in Langendorff perfused rat hearts. In addition, spe- a contribution of If, which results from its relatively slow deactivation. How- cial attention is paid to Signal:background ratios and signal kinetics of these ever some of the outward ivabradine-sensitive current may also reflect off- new dyes by epifluorescence imaging. target block of potassium currents by ivabradine. We are currently assaying Optimal peak emission wavelengths for all dyes was found between 700 nm the contribution of potassium currents to the outward ivabradine-sensitive cur- and 750 nm. The largest observed Stoke shift was found with fluorophore 1 rent. Our results suggest that If is active not only during diastole, but also family (about 250 nm). Signal:background ratios ranged from 3% (fluorophore throughout the AP upstroke and repolarization phases of the mouse SAM AP. 2) to 7% (fluorophore 3), compared to 5.5% for the usual conventional dye, Di-4-ANEPPS. 180-Plat Rapid signal decay indicative of internalization was observed for one VSD Calcium Activated Chloride Current in Mammalian Ventricular Myocytes family. However, in other two, signal stability was improved compared to 1 1 1 1 Janos Magyar , Bala´zs Horva´th , Krisztina Va´czi , Bence Hegyi , Di-4-ANEPPS. Overall, dyes made with fluorophore 3 show improved voltage 2 1 1 1 Mo´nika Go¨nczi , Beatrix Dienes , Korne´l Kistama´s , Tama´s Ba´nya´sz , sensitivity and potential for applications. Istva´n Baczko´3, Andra´s Varro´3, Gyo¨rgy Sepre´nyi4,La´szlo´ Csernoch1, Pe´ter P. Na´na´si5, Norbert Szentandra´ssy5. 182-Plat 1Dept. of Physiology, University of Debrecen Faculty of Medicine, Dynamic Blood Flow Control in Heart Debrecen, Hungary, 2Dept. of Biochemistry and Molecular Biology, Guiling Zhao, Humberto Joca, W. Jonathan Lederer. University of Debrecen Faculty of Medicine, Debrecen, Hungary, 3Dept. of BioMET and Physiology, University of Maryland School of Medicine, Pharmacology and Pharmacotherapy, University of Szeged Faculty of Baltimore, MD, USA. Medicine, Szeged, Hungary, 4Dept. of Medical Biology, University of Perfusion of the heart by blood is essential for the maintenance of physiological Szeged Faculty of Medicine, Szeged, Hungary, 5Dept. of Physiology, function. Published experiments and investigations by others reveal that the University of Debrecen Faculty of Dentistry, Debrecen, Hungary. maximum AV O2 difference that can be achieved in heart is always observed Background: Calcium activated Cl current (ICl(Ca)) mediated by TMEM16A between the coronary arteries and the venous outflow at the coronary sinus and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile over the full range of blood flow (~five fold) that is achieved. If so, exquisite of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly matching of blood flow and O2 consumption must exist even at the smallest understood. Our goal was to study the current profile under normal calcium unit of perfusion. How does such tight control occur? We have examined the cycling and AP voltage-clamp condition as well as in case of altered intracel- hypothesis put forward for the brain to determine if, in principle, it could 2þ lular calcium concentration ([Ca ]i). The expression of TMEM16A and/or work in heart. Nelson and colleagues have suggested that electrical activity Bestrophin-3 in canine and human left ventricular myocytes was examined. could be a primary regulator of blood flow in the brain. Increased neuronal ac- The possible spatial distribution of these proteins and their co-localization tivity would lead to an increase in Kþ efflux that would bathe endothelial cells with Cav1.2 was also studied. which possess inward rectifier potassium channels (Kir). Activation of Kir could Methods: Whole-cell configuration of the patch-clamp technique and action lead to hyperpolarization given the N-shaped Kir current-voltage (IV) relation- potential voltage-clamp were used to monitor ICl(Ca), detected as 9-anthracene ship that we have observed. In the current study, we examined human microvas- carboxylic acid (9-AC)-sensitive current. FURA-2-AM dye was used to measure cular endothelial cells from heart (HMVEC-C) in culture to determine how they 2þ þ þ [Ca ]i. Expression and cellular localization of Cav1.2, Bestrophin-3 and responded to elevated [K ]o. We found that extracellular increases of K (from TMEM16A was analyzed with immunocytochemistry and confocal microscopy. 5 mM to 15 mM) cause HMVEC-C hyperpolarization. This Kþ-induced mem- Results: Under AP voltage-clamp conditions the profile of ICl(Ca) contained brane hyperpolarization is dependent on Kir activation as evidenced by its an early fast outward (1.6250.06 A/F) and a late inward component blockade by extracellular Ba2þ. These findings suggest that cardiac electrical (0.1650.02 A/F), overlapping early and terminal repolarizations, respectively. activity could contribute to Kþ-dependent hyperpolarization of the endothelial Both components were reduced by ryanodine (1.0550.03 A/F; 0.0750.03 A/ cells that could hyperpolarize the pre-capillary sphincter smooth muscle cells F), while fully abolished by BAPTA (0.1350.10 A/F; 0.0850.02 A/F), but not locally and thereby contribute to activity-dependent blood flow control. 2þ EGTA (1.1750.09 A/F; 0.1350.02 A/F). Setting [Ca ]i to the systolic level 183-Plat (1.1 mM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and 2þ KChIP2 Serves Multiple Functions in Cardiac Myocytes in Splice faster stimulation rates increased the amplitude of ICl(Ca). Both L-type Ca cur- Isoform-Dependent Manner rent and ICl(Ca) were eliminated by nisoldipine. TMEM16A and Bestrophin-3 Randolph Bettinger1, Rostam Panjshiri1, Drew M. Nassal2, showed strong co-localization with one another and also with Cav1.2 channels 2 3 1 both canine myocytes and human ventricular myocardium. Isabelle Deschenes , Min Jiang , Gea-Ny Tseng . 1Physiology & Biophysics, Virginia Commonwealth University, Richmond, Conclusions: Activation of ICl(Ca) in canine ventricular cells requires calcium 2 2þ VA, USA, Medicine and Physiology & Biophysics, Case Western entry through neighboring L-type Ca channels and is only augmented by 3 2þ 2þ University, Cleveland, OH, USA, Institute of Medicine Biotechnology, SR Ca -release. Substantial activation of ICl(Ca) requires high Ca in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA. Chinese Academy of Medical Sciences, Beijing, China. K-channel-interacting-proteins (KChIPs) are EF-hand-motif-bearing Ca sensors 181-Plat that serve diverse functions. KChIP2 is the major KChIP expressed in the heart, Properties of New Voltage Sensitive Dyes in Cardiac Field that can modulate key cardiac channels (Itof,Nav,CaV and IKur) by directly inter- 1 1 1 Ndeye Rokhaya Faye , Sushmitha Raja , Richard Walton , acting with their pore-forming subunits (Kv4, Nav1.5, Cav1.2, and Kv1.5, respec- Phillipe Pasdois1, Fabien Brette1, Alan Urban2, Alexandre Hentz3, tively) on the cell surface. More recent data suggest that KChIP2 can traffic to Gihad Dargazanli3, Olivier Bernus1. nuclei and exert transcriptional activity. Nine KChIP2 splice variants have 1Electrophysiology and Heart Modelling Institute (IHU-LIRYC), Pessac, been identified, that share C-terminal EF-hand-motifs but diverge in N-termini. France, 2Neuro-Electronics Research Flanders, Imec, Leuven, Belgium, Based on N-terminal sequences, KChIP2 splice variants can be broadly divided 3Sanofi, Chilly-Mazarin, Paris, France. as palmitoylation-capable and palmitoylation-incapable. Questions: (1) Does These last years, combination of high spatiotemporal resolution techniques palmitoylation impact on KChIP2 distribution and function?(2) Is KChIP2 with the development of fluorescent voltage sensitive dyes (VSDs) have signif- splicing pattern altered in diseased heart? Methods: (1) Tag KChIP2a and 2c

BPJ 7705_7715 Sunday, February 12, 2017 37a

(palmitoylation-capable and -incapable) with mCherry, (2) Express KChIP2 iso- structural information is very limited. In addition, multiple experimental arti- forms in COS-7 cells alone or with GFP-Kv4.3, (3) Track KChIP2a/c distribu- facts can complicate the interpretation of data. However, recent advances in tion and relationship with Kv4.3 with confocal microscopy, (4) Quantify Itof smFRET experiments, particularly using Multiparameter Fluorescence Detec- channel function and subcellular distribution with patch clamp and subcellular tion (MFD), have improved its accuracy and precision, to the point that it is fractionation/immunoblots, (5) Quantify native KChIP2 and Kv4 proteins and possible to overcome this mindset. As an example, we use MFD to identify Itof channel function in ventricular myocytes from young and old spontaneously low populated conformational states of the ligand-binding domain of the hypertensive rats (SY and SO, 4-6 and 22-24 months respectively; the latter suf- N-methyl-D-aspartate (NMDA) receptors, quantify molecular dynamics in fers from severe hypertrophy/heart failure). Results: (1) When expressed alone, the sub millisecond regime and identify how conformational dynamics sheds KChIP2a is strong in vesicles, Golgi and plasma membrane (PM), while light into the mechanism of partial agonism in the GluN1 subunit of the KChIP2c is strong in nuclei and weak in PM. Pharmacological inhibition of NMDA receptor. palmitoylation directs KChIP2a from PM to nuclei, but does not alter nuclei localization of KChIP2c. (2) Kv4.3 coexpression leads to Kv4.3/KChIP2a over- 186-Symp lap in Golgi, post-Golgi vesicles and PM. KChIP2c and Kv4.3 overlap on PM. Measuring the Free Energy of ClC-ec1 Dimerization in Membranes using While both KChIP2a and KChIP2c accelerate Kv4.3 recovery from inactivation, Single Molecule Photobleaching Analysis KChIP2a but not KChIP2c increases the current amplitude. (3) Relative to SY, Janice L. Robertson. Molecular Physiology and Biophysics, University of Iowa, Iowa City, Itof density is markedly reduced in epicardial myocytes (Epi VMs) of SO hearts. Kv4.2 and Kv4.3 proteins are modestly or not lowered in Epi VMs. IA, USA. While KChIP2a protein is markedly reduced, KChIP2c protein is significantly Why do greasy membrane proteins interact with their greasy protein partners increased. Conclusions: Reversible palmitoylation confers dynamic KChIP2a instead of the similarly greasy lipid bilayer? This is a question at the root of membrane protein oligomerization and folding in membranes. Recently, we localization between PM and nuclei, while KChIP2c has a more static distribu- - þ tion. Chronic hypertension/hypertrophy differentially impacts the expression of turned to the homodimeric ClC-ec1 Cl /H antiporter, in order to develop a KChIP2a and 2c. new model system that can be used to investigate this question. The ClC-ec1 dimerization interface is large (1200 A˚ 2) and lined by ~20 non-polar residues, 184-Plat forming an interaction surface exhibiting high shape complementarity. Previ- Effect of Drugs on Repolarization of IPSCD-Cardiomyocytes ous studies showed that ClC-ec1 is folded and functional in both monomeric Mark Nowak1, Aidan Coon2, Sanjot Singh2, Shimin Wang2, and dimeric states. Using a single-molecule microscopy approach, we Randall Rasmusson3, G. Bett4. measured the photobleaching distributions across a wide range of subunit/lipid 1Cytocybernetics, Buffalo, NY, USA, 2SUNY, University at Buffalo, dilutions, with the lowest representing a sub-biological condition of 1 subunit Buffalo, NY, USA, 3Physiology and Biophysics, SUNY, University at per 40 E. coli membranes. The data shows that wild-type ClC-ec1 follows a Buffalo, Buffalo, NY, USA, 4Obstetrics and Gynecology, SUNY, University monomer to dimer reaction that is reversible and fits to an equilibrium at Buffalo, Buffalo, NY, USA. isotherm, allowing for determination of the dimerization free energy in 2:1 The FDA has proposed a Comprehensive in Vitro Pro-Arrhythmia Assay (CiPA POPE/POPG lipid bilayers (Chadda et al., eLife 2016). Next, two approaches Initiative) to address the clinical potential for cardiotoxicity during pre-clinical were used to investigate how residues at the interaction interface influence drug development in part, this involves the use of human-induced pluripotent the free energy of dimerization. First, bulky TRP residues were introduced re- adult stem cell-derived cardiac myocytes (hiPSC-CM) as a model system to sulting in a destabilization of the dimer by 2 kcal/mole per TRP. Second, sub- measure the effects of drugs on the cardiac action potential (AP). hiPSC- tractive substitutions to ALA were constructed introducing cavities at the CMs, however, have little or no IK1, resulting in unstable APs and anomalous interface. While most of the ALA substitutions appear to have no effect, behavior in the presence of some drugs. We stabilized APs from hiPSC-CMs L194A destabilizes the dimer by 2 kcal/mole in lipid bilayers while maintain- through the electronic addition of IK1 via dynamic clamp. hiPSC-CMs (iCell, ing both fold and function. These studies present ClC-ec1 as a robust model Inc., WI) were cultured according to manufacturer’s instructions, and studied system for probing the physical forces driving protein association in using the Amphotericin B perforated patch technique. Cardiac APs (1.5- membranes. 2.0 nA stimulus pulse for 1.5 ms, 0.5 Hz) were measured in the absence and presence of increasing drug concentrations. As expected, quinidine, dofetilide, 187-Symp cisapride and sotalol (known I blockers) caused dose-dependent increases in T Cell Receptor Clustering - A Mechanism of Signal Transduction KR Katharina Gaus. APD. In 10 mM quinidine APD90 increased 144512%; 1420 nM dofetilide increased APD 12853%; 103 nM cisapride increased, APD 17056%; Centre for Vascular Research, University of New South Wales, Sydney, 90 90 Australia. 324 mM sotalol increased APD90 139514%). Further, qualitative differences in potentiation were observed in that cisapride and sotalol, but not dofetilide, Antigen recognition by the T cell receptor (TCR) is a hallmark of the adaptive caused a pronounced flattening in Phase 2 of the AP. Quinidine (10 mM) also immune system. When the TCR engages a peptide bound to the restricting ma- þ jor histocompatibility complex molecule (pMHC), it transmits a signal via the decreased dV/dtmax by 68518%, which is consistent with Na channel block. 2þ associated CD3 complex. How the extracellular antigen recognition event leads Nifedipine (300nM), a Ca channel blocker, significantly decreased APD90 by 3551%. The ability to measure stable cardiac APs by the electronic addition of to intracellular phosphorylation remains unclear. We use single-molecule local- ization microscopy and novel analysis to quantify the organization of TCR- IK1 allows for more accurate comprehensive evaluation of drug effects on the ion channel components comprising the cardiac AP, particularly in the case CD3 complexes into nanoscale clusters and to distinguish between triggered of sodium channel block, and may aid in the identification of pro-arrhythmic and non-triggered TCR-CD3 complexes. For example, we found that only changes in AP morphology. TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density Symposium: Single-Molecule Membrane Protein within clusters dictates signal initiation. Both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall Dynamics phosphorylation levels. In summary, we propose a model in whcih TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by 185-Symp the CD3 complex and the formation of dense signaling-competent clusters is Submillisecond Dynamics of the NMDA Receptor a process of antigen discrimination. Hugo Sanabria. Clemson University, Central, SC, USA. Symposium: Mechanotransduction to Physiology Observation of single molecules has impacted the way we look at bio- molecular machines. Particularly, because it gave us a tool to understand 188-Symp how biomachines move as they carry out specific functions. Single molecule The Mechanism of Activation of Piezo Ion Channels fluorescence experiments, done in surface immobilized conditions or freely Jorg Grandl. diffusing, take advantage of the fact that fluorescence occurs in the nano- Duke University, Durham, NC, USA. second timescale to map a wide range of biologically relevant dynamics, Piezo ion channels mediate the conversion of mechanical touch into electrical covering over 10 orders of magnitude in time without gaps. Single molecule signals and are critical for the organism’s responsiveness to mechanical forces. Fo¨rster Resonance Energy Transfer (smFRET) experiments have evolved How Piezos detect mechanical stimuli is unknown. to the point where it is possible to work with complex systems including The apparent mechanical sensitivity of Piezo1 varies substantially across membrane proteins. However, many challenges have limited the impact on cellular environments, stimulating methods and protocols, raising the funda- the field, mostly because there is the common conception that the obtained mental questions of what precise physical stimulus activates the channel and

BPJ 7705_7715 38a Sunday, February 12, 2017 how its stimulus sensitivity is regulated. Here, we measured Piezo1 currents rupture. Genes predicted to encode MscS homologs are found in genomes evoked by membrane stretch, while simultaneously visualizing and measuring from all three kingdoms of life. We have been characterizing the structure, membrane geometry. Building on this approach, we developed protocols to function, and regulation of ten MscS-Like (MSL) proteins in the model plant minimize resting membrane curvature and tension prior to probing Piezo activ- Arabidopsis thaliana. Based on their modest homology to MscS and high topo- ity. We find that Piezo1 responds to lateral membrane tension with high sensi- logical diversity, we have proposed that MSLs might (1) sense and respond to tivity as compared to other mechanically activated channels and that resting sources of membrane tension other than environmental hypo-osmotic shock; tension can drive channel inactivation, thereby tuning overall mechanical sensi- (2) be regulated by mechanisms in addition to membrane tension; and (3) signal tivity of Piezo1. Our results explain how Piezo1 can function efficiently and in ways that are separable from ion flux. Evidence in support of all three of with adaptable sensitivity as a sensor of mechanical stimulation in diverse these hypotheses will be presented. I will also discuss our current effort to cellular contexts. engineer novel biosensors based on MSL proteins. The next step in understanding the mechanism of Piezo activation would be the identification of structures within Piezos that detect a mechanical stimulus and Symposium: Cancer Cell Biophysics that mediate channel inactivation. We hypothesized that specific structures within Piezos are highly sensitive to localized application of force, whereas 192-Symp others are less sensitive in comparison. To test this hypothesis, we developed Autocrine Role of Exosomes in Cellular Adhesion, Migration, and Invasion a novel method, where we apply highly localized force to specific protein do- Alissa Weaver. mains and simultaneously measure ion channel activity upon pressure-clamp Vanderbilt University School of Medicine, Nashville, TN, USA. stimulation. For this, we label specific domains within Piezos with magnetic Exosomes are late endosome-derived extracellular vesicles that carry multiple nanoparticles and use an external magnetic field to generate a precise mechan- motility-promoting cargos. The exact role of exosomes in promoting motility ical force that is highly localized. Simultaneously, we measure Piezo-activation under diverse circumstances is unclear. Likewise, whether the process of exo- electrophysiologically. With this approach we have identified domains that are some secretion is essential for cell migration is an open question. We have sensitive to application of localized magnetic pulling force and other domains found that exosome secretion promotes directionally persistent migration of that are completely insensitive to the same force. cancer cells through tissues. Intravital live imaging reveals that exosome secre- tion promotes a polarized cell morphology and stabilization of lamellipodial 189-Symp protrusions. Mechanistic in vitro experiments demonstrate that extracellular Mechanosensing at the Surface matrix (ECM) is an important exosome cargo that enhances motility speed. Ellen Lumpkin. Consistent with a major role for ECM as a critical motility-promoting exosome Physiology & Cellular Biophysics, Columbia University, New York, cargo, live imaging experiments reveal that exosomes are secreted at the sites NY, USA. of nascent adhesions and promote their assembly. Additional experiments in Skin is innervated by a rich variety of mechanosensitive neurons that trigger diverse in vitro environments indicate a role for additional exosome cargoes distinct sensations such as pressure, flutter and pain. A growing body of evi- in directional sensing. Altogether, our results indicate that autocrine secretion dence indicates that epithelial cells actively partipate in sensation by modu- of exosomes is a critical feedback mechanism that promotes cancer cell lating, and even directly exciting, mechanosensitive neurons in healthy skin motility. and pathophysiological conditions. This research aims to unveil how epithelial Merkel cells work in concert with the nervous system to generate discrimina- 193-Symp tive touch sensation. Using mouse genetics and optogenetics, we previously Quantitative Systems Biology Studies Reveal a Nested Relay Mechanism demonstrated that Merkel cells have dual roles: they transduce sustained firing for TGF-Beta Signaling that signals pressure, and boost firing rates of tactile afferents during dynamic Jianhua Xing. touch. Our current studies focus on defining mechanisms of neurotransmission Computational and Systems Biology, University of Pittsburgh, Pittsburgh, between Merkel cells and tactile afferents, and elucidating how connections be- PA, USA. tween Merkel cells and mechanosensory neurons remodeled during healthy Cells need to reliably sense and transmit information about extracellular stim- skin renewal. uli, and cancer cells typically have dysregulated signal transduction. In b 190-Symp this talk I will discuss TGF- induced expression of transcription factor Snail1, and the latter regulates many processes such as the epithelial-to- Role of Piezo Ion Channels in Mechanosensation b Ardem Patapoutian1,2. mesenchymal transition. A widely assumed mechanism states that TGF- 1Molecular & Cellular Neuroscience, The Scripps Research Institute, La leads to phosphorylation of Smad2 and Smad3 (pSmad2/3), which enter the Jolla, CA, USA, 2Howard Hughes Medical Institute, La Jolla, CA, USA. nucleus and activate Snail1 expression. However, both our quantitative mea- Mechanotransduction is perhaps the last sensory modality not understood at the surements and thorough parameter space analysis show that the mechanism molecular level. Proteins/ion channels that sense mechanical force are postu- does not hold. Instead we proposed and confirmed a novel nested relay mech- lated to play critical roles in sensing touch/pain (somatosensation), sound (hear- anism that a relay from pSmad2/3 to Gli1 initializes and maintains Snail1 ing), sheer stress (cardiovascular function), etc.; however, the identity of ion expression, while another relay from active to inhibitory phosphorylation of channels involved in sensing mechanical force has remained elusive. We iden- GSK3 smoothens the pSmad2/3-Gli1 transition. Our model analysis suggests that the network motif allows a cell to respond differentially to different tified Piezo1 and Piezo2, mechanically-activated cation channels that are ex- b pressed in many mechanosensitive cell types. We demonstrated that Piezos strength and duration of TGF- . To characterize this motif, we generated cells constitute an evolutionarily conserved family of mechanically-activated cation labeled with fluorescence protein reporters using the CRISPR technique, and channels. We further showed that Piezo1 is a shear stress sensor in vascular are performing live cell imaging studies. This work demonstrates the impor- endothelial cells, while Piezo2 is the major transducer of mechanical forces tance of integrated and iterative computational and quantitative experimental for touch sensation in mice. Current efforts focus on understanding structure- studies on elucidating the regulatory mechanism of a complex biological function relationship of Piezo proteins, and elucidating their physiological roles process. in various biological processes and diseases that depend on mechanotransduc- tion. Finally, we are searching for novel mechanosensors. 194-Symp Models for Cancer Cell Motility: Regulation and Signaling 191-Symp Leah Edelstein-Keshet. MscS-like Mechanosensitive Ion Channels: Modular Sensors Department of Mathematics, University of British Columbia, Vancouver, and Reporters of Membrane Tension BC, Canada. Elizabeth Haswell, Debarati Basu, Eric S. Hamilton, Grigory Maksaev, A common feature of cancer cells is aberrant motility, leading to invasion and Matthew Mixdorf, Ivan Radin, Ryan Richardson, Angela M. Schlegel, metastasis. I will survey several modeling projects in which we have explored Eric Schultz, Yanbing Wang. this aspect of malignant cells. First, I will describe the burst of actin-based pro- Biology, Washington University, Saint Louis, MO, USA. trusion that is facilitated by cofilin in mammary carcinoma cells (joint work A long-standing question is how biological systems sense and perceive with N. Tania and J. Condeelis). Next, I will discuss the intracellular signaling mechanical signals such as osmotic pressure, gravity, and touch. One well- based on Rho GTPases in melanoma cells that orchestrates their motility established molecular mechanism for force sensing is the activation of mecha- phenotype (joint with WR Holmes, JS Park, and A Levchenko). Finally, I nosensitive (MS) ion channels. The Mechanosensitive channel of Small will describe some recent efforts at understanding cell-cell signaling that coor- conductance (MscS) from E. coli functions as a hypo-osmotic safety valve, dinates the migration of mammary carcinoma cells towards blood vessels (joint opening in response to increased membrane tension and preventing cellular with H Knutsdottir and J Condeelis).

BPJ 7705_7715 Sunday, February 12, 2017 39a

195-Symp off-rates of VDP. This change in the distance between the N- and C-terminal re- Physical Interactions in Ductal Microinvasions: Integrating Histology with gions was abolished when PI(4,5)P2 was hydrolyzed, and the simultaneous VDP Computational Modeling was eliminated. Collectively, we propose that the activation of zELK channels in- Katarzyna A. Rejniak1,2. volves a rearrangement of the direct interaction between the N- and C-terminal re- 1Mathematical Oncology, Moffit Cancer Center & Research Institute, Tampa, gions, which transitions the channel to a potentiated state. FL, USA, 2Oncologic Sciences, University of South Florida, Tampa, FL, USA. 198-Plat Progression from a ductal carcinoma in situ (DCIS) to an invasive tumor is a BK Channel Gating-Ring Voltage Dependence Motions major step initiating a devastating and often lethal metastatic cascade. One Pablo Miranda1, Teresa Giraldez2, Miguel Holmgren1. 1National Institutes of Neurological Disorders and Stroke, National Institutes of the first steps in this process is the development of ductal microinvasions, 2 i.e., small cohorts of tumor cells that breach the basement membrane surround- of Health, Bethesda, MD, USA, Biomedical Sciences, Medical School, ing the duct, and migrate through the extracellular matrix (ECM). At this point, Center for Biomedical Research of the Canary Islands (CIBICAN), for the first time, the epithelially-derived tumor cells engage in a direct physical University of la Laguna (ULL), La Laguna, Spain. contact with the extracellular matrix and the stroma. We combined single cell- The open probability of Large Conductance Voltage and Calcium dependent potassium (BK) channels is regulated allosterically by change in the transmem- based model of the tumor-ECM interactions and ECM remodeling, and image- 2þ 2þ based analysis of the cellular biophysico-chemical features as determined from brane voltage and intracellular concentration of divalent ions (Ca and Mg ). patients’ histology samples of DCIS. Using this model we showed how changes BK channels are formed as tetramers of alpha subunits. The ‘‘divalent sensor’’ in the local microenvironmental niche near the DCIS edge enable initiation and resides within the gating-ring, a large tetrameric structure formed by eight Regulator of Conductance of Potassium (RCK) domains, two from each subunit. progression of ductal microinvasions. Of particular interest are the biomechan- 2þ ical interactions between the cells and the ECM fiber structure, and microenvi- Each RCK domain (RCK1 and RCK2) contains a high affinity- Ca binding site. Using Patch-clamp fluorometry, we have shown large changes in FRET signals ronmental features that define niche prone to microinvasions. These findings 2þ can be compared to the patient histology samples and help define criteria for within the gating-ring in response to Ca and voltage. Here we investigate the future development of new prognostic methods and therapeutic interventions origins of the voltage dependent motions. We tested manipulations that mostly by targeting the tumor niche. change the BK channels’ relative probability of opening and in all cases the voltage dependence of the FRET signals remained within the voltage range of Platform: Voltage-gated K Channels and wild-type. However, mutations in the voltage sensor that are known to shift the charge displacement currents have a parallel shift of the gating ring’s mo- Mechanisms of Voltage Sensing and Gating I tions. These result suggest that the conformational changes of the gating ring are coupled to the voltage sensor, and could be relevant in the allosteric modu- 196-Plat lation of the BK channels. Probing the Movement of the Ball and Chain during N-type Inactivation in Kv Channels 199-Plat Tanja Kalstrup, Roshan Pandey, Rikard Blunck. Inhibition of BK Channels by Strong Extracellular Acidification University of Montreal, Montreal, QC, Canada. Yu Zhou, Xiaoming Xia, Christopher J. Lingle. N-type inactivation is a mechanism in certain Kv channels where the N-termi- Washington University School of Medicine, St. Louis, MO, USA. nal peptide occludes the pore upon depolarization resulting in block of ionic Voltage- and Ca2þ-dependent BK-type large conductance Kþ channels regulate currents (ball-and-chain). Numerous mutational studies have characterized membrane excitability and homeostasis by providing negative-feedback control N-type inactivation functionally, while X-ray crystal structures have yet to for local [Ca2þ] in a wide variety of tissues and organelles. Some of these tissues include the ball-and-chain structure. It still remains unknown how far the N-ter- or organelles, such as distal nephron and lysosome, are exposed to extreme acid- minus travels during N-type inactivation and where it is located in the resting ity (pH <5) at physiological or pathological conditions. Such strong extracellular state. Does it reside in a fixed position near the T1 window or is it randomly acidification may interfere with BK channel function by neutralizing the negative floating in the cytosol? By incorporating a fluorescent unnatural amino acid charges of key acidic residues on the extracellular side of BK channels. How- (Anap) into the N-terminus and into receptor sites (T1 window and S4-S5 ever, little is known about the behavior of BK channels at such acidic conditions. linker) we directly tracked the movement of the ball peptide using voltage Our results show that BK activation is strongly inhibited at extracellular pH clamp fluorometry in Xenopus oocytes. Voltage dependency and kinetics of lower than 5. At pH 4 the G-V relationship of BK is positively shifted by ball-peptide movement correlated with N-type inactivating currents. Further- more than 100 mV. The inhibition is largely caused by neutralization of several more, we address the question as to whether the fluorescence changes are acidic residues in the BK voltage sensor domain (VSD), including D133, D147 caused by polarity changes or quenching, by measuring the Anap emission and D153, some of which have been identified to be important for the normal spectra at both resting and activated state. function of BK VSD. Since the transmembrane domain of BK channels shares similar overall organization with many other voltage-gated ion channels such 197-Plat as the voltage-dependent Kþ channels, the principle underlying strong extracel- Molecular Mechanisms of the Voltage-Dependent Potentiation of KCNH lular acidification-induced inhibition in BK channels may also be applicable to Potassium Channels these channels. Supported by NIH GM118114. Gucan Dai, William Zagotta. Physiology and Biophysics, University of Washington, Seattle, WA, USA. 200-Plat EAG-like (ELK, Kv12) voltage-gated potassium channels in the KCNH channel Single-Molecule Fluorescence Imaging of low Affinity Binding Interactions family are primarily and abundantly expressed in the brain. Deletion of the ELK in Pacemaker ion Channels gene leads to hyperexcitability of hippocampal neurons, epilepsy and altered Marcel P. Goldschen-Ohm1, David S. White1,2, Vadim A. Klenchin1, cognitive functions. One structural feature of KCNH channels important for chan- Randall H. Goldsmith2, Baron Chanda1. nel function is an interaction between the N-terminal EAG domain and the C-ter- 1Neuroscience, University of Wisconsin, Madison, WI, USA, 2Chemistry, minal cyclic nucleotide-binding homology domain (CNBHD). Here we studied an University of Wisconsin, Madison, WI, USA. orthologous zebrafish ELK channel (zELK) using patch-clamp recording and Molecular recognition is a cornerstone of cellular signaling processes. How- patch-clamp fluorometry (PCF). We noticed that a depolarizing prepulse potenti- ever, most observations of binding rely on ensemble measurements that ated the zELK channels in both the current amplitude and the voltage sensitivity. average over and thereby obscure the individual underlying steps. Single- We named this phenomenon voltage-dependent potentiation (VDP) and found it molecule fluorescence can reveal these dynamic events, but is traditionally developed and recovered within hundreds of milliseconds. Using direct applica- limited to low concentrations of fluorophores only suitable for studying high tion of diC8-PI(4,5)P2 and specific hydrolysis of PI(4,5)P2 by rapamycin- affinity reactions. Here, we show how a combination of fluorescence resonance recruitable lipid 5-phosphatase, we demonstrated that, PI(4,5)P2 inhibits zELK energy transfer (FRET) and zero-mode waveguide nanofabricated devices can channels, and its depletion suppresses the VDP. In addition, the VDP can be be used to study single-molecule association dynamics at the micromolar to manipulated by multiple structural perturbations including removing, mutating millimolar concentrations necessary for many relevant signaling pathways. or replacing the intracellular N-terminal EAG domain as well as the C-terminal As an exemplar system, we apply this approach to cyclic nucleotide binding CNBHD. Further, combining transition metal ion FRET and incorporation of a at its receptor domain from hyperpolarization cyclic nucleotide-gated (HCN) fluorescent noncanonical amino acid L-Anap, we measured the distances between ion channels. Our observations reveal the underlying dynamic events during positions in the EAG domain and CNBHD during voltage-dependent channel acti- cyclic nucleotide binding: an initial encounter complex when the binding site vation and deactivation. We found there was an apparent change in the distance is in its receptive state, followed by an isomerization of the bound complex between the EAG domain and CNBHD with kinetics that match the on- and that traps the ligand.

BPJ 7705_7715 40a Sunday, February 12, 2017

201-Plat experiments indicate that the C-terminal region (C-linker/CNBD) undergoes a Investigating Ligand Binding to HCN Channels by Surface Plasmon conformation change to transduce cyclic nucleotide binding into channel activa- Resonance tion, though the exact nature of this change remains unclear. To address this Purushottam Tiwari1, Aykut U¨ ren1, Tinatin I. Brelidze2. question, we first sought a stable and well-behaved bacterial CNG channel suit- 1Oncology, Georgetown University School of Medicine, Washington, DC, able for spectroscopic studies. We used fluorescence-detection size-exclusion USA, 2Pharmacology and Physiology, Georgetown University School of chromatography to screen a library of bacterial orthologs, which identified Medicine, Washington, DC, USA. several candidates that could be expressed and purified at high levels. Flux Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels studies of liposome-reconstituted channels demonstrated that these orthologs control cardiac and neuronal rhythmicity. HCN channels contain cyclic nucle- are fully functional and are activated by cyclic nucleotides. We then mutated otide-binding (CNB) domain in their C-terminal region linked to the pore- the endogenous Cys residues in these channels before introducing Cys residues forming transmembrane segment with a C-linker. The C-linker couples the throughout the C-terminal region for spin label and fluorophore attachment. conformational changes caused by the direct binding of cyclic nucleotides in Inter-subunit double electron-electron resonance (DEER) measurements will the CNB domain to the pore opening. Surface plasmon resonance (SPR) is a be used to detect large-scale structural changes in these channels induced by powerful biophysical tool for quantitatively investigating ligand-protein and cyclic nucleotide binding, while transition metal FRET (tmFRET) will be used protein-protein interactions. Here we used SPR to detect ligand binding to the to characterize subtle, localized changes to channel structure following activa- isolated C-linker/CNB domain of HCN channels. The isolated C-linker/CNB tion. Our ultimate goal is to use these two spectroscopic techniques to charac- domains of wild-type (WT) and L586W mutant HCN2 channels were immobi- terize small- and large-scale rearrangements induced by cyclic nucleotide lized on a NTA sensor chip and cAMP was injected over the protein coated sur- binding, allowing us to generate a complete structural model for CNG channel face. The mutant C-linker/CNB domain has been used before for ligand binding activation. studies based on the changes in the fluorescence of the introduced tryptophan upon ligand binding. From the cyclic nucleotide concentration dependent SPR Platform: Protein Stability, Folding, and responses, we determined the binding affinity for cAMP to be 6.3 þ 2.6 mM Chaperones I for the WT and 10.8 þ 2.3mM for the mutant C-linker/CNB domains. The bind- ing affinity determined for the mutant L586W C-linker-CNB domains deter- þ m 204-Plat mined with SPR is in agreement with the binding affinity of 13 2 M Integrated In Vivo and In Silico Studies of Cotranslational Protein Folding determined with the fluorescence-based method. These results indicate that as a Function of Translation Rate SPR is well suited for the detection of binding of known HCN channel ligands. Ian M. Walsh1, Shuxiang Li2, Adrian H. Elcock2, Patricia L. Clark1. Therefore, SPR can be used to identify novel HCN channel regulators for treat- 1Department of Chemistry & Biochemistry, University of Notre Dame, Notre ment of diseases associated with abnormal functions of these channels, such as Dame, IN, USA, 2Carver College of Medicine, University of Iowa, Iowa City, epilepsy and cardiac arrhythmias. IA, USA. In order for a protein to exhibit its proper function, it must first fold correctly. 202-Plat Most of what we know about protein folding comes from in vitro studies of Drug Interaction at the Lipid Bilayer-Potassium Channel Interface small, single-domain proteins. However, larger and more complex proteins 1 1 1 1 Nina Ottosson , Malin Silvera˚ Ejneby , Xiongyu Wu , Samira Yazdi , often fail to fold efficiently in vitro, yet fold to high yield in cells. One striking Peter Konradsson1, Erik Lindahl2, Fredrik Elinder1. 1 2 difference between protein folding in vitro and in vivo is how folding begins. Linko¨ping University, Linko¨ping, Sweden, Stockholm University, In vitro, protein folding begins from an enormous ensemble of random confor- Stockholm, Sweden. mations of full length polypeptide chains, while in vivo a protein can begin to The naturally occurring resin acid dehydroabietic acid (DHAA) opens a fold as it is synthesized by the ribosome. Vectorial appearance of the nascent voltage-gated K channel by shifting the voltage dependence of the channel acti- protein chain during translation is a universal feature of every protein in the pro- vation to more negative voltages. We synthesized 140 DHAA-derivatives and teome, yet its effect on protein folding is poorly understood. Our lab has created found that subtle alterations in the chemical structure of DHAA can have a YKB, a protein construct with two mutually exclusive native states (YK-B and large effect on the channel opening. Several of the channel-opening DHAA de- Y-KB), to study the effects of translation on folding mechanisms. Synonymous rivatives reduced excitability in dorsal root ganglion neurons, suggesting a pos- mutations to YKB alter the ratio of the YK-B and Y-KB native structures, yet sibility to act against diseases with increased cellular excitability. Here we have identical protein sequences. From this we can infer that vectorial appear- searched for the mechanism and site of action for the DHAA derivatives. We ance affects YKB folding, but the ratios provide little molecular-level detail. expressed the non-inactivating Shaker K channel in Xenopus oocytes and stud- We have developed a coarse-grain computational model to simulate YKB ied the ion currents with a two-electrode voltage clamp technique. By utilizing cotranslational folding. This model accurately reproduces experimental results the ILT-mutant, which separates the final channel-opening transition from the for YKB refolding in vitro and co-translational folding in vivo. These simula- earlier transitions, we found that the DHAA derivatives had an almost isolated tions provide us with details that cannot be learned through experiments, m effect on the channel-opening transition. 100 M of one DHAA derivative including specific molecular trajectories and the effects of altering translation affected this last transition by 65 mV. We also found that the charge profile rate on the YKB folding mechanism. Integrating information gleaned from sim- of the voltage sensor S4 was critical for the effect. Addition of positively ulations with targeted experimental studies will enable unprecedented insight charged arginines in specific positions increased the channel opening effects into in vivo folding mechanisms, particularly the effects of varying translation of the negatively charged DHAA derivatives, while addition of arginines on rate on cotranslational folding and overall folding efficiency. the opposite side of the S4 helix had opposing effects, suggesting that the DHAA derivatives support or prevent rotation of S4 to support or prevent chan- 205-Plat nel opening. In general, the DHAA derivatives affected channel closing kinetic The Ribosome Alters the Folding of a Multidomain Nascent Protein but not channel opening kinetic suggesting a rather specific effect and site of Lisa Alexander, Daniel Goldman, Ignacio Tinoco, Carlos J. Bustamante. action. In-silico docking, molecular dynamics, and mutational analyses sug- UC Berkeley, Berkeley, CA, USA. gested a binding site in the cleft between the transmembrane segments S3 Protein folding is often studied in the context of full-length polypeptides in solu- and S4. We conclude that certain resin-acid derivatives bind at the interface be- tion. However, the post-translational folding pathway may not recreate the tween the lipid bilayer and S3 and S4 of the channel’s voltage-sensor domain, pathway of folding when a protein is being synthesized by the ribosome. The ef- to electrostatically facilitate K channel opening. fects of the ribosome and the rate of translation on folding are not well understood, partially due to the difficulty of probing the nascent chain without also affecting 203-Plat the ribosome. Using optical tweezers, we can look in detail at the nascent chain Spectroscopic Studies of a Bacterial Cyclic Nucleotide-Gated Ion Channel at the single-molecule level with minimal perturbation to the ribosome. Previous Zachary M. James1, Eric G. Evans2, William N. Zagotta1. studies have shown that the ribosomal surface can act electrostatically to slow the 1Physiology & Biophysics, University of Washington, Seattle, WA, USA, kineticsof folding. Those studies useda linker tovarythe distance fromthe surface 2Chemistry, University of Washington, Seattle, WA, USA. and did not look at changes in sequence availability or multidomain proteins. To Cyclic nucleotide-gated (CNG) channels are members of the Kv channel super- further understand cotranslational folding, we are studying the folding pathway of family and play a crucial role in the phototransduction and olfactory transduction a two-domain calcium-binding protein calerythrin. Our results show that the full- pathways, where they generate the initial electrical signal following sensory length protein in solution folds robustly through a C-domain intermediate, but transduction. CNG channels are directly regulated by cyclic nucleotides, which truncated versions of the protein can fold to an N domain or a misfolded state. bind to a C-terminal cyclic nucleotide-binding domain (CNBD) connected During translation, the C-terminal residues required for native folding are not to the pore by an intervening C-linker domain. Mutagenesis and crosslinking yet available, but the other observed intermediates could fold, in principle. By

BPJ 7705_7715 Sunday, February 12, 2017 41a observing the folding at various sequence positions, and thus nascent chain Understanding the way in which proteins fold inside the cell is of fundamental lengths, we found that the ribosome can not only slow folding, but can also affect importance to both biology and medicine. In the E. Coli cell, proteins are syn- the unfolding rates, particularly increasing the unfolding rate of the misfolded thesized on the ribosome in the presence of molecular chaperones, Trigger state. The ribosome thus prevents the formation of this unproductive state via Factor and DnaK. While both the ribosome and molecular chaperones are two mechanisms: decreasing the probability of folding and decreasing the stability known to be important to protein folding in the cell, it has been nearly impos- of the folded state. This study gives further insight into the importance of the ribo- sible to isolate their independent roles in the folding process since cells die at some to regulate protein structure, and opens up new questions about the interplay temperatures over 30C when Trigger Factor and DnaK are deleted simulta- between elongation and folding. neously. In this work, we are able to access this challenging experimental con- dition and study the interplay between the ribosome and chaperones by 206-Plat synthesizing a model globin protein in a bacterial cell-free system derived Structural Investigation of an Immunoglobulin Domain on the Ribosome from a Trigger Factor- deleted cell strain in the presence of an in-house- using NMR Spectroscopy designed peptide inhibitor of DnaK. Surprisingly, we find that translation 1 1 1 Anais M.E. Cassaignau ,He´le`ne M.M. Launay , Christopher A. Waudby , through the ribosome is sufficient to grant solubility to the full-length newly 1 1 1 Tomasz Wlodarski , Maria-Evangelia Karyadi , Amy L. Robertson , synthesized protein. Ongoing studies based on time-resolved fluorescence 1 2 2 Xiaolin Wang , Carlo Camilloni , Michele Vendruscolo , anisotropy in the frequency-domain and multidimensional nuclear magnetic 3 1 1 Cheryl A. Woolhead , Lisa Cabrita , John Christodoulou . resonance yield additional details on the quality of the de novo-produced pro- 1Institute of Structural and Molecular Biology, University College London, 2 tein, and on the intriguing interplay between the ribosome and chaperones in London, United Kingdom, Department of Chemistry, University of ensuring its correct folding. Cambridge, Cambridge, United Kingdom, 3Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, 209-Plat University of Glasgow, Glasgow, United Kingdom. Trigger Factor Boosts the Work Done by Protein Folding under Force Successful protein folding is central to all biological cellular processes with a Shubhasis Haldar, Rafael Tapia-Rojo, Julio M. Fernandez. large portion of the proteome able to begin to acquire its three-dimensional biological science, Columbia University, New York, NY, USA. structure in a co-translational manner during its biosynthesis on the ribosome. Protein folding under force generates mechanical work, which is integral to The vectorial emergence of the nascent polypeptide from the exit tunnel and its several biological processes including muscle contraction, proteasomal degra- attachment to its parent ribosome results in differences between the details of dation, and protein translation. As molecular chaperones affect protein folding, the folding process of isolated polypeptides and that on the ribosome. we hypothesized that chaperones would modulate the mechanical work gener- We have developed a strategy to enable the study of co-translational folding us- ated from protein folding. Trigger Factor (TF) is a model chaperone from E. ing solution-state NMR spectroscopy, the only technique able to characterize this coli, which is found intracellularly in both ribosome-associated and free cyto- dynamic process at atomic resolution. Using isotopically-labelled ribosome- solic states. Here, we apply Magnetic Tweezers-based single molecule force nascent chain complexes (RNCs), we have determined a high-resolution, struc- spectroscopy to investigate the effect of TF in the mechanical folding of a sub- tural description of protein folding on the ribosome via snapshots that mimic the strate, protein L (PL). We find that TF increases the folding probability by up to emergence of an immunoglobulin-like domain within a multidomain protein. 40% at ~8 pN. The half-maximal TF activity, K1/2, is highly force dependent, Our NMR results reveal the structure and dynamic features of how conforma- ranging from 0.6 mM at 6.2 pN to 27.5 mM at 8.9 pN. The functional conse- tional space is sampled by a fledgling nascent polypeptide as it converts into quence of this TF-assisted mechanical folding is an increase in the effective its folded state and demonstrate that the entire immunoglobulin domain has to work generated, calculated as the product of the force, the PL folding contrac- be emerged from the tunnel before native folding is possible. These findings tion length, and the TF-dependent folding probability. The maximal activity of contrast with analogous studies of C-terminal truncations of this domain, indi- TF (at 10 mM and 8.4 pN) contributes 40 zJ to the work done by protein folding, cating significant differences between folding on the ribosome and that in bulk comparable to the energy delivered by myosin motors. 85% of the mechanical solution. The ribosome itself is shown to influence the process of folding; we contribution from TF is achieved between 500 nM and 2 mM TF, in the range of use a combination of protein engineering and NMR dynamics to describe the chaperon’s cellular concentration. This is the first study of the influence of a residue-specific sites of nascent-chain-ribosome interactions of different magni- molecular chaperone in the work done by protein folding, which could have tude of strength, and chemical shift-restrained MD simulations of RNCs to un- important consequences in the translation process, helping polypeptide synthe- derpin our spectral observations. The studies presented are providing the first sis and assisting protein folding under force. high-resolution insights of these fundamental processes and begin to shape our understanding of the energy landscape sampled by a nascent chain on the cusp 210-Plat of initiation of folding on the ribosome. Resolution of the Time Sequence of Fast Folding Transition 207-Plat by the ‘‘Transfer-Quench’’ Method Accurate Prediction of Cellular Co-Translational Folding Indicates Gil Rahamim, Dan Amir, Elisha Haas. Proteins can Switch from Post- to Co-Translational Folding Life Sciences, Bar Ilan University, Ramat Gan, Israel. Daniel A. Nissley. The rates and efficiency of the folding transition of globular protein might be Chemistry, The Pennsylvania State University, University Park, PA, USA. enhanced by early formation of few sub-domain structures. Ultrafast Fo¨rster The rates at which domains fold and codons are translated are important factors resonance energy transfer (FRET) based methods are ideal for characteriza- in determining whether a nascent protein will co-translationally fold and func- tion of the transient ensembles of refolding molecules. However, each site tion or misfold and malfunction. Here we develop a chemical kinetic model that specific labeling modification might affect rates of folding of near neighbor calculates a protein domain’s co-translational folding curve during synthesis structural elements and thus limit the ability to resolve fine differences in rates using only the domain’s bulk folding and unfolding rates and codon translation of folding of these elements. Therefore, it is highly desirable to be able to rates. We show that this model accurately predicts the course of co-translational study the rates of folding of two or more neighboring sub-domain structures folding measured in vivo for four different protein molecules. We then make using a single mutant in order to facilitate resolution of the order and interde- predictions for a number of different proteins in yeast and find that synonymous pendence of such steps. Here we report the development of the ‘‘Transfer- codon substitutions, which change translation-elongation rates, can switch Quench’’ method for measuring the rate of formation of two structural some protein domains from folding post-translationally to folding co-transla- elements using a single triple-labeled mutant. This method is based on FRET combined with fluorescence quenching. We placed the donor and tionally—a result consistent with previous experimental studies. Our approach a explains essential features of co-translational folding curves and predicts acceptor at the loop ends’ and a quencher at an -helical element involved how varying the translation rate at different codon positions along a transcript’s in the node forming the loop. The folding of the triple labeled mutant is moni- coding sequence affects this self-assembly process. tored at the acceptor emission. The formation of non-local contact (loop closure) increases the time dependent acceptor emission while the closure 208-Plat of the labeled helix turn reduces this emission. The method was applied in Protein Sculpting: Probing the Interplay between the Ribosome a study of the folding mechanism of the B domain of staphylococcal protein and Molecular Chaperones in Protein Folding in the Cell A. Only natural amino acids were used as probes and thus possible structural Rayna M. Addabbo1,2, Matthew D. Dalphin1,2, Yue Liu2, perturbations were minimized (Tyr and Trp residues as donor and acceptor at Miranda F. Mecha1,2, Silvia Cavagnero1,2. the ends of a long loop between helices I and II, and Cys residue as a quencher 1Biophysics Graduate Program, University of Wisconsin-Madison, Madison, for the acceptor). We found that the closure of the loop is formed with the WI, USA, 2Department of Chemistry, University of Wisconsin-Madison, same rate constant as the nucleation of helix II in line with the nucleation- Madison, WI, USA. condensation model.

BPJ 7705_7715 42a Sunday, February 12, 2017

211-Plat that steric pressure among crowded proteins can induce mixing of lipid do- Mechanically Unfolded Titin Immunoglobulin Domains Refold Faster and mains. However, prior to our work, the dynamics of this process had yet to More Accurately in Presence of Chaperone Alpha-B-Crystallin be investigated. The influence of protein steric pressure on dynamics is impor- Yong Li, Wolfgang A. Linke. tant for understanding time-dependent processes in cell membrane-derived bio- Institute of Physiology, Ruhr Univ. Bochum, Bochum, Germany. materials and highly transient lipid rafts in cells. Therefore we designed The spring region of the giant muscle protein titin contains many experiments that allowed us to examine the process by which protein steric immunoglobulin-like (Ig) domains, which unfold and refold under low (physio- pressure dissolved lipid membranes. Specifically, fluorescence microscopy logical) stretch forces. Small heat shock proteins such as alphaB-crystallin was used to examine the targeted binding of proteinaceous particles to (aBC) translocate under physiological or pathological stress to the titin springs. phase-separated, supported lipid bilayers. Highly localized binding of these To better understand this protective function we studied the unfolding-refolding particles within the liquid ordered (Lo) domains resulted in a highly crowded behavior of an 8-Ig-domain titin construct (I91)8 by single-molecule AFM force environment, leading to a build-up of steric pressure. The alleviation of this ste- spectroscopy, in the absence/presence of recombinant aBC (pH7; pH6; or pH5) ric pressure induced mixing of the Lo domains with the surrounding liquid or ‘control’ protein of similar size. Titin Ig domains were unfolded at 175 pN con- disordered (Ld) phase. With the development of a first-principles mass transfer stant force applied for a variable ‘‘denature’’ time (tD), then the force was set to model, the dynamics of this mixing transition were observed as a function zero for a variable ‘‘quench’’ time (tQ) to allow for domain refolding, and finally of the steric pressure among proteins. At sufficiently high steric pressure, a ‘‘probe’’ pulse (175 pN; tP = 5 s) was applied to test how many domains had re- rapid dissolution of Lo domains occurred. As steric pressure was decreased, folded. Interestingly, Ig domain unfolding kinetics were little affected by aBC. mixing became more gradual and occurred in a step-wise process where small However, upon lowering pH from 7 to 6, the refolded fraction (number of refolded lipid clusters were initially ejected from Lo domains. Time-dependent dissolu- Ig domains during tQ indexed to number of unfolded Ig domains during tD) tion data for Lo domains was in quantitative agreement with the mass-transfer decreased slightly, indicating domain misfolding. At pH5, the refolded fraction model. Diffusion coefficients derived from the model indicate that lipids un- dropped by half and ~50% of titin Ig domains showed misfolding events, an effect dergo diffusion as clusters, rather than single lipids. We also examined systems independentof tD (variation, 2 - 40 s). Importantly, aBC (10 mMor20mM) normal- in which proteins bound to the Ld phase. Our results strongly suggest that ized the refolded fraction to values observed at pH7, whereas control protein had the degree of protein crowding and the phase to which proteins are targeted no such protective effect. The refolded fraction depended strongly on tQ (variation, control the rate and mechanism of steric pressure-induced mixing. This phe- 0.5 - 10 s), under all experimental conditions. Ig domain refolding kinetics were nomena is applicable to the development of many biologically derived mate- greatly slowed by lowering pH from 7 to 5, as quantified on refolded fraction rials, including high-density arrays, microfluidic networks, and biosensors. vs. tQ plots, on which means were fit by simple exponentials. Again, aBC normal- Controlled mixing also provides fundamental insights to the impact of proteins ized the refolding kinetics to those observed at pH7 in the absence of aBC,whereas on the stability and dynamics of lipid rafts in cells. control protein had no such effect. We conclude that aBC speeds up titin Ig domain refolding (novel foldase activity!) and protects from domain misfolding, espe- 214-Plat cially under acidic stress, which is frequently encountered in muscle cells. Unique Modification of Membrane Structure by Lithium: A Molecular Dynamics Study Platform: Membrane Physical Chemistry I James Kruczek1, See-Wing Chiu2, Eric Jakobsson2, Sagar A. Pandit3. 1University of Florida, Tampa, FL, USA, 2University of Illinois, Urbana, IL, 3 212-Plat USA, University of South Florida, Urbana, IL, USA. GM1 Softens the Membrane, Induces Domains and Causes Spontaneous Lithium is profoundly different from other ions in the body in multiple ways. Tubulation in Giant Vesicles Extracellular concentrations of calcium, sodium, potassium, and hydrogen Rumiana Dimova1, Tripta Bhatia1, Raktim Dasgupta1,2, Nico Fricke1, ions must be closely regulated for well-being. On the other hand, the body Jaime Agudo-Canalejo1, Reinhard Lipowsky1. does not closely regulate lithium but rather adapts reasonably well to lithium 1Theory & Bio-Systems, Max Planck Institute of Colloids and Interfaces, concentration variation of over a thousandfold, in spite of the fact that lithium Potsdam, Germany, 2Raja Ramanna Centre for Advanced Technology, interacts with many biological molecules. This stability coupled with interac- Indore, India. tiveness implies the existence of myriad mutually compensating lithium- The ganglioside GM1 is present in neuronal membranes at elevated concentra- modulated processes, which are almost completely not understood. In this study tions with an asymmetric spatial distribution. It is known to generate curvature we explore the interaction of lithium, sodium, potassium, and rubidium with and can be expected to strongly influence the neuron morphology. To elucidate bilayer membranes comprised of POPC, using molecular dynamics simula- these effects, we incorporated GM1 into giant unilamellar vesicles (GUVs) tions. Lithium, but not any of the others, forms a long-lasting noncovalent made of POPC. We found that even a few mol% of GM1 soften the fluid bilayer bond simultaneously with the headgroup phosphate and the CO group of the significantly as measured using fluctuation analysis, vesicle electrodeformation Sn2 chain. This interaction significantly alters the structure and the surface and micropipette aspiration. At room temperature and for GM1 fractions at and area of the membrane. above ~5 mol%, we detect the formation of GM1-rich gel-like domains. We use 215-Plat fluorescence microscopy to build a partial phase diagram of the binary mixture Measuring Lipid Membrane Properties using a Mechanosensitive (Fricke and Dimova, Biophys. J. 2016, in press). After diluting the vesicle sus- Fluorescence Probe pension with the native buffer, we observe spontaneous formation of nanotubes Adai Colom Diego, Marta Dal Molin, Saeideh Soleimanpour, suggesting desorption of GM1 from the outer membrane leaflet and generation Emmanuel Derivery, Marcos Gonzalez Gaitan, Stefan Matile, Aure´lien Roux. of spontaneous curvature stabilizing the tubes. Employing electroporation of Universite de Geneve, Geneve, Switzerland. GUVs, we assess the GM1 asymmetry. The associated spontaneous curvature To measure the chemical-mechanic states of lipid membranes, once needs is measured using two approaches: micropipette aspiration of GUVs and pull- various tools, many of which being incompatible with cell biology protocols. ing of inward and outward membrane tubes via bead manipulation with optical Applying lessons from nature, we developed a mechanosensitive fluorescent tweezers (Dasgupta and Dimova, J. Phys. D. Appl. Phys. 47:282001, 2014). probe, the twisted dithienothiophene. This push-pull probe, change planariza- GM1 plays a crucial role in connection with receptor proteins. However, our tion state in function of his environment, and we have taken full advantage results that GM1 decreases bending rigidity, causes spontaneous curvature of this mechano-probe potential and we calibrated based on membrane tension, and induces phase separation in the membrane point to an additional important fluidity and different lipid composition by measuring the push-pull fluores- role of this ganglioside, namely shaping neuronal membranes. This work is part cence lifetime. Likewise, we are able to use this fluorescent probe on life cells, of the MaxSynBio consortium, which is jointly funded by the Federal Ministry for visualize differences between organelles, as well as to distinguish lipids of Education and Research of Germany and the Max Planck Society. properties among cells cultured on classic plates or in extracellular matrix. 213-Plat 216-Plat Induced Mixing of Phase-Separated Lipid Bilayers by Steric Pressure DPPC/cholesterol Revisited: Interaction Models to Explain the Excess between Adsorbed Proteins Heat Capacity in Unilamellar Vesicles Wade Zeno1, Kaitlin E. Johnson2, Darryl Y. Sasaki3, Marjorie L. Longo2. Paulo F. Almeida, Emmanuel Tejada, Faith Carter, Antje Pokorny. 1Biomedical Engineering, University of Texas at Austin, Austin, TX, USA, Chemistry, Univ North Carolina Wilmington, Wilmington, NC, USA. 2Chemical Engineering, University of California, Davis, Davis, CA, USA, The binary lipid system DPPC/cholesterol has been studied since the 1970s. 3Sandia National Laboratories, Livermore, CA, USA. Strangely enough, the nature of the interaction between the phospholipid and Our study examined the steric pressure-induced mixing and reversibility of cholesterol remains unclear. Also strangely enough, for decades, the thermody- phase-separated phospholipid regions. It has been previously demonstrated namics of these binary mixture have been investigated mainly in multilamellar

BPJ 7705_7715 Sunday, February 12, 2017 43a vesicles (MLVs). More recently, giant unilamellar vesicles (GUVs) have been asymmetric composition to quantify the incorporation of negatively charged used, which are excellent to study lipid phase separation, especially by fluores- POPG into POPC liposomes. The adjustment of cyclodextrin and lipid concen- cence confocal microscopy, but do not easily lend themselves to calorimetry. trations below the maximum lipid-binding capacity of cyclodextrins allows a However, the heat capacity of DPPC across the main phase transition is similar controlled POPG transfer without the need of donor liposomes. Furthermore, in GUVs and in large unilamellar vesicles (LUVs) but quite different from the zeta potential clearly differentiates between asymmetric and symmetric that in MLVs. Much of the attention in the thermodynamics of the phase transi- POPG incorporation, quantifies the degree of lipid asymmetry, and determines tion in DPPC/cholesterol has been concerned with understanding the heat capac- the total yield of lipid exchange. Our approach represents an easy to use method ity in MLVs. Here we turn our attention to the DPPC/cholesterol binary system for the efficient incorporation of negatively charged lipids into the outer mem- in LUVs, which we think is a much more relevant type of vesicle to understand brane leaflet of liposomes at physiologically relevant lipid contents. the molecular interactions between DPPC and cholesterol. We compare the experimental heat capacity (melting) curves in LUVs with the results of Monte 219-Plat Carlo calculations using various models of the interaction between these lipids, Investigating the Effects of the Membrane Dipole Field on the Structure including complex formation and simple pairwise interactions. and Function of a Model Membrane Protein This work has been supported by NSF grant CHE-1464769. Cari M. Anderson, Lauren J. Webb. Chemistry, University of Texas at Austin, Austin, TX, USA. 217-Plat Lipid bilayer membranes are composed of hundreds of lipids, sterols and pro- Characterization of the Physiochemical Interactions between LNPs and teins, which organize in to a heterogeneous, structural scaffold that controls the Endosomal Lipids: A Rational Design of Gene Delivery Systems critical elements of biological function. The interactions of all of these mole- Nandhitha Subramanian, Yoav Atsmon-Raz, Peter D. Tieleman. cules are mostly non-covalent and electrostatic in nature. These interactions Department of Biological Sciences & Centre for Molecular Simualtion, and the diversity of molecules, along with ordered water molecules at the University of Calgary, Calgary, AB, Canada. lipid-water interface gives rise to a large electrostatic field that traverses the Lipid nanoparticles (LNPs) are a class of materials, with each interacting in a lipid bilayer. The whole field can be broken down in to three components- complex fashion to form specialized bio-containers carrying therapeutic drugs transmembrane field, surface field and dipole field. We have extensively stud- and strands of nucleic acids such as small interfering RNA (siRNA)1. LNPs are ied the membrane dipole field, which propagates from the interior of the lipid at the forefront of the rapidly developing field of nanotechnology with several bilayer to the lipid head group-water interface, using vibrational Stark effect potential applications in drug delivery, clinical medicine, and research. Despite spectroscopy paired with molecular dynamics simulations. With our knowl- holding a significant promise for reaching the goal of controlled and site edge of how the dipole field can be altered due to a change in lipid bilayer specific drug delivery, the engineering of efficient LNPs is limited by the composition, we are exploring what role this field plays in controlling the trans- lack of knowledge about the structure of LNPs, the roles of their individual port of ions through a membrane via transmembrane protein channels (TPCs). components and the physical interactions with other biomolecules2. Because Gramicidin has been accepted as a good model for TPCs due to its ability to of this, characteristics such as the encapsulation efficiency, polydispersity, selectively transport ions with a þ1 charge through the pore it creates when and stability of LNPs are not predictable. One of the biggest problems of in its channel conformation. Using this well characterized model TPC we LNP gene therapy is the molecular details of the cellular processes that deter- hope to elucidate how the structure and function can be changed via non- mine the efficiency of intracellular drug delivery is still unclear. Studies have covalent interactions with other lipids, water molecules and sterols, specifically shown that LNPs enter cells via the endocytic pathway and are engulfed by arising from the membrane dipole field. Using VSE spectroscopy, we have endosomes. The delivery of siRNA is substantially reduced, as ~70% of the been able to begin to measure how gramicidin alters the dipole field of a internalized siRNA undergoes exocytosis through egress of LNPs from late pure phospholipid bilayer in the form of small unilamellar vesicles (SUVs). endosomes/lysosomes3. In this project, we used advanced molecular dynamics We are studying how the transport of þ1 cations through the gramicidin chan- simulations to understand the molecular basis of the structure of LNPs based on nel and the structure of the TPC are affected by both small and large perturba- their composition. Our simulations also provided the first detailed molecular- tions of the electrostatic field by intercalating sterols in to the lipid bilayer. level view of the interactions of the LNP components with endosomal lipids. Here, we also studied the change in interactions between the LNPs and endo- Platform: Kinesins and Dyneins somal lipids by varying ion concentration and altering pH. Results of our simu- lation will provide a route to the rational design of new LNPs that address 220-Plat safety concerns and ensure effective delivery to accelerate the translation of Lis1 has Two Distinct Modes of Regulating Dynein’s Mechanochemical engineering lipid-based nanoparticles towards the clinic. Cycle References Michael A. Cianfrocco, Morgan E. DeSantis, Zaw M. Htet, Phuoc T. Tran, 1. Semple, S.C., et al. (2010) Rational design of cationic lipids for siRNA Andres E. Leschziner, Samara L. Reck-Peterson. delivery. Nat.Biotechnol. 28,172-176 Cellular and Molecular Medicine, University of California - San Diego, 2. Huang, L. and Liu, Y. (2011) In vivo delivery of RNAi with lipid-based La Jolla, CA, USA. nanoparticles. Annu.Rev.Biomed. Eng. 13, 507-530 Cytoplasmic dynein-1 (‘‘dynein’’) is a minus-end-directed microtubule-based 3. Sahay G., et al. (2013) Efficiency of siRNA delivery by lipid nanoparticles is motor that couples ATP hydrolysis to force generation to move diverse cargos. limited by endocytic recycling. Nat.Biotechnol. 31,653-658. Dynein is a single-chain AAAþ ATPase that contains 6 AAAþ domains, where AAA1-4 bind nucleotide and AAA1 drives the motor. Lis1 is a conserved and 218-Plat ubiquitous dynein regulator. Previously, we showed that Lis1 binds to dynein at Tuning Membrane Asymmetry: Controlled Uptake of Negatively Charged AAA4 and causes dynein to slow down and remain attached to microtubules, Lipids into the Outer Leaflet of Liposomes even in the presence of ATP, which usually releases dynein from its track. Inter- Marie Markones1, Carina Zorzin1, Louma Kalie1, Sebastian Fiedler2, estingly, while AAA3 is occupied by ADP when dynein is walking, either the Heiko Heerklotz2,3. absence of nucleotide or the presence of ATP lead to a motor that behaves like 1Institute for Pharmaceutical Sciences, University of Freiburg, Freiburg, its Lis1-regulated state. This observation led us to hypothesize that Lis1 acts Germany, 2Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, through AAA3. To test this we determined how the nucleotide state at AAA3 ON, Canada, 3Institute for Pharmaceutical Sciences/BIOSS Centre for affects Lis1’s regulation of dynein. When AAA3 is nucleotide-free, Lis1 Biological Signalling Studies, University of Freiburg, Freiburg, Germany. increases dynein’s microtubule binding affinity, as we had previously observed, The presence of negatively charged lipids in the outer leaflets of cellular mem- Surprisingly, however, when AAA3 contains ATP, Lis1 has the opposite effect, branes is a key component of many biological membranes. For example, eukary- leading to dynein’s detachment from microtubules. High-resolution cryo-elec- otic organisms accumulate negatively charged lipids on their extracellular tron microscopy structures of dynein-Lis1 complexes revealed the basis for membrane leaflets during programmed cell death through apoptosis. As a general these puzzling effects. While a single Lis1 beta propeller (Lis1 is a dimer) binds feature, the outer leaflets of bacterial cellular membranes exhibit high contents to dynein in the AAA3-(no nucleotide) state, a second Lis1 beta propeller is of anionic phosphatidylglycerol (PG). Recently, a cyclodextrin-based lipid ex- bound to the motor in the AAA3-ATP state. This novel second site is located change assay has been introduced that transports phospholipids from the outer on dynein’s coiled coil ‘‘stalk’’, which connects dynein’s motor domain to its leaflet of donor liposomes to that of acceptor liposomes, thereby generating microtubule binding domain. Importantly, the sequence of this site is conserved model membranes of asymmetric lipid composition. In an effort to master and only in those dyneins that are regulated by Lis1. Our work revealed that Lis1 further develop the cyclodextrin method, we utilize the zeta potential of mixed can act either as a microtubule anchor or a release factor for dynein, depending 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG)/1-palmitoyl-2- upon the nucleotide occupancy at AAA3. We propose a new model for how oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes of symmetric and Lis1 serves as a dual regulator of dynein activity in cells.

BPJ 7705_7715 44a Sunday, February 12, 2017

221-Plat density and arrangement are highly controlled in vivo, it is likely that microtu- The Structure of Complete Human Dynein-1 and its Mechanism bule network organization is a key component in the regulation of cargo trans- of Activation port. To better understand how network geometry controls multi-motor cargo Kai Zhang, Helen Foster, Andrew Carter. transport, we have tracked quantum-dot molecular cargoes decorated with Structural Studies, MRC Laboratory of Molecular Biology, Cambridge, different numbers of kinesin-1 motors through microtubule networks of different United Kingdom. densities and intersection geometries. Using single molecule motility assays and Cytoplasmic dynein-1 is a 1.4 MDa motor protein, which performs essential STORM imaging of the network, we have found that cargoes with greater cargo transport to the minus end of microtubules. Long distance movement of numbers of motors travel via highly non-linear pathways, while single motor single dynein-1 molecules is induced by binding to its 23-subunit cofactor dynac- transport is confined to single filaments. These experiments help to illuminate tin and a cargo-adaptor protein. Here we present the cryo-EM structure of iso- the mechanisms by which motor number and network geometry are used in com- lated human dynein-1 in its inhibited, phi-particle conformation. We achieved bination to carefully direct transport of essential cargoes throughout the cell. aresolutionof3.8A˚ for the motor domains and 8.4A˚ in the highly-flexible tail region. We reveal the architecture of the complete complex; identify all subunits, 225-Plat build helices in the tail and a full atomic model for the human dynein-1 motor An Allosteric Gear Shift Mechanism in Eg5 Enhances Mechanochemical domains. Mutagenesis to disrupt the interface between the two motor domains Coupling and Shifts the Force-Velocity Landscape is not sufficient to activate dynein-1. Instead dynactin is required to orient the Joseph Muretta1, Babu J.N. Reddy2, Guido Scarabelli3, David D. Thomas1, motors in a parallel fashion so that they can interact correctly with microtubules, Barry Grant3, Steven Gross4, Steven Rosenfeld5. thus explaining the structural mechanism of dynein-1 activation. 1Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA, 2University of California Irvine, Irvine, CA, USA, 222-Plat 3University of Michigan, Ann Arbor, MI, USA, 4University of California The Power Stroke Distance of Human Cytoplasmic Dynein Irvine, Irvine, MI, USA, 5Cleveland Clinic, Cleveland, OH, USA. 1 2 1 Yoshimi Kinoshita , Taketoshi Kambara , Kaori Nishikawa , Microtubule motors control a diverse collection of physiologies, including cell Motoshi Kaya1, Hideo Higuchi1. 1 2 division, organelle traffic, and microtubule dynamics. Fourteen kinesin family Grad Sch of Sci, The Univ Tokyo, Tokyo, Japan, QBiC, RIKEN, members are involved in mitosis. The best studied of these is Eg5, which is a Osaka, Japan. poorly processive motor that can occupy a two head-strongly-bound state on Cytoplasmic dynein is a motor protein moving along microtubules, and plays the microtubule. It works primarily in ensembles of tetramers, cross-linking important roles in vesicle transport and mitosis. To understand the molecular parallel and anti-parallel microtubules in the maturing spindle. Chemical abla- mechanism of dynein motility, we measured the efficiency of FRET from dynein tion of Eg5 arrests mitosis. Proteomic screens have identified post-translational ring-BFP to linker-GFP, and the displacement driven by single-headed dynein modifications as potential modifiers of Eg5 activity. We investigated a novel interacting with microtubules by optical tweezers. The efficiency and the modification, acetylation of lysine 146 by mutating this residue in Eg5’s a2 he- apparent stroke displacement depend on ATP concentration. The low efficiency lix to a glutamine. Molecular dynamics simulations demonstrate that modifica- and displacement at low ATP concentration indicate no conformational change tions of this lysine disrupts critical ionic interactions between the a2 and a1 of dynein at no ATP binding (apo) state that is predominant at low ATP concen- helices, increasing the allosteric coupling between the nucleotide binding site tration. With increasing ATP concentration, population of apo state decreases and Eg5’s mechanical element, the neck-linker. We tested these predictions us- and that of the pre-power stroke state such as dynein ADP-Pi should increase. ing structural kinetics measurements made by transient time-resolved FRET Dynein at the pre-power stroke state will generate the power stroke at binding detection of neck-linker docking and switch-1 closure during ATP binding to to microtubule. Therefore, the displacement increased with ATP concentration. rigor Eg5 attached to microtubules. We also investigated the mechanical con- This is the first obtained interesting result and has not been reported for myosin sequences of this charge mutant by investigating processive motility of fluores- and kinesin power strokes. High FRET efficiency and distance (8-9 nm) at satu- cently labeled Eg5 dimers in the presence and absence of applied load. These rated concentration of ATP indicate that the 8-9 nm power stroke distance is tests show that the K146Q acetylation-mimic mutation increases the coupling generated by swing of linker. This is supported by the result that the dynein between the neck-linker and nucleotide binding site, accelerates ATP driven deleted at the loops interacted with the linker did not generate the power stroke. neck-linker docking, tightens the force dependence of stepping, and increases The power stroke driven by the conformational change of dynein linker will be processive run-length and stall force in the presence of load, all indicating the fundamental mechanism of dynein motility. that the modification enhances mechanochemical coupling in Eg5. After modi- 223-Plat fication of this residue, Eg5 behaves much more like kinesin-1, a processive Intracellular Cargo Transport by Single-Headed Kinesin Monomers motor with head-head gating. We are investigating the physiologic implications Kristin I. Schimert1, Breane G. Budaitis2, Kristen J. Verhey1,3. for these changes. 1Biophysics Program, University of Michigan, Ann Arbor, MI, USA, 2Cellular and Molecular Biology Program, University of Michigan, Ann 226-Plat Arbor, MI, USA, 3Department of Cell and Developmental Biology, Single Molecule Characterization of Mitotic Kif15 Reveals Capability to University of Michigan, Ann Arbor, MI, USA. Generate Force in Anti-Parallel Microtubules 1 2 1 Kinesins are motor proteins that transport cargoes along microtubules in eu- Dana N. Reinemann , Emma G. Sturgill , Dibyendu Kumar Das , Ryoma Ohi2, Matthew J. Lang1,3. karyotic cells. The canonical picture of kinesin motility is that the two motor 1 domains of a dimer step hand-over-hand in a tightly coordinated manner. How- Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN, USA, 2Cell and Developmental Biology, Vanderbilt University, ever, it remains controversial whether kinesin-3 family motors function as 3 monomers or dimers. We compared truncated, single-headed monomeric mo- Nashville, TN, USA, Molecular Physiology and Biophysics, Vanderbilt tors to their dimeric forms using cargo transport assays in cells. We show University, Nashville, TN, USA. that surprisingly, teams of monomeric motors across different kinesin families Proper formation of the mitotic spindle is essential for chromosome separation are able to transport cargoes against high load. Strikingly, monomeric transport and cell division. Dimeric Kinesin-12, or Kif15, has been shown to serve as a capability varies widely even within a single kinesin family. Future work will backup mechanism for cell division, promoting spindle assembly in the absence investigate the structural and mechanical features that enable select monomeric of tetrameric Kinesin-5, or Eg5. The mechanism by which Kif15 aids in the as- motors to cooperatively transport cargoes. These findings will provide insight sembly of the spindle is unknown. Here, single molecule motility assays were into the minimal requirements for collective kinesin motility and intracellular performed using optical tweezers to evaluate the mechanistic behavior of Kif15 cargo transport. on microtubules (MTs). Constructs containing various sub-components of the motor were utilized to determine the role of each domain, including the motor 224-Plat heads, stalk, MT binding domain (N700) and the full length motor (FL). Step Kinesin-1 Cargo Transport through Dense Microtubule Networks size, dwell times, and stall forces were measured. Rupture assays were also per- Joelle A. Labastide1, Reilly K. Curtin2, Jennifer L. Ross1. formed on the MT binding domain (Coil-1) to determine the binding strength 1Phyiscs, University of Massachusetts Amherst, Amherst, MA, USA, and the nature of the Coil-1/MT interaction. Motility and rupture assays 2Biology, University of Massachusetts Amherst, Amherst, MA, USA. were repeated on subtilisin-digested MTs (dMT) in which the negatively- Essential motor transport processes occurring in living cells rely heavily on traf- charged carboxyl-terminal tail of MTs, called the E-hook, is removed. We ficking of cargoes by kinesin motors through dense microtubule networks. found that the Coil-1 was stronger than the stall force of the motor due to its Kinesin-1 motors in particular have been shown to walk unidirectionally along interaction with the E-hook, revealing that Kif15 has the ability to build up me- single filaments, and to fall off their tracks upon encountering obstacles, lattice chanical strain and slide MTs apart. In order to determine the importance of MT defects, and network intersections. Knowing that both microtubule network orientation, an in vitro optical trapping and fluorescence assay was developed

BPJ 7705_7715 Sunday, February 12, 2017 45a to determine the force generating capabilities of Kif15 in different bundle en- 229-Plat vironments. We determined that Kif15 slides anti-parallel MTs apart while par- Spoton: A Machine-Learning Approach for Hot-Spot Determination allel bundles remain stationary with a small amount of antagonizing force Irina S. Moreira1,2, Panos Koukos2, Rita Melo1,3, Jose G. Almeida1, generation. Our studies provide insight regarding how Kif15 is able to rescue Antonio J. Preto1, Jorg Schaarschmidt2, Mikael Trellet2, Zeynep H. Gumus4, bipolar spindle assembly in the absence of Eg5. Joaquim Costa5, Alexandre M.J.J. Bonvin2. 1Center for Neurosciences and Cell Biology (CNC.IBILI), Coimbra, 227-Plat Portugal, 2Bijvoet Center for Biomolecular Research, Utrecht University, Measuring Forces Generated by Ensembles of Kinesin-5 Crosslinking Two Utrecht, Netherlands, 3Centro de Cieˆncias e Tecnologias Nucleares, Lisbon, Microtubules Portugal, 4Department of Genetics and Genomics and Icahn Institute for Scott Forth1, Yuta Shimamoto2, Tarun Kapoor3. Genomics and Multiscale Biology, Icahn School of Medicine at Mount Sinai, 1Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY, USA, New York, NY, USA, 5CMUP/FCUP, Centro de Matema´tica da 2National Institute of Genetics, Mishima, Japan, 3Laboratory of Chemistry Universidade do Porto, Porto, Portugal. and Cell Biology, Rockefeller University, New York, NY, USA. The identification of protein complexes and interactions is crucial for the un- The proper organization of the microtubule-based mitotic spindle is proposed derstanding of cellular organization and machinery. Due to the high difficulty to depend on nanometer-sized motor proteins generating forces that scale in attaining experimental data about such an important subject, computational with a micron-sized geometric feature, such as microtubule overlap length. tools and methodologies are emerging as reliable alternatives. It is especially However, it has been unclear whether such regulation can be achieved by true that Machine-Learning (ML) algorithms hold an incredible promise for any mitotic motor protein. To address this knowledge gap, we have employed protein interaction research by identifying biological relevant patterns. In this an assay that combines an optical trap and total internal reflection fluorescence work, we have developed and applied ML techniques that went beyond the cur- (TIRF) to show that ensembles of kinesin-5, a conserved mitotic motor protein, rent state-of-the-art, leading to quantitative and reliable molecular-level predic- can push apart overlapping antiparallel microtubules to generate a force whose tions of Hot-Spots at protein-protein complexes. Our model was trained on a magnitude scales with filament overlap length. We also find that kinesin-5 can large number of complexes and on a significantly larger number of different produce overlap-length-dependent ‘‘brake-like’’ resistance against relative structural- and evolutionary sequence-based features. We used 54 algorithms microtubule sliding in both parallel and antiparallel geometries, an activity from a simple linear-based function to support-vector machine models with that has been suggested by cell biological studies but had not been directly different cost functions and compared their performance in 6 different pre- measured. Finally, we performed numerical simulations which reveal the processing sets. The best model was achieved by the use of the C5.0 tree- bio-mechanical properties of this motor protein that allow for efficient force based algorithm with a dataset pre-processed by the normalization of features transmission within crosslinked microtubule networks. Together, our results and with up-sampling of the minor class. The method showed an overall accu- reveal how a motor protein can function as an analog converter, ‘‘reading’’ sim- racy of 0.88, a sensitivity of 0.91 and a specificity of 0.84 for the independent ple geometric and dynamic features in cytoskeletal networks to produce regu- test set. Due to the theme’s relevance to the large scientific community working lated and scalable force outputs. on structural biology, we have assembled a freely available web-server that can be found at: http://milou.science.uu.nl/services/SPOTON/ Platform: Computational Methods and 230-Plat Bioinformatics Morphodynamic Profiling of Cell Protrusion Based on Spatiotemporal Spectrum Decomposition and Unsupervised Clustering 228-Plat Xiao Ma1, Onur Dagliyan2, Klaus Hahn2, Gaudenz Danuser1. Elucidating the Role of Tumor Microenvironmental Heterogeneity with a 1Bioinformatics, University of Texas Southwestern Medical Center, Dallas, Computational Model of 3D Breast Spheroids TX, USA, 2Pharmacology, UNC-Chapel Hill School of Medicine, Chapel Aleksandra Karolak1, Branton Huffstutler1, Dmitry A. Markov2, Hill, NC, USA. Lisa J. McCawley2, Katarzyna A. Rejniak1. Cellular morphogenesis is governed and regulated by complex interactions be- 1Integrated Mathematical Oncology, H. Lee Moffitt Cancer Center and 2 tween the cytoskeleton and molecular pathways. Due to their central roles in Research Institute, Tampa, FL, USA, Biomedical Engineering & Cancer the differentiation, embryogenesis and homeostasis process, cell morphology Biology, Vanderbilt University, Nashville, TN, USA. and morphodynamics are often used for qualitative and quantitative assessments Tumor development and its response to therapies are highly affected by hetero- of cell state. Here, we propose a framework to quantitatively profile cellular mor- geneous microenvironments, including fluctuations in chemical and physical phodynamics based on an adaptive spectrum decomposition method. This method cues, as well as in the composition of the surrounding extracellular matrix. decomposes the motion of cell boundary into analytical instantaneous frequency For example, the abnormal topology of tumor vasculature and tumor tissue leads spectrum through Hilbert-Huang transform (HHT). Subsequently, we computed to disruption in oxygen transport and in the emergence of regions of low oxygen for each decomposed signal the distribution of instantaneous frequency and ampli- (hypoxic conditions), and in accumulation of acidic products and decreased pH tude, from which we compiled the HHT spectrum. For spontaneously protrusive (acidic conditions). In addition, the elevated mechanical tension is commonly Cos7 cells, the instantaneous magnitudes of protrusion were found to greatly vary observed in the tumor connective tissue (high tension conditions). All these fac- across different cells with nearly identical genetic and molecular backgrounds, tors affect tumor growth and efficacy of anticancer drugs. Moreover, the micro- whereas the frequency spectra were remarkably consistent. Meanwhile we exam- environmental conditions can dynamically evolve after treatment that is difficult ined Cos7 cells in which the Vav2 Guanine Exchange Factor (GEF) activity was to investigate in vivo in a systematic way. To represent the complexity and dy- acutely perturbed by optogenetic tools, and found significant shifts in the fre- namics of tumor microenvironment we developed a computational model for the quency distribution for specific signals. We thus concluded that edge motion ve- growth of 3D organotypic cultures integrated with experimental organ-on-the- locity is an arbitrary parameter of cell morphogenesis whereas the frequency chip 3D cultures. The computational model was validated based on the data spectrum encodes the molecular state of regulation. The features extracted from collected from cultured breast spheroids, including the non-tumorigenic epithe- the HHT spectrum could be used as a classifier to group subcellular regions into lial MCF-10A cell line, mildly tumorigenic c-Ha-ras oncogene transfected MCF- numerous characteristic sectors with specific molecular states through Statistical 10AT1 cell line, and a metastatic line MCF-10CA-1a. Based on the differences Region Merging (SRM) based clustering. Finally, we monitored the Rac1 activity in these cell lines genetic profiles, cell mechanical properties, and image-guided downstream of Vav2 expressed in Cos7 cells and found that those clustering sec- variations in organotypic morphologies, the computational model was able to tors distinguished by morphodynamic parameters indeed reflect regions with reproduce in a quantitative way the dynamics of 3D tumor spheroids’ growth un- different local signaling. Thus, cell morphodynamic behavior can be used todetect der various microenvironmental conditions. This calibrated model was subse- different modes of operations or perturbations in cellular signaling. quently applied to predict tumor growth and its response to chemotherapy in heterogeneous microenvironments consisting of dynamically changing gradients 231-Plat of oxygen and pH, as well as tension of the surrounding microenvironment. The MechStiff: A New Tool for Evaluating Stress-Induced Dynamics overarching aim of our project is to use this integrated (in silico-organ and organ- and Application to Cell Adhesion Proteins on-the-chip) approach to target the treatment of tumor 3D organotypic cultures Karolina Mikulska-Ruminska1,2, Andrzej J. Kulik3, Cihan Kaya1, with drugs that display different mechanism of action, such as cell cycle arrest, Carine BenAdiba3, Giovanni Dietler3, Wieslaw Nowak2, Ivet Bahar1. initiation of apoptosis, or treatment combinations. Furthermore, this approach 1Computational and Systems Biology, Pittsburgh Univesity, Pittsburgh, PA, will allow us to propose new treatment schedules and dosages that will be tested USA, 2Physics, Astronomy and Applied Informatics, Niculaus Copernicus in the organ-on-the-chip experiments. The progress on this integrated model Universiy, Torun, Poland, 3Laboratory of Physics of Living Matter, Ecole development will be presented. Polytechnique Fe´de´rale de Lausanne, Lausanne, Swaziland.

BPJ 7705_7715 46a Sunday, February 12, 2017

Based on recent findings, there is growing evidence that many biological systems A molecular model using single chain mean field theory (SCMFT) is used to scan in the brain integrate biochemical information with mechanical signals making the wide range of possible design parameters. The molecular theory properly ac- critical decisions about cell differentiation, synaptic regulation, axonal growth, counts for the highly non-additive coupling of molecular interactions among all neuronal migration or proliferation. These events seem to be controlled by the the species. The size, shape, electrical properties and physical conformations of mechanical behavior of intervening proteins in addition to their well-studied the polymer, drug and solvent are taken into account. The binding of PD-166793 biochemistry; and therefore to obtain a quantitative understanding of the me- with polymer is modeled by a ligand-receptor binding mechanism. The model is chanical events implicated in neurosignaling events is becoming increasingly used to study the variation of this binding with changing pH, salt concentration, important. We have developed to this aim the MechStiff module that we imple- grafting density and length of the polymer. Experimental studies have shown that mented in ProDy API (1), based on an earlier comparison (2) of anisotropic this system is capable of retaining PD-166793 at more than 100 times the inhib- network model predictions with single molecule atomic force microscopy itory concentration against MMP-2 with a particle concentration of 2.5mg/mL. (smAFM) data. We further used MechStiff module to evaluate the effective resis- The model is used as a tool for continual improvements in binding of PD- tance of individual domains in the neuronal adhesion protein, contactin, to uni- 166793 by providing valuable feedback on how the variations of system param- axial tension. Contactins play an important role in maintaining the mechanical eters affect the binding efficiency. integrity and signaling properties of chemical synapses in the brain. To validate the predictions on the the molecular-level stress-strain behavior of contactin, 234-Plat we compared MechStiff predictions with smAFM measurements as well as re- Systematic Analysis of Symmetry in Membrane Proteins sults from steered molecular dynamics simulations. The multiscale approach Antoniya A. Aleksandrova. combining anisotropic network model with molecular simulations emerges as CSB, NINDS - NIH, Bethesda, MD, USA. a useful tool for interpreting experimental data and characterizing contactin Membrane proteins are encoded by around one third of a given genome, and nanomechanics. It also helps reveal the stress-induced conformational changes play key roles in transmission of information and chemicals such as neurotrans- being accommodated by cell adhesion proteins and can be readily extended to mitters into the cell. Available membrane protein structures have revealed an elucidating the stress-induced dynamics of multicomponent or modular proteins. abundance of symmetry and pseudo-symmetry, which are observed not only 1.Bakan, A., A. Dutta, W. Mao, Y. Liu, C. Chennubhotla, T. R. Lezon, and I. in the formation of multi-subunit assemblies, but also in the repetition of inter- Bahar. 2014. Evol and ProDy for bridging protein sequence evolution and nal structural elements. Secondary active transporters provide striking exam- structural dynamics. Bioinformatics 30:2681-2683. ples of the functional significance of symmetry. For instance, the structures 2.Eyal, E., and I. Bahar. 2008. Toward a molecular understanding of the aniso- of many transporters consist of two pseudo-symmetric repeats with opposite tropic response of proteins to external forces: insights from elastic network transmembrane orientations. These repeats can form asymmetric conforma- models. Biophys. J. 94:3424-3435. tions to create a pathway to one side of the membrane, consistent with the so-called alternating access hypothesis. By having one repeat adopt the confor- 232-Plat mation of the second repeat and vice-versa, the protein can create a new asym- Optimal Temperature Set for Replica Exchange Sampling metric structure that is open to the opposite side of the membrane. This Dominik Gront. ‘‘asymmetry exchange’’ underlies rocking-bundle or elevator-like movements Faculty of Chemistry, University of Warsaw, Warsaw, Poland. that result in the transport of a substrate. In this context, a systematic study of Replica Exchange Monte Carlo method has been introduced to improve sam- symmetry should provide a framework for a broader understanding of the pling of a rugged energy landscape for such systems as polymers, biopolymers mechanistic principles and evolutionary development of membrane proteins. and spin glasses. Efficiency of the method however critically depends on the set However, existing analyses lack the detail and breadth required for such a sys- of replica temperatures used for simulations. A novel method selecting these tematic study. Therefore, in this project we aim to quantify both the extent and parameters has been recently proposed1, which numerically evaluates the prob- diversity of symmetry relationships in known structures of membrane proteins. ability of replica swap between temperatures based on estimated density of To achieve this task, we combine the output of two programs for symmetry states for a system under study. Here we extend this method and prove it pro- detection, namely SymD and CE-Symm, each of which has certain limitations. vides the optimal set of temperatures i.e. temperatures that guarantee the fastest Our approach also allows us to explore the characteristics that discriminate flow of replicas from the lowest to the highest temperature. symmetric from pseudo-symmetric or asymmetric structures. We anticipate During an initial phase of the protocol, energy distributions are collected at that this analysis will provide a valuable foundation for addressing a wide range different temperatures. Based on these observations, the density of states for of questions relating to the function and evolution of these important proteins. the system is computed by the multihistogram method. Knowing the density, improved temperature set is established by minimizing the mean first passage 235-Plat time of replicas in the temperature space. The procedure has been illustrated Membrane Recruitment can Increase the Number of Protein Assemblies with a coarse-grained protein folding simulation and all-atom dynamics in by Many Folds: Insights from Theory and Reaction-Diffusion Simulation AMBER force field. The method has been implemented in BioShell package.2,3 Osman N. Yogurtcu, Margaret E. Johnson. 1 D. Gront and A. Kolinski ‘‘Efficient scheme for optimization of parallel Biophysics, Johns Hopkins University, Baltimore, MD, USA. tempering Monte Carlo method’’ Journal of Physics: Condensed Matter 2007 A significant number of the cellular protein interaction networks, such as 19(3) 036225 http://dx.doi.org/10.1088/0953-8984/19/3/036225 receptor-mediated signaling and vesicle trafficking pathways, includes reac- 2 D. Gront and A. Kolinski ‘‘BioShell - a package of tools for structural biology tions that involve membranes as a molecular assembly platform. Membranes computations’’ Bioinformatics 2006 22(5):621-622 both reduce the search space and induce a cooperative binding effect for sta- 3D. Gront and A. Kolinski ‘‘Utility library for structural bioinformatics’’ Bio- bilizing complexes with multiple membrane recruiter molecule binding sites. informatics 2008 24(4):584-585 Mathematical models and computer simulations provide insight into the dy- namics of complex formation and help identify general principles that govern 233-Plat successful recruitment and assembly on membranes. However, sufficiently Molecular Design of a Nanoparticle-Polymer Conjugated Drug Delivery long and physically accurate simulations of protein assemblies are quite chal- System for PD-166793 in Cardiovascular Repair lenging. In this work, using equilibrium theory and a very efficient in-lab Merina Jahan1, Stephen K. Roberts2, Andrew B. Greytak2, Mark J. Uline1. developed single-molecule scale stochastic simulation software, we provide 1Chemical Engineering, University of South Carolina, Columbia, SC, USA, simple formulas for quantifying how the ratio of membrane-to-solution, 2Department of Chemistry and Biochemistry, University of South Carolina, both in vitro and in vivo, can change the observed protein-protein interaction Columbia, SC, USA. strengths of peripheral membrane proteins by orders of magnitude. We show Overexpression of matrix metalloproteinases (MMPs) following myocardial that the magnitude of complex formation enhancement has a simple functional infarction (MI) is linked to deleterious left ventricle remodeling and heart failure. form that applies whenever membrane recruiter concentrations are sufficiently Current research has focused on introducing a therapeutically relevant concentra- high, and surprisingly, is independent of the protein binding strength. We pro- tion of effective MMP inhibitor to the MI site to mitigate the harmful tissue re- pose that membrane localization works as a mechanism that ensures assembly modeling. Theoretical molecular level studies provide an effective platform for only at specific times (after recruitment to surfaces) but does not precisely designing novel delivery systems for MMP inhibition that can provide valuable regulate the proteins involved since they benefit equally from surface restric- insights for experimental researchers. This study focuses on developing a drug tion. This robust strategy is employed by adaptor proteins involved in delivery pathway using PD-166793 that has shown great promise as a broad clathrin-mediated endocytosis in both yeast and mammalian cells, where their spectrum MMP inhibitor in recent years. In this system, PD-166793 is bound relatively weak binding interactions with one another prevents protein coat as- to poly methyl acrylic acid (PMAA) polymer and one end of the polymer is teth- sembly in solution, but transitions to a rapid assembly on the plasma ered to a spherical silica nanoparticle surface to carry the drug to the desired site. membrane.

BPJ 7705_7715 Sunday, February 12, 2017 47a

Posters In archaea, the functional roles of Sm proteins remains an open question. Sm proteins assemble into cyclic oligomers of 5, 6, 7, or 8 subunits, and the assem- blies can be either homo- or heteromeric. Bacterial Sm proteins have only been Protein Structure and Conformation I found as homo-hexamers, while eukaryotic Sm proteins typically assemble into hetero-heptamers. Archaeal Sm proteins have been found as homomeric hex- 236-Pos Board B1 amers, heptamers, and octamers. Despite this variation in quaternary structure, A Discrete Loop of the SERCA N-Domain Interacts with Phospholamban all Sm monomers exhibit nearly identical tertiary structures. How can this be? and Stabilizes a Compact Conformation of the SERCA Cytosolic What is the origin of this oligomeric plasticity, if not encoded in the monomer? Headpiece We have used a systematic array of molecular dynamics simulations to Olga N. Raguimova, Nikolai Smolin, Daniel Blackwell, Elisa Bovo, examine the interfaces between Sm subunits, and have developed several quan- Aleksey Zima, Seth Robia. titative relations that link the results of dimer simulations to the behavior of Cell and Molecular Physiology, Loyola, Maywood, IL, USA. complete rings. The simulations reveal that Sm oligomers are remarkably flex- The cardiac sarco/endoplasmic reticulum (ER) calcium ATPase (SERCA) and ible. Sm dimers can adopt multiple conformations, and Sm rings are distinctly its inhibitor phospholamban (PLB) are key regulators of calcium levels in car- asymmetric. In particular, the octameric ring of an Sm-like archaeal protein diac cells. In previous molecular dynamics (MD) simulations of SERCA spon- deforms drastically, and dimers of that protein appear to adopt a non-Sm-like taneous headpiece closure, we identified a discrete N-domain beta 5,6-loop that tertiary structure. For a dimer of the E. coli Sm protein, our simulations directs the closure of SERCA headpiece. Here we use additional MD simula- show one monomer twisting nearly fifteen degrees from its position in the crys- tions to pinpoint specific negative residues that facilitate SERCA headpiece tal structure. The surprising flexibility of Sm oligomers may be related to the closure. In addition, fluorescence resonance energy transfer (FRET) measure- dynamical effects of RNA binding and we are currently investigating these ments showed that mutation of these key residues slows down calcium- effects in a variety of Sm systems. dependent SERCA headpiece closure, as predicted from MD calculations. The functional significance of impaired SERCA headpiece closure was demon- 239-Pos Board B4 strated by a 60% decrease in SERCA ATPase activity with no detectable An NMR Study of Pin1- Histone H1 Interactions change in calcium sensitivity. Additionally, loop mutations reduced transport Dinusha S. Jinasena, Hawa Gyamfi, Nicholas C. Fitzkee. of calcium as measured by a low affinity calcium sensor (R-CEPIA1er) in Chemistry, Mississippi State University, Starkville, MS, USA. the ER of HEK cells. Interestingly, MD simulations of the SERCA-PLB regu- Pin1 is an essential Peptidyl-prolyl isomerase (PPIase) that catalyzes cis-trans latory complex model showed that PLB phosphorylation on Serine 16 stabi- prolyl isomerization in proteins containing pSer/Thr-Pro motifs. It has an N-ter- lized a compact, ordered SERCA headpiece through an interaction with the minal WW domain and a C-terminal PPIase domain. Pin1 targets pSer/Thr-Pro beta 5,6-loop. Fluorescence lifetime imaging microscopy (FLIM) measure- motifs by its WW binding domain and catalyzes isomerization through its ments also detected a conformational change of the SERCA-PLB regulatory PPIase domain. Recently, Pin1 was shown to modify the conformation of phos- complex after phosphomimetic mutation of PLB. Overall, the data suggest phorylated Histone H1 and to stabilize the chromatin-H1 interaction by that this discrete loop is important for SERCA transport activity and functional increasing its residence time. The Pin1/Histone H1 interaction plays a key regulation by PLB. This novel structural element may represent a target for role in pathogen response in infection and cell cycle control; therefore, anti- modulation of SERCA activity. Pin1 therapeutics is an important target for treating infections as well as cancer. The H1 Histones (H1.0-H1.5) each contain several potential Pin1 recognition 237-Pos Board B2 (pT/pS)-P motifs. To understand this interaction fully, we have investigated Preparation of a Orexin Precursor Protein by Chemical Digestion how both the isolated WW domain and full length Pin1 bind to these H1 Natsumi Mitsuoka, Shigeru Shimamoto, Yuji Hidaka. Histone substrates. NMR spectroscopy has been used to measure the binding Kindai University, Higashi-Osaka, Osaka, Japan. affinities of Pin1 with select H1 sequences (H1.1, H1.4 & H1.5), and the Endogenous peptide hormones are often produced in vivo in the form of precur- inter-domain dynamics upon binding theses sequences have been measured. sor proteins that contain pre- and pro-leader sequences, which are subsequently We observe different Kd values depending on the histone binding site, suggest- processed into the biologically active mature peptide. Therefore, it is important ing that energetics play a role in guiding the Pin1-Histone interaction. Relaxa- to study the folding of precursor proteins to understand the mechanisms of post- tion data has revealed that bound Pin1 can behave as two independent domains translational modifications and the formation of the active conformation of the connected by a flexible linker, or as a single rigid molecule, depending on the mature peptide. However, it is generally difficult to obtain precursor proteins Histone H1 sequence. In the long term, we believe these studies will help to from intact organs because only small amounts are produced in vivo. To over- reveal not only the specifics of the Pin1-Histone H1 interaction, but also how come this issue, an E. coli expression system is employed to prepare recombi- Pin1 interacts with its substrates in general. nant proteins. Pro-orexin consists of orexin A, B, and a C-terminal peptide. To investigate the 240-Pos Board B5 mechanism responsible for the processing and post-translational modifications Nanomechanical Properties of Polymorphic Human Islet Amyloid of orexin peptides, we tried to express the recombinant pro-orexin in E. coli Polypeptide Protofibrils in the Multiple Physiological and Mechanical cells. However, recombinant pro-orexin was not expressed in E. coli cells. In Conditions addition, it was also difficult to obtain pro-orexin, even though Trx-fused Myeongsang Lee. pro-orexin was readily produced in E. coli cells because the digestion of Department of Mechanical Engineering, Korea University, Seoul, Korea, Trx-fused pro-orexin with an enzyme yielded significant amounts of non- Republic of. specific digests. Therefore, to solve this issue, we employed a chemical method Fibrillar and oligomeric amyloids are the origin of neuro-degenerative and to release pro-orexin from fusion proteins. degenerative diseases as type II diabetes, Alzheimer’s diseases, and so forth. K. Pane et al. recently reported a chemical method to obtain a target protein The common properties of amyloids are located near functional cells, not easily from an onconase-fused (ONC-fused) protein. They applied a chemical cleav- degraded, and disrupt their own functions. These amyloids had polymorphic age of the fusion protein at the Asp-Pro sequence with acetic acid. We em- characteristics under physiological conditions such as thermal effect, pH, ionic ployed this chemical method to produce recombinant pro-orexin. ONC-fused strength, metal ions and internal fluctuations. Developed from denatured and pro-orexin was successfully over-expressed as an inclusion body in E. coli misfolded amyloid monomers, polymorphic amyloids are mainly existed as BL21(DE3) cells and treated with acids under several sets of conditions to parallel or antiparallel composition along to fibril axis and lateral thickness cleave the Asp-Pro sequence of ONC-fused pro-orexin. The results will be dis- composition by adding unit amyloid protofilament through the experimental cussed in this paper. techniques. Despite experimental effort of polymorphic structures of amyloid proteins, there are some lacks of knowledge that relationship between structural 238-Pos Board B3 composition of polymorphic amyloids and their stabilities in detail. The me- A Simulation Based Analysis of the Oligomeric Plasticity of Sm Protein chanical characterization as bending, stretching can give insight for under- Assemblies standing the structure-property relationship of amyloid protofilaments. Charles E. McAnany, Berk Ekmekci, Peter Randolph, Cameron Mura. Considering that amyloids are developed through the repetitive fragmentation Chemistry, University of Virginia, Charlottesville, VA, USA. and elongation mechanisms, it is crucial to understand the structural stabilities RNA-associated Sm proteins can be found in all three domains of life. In eu- of polymorphic amyloid protofibrils. Here, we reported the polymorphic char- karya, Sm proteins are well-studied in connection with their roles mRNA acteristics of hIAPP amyloid protofibrils in mechanical insight via equilibrium splicing. In bacteria, the Sm protein Hfq acts as an RNA chaperone, playing and steered molecular dynamics (MD and SMD). Through the principal vital roles in mRNA-sRNA annealing and RNA-based regulatory networks. component analysis of polymorphic hIAPP protofibrils after equilibrium MD

BPJ 7716_7719 48a Sunday, February 12, 2017 simulations, we found the different deformation mode (i.e. bending, stretching activator binding for transactivation. In apo form, helix 11 bends outward by and torsion) of polymorphic hIAPP protofibrils. Using eigenvalue analysis, a kink-centered hinge-bending motion, and the motion creates wide entrance antiparallel models had considerable structural stability than parallel models of the ligand-binding pocket. In antagonist-binding form, loop 11-12 remark- due to the asymmetric hydrogen bond network composition. Furthermore, by ably flexible compared to that in apo form. applying the constant force bending and constant velocity tensile SMD simula- tions, we found the dominant material characteristics that homo composition 243-Pos Board B8 had ductile feature, while hetero composition had brittle characteristics. We Conformational Fluctuations as an Intrinsic Mechanism of Action: The Pseudomonas Aeruginosa provide the relationship between structural composition and their properties, Lipase-Specific Foldase of Studied by Hybrid which are related to growth mechanism of amyloids. Our study also provides Fluorescence Spectroscopy and MD Simulations 1 2 3 3 the possibilities for the basic template for functional biomaterials in the future. Jakub Kubiak , Neha Verma , Peter Dollinger , Filip Kovacic , Karl-Erich Jaeger3, Holger Gohlke2, Claus A.M. Seidel1. 1 € 241-Pos Board B6 Institute for Molecular Physical Chemistry, Heinrich-Heine-Universitat € € 2 Protein Structure Determination by High-Precision FRET and Molecular Dusseldorf, Dusseldorf, Germany, Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-Universit€at Dusseldorf,€ Dusseldorf,€ Modeling 3 Mykola Dimura1, Thomas Peulen1, Holger Gohlke2, Claus A.M. Seidel1. Germany, Institute of Molecular Enzyme Technology, Heinrich-Heine- € € € 1Molecular Physical Chemistry, Heinrich-Heine-Universit€at Dusseldorf,€ Universitat Dusseldorf, Dusseldorf, Germany. Dusseldorf,€ Germany, 2Pharmazeutische und Medizinische Chemie, The description of protein function requires structural information, not only Heinrich-Heine-Universit€at Dusseldorf,€ Dusseldorf,€ Germany. from static structural models but also of flexibility and dynamics. An investiga- A comprehensive methodology for FRET based modeling of biomolecules is tion of highly flexible proteins or IDPs requires techniques sensitive to a variety presented. Conformations of six multi-state model proteins are determined of time- and distance-scales. Fluorescence (FRET, FCS) is known to provide based on synthetic FRET data in the given benchmark. The workflow consists good temporal and spatial coverage but it is a label-based technique and lacks of five steps: atomistic-details. All-atom MD, on the other hand, is challenged by simulation 1. Initial conformational ensemble generation with NMSim geometric sim- of slow processes and accurate description of all interactions. The lipase- ulations utilizing available prior knowledge like homology model or crystal specific foldase (Lif) from Pseudomonas aeruginosa is a highly flexible protein structure of the one of the states. with residual secondary and tertiary structure. It is mandatory to produce lipase 2. Identification of the most informative labeling positions and FRET pairs A (LipA) in an enzymatically active conformation. Using a combination of for fluorescence measurements based on proposed ensemble generated at first fluorescence techniques (single molecule FRET, fluorescence lifetime analysis step. and fFCS) and all-atom MD we propose a description of structure and dynamics a 3. State-representative conformer identification, based on their agreement of Lif in relation to LipA. In the Lif:LipA complex, Lif forms flexible -helical with experiment, using FRET-screening and Accessible Volume simulations. scaffold embracing LipA in headphone-like structure. In the unbound form, Lif 4. Expansion of the initial conformational ensemble with FRET-guided does not stay in the hollow ‘‘headphone’’ conformation but rather exhibits a NMSim Markov Chain Monte Carlo simulations based on FRET data. As a large-scale conformational dynamics, where its -helical structure undergoes result conformations with better FRET-agreement are identified. reversible collapses and extensions as well as unfolding, on the sub- 5. Precision and accuracy assessment and quality control to outline the capa- microsecond to sub-millisecond timescale. This process allows Lif to bind bilities and limitations of the shown methodology. LipA despite the fickleness of structure. We show how the multitude of states Using these steps, conformations of the model proteins were determined accu- in fast exchange provides a basis for binding. In addition we show how this rately. Confidence levels were determined together with the corresponding con- hybrid approach benefits FRET (insight into the molecular structure) and fidence levels and accuracy measures. Performance capabilities and limitations MD (sampling). of the methodology are rigorously assessed. The software package is presented 244-Pos Board B9 on site. Does Cas9-Catalyzed DNA Cleavage Generate Blunt Ends or Staggered Ends? Insight from Molecular Dynamic Simulations 242-Pos Board B7 Zhicheng Z. Zuo, Jin Liu. Apo- and Antagonist-Binding Structures of Vitamin D Receptor Ligand- UNT Health Science Center, Fort Worth, TX, USA. Binding Domain Revealed by a Combination Andlysis of MD Simulations The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes and SAXS Experiments (spCas9) along with a single guide RNA (sgRNA) has merged as a versatile 1 2 3 1 Yasuaki Anami , Nobutaka Shimizu , Toru Ekimoto , Daichi Egawa , toolbox for genome editing. Despite recent advances in the mechanistic studies Toshimasa Itoh1, Mitsunori Ikeguchi3, Keiko Yamamoto1. 1 2 on spCas9-sgRNA-mediated target double-stranded DNA (dsDNA) recogni- Showa Pharmaceutical University, Tokyo, Japan, Photon Factory, Institute tion and cleavage, it is still unclear how the catalytic Mg2þ ions induce the of Material Structure Science, High Energy Accelerator Research 3 conformation changes toward the catalytic active state. It also remains contro- Organization, Tsukuba, Japan, Yokohama ciry university, Yokohama, versial whether Cas9 generates blunt-ended or staggered-ended breaks with Japan. overhangs in the target DNA. To investigate these issues, here we performed Vitamin D receptor (VDR) is a member of the nuclear receptor (NR) family and the first all-atom molecular dynamics simulations of the spCas9-sgRNA- regulates the expression of genes related to calcium homeostasis , immunomo- dsDNA system with and without Mg2þ bound. The simulation results show dulation, and cell differentiation and proliferation. The VDR and other NRs are that binding of two Mg2þ ions at the RuvC domain active site could lead to involved in human diseases, therefore, understanding of their regulation by structurally and energetically favorable coordination ready for the non-target ligand binding is a pharmaceutical demand for structure-based drug design. DNA strand cleavage. Importantly, we argue that Cas9-catalyzed target DNA According to X-ray crystallographic analysis of NRs and other experimental re- cleavage produces 1-bp staggered ends rather than generally assumed blunt sults, the ligand-binding domain (LBD) undergoes conformational change ends. upon ligand binding, and the local conformational change around helix 12 is key to regulating agonism/antagonism. 245-Pos Board B10 Many crystal structures of agonist/antagonist-binding VDR-LBD have been Methionine Sulfoxide Formation by Cigarette Smoke is Associated with solved so far. However, all the crystal structures are almost identical, regardless the Degradation of Plasma Proteins of agonist/antagonist binding, and those crystal structures are considered as the Abdullah Qassab, Rohana Liyanage, Wesley Stites. agonist form. Because the conformation of helix 12 is key, the crystal structures Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA. do not provide structural insight into the mechanism of antagonist activity. In Background: High level of protein carbonyls have been found in plasma pro- addition, no crystal structures of apo form has been reported, and the exact teins in smokers compared to non-smokers. While oxidation may directly inter- conformation of apo form remains unknown. fere with activity, the extent to which oxidation affects protein turnover is less To reveal the apo and antagonist-binding forms of VDR-LBD, we analyze them clear. by a combination approach of small-angle X-ray scattering (SAXS) and molec- Objectives: To determine levels of oxidized serum proteins cleared in the urine ular dynamics (MD). We obtained SAXS profiles of both forms, however, the of smokers and non-smokers with focus on methionine sulfoxide (MSO) forma- profiles were different from the theoretical profiles calculated from crystal tion in Human Serum Albumin (HSA) and to determine the effect of methio- structures. To clarify the solution structures at atomic resolution, we conducted nine oxidation on the turnover of HSA. MD and collected each structural ensemble. Comparing the SAXS profiles and Method: 100 mL of urine were obtained from smoker and non-smokers. the MD results, we report a reliable structure for each form. In both forms, helix proteins were concentrated by reducing the sample size to 1.5% of the original 12 is partially unraveled, and does not adopt the active form, preventing co- volume. 200 mL of the concentrate then were separated by SDS-page gel

BPJ 7716_7719 Sunday, February 12, 2017 49a electrophoresis. The band with intact HSA was cut out and the remainder of the stability against complex proteolytic environment. Regardless of the site of gel was cut into four different pieces. Gel sections then were digested with modification, the two isoforms showed a remarkable 7-fold improvement in trypsin. Levels of MSO in the resulting peptides were assessed by LC-MS/ the larvicidal activity of BinA protein against 3rd instar Culex larvae, compared MS and data analysis was performed using the Skyline software package. to the unmodified protein. The PEGylation of recombinantly produced BinA Results: A group comparison between non-smokers (control) and smokers can be achieved easily. It promises a judicious approach towards mosquito pop- showed a slight increase in the levels of MSO found in intact HSA of smokers ulation control. relative to non-smokers. Regions of gels with proteins of lower mass than intact HSA showed that degraded fragments of HSA in urine of both smokers and 248-Pos Board B13 non-smokers have higher levels of MSO than are found in intact HSA. A Novel Electrostatic Regulatory Mechanism in a Calcium Binding Pro- Conclusions: HSA in smokers has statistically significant higher levels of MSO tein, L-plastin than HSA in non-smokers. However, the higher levels of oxidation in smokers Jonn Keenan Fanning, Van A. Ngo, Hiroaki Ishida, Hans Vogel, are concentrated in partially degraded HSA. At the moment it is not possible to Sergei Yu. Noskov. say unequivocally whether oxidized HSA is more likely to be cleaved and Biological Sciences, University of Calgary, Calgary, AB, Canada. cleared, if cleaved protein is more likely to be oxidized before clearance, or Plastins are a group of highly conserved actin binding proteins. L-plastin is a hu- both. man isoform of this protein found active in hemopoietic cells. This isoform is also expressed in cancer cells. The N-terminal of this isoform contains a 246-Pos Board B11 Calcium-binding EF-hand domain that allosterically regulates this protein for Dynamics and Energetics of Elongation Factor SelB in the Ternary binding to its target. It is, however, very difficult to delineate the effects that un- Complex and the Ribosome derlie the allosteric mechanism. To investigate the regulatory mechanisms of the Lars V. Bock1, Niels Fischer2, Holger Stark2, Holger Stark2, activation of this protein domain, we have used both computational and exper- Helmut Grubmuller€ 1. imental methods. Working with calcium in MD simulations has previously 1Theoretical and Computational Biophysics, MPI for biophysical chemistry, caused significant problems as classical force fields are not well equipped to Goettingen, Germany, 2Structural Dynamics, MPI for biophysical chemistry, deal with calcium. This is because calcium is a divalent ion, which can induce Goettingen, Germany. a strong local electrostatic field, and is capable of charge transfer. Although clas- SelB is an elongation factor specialized to deliver the selenocysteine (Sec) sical force fields are insufficient, a new polarizable force field now known as a tRNA to the ribosome by recoding the UGA stop codon on the mRNA. Initially Drude force field, provides a more accurate solution to this problem. We the tRNA is in complex with selB and GTP forming the ternary complex (TC). describe results from simulations with both Classical and Drude force fields High-resolution cryo-EM structures of intermediates of the Sec incorporation for the calcium-sensitive regions of L-plastin. These ongoing simulations pathway uncover large-scale conformational changes of the ribosome and the have already provided valuable insight an unexpected and previously unseen TC. To complement the structural information with energetics and rapid dy- electrostatic regulatory mechanism of L-plastin. In addition to computational namics, we performed extensive all-atom molecular dynamics simulations of simulations, to further validate this novel mechanism we obtained promising the ribosome with bound TC as well as of the free TC in solution. The simula- experimental validation using a number of biophysical methods including tions of the free TC were started after extracting the TC from the ribosome- isothermal calorimetry (ITC) and differential scanning calorimetry (DSC). bound cryo-EM structures. The TC was found to rapidly interconvert between Although we are looking into this mechanism in L-plastin, the conserved nature the different conformations allowing us to construct the free-energy landscape of this protein may indicate that this mechanism is present in a wide range of of the involved motions. This free-energy landscape indicates that the intrinsic related proteins, and may help provide us with a deeper understanding of the large-scale conformational changes of the tRNA and SelB during the delivery mechanisms associated with calcium activation and deactivation. to the ribosome are not rate-limiting to the process. In simulations of the free TC started from the GTPase-activated ribosome-bound conformation, the TC 249-Pos Board B14 rapidly transitions into an inactivated conformation, showing that the Structural Destabilization of Tropomyosin Induced by a Cardiomyopathy- GTPase-activated state is strongly stabilized by the ribosome. The simulations Linked Mutation 1 2 3 1 of the full ribosome with bound TC in the intermediate states allow us to iden- Thu N. Ly , Inna Krieger , Young-Ho Yoon , Dmitri Tolkatchev , 3 1 tify the motions that are rate-limiting to the process of tRNA delivery and to Fadel A. Samatey , Alla S. Kostyukova . 1Chemical Engineering and Bioengineering, Washington State University, identify the molecular mechanism of the domain closure of small ribosomal 2 subunit upon tRNA decoding. Pullman, WA, USA, Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX, USA, 3Trans-Membrane Trafficking 247-Pos Board B12 Unit, Okinawa Institute of Science and Technology, Okinawa, Japan. Polyethylene Glycol Conjugation Enhances Mosquito-Larvicidal Activity In striated muscle, thin filaments, composed of F-actin, and thick filaments of Lysinibacillus Sphaericus BinA Protein constitute the basic contractile units called sarcomeres. Tropomyosin (Tpm), Mahima Sharma1,2, Ramesh S. Hire2, Ashok B. Hadapad2, a two-stranded coiled-coil protein, binds along the actin filaments through Gagan Deep Gupta2, Vinay Kumar2,3. head-to-tail polymerization, protects and stabilizes the thin filaments. The 1Life science, Homi Bhabha National Institute (HBNI), Mumbai, India, N-terminus of Tpm orients toward the pointed end of thin filament, where it in- 2Bhabha Atomic Research Centre, Mumbai, India, 3Homi Bhabha National teracts with pointed end-binding proteins such as tropomodulin (Tmod) and Institute (HBNI), Mumbai, India. leiomodin (Lmod) to maintain the uniform filament length critical for proper Mosquitoes are known to spread human diseases like West Nile fever, dengue, sarcomeric functions. Recently, a hypertrophic cardiomyopathy-associated mu- malaria, zika etc. accounting for millions of deaths worldwide. Lysinibacillus tation, R21H, has been identified in striated muscle Tpm (Tpm1.1) with molec- sphaericus, a gram positive, spore producing commensal soil bacterium, has ular mechanism of perturbing muscular function unknown. We designed, been used worldwide for controlling mosquito population like Culex and expressed, and purified the Tpm chimeric peptide aTM1a1-28Zip. The peptide Anopheles, and is regarded as safe against non-target organisms. Binary toxin, consists of 28 N-terminal residues of Tpm1.1 followed by 18 C-terminal resi- composed of BinA (41.9 kDa) and BinB (51.4 kDa) component proteins, is dues of the GCN4 leucine zipper domain. The peptide was crystallized and its responsible for the high larvicidal activity of several L. sphaericus strains. structure was solved. To study how this mutation affects Tpm1.1, we intro- The two proteins exert high toxicity when administered together. BinA alone duced the mutation R21H in the peptide. An effect of the mutation was studied displays larvicidal activity, in the absence of BinB, albeit at reduced levels. in silico using molecular dynamics simulation (MDS) and in vitro by circular But instability, shorter half-lives and rapid proteolytic digestion can limit their dichroism (CD). Temperature measurements using CD were conducted to use as an effective insecticide. We for the first time demonstrate the beneficial characterize the effect of the mutation R21H on thermal stability of the effect of PEGylation (covalent attachment of polyethylene glycol) on aTM1a1-28Zip peptide alone and its complexes with Tmod and Lmod frag- mosquito-larvicidal activity of BinA protein. PEG-protein conjugates were ments containing Tpm-binding sites. CD data showed that the mutation synthesized using PEG-isocyanate polymer. The resulting bio-conjugates R21H caused a significant decrease in the helical content and structural stability were purified to homogeneity by column chromatography methods. These of aTM1a1-28Zip. Complexes formed between the aTM1a1-28Zip[R21H] pep- were characterized by various biophysical methods like MALDI-TOF, DLS, tide and Tmod or Lmod fragments were less stable than those formed with DSF and CD. Two different isoforms of PEG-BinA conjugates are expected wild-type aTM1a1-28Zip. All CD data were in agreement with MDS results from biophysical analysis, which appear to be mono-PEGylated but may differ which showed that the mutation R21H significantly altered the coiled-coil in the site of PEG attachment to BinA protein. The PEGylated proteins dis- structure of aTM1a1-28Zip. We suggest that the mutation R21H destabilizes played preservation of protein’s native structure and exhibited improved ther- Tpm structure by disrupting local salt bridges formed between residues mal stability by about 3-5 oC. The PEGylated proteins were also checked for Arg21 and Glu26 on opposite strands.

BPJ 7716_7719 50a Sunday, February 12, 2017

250-Pos Board B15 253-Pos Board B18 Probing Conformational and Functional Substates of Calmodulin by High- Regulation of Folding of de novo Designed Peptides by a-Helix Formation Pressure FTIR Saya Nishihara, Kosuke Toyama, Shigeru Shimamoto, Yuji Hidaka. Nelli Erwin, Satyajit Patra, Roland Winter. Kindai University, Higashi-Osaka, Japan. TU Dortmund, Dortmund, Germany. Uroguanylin folds into its correct conformation with assistance by the propep- In response to intracellular Ca2þ-concentration changes, the highly dynamic tide region. Our previous study of the disulfide-coupled folding of pro- and flexible Ca2þ-sensing protein calmodulin (CaM) interacts with more than uroguanylin suggested that a mis-bridged disulfide isomer is kinetically 300 diverse target proteins that are involved in numerous signaling pathways stabilized by the a-helical structure at the processing site between the propep- in eukaryotic cells. This unique promiscuous target binding behavior and the tide region and the mature region at the early stage of the folding reaction. The underlying functional versatility of CaM is a result of its structural flexibility. mis-bridged form is then converted to the native conformation via the forma- CaM spans multiple conformational substates in solution providing adaptable tion of a b-sheet structure between the mature and the propeptide region along binding surfaces for different target proteins. K-Ras4B, known as a key regu- with disulfide exchange reaction. lator in the ERK pathway, was identified as specific binding partner of CaM, To further investigate and utilize the intra-molecular chaperone function of the causing the dissociation of the lipidated K-Ras4B from the plasma membrane propeptide, we designed a de novo peptide (NDD hybrid peptide) which folds and thus reducing its activity. However, the interaction of K-Ras4B and CaM into its bioactive conformation (G-type) with difficulty, and also designed a has not been elucidated in detail until now. In order to evaluate the conforma- de novo protein (pro-NDD hybrid protein), which is able to fold into only a tional space of CaM and shed more light on the mechanism of subsequent target G-type, via the fusion of the propeptide region of uroguanylin. Our previous recognition and protein function, identification and characterization of func- results for their refolding reactions suggested that the G-type conformation is tionally relevant conformational substates is mandatory. Applying pressure in formed via the mis-bridged disulfide isomer (R-type). Considering these results combination with spectroscopies such as FTIR spectroscopy enables to popu- and the propeptide-mediated folding of pro-uroguanylin, we proposed that the late and probe otherwise transient low-lying excited conformational substates formation of the a-helical structure at the processing site is important in form- of CaM close in energy to ground state, but with a smaller partial volume. ing the folding intermediate. Evidence has grown that these states are functionally relevant, for instance in In this study, to validate this hypothesis, a series of the de novo peptide analogs recognition and ligand binding events. The pressure-induced conformational containing extended N-terminal sequences was prepared and their folding reac- changes of CaM were studied in its Ca2þ-free and Ca2þ-bound state and in tions were examined under kinetic or thermodynamic control. The results indi- the presence of the C-terminal K-Ras4B peptide. We show that not only cated that the formation or stabilization of the a-helical structure regulates the Ca2þ-binding, but also the presence of the target peptide has a drastic effect formation of the disulfide isomer as the proper intermediate under kinetic con- on the conformational dynamics of the protein. trol. The results of our studies will be discussed in this paper. 251-Pos Board B16 254-Pos Board B19 Obscurin Acts as a Variable Force Resistor Characterization of Protein Kinase a Free Energy Landscape by NMR- Nathan T. Wright, Aidan M. Willey. Restrained Metadynamics Chemistry and Biochemistry, James Madison University, Harrisonburg, Yingjie Wang1,2, Carlo Camilloni3, Jonggul Kim1,2, Michele Vendruscolo3, VA, USA. Jiali Gao1, Gianluigi Veglia1,2. Obscurin, a giant cytoskeletal protein, functions in part to connect distal ele- 1Department of Chemistry, University of Minnesota, Minneapolis, MN, ments within a large variety of cell types. Due to this cellular role, obscurin USA, 2Department of Biochemistry, Molecular Biology and Biophysics, likely changes shape as its molecular targets move throughout the cell. In University of Minnesota, Minneapolis, MN, USA, 3Department of this way, obscurin must intrinsically resist force. Here, we describe the molec- Chemistry, University of Cambridge, Cambridge, United Kingdom. ular etiology of force resistance through the study of multiple tandem obscurin The free-energy landscape of a protein underlies the conformational transi- Ig domains. Using a combination of experimental and computational tech- tions that are vital to its biological function. Recent advances in experimental niques, we find that obscurin acts as a differential force resistor. Differences and computational methods are making it possible to characterize these free in tandem Ig domain flexibility are dictated primarily by linker sequence and energy landscapes. In particular, the use of enhanced sampling techniques not linker length. These studies provide insight into the basic mechanisms of in molecular dynamics simulations, including the replica average metadynam- how cells respond to and resist stretch force. ics (RAM) method, have partly alleviated the sampling bottleneck and bridged the gap between simulations and experiments. Here we applied 252-Pos Board B17 RAM to study the free energy landscape of the catalytic subunit of protein ki- Structural and Functional Insight into Recombinant Lung Surfactant nase A in the apo, binary (with ATP), and ternary (with ATP and pseudosub- Protein B (rSP-B) strate, PKI5-24) forms. We used backbone NMR chemical shift restraints in all Tadiwos G. Asrat, Valerie Booth. three states to bias the conformational search toward conformations more Biochemistry, Memorial University of Newfoundland, St. John’s, NL, reflective of experimental observables. Through this rigorous approach, we Canada. were able to characterize the rugged free energy landscape of protein kinase Surfactant associated-protein B (SP-B) is the most essential protein component A and identify its modulation by ligand binding: whereas the apo state ex- of lung surfactant; its absence is fatal. Lung surfactant associated proteins, hibits heterogeneous conformations, nucleotide binding partly reduces the especially SP-B, assist in the organization and rearrangement of the lipid mono- conformational plasticity, and subsequent inhibitor binding further quenches layers and multilayers that coat the air-water interface of the alveolus, and so the fluctuations. We conclude that NMR-Restrained Metadynamics is a help to reduce the work of breathing and prevent lung collapse. Due to promising approach to describe the free energy landscapes of complex pro- SP-B’s unique hydrophobic nature however, the overall three dimensional teins at atomic resolution through the integration of experiments and structure of the protein is not yet determined, therefore the molecular basis simulations. for its activity is not clearly understood. Recently, our lab has managed to pro- duce the protein in bacteria using recombinant DNA technology. Recombinant 255-Pos Board B20 SP-B (rSP-B) is shown to retain the expected alpha helical conformation in Thermal Response of Inner and Outer Transmembrane Segments of Cora different membrane mimetic conditions, over a range of temperatures as Protein by a Coarse-Grain Monte Carlo Simulation observed by far UV circular dichroism (CD) spectroscopy. Dynamic Light Ras Pandey1, Sunan Kitjaruwankul2, Channarong Khrutto3, Scattering (DLS) of rSP-B suspended in detergent micelles indicates two pop- Pornthep Sompornpisut3, Barry Farmer4. ulations, one with a hydrodynamic size of ~3 nm and the other at ~100 nm. 1University of Southern Mississippi, Hattiesburg, MS, USA, 2Kasetsart Nanoparticle Tracking Analysis (NTA) confirmed the ~100 nm species to University, Sriracha Campus, Thailand, 3Chulalongkorn University, be prominent. Preliminary 1D solution NMR experiments have also been car- Bangkok, Thailand, 4Air Force Research Laboratory, Dayton, OH, USA. ried out. Inner (i.corA) and outer (o.corA) transmembrane (TM) components of CorA Moreover, rSP-B also shows promising results in in vitro measures of function- protein perform specific functions in transport of Mg2þ across the ion channels. ality when tested with a Langmuir-Blodgett trough. Lipid films in the presence The monomer protein (with 351 residues) consists of its outer (o.corA, residues of rSP-B are able to promote multilayer formation when compressed to a suf- 1-290) and an inner (i.corA, residues 291-351) segments that form the pore. We ficiently high surface pressure, comparable to that of clinical lung surfactants. investigated the structure and dynamics of CorA protein and its inner (i.corA) What is more, films in the presence of rSP-B demonstrate superior compress- and outer (o.corA) TM components as a function of temperature by a coarse- ibility and film recovery when compared to films compressed without rSP-B. grained Monte Carlo simulation in an implicit solvent. We found that the

BPJ 7716_7719 Sunday, February 12, 2017 51a thermal response of i.corA differed considerably from that of the outer compo- 258-Pos Board B23 nent o.corA. Analysis of the radius of gyration revealed that the inner TM Biophysical and Structural Characterization of Antibody Responses to component underwent a continuous transition from a globular conformation Malaria Antigens to a random coil structure on raising the temperature. In contrast, the outer Stephen Scally1, Alexander Bosch1, Brandon McLeod1, Gianna Triller2, transmembrane component exhibited an abrupt (almost discontinuous) thermal Katharina Imkeller2, Rajagopal Murugan2, Sebastian R€amisch3, Rick King4, response in a narrow range of temperature. Scaling of the structure factor William Schief3, Hedda Wardemann2, Jean-Philippe Julien1. showed a globular structure of i.corA at low temperature with an effective 1Molecular Structure & Function, The Hospital for Sick Children Research dimension D~3and random coil at high temperature with D~2. The residue Institute, Toronto, ON, Canada, 2Max Planck Institute for Infection Biology, distribution in o.corA is slightly sparser than that of i.corA in a narrow thermos- Heidelberg, Germany, 3The Scripps Research Institute, San Diego, CA, USA, responsive regime. The difference in thermo-response characteristics of these 4Malaria Vaccine Inititive, Washington, DC, USA. components (i.corA, o.corA) may reflect their unique transmembrane func- Malaria is a global health priority: 214 million malaria cases were reported in tions. Attempts are being made to incorporate such realistic features as explicit 2015 alone, predominantly in Africa and resulting in 438,000 deaths - 70% of solvent and membrane; corresponding results may also be presented as data which were in children below five years of age. Reverse vaccinology holds become available. promise to design effective immunogens for the development of malaria vac- cines. This concept is based on interrogating the B cell repertoire of vaccinated 256-Pos Board B21 or infected subjects to identify protective antibodies that will guide immunogen The Competition between Electrostatic-Steering and Conformational design. Our efforts have focused on plasmodium falciparum targets associated Dynamics in the Diffusion-Limited Association of Calcineurin and with the development of pre-erythrocytic and transmission-blocking vaccines. Calmodulin We isolated B cells from individuals naturally exposed to plasmodium falcip- Peter M. Kekenes-Huskey1, Bin Sun1, Eric C. Cook2, Trevor P. Creamer3. arum, and from vaccinated animals and humans having undergone controlled 1Chemistry, UK, Lexington, KY, USA, 2Biochemistry, University of infection. We performed extensive binding experiments to characterize affin- Kentucky, Lexington, KY, USA, 3Chemistry, University of Kentucky, ities and competition of dozens of antibodies by isothermal titration calorimetry Lexington, KY, USA. and biolayer interferometry. These studies uncovered several epitope bins, Calcineurin (CaN) is a serine/threonine phosphatase that regulates a variety of which give biophysical insights into immunodominant and protective B cell physiological and pathophysiological processes in most mammalian tissue. In responses. X-ray crystallography, small-angle X-ray scattering and single- its inactive state, the CaN catalytic domain is inhibited by the auto-inhibitory particle electron microscopy were used and integrated to define antibody recog- domain (AID); the active state is obtained upon CaM binding to the CaN reg- nition at the molecular level. Our structural delineation of protective epitopes ulatory domain (RD). It has been established that the RD is highly disordered provides the blueprints to engineer optimized antigens that can be formulated when inhibiting CaN, yet it undergoes a disorder-to-order transition upon and tested as pre-erythrocytic and transmission-blocking malaria vaccines. binding calmodulin (CaM) to activate the phosphatase. Given that the RD 259-Pos Board B24 is richly populated with polar and charged amino acids, arguably electrostatic Structural Analysis of the Precursor Protein of Atrial Natriuretic Peptide interactions may influence the rate of CaM association. However, it is likely Sumika Futori, Satomi Higashigawa, Shigeru Shimamoto, Yuji Hidaka. that properties of the RD conformational ensemble, such as its ‘effective vol- Kindai university, Higashi-Osaka, Osaka, Japan. ume’ and accessibility of its CaM binding motif, influence the CaM/CaN Atrial natriuretic peptide (ANP) is produced in the atrium and functions as a association rate. In present study, we investigated via computational modeling vasodilator. ANP is expressed in the form of a precursor, prepro-ANP, and is the extent to which electrostatics and structural disorder co-facilitate or hinder then processed into pro-ANP, which consists of the pro-peptide (98 residues) CaM/CaN binding kinetics. We examined several peptides containing the and mature ANP (28 residues, one disulfide bond) regions, in the endoplasmic CaM binding motif, for which lengths and amino acid charge distributions reticulum. The biological role of ANP has been studied extensively. However, were varied, to isolate the contributions of electrostatics versus conforma- our knowledge of the role of the pro-peptide region of pro-ANP is still unclear. tional diversity to predicted, diffusion-limited association rates. These rates Therefore, to study the processing mechanism of pro-ANP and the role of the were predicted using Replica Exchange Molecular Dynamics (REMD) pro-peptide region, recombinant pro-ANP was prepared by an E. coli expression and Brownian Dynamics (BD) simulations. Our results indicate that associa- system and its conformation examined by means of circular dichroism (CD). tion rates vary as a function of increasing CaN RD length (beyond the The recombinant pro-ANP, which was readily over-expressed as a soluble form required CaM recognition sequence), thus indicating that RD conformational in E. coli cells, was purified by ion exchange chromatography, and identified by ensemble properties influence CaM binding. Second, we found that increasing MALDI-TOF/MS. the solvent ionic strength generally depressed CaM/CaN association rates, To obtain structural information regarding pro-ANP, CD measurements were car- owing to the attenuation of long-range electrostatic interactions that would ried out in 20mM Tris/HCl buffer (pH7.4). However, the results suggested that normally accelerate protein-protein association. Finally, CaN peptides with pro-ANP does not possess specific secondary structures similar to other intrinsi- positively-charged amino acids substituted at native negatively-charged sites cally disordered proteins. Therefore, to further investigate the tertiary structure of had complex effects on the predicted association rate, owing to ‘off-target’ in- pro-ANP, we employed trifluoroethanol (TFE) as a structural mediator and teractions that in some cases competed with the intending binding site. Our collected CD spectra of pro-ANP under several sets of conditions. The results findings detail the interplay between conformational diversity and indicated that TFE dramatically induced the formation of an a-helical structure electrostatically-driven protein-protein association involving CaN, which in pro-ANP, suggesting that the pro-peptide region of pro-ANP is able to form are likely to extend to wide-ranging processes regulated by intrinsically- an a-helical structure under a hydrophobic environment. Therefore, we propose disordered proteins. that the tertiary structure of pro-ANP may be stabilized by interacting with hydro- phobic compounds, such as serum albumin or membrane lipids, in vivo. 257-Pos Board B22 Equilibrium Molecular Dynamics of the Monomer and Dimer Units of 260-Pos Board B25 Streptococcus Pnuemonae and Corynebacterium Diphtheriae Pili Inhibition of Aggregation in B-Sheet Model Peptide by PPII Helix Capping Emmanuel Naziga, Jeff Wereszczynski. Heng Chi1, Min Zhou1, Timothy A. Keiderling2. Illinios Institute of Technology, Chicago, IL, USA. 1pharmacy, Jiangsu Food and Pharmaceutical Science College, Huaian, Pili are elongated protein structures that enhance the adhesive abilities and China, 2Chemistry, University of Illinois at Chicago, Chicago, IL, USA. virulence of bacteria. As a result, it is important to understand their structure A 12mer peptide (Beta, SWTVEGNKYTYK-NH2) was designed based on the and mechanical properties. Pili are constructed by joining monomeric units (or Trpzip model. Beta can form an aggregate with fibril like morphology imaged pilins) into polymeric fibers via intermolecular isopeptide bonds. Interest- by TEM. The secondary structure of the fibril was characterized to be anti- ingly, the pilins additionally possess intramolecular isopeptide bonds that parallel b-sheets by FT-IR spectra. VCD (Vibrational Circular Dichroism) are formed between the sidechains of lysine and asparagine residues in the spectrum demonstrated the supramolecular chirality of the fibril and UV-CD same domain of the protein, which are believed to impart mechanical stability spectrum of this peptide confirmed the tertiary contact between aromatic resi- to the pili. In this work, we use all atom molecular dynamics simulations to dues in the sequence. Upon mutation with an added PPII helix inducing combo study the conformational dynamics of monomeric and dimeric forms of the to the C-terminal of this peptide, a new peptide BP (SWTVEGNKYTYKN pilins of streptococcus pnuemonae (RrgB) and corynebacterium diphtheriae GAPPPK-NH2) was synthesized. BP did not enhance ThT fluorescence, its sec- (spaA) in the presence and absence of intramolecular isopeptide bonds. The ondary structure was characterized to be unordered by UV-CD and FT-IR results reveal how the energetics and dynamics of these subunits are affected spectra. Further FRET analysis with a dansylated version of BP shows that by intramolecular isopeptide bonds, and demonstrate how they affect pili the end to end distance is consistent with the proline mutations disrupting the stability. aromatic contacts and thus stabilized the unordered conformation. In contrast,

BPJ 7716_7719 52a Sunday, February 12, 2017

1:1 ratio mixture of a 9mer PPII conformation peptide and Beta did not change Cells rapidly change their volume in both optimal conditions and under the aggregation behavior of Beta. This study points out that sequence mutation duress, causing changes to their internal solute concentrations. Proteins is crucial in order to change the propensity of aggregation in peptides. are known to respond to such fluctuations in solution concentrations even faster, well before the cell can initiate regulatory response. We take advan- 261-Pos Board B26 tage of this response to probe protein conformation and interactions in live Protein Folding as a Resonance Phenomenon, with Folding Free Energies cells, under biologically relevant conditions. We induce a cellular volume Determined by Protein-Hydration Shell Interactions change in cells by subjecting them to a jump in media osmotic pressure. Sungchul Ji. Protein dynamics are observed by FRET signal collected on an epifluores- Pharmacology and Toxicology, Rutgers University, Kendall Park, NJ, USA. cent microscope from adherent cells expressing mCherry/GFP tagged pro- The single-molecule enzyme-turnover-time histogram of cholesterol oxidase teins. Our experiments reveal protein conformational changes, as well as [1] resembles the blackbody radiation spectrum at 4000 K. This observation binding of weakly bound protein complexes, depending on the labeling motivated the author to generalize the Planck radiation equation (PRE), Sl = scheme used. We observe that proteins can bind and unbind, and structures p l5 hc/lkT (8 hc/ )/(e 1), by replacing the universal constants and temperature can expand and contract reversibly when the cell volume increases or de- by free parameters, resulting in the Planckian Distribution Equation (PDE), creases. Our results indicate that some proteins structures are more sensi- þ 5 C/(x þ B) y = (A/(x B) )/(e 1) [2]. Since the first factor in PRE reflects tive than others to cellular volume change, and that altering the internal the number of standing waves generated in the blackbody and the second factor concentrations within the cell can alter protein processes, including folding the average energy of the standing waves [3], it was postulated that any mate- and binding. rial system that generates data fitting PDE can be interpreted as implicating standing waves with associated average energies [2]. PDE has been found to fit the long-tailed histogram of the folding free-energy changes measured Protein Structure, Prediction, and Design I from 4,300 proteins isolated from E. coli [4]. One possible interpretation of this finding is (i) that proteins (P) and their hydration shells (HS) are organized 264-Pos Board B29 systems of oscillators with unique sets of natural frequencies, (ii) Ps assume Understanding the Role of Chain Flexibility in Amyloid Protein Aggrega- their conformations whose standing waves are frequency-matched (or resonate) tion through Rationally Designed Protein Sequences 1 2 1 1 with the standing waves of their HSs, and (iii) the folding free energies are Steven Z. Vance , Xavier Redmon , Rachel Hall , Colman Moore , 2 2 1 determined by the resonance frequencies of the P-HS complexes. Gram Booth , Christa Hestekin , Melissa Moss . 1Biomedical Engineering, University of South Carolina, Columbia, SC, USA, [1] Lu, H. P., Xun, L. and Xie, X. S. (1998). Single-Molecule Enzymatic Dy- 2 namics. Science 282, 1877-1882. Chemical Engineering, University of Arkansas, Fayetteville, AR, USA. [2] Ji, S. (2012) Isomorphism between Blackbody Radiation and Enzyme The aggregation of amyloid proteins is associated with a myriad of medical Catalysis. In: Molecular Theory of the Living Cell: Concepts, Molecular Mech- conditions including Alzheimer’s disease (AD), diabetes, and Parkinson’s anisms and Biomedical Applications. Springer, New York, pp. 343-368. http:// disease. While attributable to different amyloid proteins, these proteins www.conformon.net/wp-content/uploads/2012/11/Isomorphism_blackbody_ all share a common feature: a periodic glycine motif (GxxxG). This radiation_enzymic_catalysis_p343_p368.pdf glycine motif, associated with increased backbone flexibility, is extended [3] Blackbody radiation. http://hyperphysics.phy-astr.gsu.edu/hbase/mod6. in a number of familial mutations that significantly increase severity of html AD. A better understanding of the role that chain flexibility plays in pro- [4] Dill, K. A., Ghosh, K. and Schmidt, J. D. (2011) Physical limits of cells and tein aggregation will allow for new and innovative protein engineering proteomes. PNAS 108:17876-82. strategies for nanotech development as well as give insight into therapeutic strategies. 262-Pos Board B27 In this study, the glycine motif is targeted via either extension, by introduction Reconstruction of Primordial P-loop NTPase Precursors of additional periodic glycine, or contraction, by replacement of glycine with Maria Luisa Romero-Romero. a bulky or constrained amino acid. Modifications that extend periodic glycine Weizmann Institute of Science, Rehovot, Israel. include those that align with familial AD mutations. To examine how these In collaboration with: Agnes Toth-Petroczy, Alexander Goncearenco and Igor alterations to the glycine repeat motif impact aggregation kinetics, aggrega- Berezovsky (inference of the ancestral P-loop sequence) and Lin Yu-Ru and tion was monitored via thioflavin-T fluorescence and fit using a novel kinetic David Baker (computational protein design). equation that accounts for unique features observed at late stages of aggre- Short functional peptides are likely to have served as crucial intermediates be- gation. Aggregation products were visualized using transmission electron mi- tween a primordial RNA world and the extant protein world. Our working hy- croscopy to examine morphological features. In addition, aggregates were pothesis is that relics of these ancestral peptides still exist in the form of key fractionated by size exclusion chromatography for size analysis via light scat- motifs in active sites of present-day proteins. One such motif, probably the tering and morphology analysis via surface hydrophobicity. Results indicate most obvious one, is the P-loop (Walker A motif) that typically binds the trans- that increased chain flexibility correlates with faster nucleation as well as a ferred phosphate moiety of ATP. The P-loop’s extended motif comprises a larger quantity of small, intermediate aggregates that exhibit reduced fibril b-strand and an a-helix connected by the P-loop – a glycine-rich phosphate- morphology with unchanged surface hydrophobicity. Taken together with binding loop. The P-loop containing nucleoside triphosphate hydrolase fold observations that smaller aggregates are more physiologically active, these includes this motif, and is one of the most ancient folds and by far the most results support the hypothesis that increases in protein chain flexibility, abundant (10-18% of all ORFs). In the present work, the sequence of a primor- including that associated with familial mutations, may contribute to disease dial b-Ploop-a motif was inferred. We further showed that this b-Ploop-a motif progression. (~25 amino acids) could have served as a building block for the creation of Future work will extend studies to other amyloid proteins, such as amylin and ancestral globular domains by means of duplication and fusion. Computational chaplin H. designed proteins were generated consisting of 2 repetitions of the ancestral motif grafted into an ideal b/a- repeat protein. Our results indicate that these 265-Pos Board B30 simple, repetitive proteins avidly bind ssDNA and RNA (possibly a crucial Probing Interactions between the Curli Accessory Protein CsgE and step in the transition from the RNA world to a protein world). Further, a Human Islet Amyloid Polypeptide b-a-b fragment of this protein (< 40 amino acids) was found to self- Tanya J. Espino, Sharon Patray, Isamar Aranda, Karen Guerrero, assemble and thereby confer ssDNA binding. Overall, our results show that Sajith Jayasinghe. P-loop NTPases could have emerged from a relatively short P-loop containing Biochemistry, CSUSM, San Marcos, CA, USA. peptide and that self-assembly played a key role in endowing biochemical func- Curli fibers are known to be involved in bacterial adhesion to surfaces, cell ag- tion despite limited size and complexity. gregation, and biofilm formation. Curli belong to a class of fibers known as am- yloids. Curli assembly requires 5 proteins: CsgA, CsgB, CsgE, CsgF, and 263-Pos Board B28 CsgG. CsgA and CsgB are the major structural components of curli. CsgG is Probing Protein Folding and Interaction in Live Cells using Osmotic an outer membrane protein responsible for the secretion of CsgA and CsgB Perturbations to the extracellular surface. CsgE and CsgF are thought to be chaperon proteins Shahar Sukenik1, Martin Gruebele2. which are responsible for the transport of CsgA and CsgB in the periplasm. It 1Center for Physics of Living Cells and the Department of Chemsitry, has been shown that CsgE inhibits the aggregation of CsgA, the main compo- University of Illinois, Urbana-Champaign, Urbana, IL, USA, 2Department of nent of curli fibers. However, not much is known about the specific interactions Physics and Department of Chemistry, University of Illinois, Urbana- between these two proteins. We employed a series of single cysteine mutants of Champaign, Urbana, IL, USA. CsgE and fluorescence quenching to investigate the interaction of CsgE with

BPJ 7716_7719 Sunday, February 12, 2017 53a

CsgA and with human islet amyloid polypeptide another amyloidogenic protein PloS one, 11 (6), e0158372], Atomic Force Microscopy and Optic Spectros- unrelated to curli formation. copies will be also discussed.

266-Pos Board B31 268-Pos Board B33 Non-Standard Protein Engineering at the Boundary of Molecular Molecular Determinants of Specificity in the Dpr-DIP Interaction Mechanics and Quantum Chemistry: Halogen-Based Design of Insulin Network 1 2 3 3 Analogs Aiman Sherani , John M. Jumper , Engin Ozkan , Tobin R. Sosnick , 3 1 1 2 Benoit Roux . Michael A. Weiss , Nelson F. Phillips , Faramarz Ismail-Beigi , 1 1 1 1 Biophysical Sciences, University of Chicago, Chicago, IL, USA, Vijay Pandyarajan , Yanwu Yang , Yen-Shan Chen , 2 3 Nalinda Wickramasinghe1, Brian Smith3, John G. Menting4, Chemistry, University of Chicago, Chicago, IL, USA, Biochemistry and Michael C. Lawrence4, Krystel El-Hage5, Markus Meuwly5. Molecular Biology, University of Chicago, Chicago, IL, USA. 1Biochemistry, Case Western Reserve University, Cleveland, OH, USA, Understanding the physical and chemical principles governing specificity in 2Medicine, Case Western Reserve University, Cleveland, OH, USA, protein—protein binding is important both for revealing mechanisms of molec- 3Chemistry & Physics, La Trobe University, Melbourne, Australia, ular recognition and for designing novel biomolecular systems. The protein 4Structural Biology, Walter and Eliza Hall Institute of Medical Research, families Dprs and DIPs, comprised of 21 and 9 immunoglobulin superfamily Parkville, Australia, 5Chemistry, University of Basel, Basel, Switzerland. (IgSF) proteins, respectively, mediate neural cell adhesions and are critical The broad utility of halogens in medicinal chemistry has motivated applica- for synapse formation specificity during development in arthropods. Previous tion of hybrid quantum- and molecular-mechanical methods. Extending these research has recently characterized the Dpr-DIP interaction network and found concepts to a therapeutic protein (insulin), iodination of a conserved tyrosine that each Dpr selectively interacts with specific DIPs; the synaptic connectivity at position B26 was recently shown to enhance key properties of a rapid- pattern in the fly brain is encoded by this molecular interaction network. The acting clinical analog, including its thermodynamic stability and resistance goal of this project is to determine the molecular basis of the specificity of these to fibrillation. Here, we (i) describe quantitative atomic-level simulations of interactions. To achieve this goal, we are computing the binding free energies the mono-iodininated insulin to predict its structural features and (ii) test these of each possible Dpr-DIP interaction using both the Molecular Mechanics- predictions by X-ray crystallography and multidimensional NMR spectros- Poisson Boltzmann Surface Area (MM-PBSA) approach and potential of copy. Using an electrostatic model of the modified aromatic ring based on mean force (PMF) based framework, constructed from all atom molecular dy- quantum chemistry, the calculations suggested that the analog—as a dimer namics (MD) simulations. We are also employing a new near-atomic level MD and hexamer—exhibits subtle differences in aromatic-aromatic interactions program, Upside, to rapidly sample possible poses. Due to the lack of high- at the dimer interface. Eight aromatic rings at this interface (residues B16, resolution crystal structures for most of these interactions, homology modeling B24-B26 and their dimer-related mates) must reorganize to enable packing was used in order to generate the initial structures. The calculated binding free of the hydrophobic iodine atoms within the core of each monomer. Strikingly, energies will be used to predict possible Dpr-DIP interactions. Ultimately, we these features were observed in the crystal structure of the mono-iodonated hope to use our current approach to predict potential protein—protein interac- insulin analog (determined as a zinc hexamer at 2.3 A˚ resolution). Given tions between other IgSF proteins, which make up the second largest protein that residues B24-B30 detach from the core on receptor binding, the environ- family in the human proteome, and are crucial for the development and func- ment of the 3-iodo-tyrosine at position B26 in a hormone-receptor complex tion of the nervous and immune systems. must differ from that in the free hormone. Based on the recent structure of 269-Pos Board B34 a ‘‘micro-receptor’’ complex, we predict that more marked reorientation of Prediction of Protein and RNA Structures by Co-Evolution: Going Beyond the modified tyrosine at the receptor interface enables directional halogen Anecdotal Cases towards Large-Scale bonding and halogen-directed hydrogen bonding: favorable electrostatic Guido Uguzzoni1, Shalini John Lovis2, Francesco Oteri1, Hendrik Szurmant3, s interactions exploiting, respectively, the halogen’s electron-deficient -hole Weigt Martin1, Alexander Schug2. and electronegative equatorial band. Thus, inspired by quantum chemistry 1Sorbonne Universite´s, UPMC, Institut de Biologie Paris-Seine, CNRS, and molecular dynamics, such ‘‘halogen engineering’’ promises to extend Paris, France, 2SCC, Karlsruhe Institute of Technology, Karlsruhe, Germany, principles of medicinal chemistry to proteins. Extensions of this approach 3College of Osteopathic Medicine of the Pacific, Western University of to fluoro-aromatic, chloro-aromatic systems, and bromo-aromatic systems in Health Sciences, Los Angeles, CA, USA. non-standard protein design and their potential therapeutic applications will Structural characterization of many important proteins and protein complexes - also be discussed. typically preceding any detailed mechanistic exploration of their function- re- mains experimentally challenging. Novel statistical tools such as Direct 267-Pos Board B32 Coupling Analysis (DCA) take advantage of the explosive growth of sequential Sweeter and Stronger: Structural-Driven Molecular Design to Enhance databases and trace the co-evolution of amino acids to predict secondary and Sweetness and Stability of the Single Chain Monellin MNEI tertiary contacts for proteins [1] and RNAs [2]. These contacts can be exploited 1 1 1 Serena Leone , Andrea Pica , Federica Donnarumma , as spatial constraints in structure prediction workflows leading to excellent 1 1 2 Alessandro Emendato , Rocco Di Girolamo , Roberta Spadaccini , quality predictions [1,2,3,4]. We demonstrate for two-component signal trans- 3 4 Piero A. Temussi , Delia Picone . duction systems (TCS), a ubiquitous signal response system, how different sub- 1Dept. of Chemical Sciences, University of Naples Federico II, Napoli, Italy, 2 families of TCS can be identified based on genomic data [unpublished data]. Department of Science and Technology, University of Sannio, Napoli, Italy, Going beyond anecdotal cases of a few protein families, we have applied our 3Department of Basic and Clinical Neurosciences, King’s College London, 4 methods to a systematic large-scale study of nearly 2000 PFAM protein fam- Napoli, United Kingdom, Department of Chemical Sciences, University of ilies of homo-oligomeric proteins [unpublished data]. Also, we can apply Naples Federico II, Napoli, Italy. DCA to infer mutational landscapes by capturing epistatic couplings between Sweet proteins are a family of proteins with no structural homology, able to residues and can assess the dependence of mutational effects on the sequence elicit a sweet sensation by interacting with the dimeric T1R2-T1R3 sweet re- context where they appear [5]. ceptor [Picone, D. and Temussi, P.A., 2012, Plant science, 195, 135-142]. References Our studies focus on MNEI, a single chain derivative of the natural protein [1] Weigt M et al., Proc Nat Acad Sci USA (2009) 106, 67-72; F. Morcos et al., monellin, representing one of the sweetest molecules known to date. MNEI Proc Nat Acad Sci (2011) 108, E1293-E1301. stimulates a strong interest for its biological properties, but is also a well [2] E. De Leonardis et al., Nucl Acids Res (2015), gkv932. accepted model for protein folding and aggregation studies. In the framework [3] Schug A et al., Proc Nat Acad Sci USA (2009) 106, 22124-22129. of the ‘‘wedge model’’ [Temussi, P.A., 2009, TIBS, 34, 296-302], we have in- [4] Dago A et al., Proc Nat Acad Sci USA (2012), 109: E1733-42. tegrated computational and experimental techniques to enhance sweetness and [5] M. Figliuzzi et al., Mol. Bio. Evol. (2016), 33:268-280, msv211. stability of MNEI against physical and chemical agents [Leone S., et al., 2016, Scientific Reports, 6, 34045]. Based on molecular modeling, NMR [Spadaccini, 270-Pos Board B35 R., et al., 2016, FEBS Lett, (in press)], and X-ray crystallography, we designed Protein-Protein Complex Structure Prediction using the Solution Theory the sweetest protein ever obtained, which combines a sweetness threshold of in the Energy Representation about 25 nM with a very high and pH-independent thermal stability. Docking Kazuhiro Takemura1, Akio Kitao1, Nobuyuki Matubayasi2. studies have provided a rationale basis to explain these properties, hinting at a 1IMCB, University of Tokyo, Tokyo, Japan, 2Grad. Sch. Eng. Sci, Osaka previously unpredicted role of plasticity in the interaction between this protein Univ., Osaka, Japan. and the sweet receptor. Additional aspects of protein aggregation and unfolding Proteins conduct their functions through interactions with other molecules. properties studied by Molecular Dynamics [Leone S. and Picone, D., 2016, Thus, accurate prediction of protein complex is a key to understand functional

BPJ 7716_7719 54a Sunday, February 12, 2017 mechanisms. We have developed a method to evaluate binding free energy dif- 273-Pos Board B38 ferences of complex models generated by docking prediction using the all-atom Using Molecular Dynamics Simulations to Understand Pattern Formation molecular dynamics simulation and solution theory in the energy representa- in Polymers tion. By evaluating binding free energy difference with the method, we previ- Anna Vernon1, Paul Fenimore1, Charlie Strauss2, Chang-Shung Tung1, ously show that ‘‘near-native’’ models similar to crystal structure are Daan Frenkel3, Eugene Terentjev4. successfully selected as the lowest energy structures in two protein-protein 1Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los complexes, bovine trypsin with CMTI-1 squash inhibitor and RNase SA with Alamos, NM, USA, 2Bioscience Division, Los Alamos National Laboratory, barstar [Takemura, J. Chem. Phys. 2012]. The method requires relatively short Los Alamos, NM, USA, 3Department of Chemistry, University of MD simulations (2 ns) and can calculate binding free energy difference among Cambridge, Cambridge, United Kingdom, 4Cavendish Laboratory, several hundreds of complex models. As a protein-protein complex structure University of Cambridge, Cambridge, United Kingdom. prediction method, we combined the evaluation method with CyClus [Omori, We use Molecular Dynamics simulations to study single chain polymer dy- Proteins, 2013] that performs fast clustering/reranking using a cylindrical namics and collapse to imitate the early stages of protein folding, as well approximation of interface and improves the results of the rigid-body docking. as polymer brush collapse, to understand the physics and morphology of The initial complex models were prepared using the protein-protein rigid body collapsing polymer brushes. Simulations of this nature can help us understand docking program, ZDOCK. The generated complex models were then clustered complex phenomena such as glass transition, molecular crowding and com- and reranked using CyClus. Free energy analysis for top 100 or 300 complex plex higher order organisation. As an example, we demonstrate a study of models returned by CyClus were conducted using conformational energies, so- the physical origin of the polymer glass transition from the point of view lute entropies, and solvation free energies calculated with the solution theory in of marginal rigidity, which is achieved above a certain number of intermolec- the energy representation. Our analysis improved the results of CyClus and the ular contacts. We find that when the average number of contacts per monomer complex models similar to the native structure have the lowest binding free en- (covalent and non-covalent) exceeds the critical value z*=4, the system be- ergies, suggesting that this procedure is effective in protein-protein complex comes solid and the dynamics arrested - a state that we declare the glass. structure predictions. We further improved the procedure by placing interface We also look at polymer brushes and describe spinodal decomposition-like waters into protein-protein interface before free energy evaluations. processes that take place upon brush collapse. It is often impossible to simulate large systems without a certain level of sophisticated coarse graining. 271-Pos Board B36 We introduce a possible model for studying higher order organisation of Refinement of Protein Docking with Atom-Atom Contact Potentials, Back- chromatin, a biological complex of DNA and histone proteins within the nu- bone Flexibility and Side-Chain Repacking cleus of a cell. Chromatin organisation is a fascinating topic that resembles Taras Dauzhenka, Ivan Anishchenko, Petras J. Kundrotas, Ilya A. Vakser. protein folding, however, it is much more complex due to multiple levels at Center for Computational Biology, University of Kansas, Lawrence, which chromatin folding and dynamics have to be understood. Using a simpli- KS, USA. fied force field, we also study helical structures formed by peptoids, small Protein-protein docking is a challenging task for proteins with large conforma- ˚ peptidomimetic molecules that are of importance to the pharmacological tional changes upon binding (unbound/bound interface RMSD > 3 A). The industry. protein-protein docking approaches typically involve a refinement stage that follows the lower-resolution global rigid-body search. Most refinement proto- 274-Pos Board B39 cols utilize the same rotational/translational degrees of freedom as at the global Molecular Interactions of Cannabinoid Receptor Interacting Protein 1 A scan stage, with a more thorough sampling and sophisticated scoring. The and B with Cannabinoid Receptor 1 RosettaDock protocol also employs side-chain torsional degrees of freedom Pratishtha Singh1, Anjali Ganjiwale2, Allyn C. Howlett3, Sudha M. Cowsik4. in the form of rotamers, and simultaneously optimizes the side-chain conforma- 1School of Life Sciences, Jawaharlal Nehru University, New Delhi, India, tions and the rigid-body position. In our approach, we incorporate rigid-body 2Institute of Bioinformatics and Applied Biotechnology, Bangalore, India, rotational/translational and side-chain and backbone loop torsional degrees of 3Department of Physiology and Pharmacology, Wake Forest School of freedom into a single automated pipeline, that utilizes (i) our previously devel- Medicine, Winston-Salem, NC, USA, 4Jawaharlal Nehru University, oped semi-empirical atom-atom contact potentials for the rigid-body adjust- New Delhi, India. ment of the protein positions; (ii) a neighbor-dependent Ramachandran Cannabinoid receptor interacting protein isoform 1a (CRIP1a) modulates probability distributions for the backbone loops torsional degrees of freedom; the Cannabinoid receptor 1 (CB1R) activity, which binds to D9-tetrahydro- and (iii) our library of protein-protein interface side-chain rotamers. The pro- cannabinol (main active ingredient of marijuana).The CB1R has important cedure was benchmarked on the Dockground (http://dockground.compbio.ku. physiological roles in synaptic plasticity, analgesia, appetite, and neuro- edu) X-ray unbound set 4.0, which contains 396 co-crystallized protein-protein protection. Studies indicate that CRIP1a suppresses tonic inhibition of complexes and corresponding unbound structures for both proteins. The pro- þ voltage-gated Ca2 channels without altering its agonist dependent activ- cedure was evaluated by CAPRI assessment criteria for its ability to refine ities. There is another isoform of CRIP, known as CRIP1b (128 amino acids) docking predictions with ligand RMSD < 10 A˚ , classified as incorrect, to the shares same functional domain (residue in 34-110 region) with CRIP1a (164 acceptable or better quality category, using only the atom-atom contact poten- amino acids). Although CRIP1b also interacts with CB1R, but its functional tials and the rotational/translational degrees of freedom. significance is not known. As not experimental structure is not available for 272-Pos Board B37 both, here we have attempted to investigate structures and interactions of Protein Folding upon Binding Revealed by Molecular Dynamics CRIP1a and b by in-silico methods. The intense homology searches did Simulation not suggest any PDB structures with significant identity, but Fold Domain Chris Neale1,Re´gis Pome`s2, Rachel Sterne-Marr3, Angel Garcı´a1. Recognition suggested Rho GDP-Dissociation Inhibitor 2(pdb id 1DS6_B) 1Center for Nonlinear Studies, Los Alamos National Laboratory, Los as suitable template for both the targets i.e., CRIP1a and CRIP1b. The Alamos, NM, USA, 2Biochemistry, University of Toronto, Toronto, ON, CRIP1a/b sequences were aligned manually using Discovery Studio2.5, fol- Canada, 3Biology, Siena College, Albany, NY, USA. lowed by model calculations. The outlier residues were indentified and by Macromolecules frequently associate in living cells and tissues, where specific loop refinement protocol. The models were optimized by energy minimiza- binding interactions based on dynamic conformation regulate molecular local- tion using Gromacs, and showed favorable Ramachandran plot statics with ization and activity. Despite the rapidly expanding experimental characteriza- all the residues in favorable regions. Both the models were also supported tion of the structural proteome, the combinatorial explosion of its interactome by their respective secondary structure data. The CRIP1a and 1b both fold provides a strong motivation for the development of new tools to predict into beta sandwich fold model having a beta sheets by loops. The obtained macromolecular binding modes. Molecular simulation is one such promising structure of CRIP1a contains nine beta sheets while CRIP1b contain eight approach, where advances in hardware, software, and parameterization are beta sheets. Both the models were subjected to molecular docking studies driving increasingly accurate models of protein folding. Here, we use temper- with CB1R-C terminus peptide (S464TDTSAEAL472), which is minimal re- ature replica-exchange simulations to study folding at a protein-protein inter- gion involved interactions with CRIP1a/b. Both the complexes were sub- face and reveal the disorder-to-order transition that is necessary for binding jected to molecular dynamics simulations, followed by interaction between a G protein-coupled receptor and either a G protein or a regulatory ki- analysis.Both CRIP1a and CRIP1b shows multiple hydrogen bonds with nase. Specifically, we recapitulate a known G protein binding mode and predict CB1R peptide, revealing the key residues involved in interaction. Since the experimentally uncharacterized binding mode of G protein coupled receptor no crystal/solution structures are available for CRIP1a and CRIP1b, the kinase 2. The macromolecular signaling assembly that we conformationally binding sites revealed from this study can be utilized for site directed muta- define has important implications for kinase function and provides new insight genesis and can serve as major target for diseases related to central nervous into biased signaling from these receptors. system.

BPJ 7716_7719 Sunday, February 12, 2017 55a

275-Pos Board B40 thousands of sequence homologs. To improve contact prediction for small- Polarizable Amoeba Force Field Metadynamics with Minimization Pre- sized protein families, we employ the emerging deep learning technique dicts Missing Protein Loops from Computer Science, a powerful technique that can learn complex patterns Armin Avdic1, Mallory R. Tollefson2, Nicole Tatro2, Stephen D. LuCore2, from large datasets and has revolutionized object and speech recognition, ma- Jacob M. Litman3, Timothy D. Fenn4, Michael J. Schnieders2,3. chine translation and the GO game. Our deep learning model for contact pre- 1Carver College of Medicine, The University of Iowa, Iowa City, IA, USA, diction is formed by two deep residual neural networks. The first one learns 2Biomedical Engineering Department, The University of Iowa, Iowa City, relationship between contacts and sequential features (residue conservation IA, USA, 3Biochemistry Department, The University of Iowa, Iowa City, IA, and predicted secondary structure) from thousands of protein families, while USA, 4Boehringer Ingelheim, Ridgefield, CT, USA. the second one learns the occurring patterns of contacts and their relationship Computational methods developed to find the global free energy minimum of with pairwise features such as contact potential, residue co-evolution strength amino acid sequences are increasingly successful, but limitations in both accu- and the output of the first network. Experimental results suggest that our deep racy and efficiency remain. Optimization algorithms are typically focused on learning method greatly improves contact prediction and contact-assisted proteins of modest size (i.e. of approximately 100 residues) and utilize potential folding, especially for small-sized protein families. Tested on 579 proteins dis- energy functions based on fixed charged force fields, statistical or knowledge similar to training proteins, the average top L (L is sequence length) long-range based potentials, and/or potentials incorporating experimental data. Although prediction accuracy of our method, the representative direct evolutionary the aforementioned methods are widely used, known limitations include 1) coupling method CCMpred and the CASP11 winner MetaPSICOV is 0.47, search protocols that are inefficient or not deterministic due to rough energy 0.21 and 0.30, respectively; their average top L/10 long-range accuracy is landscapes characterized by large energy barriers between multiple minima 0.77, 0.47 and 0.59, respectively. Even without using force fields, our predicted and 2) use of a target function whose global minimum does not correspond contacts allow us to correctly fold 203 test proteins, while MetaPSICOV and to the actual free energy minimum. To overcome the first limitation, this CCMpred contacts can do only 79 and 62 proteins, respectively. In the three work describes a global optimization approach based on metadynamics to drive weeks of blind test with the weekly benchmark CAMEO (http://www. the search of conformational space toward unexplored regions by adding a cameo3d.org/), our method successfully folded three large hard targets with time-dependent bias to the objective function. To overcome the second limita- a new fold and only 1.3L-2.3L sequence homologs while all template-based tion, a hybrid objective function is defined as the sum of the polarizable methods failed. AMOEBA polarizable force field and an experimental X-ray crystallography target. As metadynamics drives the search, periodic quenching via local mini- 278-Pos Board B43 mization is used to access structure quality via evaluation of R . Thus, the Next Generation Evolutionary Sampling and Energy Function Guided work ab initio overall method is called AMOEBA Metadynamics with Minimization Protein Structure Prediction (AMM), and is suitable for optimization of side-chains, ligand binding poses, Avdesh Mishra, Md Tamjidul Hoque. protein loops or even protein complexes. Here we focus on characterizing Computer Science Department, University of New Orleans, New Orleans, the ability of AMM to elucidate the structural details of missing protein loops, LA, USA. which are often excluded from experimental X-ray crystallography structures The conformation of a protein is vital to understand its function. Homology and due to conformational heterogeneity and/or limitations in the resolution of ab initio modeling are the two major strategies to solve the protein structure the data. We first show that the correlation between experimental data and prediction from sequence. The homology methods are not applicable in the AMOEBA structural minima is stronger than that for OPLS-AA/L (i.e. a fixed absence of homologous sequences. This makes the ab initio modeling unavoid- charge force field). Next, missing protein loops are optimized using 5 nsec of able. The development of ab initio method hinges on the effective conforma- sampling for both AMM and simulated annealing with OPLS-AA/L. The tional space sampling and the accurate energy function to guide the search AMM procedure provides more accurate structures in terms of both experi- process. In addition, recent studies demonstrates that it is possible to sample and predict good quality protein structure without using native fragments mental (i.e. lower Rfree values) and structural metrics (i.e. MolProbity). In addi- tion to providing more accurate loop conformations, AMM converged faster from known protein structures. Towards this goal, we developed an ab initio than the simulated annealing protocol. Overall, this work suggests that AMM method that applies a memory assisted Evolutionary Algorithm (EA) to sample is well-suited to refine or predict the coordinates of missing amino acid residues the energy hyper-surface of the protein folding process, looking for the global and/or protein loops due to both the increased accuracy of the target function minimum or the native fold of the protein. Sampling of the energy hyper- relative to OPLS-AA/L and more rapid convergence of the metadynamics surface of the protein is achieved by novel mutation and crossover operations driven search compared to simulation annealing. based on angular rotation and translation capabilities. Furthermore, the cross- over operations in current generation are enhanced by the use of the best parent 276-Pos Board B41 selected from previous generation. In addition, we developed and employed a Improving 3D Structure Prediction of Beta-Barrel Membrane Proteins knowledge-based novel energy function, called 3DIGARS, which can dif- Wei Tian1, Hammad Naveed2, Jie Liang1. ferentiate the native structure that corresponds to the most thermodynamically 1University of Illinois at Chicago, Chicago, IL, USA, 2Toyota Technological stable state, compared to the possible decoy structures most effectively. The Institute at Chicago, Chicago, IL, USA. 3DIGARS energy function is an optimized combination of crucial properties Beta-barrel membrane proteins are found in the outer membrane of gram- such as hydrophobic versus hydrophilic properties, sequence-specific predicted negative bacteria, mitochondria, and chloroplasts. They carry out diverse bio- accessibility and ubiquitous phi-psi angular characterization. The samples logical functions, including pore formation, membrane anchoring, enzyme obtained from the sampling of the energy hyper-surface are scored using activity, and bacterial virulence. In addition, beta-barrel membrane proteins 3DIGARS and passed through the EA operators for natural selection and pro- increasingly serve as scaffolds for bacterial surface display and nanopore- cessing. The merits of the proposed approach has been rigorously examined and based DNA sequencing. Due to difficulties in experimental structure determi- found to be effective. nation, they are sparsely represented in the protein structure databank and computational methods are required to understand their biophysical principles. 279-Pos Board B44 We have developed a novel 3D structural template for the construction of beta- A Predicted Structure of the Angiomotin Lipid Binding Domain barrel membrane proteins, which captures major geometric properties of these Ann C. Kimble-Hill, Cameron J. Peck, Piiamaria S. Virtanen. proteins. With the help of the prediction procedure of strand registers that we Biochemistry & Molecular Biology, Indiana University School of Medicine, developed, we have achieved a high accuracy for the structure prediction of Indianapolis, IN, USA. beta-barrel proteins. In addition, for the beta-barrel proteins with irregular bar- Amots are a family of adapter proteins that modulate cellular polarity, differ- rel shape, we have further developed an elastic model, which characterizes entiation, proliferation, and migration. Amot family members also have a char- bending and curvatures of the barrels, to improve the structure prediction of acteristic lipid-binding domain, the coiled coil homology (ACCH) Domain that these proteins. selectively targets the protein to membranes, which has been directly linked to its regulatory role in the cell. Therefore, we endeavored to understand the 277-Pos Board B42 structure-function relationship of this domain with the desire to find ways to De Novo Protein Structure Prediction by Big Data and Deep Learning modulate these signaling pathways. After many failed attempts to crystallize Sheng Wang, Jinbo Xu. the ACCH domain of each of the Amot family members for structural analysis, Toyota Technological Institute at Chicago, Chicago, IL, USA. we decided to pursue homologous models that could be refined using small Recently ab initio protein folding using predicted contacts as restraints has angle x-ray scattering data. Theoretical models were produced using the Zhang made some progress, but it requires accurate contact prediction, which by ex- suite programs I-TASSER and LOMETS and then refined and analyzed using isting methods can only be achieved on some large-sized protein families with Coot and PyMol modeling software based on RMSD, C-score, TM-score, and

BPJ 7716_7719 56a Sunday, February 12, 2017 template redundancy. Top models were then compared to SAXS data for uate the complexity of the algorithm on real-case of protein structure determi- further model selection and refinement. As a result, we present a theoretical nation. Several procedures of acceleration will be used in order to allow a model of the domain that is driven by alpha helices and short random coil re- complete exploration of the tree describing the molecular Distance Geometry gions. These alpha helical regions form a classic dimer interface followed by problem. The efficiency of the exploration will be evaluated using the self- two wide spread legs that we predict to be the lipid binding interface. Finally, organizing map (SOM) clustering approach, and the quality of obtained confor- we validate the model presented with several lipid binding assays, which leads mations with respect to Ramachandran plots and steric clashes will be to a suggested mechanism that links ACCH lipid binding, membrane deforma- evaluated. tion, and vesicle fusion functions. References: Cassioli A, Bardiaux B, Bouvier G, Mucherino A, Alves R, Liberti L, Nilges M, 280-Pos Board B45 Lavor C and Malliavin TE. An algorithm to enumerate all possible protein con- Self-Association and Conformational Stability of NAMPT Protein formations verifying a set of distance constraints. BMC Bioinformatics, Trivikram R. Molugu1, Udeep Chawla1, Annie Huang1, Radu C. Oita2, 28;16:23 (2015). Ting Wang2, Michael F. Brown1,3, Joe G.N. Garcia2. Lavor C., Alves R., Figueiredo W., Petraglia A., Maculan N. Clifford Algebra 1Chemistry and Biochemistry, University of Arizona, Tucson, AZ, USA, and the Discretizable Molecular Distance Geometry Problem. Adv. Appl. Clif- 2Department of Medicine, University of Arizona, Tucson, AZ, USA, ford Algebras 25 (2015), 925-942. 3Department of Physics, University of Arizona, Tucson, AZ, USA. Nicotinamide phosphoribosyltransferase (NAMPT), also known as Visfatin Protein Stability and Pre B-cell colony enhancing factor (PBEF), is a rate-limiting enzyme in the salvage pathway required for nicotinamide adenine dinucleotide (NAD) 282-Pos Board B47 biosynthesis. It is a highly conserved 52-kDa protein, found in living species Biological Roles of Protein Hyperstability: Implications for Biotechnology from bacteria to humans [1]. The protein serves as a cytokine and is involved Wilfredo Colo´n, Ke Xia, Jennifer Church, Jayeeta Sen, Jane Thibeault, in cellular regulation influencing cancer, ischemia, obesity, and type-II diabetes Hannah S. Trasatti. [2, 3]. Despite being involved in many regulatory pathways, there is a paucity Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, of information concerning the function of NAMPT, due to the limitations of NY, USA. in vivo assays, and lack of expression systems for the protein. Here, we success- Although most proteins in nature are marginally stable, some proteins are hy- fully expressed the NAMPT protein using the pET-SUMO expression vector in perstable, as indicated by their resistance to degradation, even under relatively E.coli strain SHuffle containing a hexa-His tag for protein purification. Activity harsh conditions. The hyperstability of such proteins is usually under kinetic assays demonstrated functionality of the protein. Moreover, initial biophysical control due to a high-energy barrier for unfolding that virtually traps them in characterization of the protein using circular dichroism revealed secondary a specific conformation. Although the protective role of protein kinetic stability structural elements consistent with crystallographic data. Dynamic light scat- is well established, relatively little is known about the extent of biological roles tering showed the protein exists as large oligomeric units potentially involved related to this biophysical property. We have demonstrated a correlation be- in the NAMPT signal amplification pathway. Hydropathy analysis indicated tween kinetic stability and a protein’s resistance to the denaturing detergent possible hydrophobic patches on the protein surface that explains the native SDS, and have developed electrophoresis assays that in combination with pro- oligomeric state. Most striking, we discovered that NAMPT can be solubilized teomics analysis allow the identification of kinetically stable proteins in in n-dodecyl-b-D-maltopyranoside detergent in monomeric form. These find- complex biological samples. We have applied these methods to discover hyper- ings open opportunities for further structural and functional investigations. stable proteins in diverse systems, including mesophilic and thermophilic bac- Presently we are optimizing conditions for NMR experiments on NAMPT pro- teria, beans, and human plasma. The results of these studies have revealed tein. These methods [4] are complementary to X-ray crystallography, and pro- novel insight about the biological significance and roles of protein kinetic sta- vide valuable information on the structure and dynamics, offering an important bility. In addition, the analysis of a growing list of SDS-resistant proteins gener- tool for understanding biological functioning. ated from these studies is enhancing our understanding of the structural basis of [1] T. Wang et al. (2006) Nat. Struct. Mol. Biol 13, 661. protein kinetic stability. [2] S. M. Camp et al. (2015) Sci. Rep. 5, 13135. 283-Pos Board B48 [3] A. Fukuhara et al. (2005) Science 307, 426. Volumetrically Derived Thermodynamic Profile of Interactions of Urea [4] T. R. Molugu et al. (2016) Chem. Rev. (in press). with a Native Protein Ikbae Son, Tigran Chalikian. 281-Pos Board B46 University of Toronto, Toronto, ON, Canada. The Interval Branch-And-Prune Algorithm for the Protein Structure We report the first experimental characterization of the thermodynamic pro- Determination file of urea binding to a native protein. We measured the volumetric param- 1 1 1 The´re`se E. Malliavin , Bradley Worley , Benjamin Bardiaux , eters of lysozyme at pH 7.0 as a function of urea within a temperature range 1 1 2 3 Guillaume Bouvier , Mohamed Machat , Andrea Cassioli , Carlile Lavor , of 18 to 45 C. At neutral pH, lysozyme retains its native conformation 2 1 Leo Liberti , Michael Nilges . between 0 and 8 M urea over the entire temperature range studied. Conse- 1Institut Pasteur, Paris, France, 2Ecole Polytechnique, Paris, France, 3 quently, our measured volumetric properties solely reflect the interactions University of Campinas, Campinas, Brazil. of urea with the native protein and do not involve contributions from A general trend of structural biology is the switch of a rigid description of pro- urea-induced conformational transitions. We treated our data within the tein structures, as in the first X-ray structures in the 50’s, to a flexible descrip- framework of a statistical thermodynamic analytical model in which urea- tion, in which each protein populate several distinct conformations or even a protein interactions are viewed as solvent exchange in the vicinity of the continuum of conformations. This flexibility was shown in numerous cases protein. The van’t Hoff analysis of the temperature dependence of the equi- to have a crucial importance in the function of proteins. Most of the methods librium constant, k, for the urea-protein binding reaction produced changes for bio-molecular structure calculations are, up to now, based on a combination in free energy, DG, enthalpy, DH, and entropy, DS, accompanying the of sampling and optimization, which does not allow a systematic exploration of binding. The thermodynamic profile of urea-protein interactions, in conjunc- the conformational space. But, in the case of highly flexible bio-molecules, a tion with published MD simulation results, is consistent with the picture in systematic (or global) exploration of the conformational space would be very which urea molecules, being underhydrated in the bulk, form strong, en- welcome. thalpically favorable interactions with the surface protein groups while On the other hand, the interval branch-and-prune (iBP) approach has been paying a high entropic price. We discuss ramifications of our results for proposed as a method for allowing a global optimization of molecular struc- providing insights into the combined effect of urea, temperature, and pres- ture under distance restraints, the so-called Distance Geometry problem. A sure on the conformational preferences of proteins. recursive implementation of this algorithm has permitted to apply this approach on small structures of proteins in alpha-bundles for which few 284-Pos Board B49 long-range distance restraints were known. Here, we are going to present Sub-State Conformations of the Mesophilic and Psychrophilic Lactate the results obtained on a set of protein structures with sizes from 24 to Dehydrogenases Preceding Irreversible Thermal Inactivation 100 residues, displaying various secondary structures and topology. The dis- Sergei Khrapunov, Eric Chang, Robert Callender. tance restraints present on these structures were chosen to contain exclu- Biochemistry, Albert Einstein College of Medicine, Bronx, NY, USA. sively short-range information. The thermodynamics of oxamate binding and the temperature stability of The results obtained with various sets of distance restraints, including exact the glycolytic enzyme lactate dehydrogenase from porcine heart, phLDH values and interval of values, will be presented, in order to experimentally eval- (mesophilic Sus scrofa) and from mackerel icefish, cgLDH (psychrophilic

BPJ 7716_7719 Sunday, February 12, 2017 57a

Chamapsocephalus gunnari) have been investigated. A novel fluorescence was still able to form native disulfide bonds even at 80% HMIM or dioxane assay was utilized that simultaneous monitors changes to the global protein although prouroguanylin was not able to fold into its native structure under structure, structural changes near the active site, and aggregation of the enzyme denaturing condition using urea or guanidine hydrochloride. Therefore, the re- in response to increasing temperature and increasing concentration of the nat- sults indicate that those solvents are suitable candidate solvents for use in pro- ural osmolyte, trimethyl amine N-oxide (TMAO), a stabilizer of protein struc- tein folding studies. The results will be discussed in this paper. ture. Using this assay, the reverse changes of stability and affinity for oxamate were established for both, phLDH and cgLDH. Importantly, a low-temperature 287-Pos Board B52 (pre-denaturation) structural transition was found that precedes the high-tem- Glycine Betaine Reverses Osmotic Shock Induced Protein Destabilization perature (denaturation) transition for both LDHs and coincides with increasing in Living Cells enzymatic activity. The structural transitions of the global protein structure and Samantha S. Stadmiller, Gary J. Pielak. the active site are concerted for the rigid (phLDH) and not concerted for the Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, flexible (cgLDH) LDHs. The profound contribution of entropy to G along NC, USA. with the higher structural flexibility increases functional plasticity of the psy- Cells utilize several mechanisms for adapting to changes in osmotic pressure. chrophilic cgLDH. TMAO increases stability and shifts all structural transitions Bacteria, including Escherichia coli, can grow in a wide range of osmolarities. to the higher temperatures for both orthologs and simultaneously reduces their Increasing the external osmolarity (i.e., hyperosmotic shock) causes water catalytic activity. The multiple active and inactive along with intermediate sub- efflux, reduction in cell volume and accumulation of osmolytes such as glycine state conformations of the enzyme exist in equilibrium at the stage preceding betaine. This volume reduction increases the crowded nature of the cytoplasm, irreversible thermal inactivation. This equilibrium is an essential selective fac- which is expected to affect protein stability. In contrast to traditional theory, tor for the adaptation of an enzyme to the environmental temperature. It seems which predicts that more crowded conditions can only increase stability, recent also possible that thermal adaptation of proteins may be complemented by evo- work shows that crowding can destabilize proteins through transient attractive lution of the cellular milieu. interactions. Here, we quantify protein stability in living E. coli cells before and after osmotic shock in the presence and absence of glycine betaine. The 7-kDa 285-Pos Board B50 N-terminal SH3 domain of Drosophila signal transduction protein drk (SH3) is Denatured State Loop Formation Thermodynamics of a Hybrid used as the model protein because it exists in an equilibrium between a folded Polypeptide state and an unfolded ensemble. Labeling SH3 with a fluorine on its sole tryp- Moses Leavens, Bruce E. Bowler. tophan facilitates NMR-based detection of both states simultaneously, allowing University of Montana, Missoula, MT, USA. quantification of the free energy of unfolding in vitro and in living E. coli cells. Previous work with the four-helix bundle protein cytochrome c’inRhodopseu- We find that hyperosmotic shock decreases SH3 stability, consistent with the domonas palustris using histidine-heme loop formation thermodynamic idea that weak interactions are important under physiologically relevant methods revealed fold-specific deviations from random coil character in its de- crowded conditions. Subsequent uptake of glycine betaine returns SH3 to the natured state ensemble. To examine the generality of this finding, we extend stability observed without osmotic shock. These results highlight the effect this work to a three-helix bundle polypeptide, the human DNA excision repair of transient interactions on protein stability in cells and provide a new explana- protein’s second ubiquitin-associated (UBA) domain, UBA(2). We use yeast tion for why stressed cells accumulate osmolytes. iso-1-cytochrome c as a scaffold, fusing the UBA(2) domain to the N-terminus 288-Pos Board B53 of iso-1-cytochrome c. Using site-directed mutagenesis, we have engineered Fluorescence Evidences for Non-Homogeneity and Residual Structure of histidine into solvent accessible surface residue positions within the all-alpha Denatured States fold, creating eight single histidine variants. Isothermal equilibration denatur- Katherina Hemmen1, Dmitro Rodnin1, Igor Markovic1, ation studies reveal that the fusion protein unfolds in a 3-state process, Thomas Otavio Peulen1, Suren Felekyan1, Ralf Kuehnemuth1, commencing with iso-1-cytochrome c followed by UBA(2). Thermodynamic Hugo Sanabria2, Claus A.M. Seidel1. stability experiments also demonstrate that the histidine residues in the 1Institute of Molecular Physical Chemistry, Heinrich-Heine-University, UBA(2) domain strongly destabilize iso-1-cytochrome c. Furthermore, histi- Dusseldorf,€ Germany, 2Department of Physics & Astronomy, Clemson dine-heme loop formation equilibria show lower apparent pKa’s compared to University, Clemson, SC, USA. the pseudo-wild type variant, indicating significant interactions in the dena- About 30 % of human proteins do not fold into a stable 3D arrangement of sec- tured state. We will compare the degree of deviation of loop stability versus ondary structure elements, but stay predominantly unfolded -similar to proteins loop size, relative to predictions of the Jacobson-Stockmayer relationship under highly denaturing conditions. These proteins are involved in many cell used in our previous work on cytochrome c’. This comparison will allow eval- signaling processes. Their characterization poses a great challenge for current uation of sequence-based conformational bias in the denatured state of this experimental methods as they consist of an ensemble of rapidly interconverting protein. conformations. Intense debate exists on the possibility that they show, to certain extent, residual structure, which might facilitate folding or enhance ligand 286-Pos Board B51 binding. To study the unfolded state conformational heterogeneity using Regulation of Protein Folding using Organic Solvents and Ionic Liquids Fo¨rster resonance energy transfer (FRET), we used the lysozyme from the Yuji Hidaka, Ryosuke Nishimura, Shigeru Shimamoto. phage T4 (T4L) in denaturing conditions as a model system. We built an elastic Kinki University, Higashi-Osaka, Japan. network model that spans T4L’s topology in order to evaluate local and global The Escherichia coli expression system is frequently used to prepare various conformational changes by combining ensemble (ensemble time-resolved proteins in a highly efficient manner using information obtained by the Human fluorescence lifetime and anisotropy) and single-molecule spectroscopic Genome Project. However, recombinant proteins are often expressed as inclu- (multiparameter fluorescence detection, photon distribution analysis, (filtered) sion bodies which are biologically inactive, and a refolding reaction is abso- fluorescence correlation spectroscopy) methods. Through extensive compari- lutely required to form the correct tertiary structure. For this purpose, in vitro son of models, we identified regions with apparent residual structure under refolding reactions of recombinant proteins are generally performed in aqueous highly denaturing conditions, which might serve as folding nuclei; and solutions. However, it is still difficult to efficiently fold recombinant proteins additionally we showed that chemically denatured T4L is not a random coil into their biologically active form in aqueous solutions because aggregates as previously thought. By using obtained distance restraints we determined are formed as the result of the hydrophobic folding intermediates. Ionic liquids that denatured T4L shows a native-like mean structure, albeit larger in size have recently been employed for refolding reaction of proteins in place of compared to the native state. We demonstrate here the necessity of careful aqueous solutions and successfully folded into their native conformations can data interpretation, but also the potential of a multidimensional approach to be accomplished, although the folding yield was still low. In addition, organic characterize an ensemble of states, which can be applied generally to unstruc- solvents, such as trifluoroethanol, are generally used for protein folding to tured or denatured proteins. induce a-helix formation and may reduce the extent of hydrophobic interac- tions. Therefore, to regulate or suppress hydrophobic interactions during 289-Pos Board B54 the refolding reaction, organic solvents and ionic liquids were examined in at- Selection Maintaining Protein Stability at Equilibrium tempts to construct the native conformation or to produce a proper folding Sanzo Miyazawa. intermediate. Gunma Univ., Kiryu, Japan. Dioxiane and 1-hexyl-3-methylimidazolium chloride (HMIM) were examined Recently it was indicated that fitness costs due to misfolded proteins are a deter- for the refolding reaction of prouroguanylin, as a model protein, which contains minant of evolutionary rate and selection originating in protein stability is a three disulfide bonds. Prouroguanylin was able to fold into its native conforma- driving force of protein evolution. Here we examine protein evolution under tion at a low concentration of HMIM or dioxane. Surprisingly, prouroguanylin the selection maintaining protein stability.

BPJ 7716_7719 58a Sunday, February 12, 2017

Protein fitness studied is a generic form of fitness costs due to misfolded pro- formation as well as metabolic diseases. Compared to in vitro conditions, the teins; s = k exp(DG / kT) ( 1 - exp(DDG / kT)), where s and DDG are selective cellular milieu is crowded by large biopolymers resulting in intermolecular in- advantage and stability change of a mutant protein, DG is the folding free teractions and excluded-volume effects affecting each biopolymer inside the energy of the wild-type protein, and k is a parameter representing protein cell. abundance and indispensability. The distribution of DDG is approximated to To understand the stability of this large enzyme (70 kDa), we conducted bio- be a bi-Gaussian distribution, which represents structurally slightly- or physical studies on the individual domains, APS kinase and ATP sulfurylase, highly-constrained sites. Also, the mean of the distribution is negatively pro- as well as the full length protein. Using Fast Relaxation Imaging we studied portional to DG. disease-relevant mutants of PAPSS2 directly within the cell. The data provide The evolution of this gene has an equilibrium point DGe, the range of which is novel thermodynamic insights into PAPSS2 stability inside the cellular milieu consistent with observed values in the ProTherm database. The probability dis- and its influence on the naturally destabilized protein. These results also un- tribution of Ka/Ks, the ratio of nonsynonymous to synonymous substitution rate ravel new insights into disease mechanisms of PAPSS2 mutations. per site, over fixed mutants in the vicinity of the equilibrium shows that nearly neutral selection is predominant only in low-abundant, non-essential proteins of 293-Pos Board B58 DGe > 2.5 kcal/mol. In the other proteins, positive selection on stabilizing Effects of Salt or Cosolvent Addition on Thermal Stability of a Protein: mutations is significant to maintain protein stability at equilibrium as well as Relevance to those on Solubility of a Hydrophobic Solute in Water 1 2 2 random drift on slightly negative mutations, although the average is less Shota Murakami , Tomohiko Hayashi , Masahiro Kinoshita . 1Graduate School of Energy Science, Kyoto University, Uji, Kyoto, Japan, than 1. Slow evolutionary rates can be caused by high protein abundance/indis- 2 pensability and large effective population size, which produce positive shifts of Institute of Advanced Energy, Kyoto University, Uji, Kyoto, Japan. DDG through decreasing DGe, and by strong structural constraints, which The thermal stability of a protein is changed upon addition of a salt or cosol- directly make DDG more positive. Protein abundance/indispensability more vent. The solubility of a hydrophobic solute (e.g., argon or methane) in water affect evolutionary rate for less constrained proteins, and structural constraint is also influenced by the addition. Interestingly, the addition which decreases for less abundant, less essential proteins. the solubility usually enhances the thermal stability. This suggests that the hy- (Reference: J. Theor. Biol., 391, 21-34, 2016.) drophobic effect is a principal factor governing the stability change, because the decrease and increase in the solubility, respectively, are ascribed to 290-Pos Board B55 enhancement and reduction of the effect. However, urea decreases the solubi- Effects of Flanking Disorder on the Behaviour of Ordered Domains lity but lowers the stability. Bromide and iodide ions decrease the solubility but Katie R. Kemplen1, Petur O. Heidarsson2, Lasse Staby1, Charlotte O’Shea1, lower the stability of a protein with a large, positive net charge. In these cases, Karen Skriver1, Birthe B. Kragelund1. the stability change is influenced by the changes in not only the hydrophobic 1Biocenter, University of Copenhagen, Copenhagen, Denmark, 2University effect but also other physical factors. We show for hydrophobic solutes that of Zurich, Zurich, Switzerland. the integral equation theory where the solute and solvent particles are modeled Both structured proteins and those containing intrinsic disorder have been the as hard spheres with different diameters can reproduce the experimental data subject of much investigation; especially as disorder relates to protein-protein for the following items: salting out by an alkali halide and salting in by tetra- interactions. However, there has been little exploration into the influence of methylammonium bromide, increase in solubility by a monohydric alcohol, and these elements on each other. Do IDPs regulate the stability and folding mech- decrease in solubility by sucrose or urea. The orders of cation or anion species anisms of connected structured regions and vice versa do ordered domains in terms of the power of decreasing the solubility can also be reproduced for affect the affinity of IDRs for their ligands e.g. by affecting the association ki- alkali halides. With our model, the analyses are focused on the roles of entropy netics? To try and answer these questions we use biophysical methods and pro- originating from the translational displacement of solvent particles. As the gressive deletions to vary the length of disordered regions flanking ordered products, we clarify the pivotal physical origin of the hydrophobic effect and domains in two model systems of one and two globular domains, respectively. present a new view on the Hofmeister series. We show how the series is ex- pressed when the hydrophobic effect dominates and how it is modified when 291-Pos Board B56 other physical factors are also influential. The Effect of Polydisperse Crowding on Protein Stability Alan van Giessen, Anastasia Osti. 294-Pos Board B59 Chemistry, Mount Holyoke College, South Hadley, MA, USA. Residual Structure in the Denatured State of a Three-Helix Bundle Protein The dense, heterogeneous cellular environment is known to affect protein sta- Dustin Becht, Klara Briknarova, Bruce Bowler. bility through interactions with other biomacromolecules. The effect of University of Montana, Missoula, MT, USA. excluded volume due to these biomolecules, also known as crowding agents, The denatured state of a protein is canonically described as an ensemble of non- on a protein of interest, or test protein, has long been known to increase the sta- interacting random coil conformations. By studying a helical bundle protein un- bility of a test protein. The cellular environment is heterogeneous not only in der denaturing conditions, we have found that residual structure persists in its terms of its chemical composition, but also in terms of the sizes of the denatured state. Our work uses the upstream Ubiquitin-Associated domain, biomacromolecules, or crowding agents, present. It has been shown experimen- UBA(1), of the HHR23A protein as a model system to study residual structure tally that the effect of polydisperse or mixed crowding agents has a non-addi- in the denatured state ensemble. Multi-dimensional nuclear magnetic resonance tive effect, i.e. that there is an optimal mixing ratio where the effect of the (NMR) experiments 1H-15N HSQC, HNCO, and HNCA were used to find back- crowding agents is larger than that of monodisperse systems of each crowder. bone atom secondary chemical shifts which correspond to protein secondary Here we investigate the role of polydisperse crowding on two small test pro- structure with amino acid resolution. By comparing chemical shifts in moder- teins: the helical trp-cage and the beta-hairpin GB1m3. For each test protein, ate- to highly denaturing conditions of 4M, 5M, and 6M guanidine HCl to a a series of simulations using crowding agents of two different sizes in various reference state of 7M GdnHCl, UBA(1) shows residual helical content among ratios were conducted. Crowding agents used were either spherical excluded- all three helices. In contrast, the helical propensity is predicted by the program volume only crowders or proteins with fixed backbones. In particular, we relate Agadir to be higher in helix 2 and negligible elsewhere, with the assumption the non-additivity to the excluded volume of the crowding agents. We also that helices do not interact. The presence of relatively uniform helical content show that protein-crowder interactions can play a large role in either enhancing among all three helices may indicate stabilizing tertiary interactions despite or offsetting the effect of the crowding agent excluded volume. highly denaturing conditions. To further probe tertiary interactions, single-res- idue mutants of UBA(1) will be used to determine if residues in the hydropho- 292-Pos Board B57 bic core or turn regions are contributing to stability of residual structure in the PAPS-Synthase: Dissecting Folding of a Large and Naturally Fragile denatured state. Protein In Vitro and In Cellulo Oliver Brylski1, Jonathan Wolf Mueller2, Simon Ebbinghaus1. 295-Pos Board B60 1Physical Chemistry II, Ruhr-University Bochum, Bochum, Germany, Phosphorylation Induced Global Structural Destabilization of a Small 2Institute of Metabolism and Systems Research, University of Birmingham, Protein Domain Birmingham, United Kingdom. Ashleigh Bachman, Radwan Ebna Noor, Dimitra Keramisanou, PAPS synthases are bifunctional enzymes providing the cell with the sulfate Ioannis Gelis. donor PAPS (3’-phosphoadenosine-50-phosphosulfate), which is further used Chemistry, University of South Florida, Tampa, FL, USA. by sulfotransferases for modification of several biomolecules (e.g. steroids). Protein phosphorylation at a single or multiple sites is utilized to regulate pro- Isoform PAPS synthase 2 (PAPSS2) has been shown to be fragile within tein functional outcomes and overall cellular activities through signaling path- in vitro experiments, but is stabilized by binding of its endogenous ligands. Mu- ways. At a molecular level, the addition of a phosphoryl group may alter the tations affecting its activity lead to disease states like bone and cartilage mal- function of a protein through distinct and versatile mechanisms. These include

BPJ 7716_7719 Sunday, February 12, 2017 59a allosteric structural and dynamic changes, direct positive or negative modula- a protein that functions as a molecular chaperone, to be less stable. Here, we tion of affinities, masking or unmasking of cofactor binding sites, autoinhibi- investigated the effect of GTP on the stability of H. pylori Hsp60 by using fluo- tion and local disorder-to-order or order-to-disorder transitions. Here we rescence. Hsp60 does not contain any tryptophans. Therefore, a mutant of present evidence from NMR spectroscopy and other biophysical techniques Hsp60 containing a tryptophan replacement at residue 202 (Y202W) was that protein phosphorylation may lead to a global domain destabilization. used for fluorescence studies. Samples of 1 mL were prepared containing Upon phosphorylation a small protein domain acquires a molten globule state, 50 mM Tris-HCl pH 7.5, 1 uM Y202W, 0.25 mM GTP, and varying urea con- where at least two stable folding intermediates exist in equilibrium with a het- centrations (0-6 M). All aliquots were incubated for 24 h before their fluores- erogeneous conformational ensemble. This conformational transition modu- cence was measured between 310 nm and 370 nm. Samples were excited at lates in turn the affinity for protein partners but also permits rapid domain 280 nm. The native state of the Hsp60 mutant displayed a maximum fluores- refolding upon removal of the phosphoryl group. cence intensity at 331 nm. The Hsp60 mutant appeared to be denatured with 4 M urea or higher concentrations. The denatured state of the Hsp60 mutant dis- 296-Pos Board B61 played a maximum fluorescence intensity at 353 nm. Fluorescence was then Promiscuous Contacts and Heightened Dynamics Increase Thermosta- used to determine whether the binding of GTP affected the stability against bility in an Engineered Variant of the Engrailed Homeodomain urea denaturation. Our results show that Hsp60 has a relatively low stability Michelle E. McCully1,2, Valerie Daggett2. against urea denaturation (C1/2 = 2.75 M) and that the binding of GTP had a 1Biology, Santa Clara University, Santa Clara, CA, USA, 2Bioengineering, small destabilizing effect. A C1/2 of 2.50 M was observed for the Hsp60 mutant University of Washington, Seattle, WA, USA. in the presence of GTP. This nucleotide did not have any significant effect on The Engrailed Homeodomain (EnHD) is a three-helix-bundle transcription the thermal stability of Hsp60. factor that is popular within the protein folding community due to its ultra- fast un/folding kinetics. In 2007, the Mayo group used EnHD as a backbone template for their repacking algorithm and designed UVF as a proof of Folding Pathways principle. UVF not only folded as designed but was extremely stable; its Tm 299-Pos Board B64 is >99 C whereas the Tm of EnHD is 52 C. Here, we present a structural and dynamical explanation for UVF’s increased thermostability based on In Vitro Studies of the Folding and Assembly Mechanism of Hemoglobin 1 ms of all-atom, explicit-solvent molecular dynamics simulations of the two Premila P. Samuel, William Ou, George N. Phillips, John S. Olson. proteins at room temperature (25C) and high temperature (100C). UVF Biosciences, Rice University, Houston, TX, USA. had heightened dynamics at room temperature, relative to EnHD, as measured Deriving the pathway for human hemoglobin assembly is important both for by Ca motion and interchangeability of side-chain contacts. UVF maintained developing treatments for hemoglobinopathies and for designing robust acel- these dynamics at 100C whereas EnHD began to unfold. UVF’s binary distri- lular hemoglobin oxygen carriers. The adult human hemoglobin (HbA) com- a b a b bution of amino acids, with only hydrophobic residues at buried positions prises two globin heterodimers, ( 1 1)( 2 2), with each subunit containing a and only hydrophilic residues on the surface, allowed buried residues to heme group for coordination of oxygen and having a tertiary structure very move independently from the surface, effectively acting as an entropy sink. similar to the monomeric paralog, myoglobin. Hydrogen bonds and salt bridges between buried and surface residues of In order to determine the complete folding mechanism, we have measured and EnHD coupled core motion to surface motion, resulting in EnHD denaturing analyzed guanidinium-induced unfolding curves for both apo- and holo- at 100C. Based on our simulation data, we hypothesize that UVF benefits hemoglobin. This approach expands on previous work done in our lab with from more favorable entropy of folding in addition to its rationally designed, myoglobin and has allowed us to deconvolute the globin unfolding pathway more-favorable enthalpy of folding. Future work will investigate this hypoth- and the heme-binding effects for the various folding states. When heme is ex- a b esis experimentally and determine how the heightened dynamics effect native tracted from human Hb, the resulting apohemoglobin dissociates to an 1 1 het- DNA-binding function. erodimer. Unfolding occurs in two major phases. The first involves melting of the heme pockets to generate a dimeric molten globule, followed by dissocia- 297-Pos Board B62 tion into almost completely unfolded monomers. Structural identification of Rational Design of a Synthetic Peg-Like Polymer for Protein Stabilization these phases was achieved by comparing the unfolding curves for native Christopher DelRe1, Brian Panganiban1, Tim Li1, Charley Huang1, HbA, recombinant HbF, a N-to-C termini linked a Hb variant, and a series Monica Olvera de la Cruz2, Patrick Dennis3, Ting Xu1. of Hb mutants with large, hydrophobic residues replacing the distal histidine 1University of California, Berkeley, Berkeley, CA, USA, 2Northwestern and valine in the a and b heme pockets. University, Evanston, IL, USA, 3Air Force Research Laboratory, Dayton, Previously, we showed that monomeric apoglobin stability, and not hemin af- OH, USA. finity, is the determining factor for myoglobin expression. In the case of hemo- Organophosphorus hydrolase (OPH) is an enzyme that can deteriorate acutely globin, the individual monomers are unstable, but associate strongly to form a toxic chemicals known as organophosphates, which are ubiquitously employed stable molten globule dimeric state, which binds hemin and facilitates the over- as pesticides and are thus considerable environmental hazards. However, the all assembly of the hemoglobin. proclivity of OPH to aggregate in aqueous media severely hinders its biological Supported by the NIH Grant HL110900 and by Grant C-0612 from Robert A. activity. Inspired by natural chaperones, we have designed and synthesized het- Welch Foundation. eropolymers to encapsulate and stabilize OPH. When OPH is complexed with the designed heteropolymer, it exhibits a drastic increase in biological activity 300-Pos Board B65 in a buffer solution - approximately 15 times greater than the native biological An Evolutionary Trend towards Kinetic Stability in the Folding Trajec- activity of pure OPH in the same environment. We have confirmed via dynamic tory of RNases H Shion An Lim1,2, Kathryn M. Hart3, Michael J. Harms4,5, Susan Marqusee1,2. light scattering that while pure OPH aggregates in the buffer solution, the het- 1 eropolymer prevents aggregation from occurring. Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA, 2Institute of Quantitative Biosciences (QB3), Two model proteins, bovine serum albumin and lysozyme, have also been stud- 3 ied with the heteropolymer to aid our understanding of the protein-polymer in- University of California Berkeley, Berkeley, CA, USA, Department of Chemistry, University of California Berkeley, Berkeley, CA, USA, 4Institute teractions that occur in our system. The structure and particle size of the 5 different protein-polymer mixtures at several temperatures have been probed of Molecular Biology, University of Oregon, Eugene, OR, USA, Department via dynamic light scattering, circular dichroism, and Fourier transform infrared of Chemistry and Biochemistry, University of Oregon, Eugene, CA, USA. spectroscopy. These characterization techniques have elucidated the mecha- Proper folding of proteins is critical to producing the biological machinery nisms of interactions between the polymer and proteins. essential for cellular function. Over the course of evolution, the rates and ener- getics of a protein’s folding landscape must be maintained such that the protein 298-Pos Board B63 folds and remains folded over its biological lifetime. Developing a comprehen- Stability of Hsp60 from Helicobacter Pylori: Effect of GTP Binding sive understanding of how a protein’s folding process is modulated during evo- Karina Guadalupe, Jose Mendoza. lution is critical to our understanding and engineering of protein biophysical California State University of San Marcos, San Marcos, CA, USA. properties. In this study, we characterized the folding trajectories of ancestral The heat shock protein Hsp60 is one of the most abundant proteins observed in proteins of the ribonuclease H (RNase H) family by using ancestral sequence H. pylori. Since the sequence of Hsp60 is similar to that of the chaperonin from reconstruction to access the evolutionary history between RNases H from mes- E. coli, GroEL, it is hypothesized that their functions are similar as well. How- ophilic and thermophilic bacteria. We find that the overall folding pathway of ever, unlike GroEL which binds to ATP, Hsp60 from H. pylori also binds to RNase H is preserved over billions of years of evolution. Although thermody- GTP. It was previously shown that the binding of GTP caused alpha-crystallin, namic stabilities diverge between the mesophilic and thermophilic lineages,

BPJ 7716_7719 60a Sunday, February 12, 2017 kinetic stability increases along both, with the last common ancestor folding 304-Pos Board B69 and unfolding faster than the modern descendants. The conserved folding inter- Folding Pathways of Evolutionarily Related Proteins Probed by Hydrogen mediate permits this paradoxical uncoupling of thermodynamics and kinetics, Exchange Mass Spectrometry and allows for the folding landscape to independently respond to different se- Eric Bolin, Shion An, Susan Marqusee. lective pressures on global stability and kinetic barriers. UC Berkeley, Berkeley, CA, USA. The process by which proteins adopt their native structure has been an active 301-Pos Board B66 field of study for decades. However, many basic questions about this protein Interpreting Phi-Values using Protein Folding Transition Paths folding process remain unanswered. For instance, the role of sequence in Robert Best1, Gerhard Hummer2. determining the pathway through which a protein folds has been difficult to 1Laboratory of Chemical Physics, National Institutes of Health, Bethesda, 2 characterize since most techniques to characterize pathways are slow, require MD, USA, Department of Theoretical Physics, Max Planck Institute of large amounts of protein, or only probe folding in a small region of the pro- Biophysics, Frankfurt, Germany. tein. Hydrogen exchange measured by mass spectrometry (HX/MS) has It is now possible to watch proteins folding and unfolding spontaneously in mo- proven to be a useful tool that can quickly determine folding pathway of a lecular dynamics simulations using all-atom force fields. Nonetheless, recent protein by measuring the buildup of hydrogen bonding along the protein back- results have shown that several force fields may be able to fold the same pro- bone. In addition to its relative speed, HX/MS is able to measure to probe tein, but with very different mechanisms. Distinguishing which of these is cor- folding across the entire protein sequence allowing a pathway to be deter- rect by comparison to experiment is tricky due to the transient nature of folding mined from a single experiment and uses nanomolar quantities of protein. transitions. Very useful information is furnished by protein folding phi-values, Using HX/MS to determine the folding pathway for the RNases H from which yield residue-resolved information on the relative effect of mutations on E. coli and T. thermophilus shows that despite folding to the same final protein folding rates compared to protein stability. Since computing phi-values structure these proteins fold through distinct pathways. Applying this tech- by brute force is very inaccurate and computationally costly, we have devel- nique to proteins from an ancestral sequence reconstruction of the RNase H oped a theory for estimating phi-values based on folding transition-paths. family further refined the sequence changes responsible for this shift in Applying the theory to all-atom folding simulations by DE Shaw, we find folding pathways. This has led to a model that intrinsic helicity of stretches that in many cases the agreement with experimental data is reasonable. We of the RNase H sequence may determine the earliest steps in the folding are able to resolve a long-standing controversy in phi-value interpretation, pathway. i.e. do fractional phi-values come from parallel pathways or partially formed contacts? The answer is some of each. We show that the diversity of folding 305-Pos Board B70 pathways can be probed by varying the strength of the perturbing mutation at How Hydrodynamic Interactions affect the Folding Rate of Proteins a given residue. Lastly, we have compared the results directly with all-atom Fabio C. Zegarra1,2, Dirar Homouz1,3, Margaret S. Cheung1,2. folding simulations using a Go model in order to assess the importance of 1Department of Physics, University of Houston, Houston, TX, USA, 2Center non-native interactions in protein folding pathways. for Theoretical Biological Physics, Rice University, Houston, TX, USA, 3 302-Pos Board B67 Department of Applied Math and Sciences, Khalifa University, Abu Dhabi, Reconstructing the Folding of Luciferase to Elucidate the Vectorial United Arab Emirates. Folding Pathways of Large, Multidomain Proteins Hydrodynamic interaction (HI) arises by the solvent flow generated by the Zackary N. Scholl1, Weitao Yang2, Piotr Marszalek2. movement of a particle that affects other particles. This long-range interaction 1Program in Computational Biology and Bioninformatics, Duke University, is particularly important in the dynamics of proteins that are immersed in the Durham, NC, USA, 2Duke University, Durham, NC, USA. crowded milieu of cells. Therefore, there is a need for a computationally and Proteins obtain their final functional configuration through incremental folding theoretical quantitative evaluation of its role in protein folding. Using with many intermediate steps in the folding pathway. If known, these interme- coarse-grained molecular simulations and theoretical calculations according diates could be valuable new targets for designing therapeutics and could pro- to the Energy Landscape Theory of protein folding, we assess the impact of hy- vide information on the mechanism of chaperones. However, determining these drodynamic interaction in the folding of two model proteins, the 64-residue intermediate steps is hardly an easy feat, and has been elusive for most proteins, protein chymotrypsin inhibitor 2 (CI2), and the 57-residue alpha-spectrin especially large, multidomain proteins. Here, we effectively map out the major- Src-homology 3 (SH3) domain. In our study, we included hydrodynamic inter- ity of a folding pathway for the model large multidomain protein, Luciferase, action into the equations of motion from Brownian dynamics by computing the by combining coarse-grained simulation and single-molecule force-spectros- diffusion correlation matrices between each residue. We investigated the copy experiments. Simulations indicate that there are several consistent and sta- folding kinetics as well as the thermodynamics of each protein in the presence ble core structures of various sizes nucleating in different regions of Luciferase, and in the absence of hydrodynamic interaction. Our results suggest that the each of which has different propensities for propagating to the final folded impact from hydrodynamic interaction is dependent on the topology of a pro- native state. We identified, using Monte Carlo simulations of Markov chains tein, and temperature. generated from simulation, that Luciferase most often folds along a pathway originating from the nucleation of the N-terminal domain, and that this pathway 306-Pos Board B71 is the least likely to form non-native structures. We engineered truncated var- Folding Mechanisms of Small Proteins GB1 and LB1 1 1 1 2 iants of Luciferase whose sequences corresponded to the putative nucleated Qianyi Cheng , Insuk Joung , Keehyoung Joo , Kunihiro Kuwajima , 1 cores and using atomic force spectroscopy we determined their unfolding Jooyoung Lee . 1School of Computational Sciences, Korea Institute for Advanced Study, and stability. The experimental results corroborate the structures predicted 2 from the folding simulation and strongly suggest that they are intermediates Seoul, Korea, Republic of, Department of Physics, University of Tokyo, along the folding pathway. The simulation also identified non-native structures Tokyo, Japan. originating mainly from the C-terminal domain. Taken together, our results The B1 domains of protein G (GB1) and protein L (LB1) are two small proteins suggest a pathway for cotranslational folding of Luciferase and also suggest that binds to antibody immunoglobulin G (IgG). GB1 and LB1 are similar in a mechanism that chaperones may exploit to prevent misfolding. size (about 60 residues), and also have an overall similar structure (b-hairpin–a-helix–b-hairpin). However their sequences are very different, 303-Pos Board B68 possessing only 15% identity in a structure-based alignment. Therefore, there Using Single Molecule Force Spectroscopy to Detect High-Energy Inter- are interesting similarity and differences in their folding mechanisms. Experi- mediates on Protein Folding Pathway mental evidence indicated that LB1 folds in a two-state manner; while GB1 Ha H. Truong, Emily J. Guinn, Susan Marqusee. folds in a more complex way – an early stage intermediate may exist in the California Institute for Quantitative Biosciences, University of California, folding path. Till now, the folding mechanisms are still under extensive exper- Berkeley, Berkeley, CA, USA. imental and computational study. Structure-based modeling is one of the less Single-molecule force spectroscopy allows for molecular insight into the costly computational methods. It has a simple formulated potential energy behavior under force and the folding mechanism of proteins. High-energy function summing over various geometrical restraints from one or more tar- on-pathway intermediates do not accumulate significantly under native condi- geted structures. Here, we used a new all-atom structure-based method to inves- tions, but may be populated at high force. Here, we use optical tweezers to tigate the folding mechanisms of GB1 and LB1. In this approach, folded investigate a high-energy intermediate and the force dependent movement of structures of the two proteins were used to construct the restraints and they the transition state ensemble. We also apply different pulling geometries to are stabilized by Lorentzian attractive term instead of conventional harmonic determine the effects of the direction and magnitude of the applied force on un- term.3 Our model is able to identify two-state and non-two-state proteins, folding trajectories and compare those results to computational predictions. and gives us more insights of the their folding pathways.

BPJ 7716_7719 Sunday, February 12, 2017 61a

1. Scalley, M. L., Yi, Q., Gu, H., McCormack, A., Yates, 3rd, J. R., and Baker, axial heme ligands. Four species exist in solution: the native HM state (His18/ D. Biochemistry 36: 3373-82, 1997. Met80), the partially folded HW (His18/water) and HH (His18/His33) interme- 2. McCallister, E., Alm, E., and Baker, D. Nat. Struct. Biol. 7: 669-673, 2000. diates, and the 5C (water) unfolded state. The results indicate that the thermal 3. Lee, J., Joo, K., Brooks, B., and Lee, J. J. Chem. Theory Comput. 11: 3211- and chemical denaturization pathways are not the same and that both involve 3224, 2015. significant backbone rearrangement. The relative populations of the conforma- tional states depend on how the protein is denatured. For the same concentra- 307-Pos Board B72 tion, guanidinium causes more unfolding than does urea. The thermal unfolding A Novel Trp Cage Conformer Revealed by Combining High Pressure pathway appears to involve a more gradual unfolding of the protein and a NMR and MD Simulations different sequence of change in the heme iron axial ligands. Additionally, it 1 1 2 3,4 Martin J. Fossat , Soichiro Kitazawa , Scott McCallum , Angel Garcia , was found that addition of multiple ions changed the protein’s stability in an Catherine Royer1. 1 2 additive manner. These results are discussed in terms of the hydrophobic effect, Biology, Rensselaer Polytechnic Institute, Troy, NY, USA, Center for partitioning of the ions to the protein surface, and an altered water structure Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic around the protein. Institute, Troy, NY, USA, 3Department of Physics, Rensselaer Polytechnic 4 Institute, Troy, NY, USA, Center for NonLinear Studies (CNLS), Los 310-Pos Board B75 Alamos National Laboratory, Los Alamos, NM, USA. Uncovering Conformational Substates and Kinetic Constants by Pressure We have used the tryptophan cage variant Tc5b, a widely used model protein Modulation for benchmarking of force fields and water models, to probe pressure effects on Roland Winter. proteins using a combination of high pressure 2D proton NMR TOCSY and TU Dortmund University, Dortmund, Germany. molecular dynamics simulations. Understanding the key factors of the pressure Regulation of protein function is often linked to a conformational switch trig- stability of protein is an essential step towards the development of better force gered by chemical or physical signals. To evaluate such conformational fields and increased predictive power of simulation. We show that results of changes and to elucidate the underlying molecular mechanisms of subsequent MD simulations on Tc5b as a function of pressure using the Amber 99sb force protein function, experimental identification of conformational substates and field show remarkable agreement with the pressure-dependent NMR data. Pres- characterization of conformational equilibria and reaction constants are manda- sure perturbation in different solvent conditions yielded novel information on tory. We applied pressure modulation in combination with various spectros- the structural changes in Tc5b induced by pressure. We identified a sub- copies and scattering experiments to reveal equilibria between substates of ensemble in the folded basin that is strongly destabilized by pressure. Hence, biomolecular systems, such as of the lipidated signaling protein Ras and its as- most of the pressure effect on Tc5b between 1-2500 bar arises from modulation sociation with membrane systems. We show that not only nucleotide binding of the sub-populations of the folded state. The residues observed in the NMR but also the presence of the membrane has a drastic effect on the conforma- experiments to be strongly affected by pressure coincided precisely with those tional dynamics and selection of conformational substates of the protein, and implicated in the pressure-dependent change in Tc5b secondary structure re- a new substate appearing upon membrane binding could be uncovered. In a sec- vealed by the simulations. The relative populations of two folded state con- ond example, we explored the effect of pressure perturbation on phospholipase formers, in which the central 3-10 helix and the following bend are inverted, A2, which catalysis the hydrolysis reaction of sn-2 fatty acids of membrane changed upon pressurization, while the total secondary structural content re- phospholipids, to reveal new mechanistic information about the membrane mained the same. These results highlight the existence of significant plasticity association and subsequent enzymatic reaction. To this end, high-pressure in the secondary structure of folded proteins. Fourier-transform infrared and high-pressure stopped-flow fluorescence spec- 308-Pos Board B73 troscopies were employed. Finally, we discuss the combined effects of temper- Microfluidic Turbulent Mixers, Time Resolved SAXS and Folding Inter- ature, pressure, confinement and osmolytes on the kinetics of enzymatic mediates of CheY reactions (e.g., fibril-based enzymes) and polymerization processes (e.g., of Sagar V. Kathuria1, Osman Bilsel1, Srinivas Chakravarthy2, actin) using high-pressure stopped-flow experiments in combination with rapid C Robert Matthews1. fluorescence and UV/Vis absorption detection. We show that the compatible 1Biochemistry and Molecular Pharmacology, University of Massachusetts osmolyte trimethylamine-N-oxide (TMAO) is not only able to compensate Medical School, Worcester, MA, USA, 2BIOCAT, Argonne National for the strongly retarding effect of chaotropic agents such as urea on actin poly- Laboratories, Argonne, IL, USA. merization, it is also able to largely offset the deteriorating effect of pressure on The process of folding a polypeptide chain from an unstructured random coil in actin polymerization, thereby allowing biological cells to better cope with denaturant to a functional 3 dimensional form is a rapid process that is perhaps extreme environmental conditions. Hence TMAO serves also as efficient coun- biased very early on during the initial steps, even as the solvent conditions are teractant of pressure-induced depolymerization reactions, thereby justifying its transitioning from favoring denaturation to favoring folding. Access to kinetics label piezolyte. in the microseconds to milliseconds time scale by multiple spectroscopic probes is crucial to interpreting these initial steps of folding. Time resolved 311-Pos Board B76 small angle X-ray scattering (trSAXS) is a powerful technique for studying 3 Investigating Cotranslational Folding in Membrane Proteins using dimensional shapes of proteins as they transition from unfolded to native struc- Fragment-Based Structure Prediction 1 1 2 2 tures. By interfacing microfluidic devices with powerful x-ray sources we can Eleanor C. Law , Saulo H.P. de Oliveira , Sebastian Kelm , Jiye Shi , 1 access timescales in the sub-hundred microseconds range. Structural details Charlotte M. Deane . 1Department of Statistics, University of Oxford, Oxford, United Kingdom, determined using trSAXS of the kinetic refolding process of CheY reveal a 2 burst phase in the sub 50-microsecond time-scale, that is perhaps structured Department of Informatics, UCB Pharma, Slough, United Kingdom. around a local-in-sequence cluster of hydrophobic residues. There is experimental evidence that both soluble and membrane proteins start to fold during the process of translation, while still attached to the ribosome. In 309-Pos Board B74 the case of most alpha-helical membrane proteins, their insertion into the Thermal and Chemical Unfolding of Cytochrome C in the Presence of membrane is simultaneously carried out by the Sec complex. We have found Hofmeister Ions evidence to suggest that membrane proteins are also forming tertiary interac- Eric S. Peterson, Collin A. O’Leary, Sean J. Steinke, Mikayla J. Freese. tions cotranslationally. We have tested two structure prediction protocols using Chemistry and Biochemistry, University of Northern Iowa, Cedar Falls, our software SAINT2: one to imitate cotranslational folding which grows the IA, USA. nascent peptide from the N-terminus, and one to imitate in vitro folding which The ferric cytochrome c (Cyt c) (un)folding mechanism in the presence of ions starts from an extended chain. The cotranslational method generates more ac- from the Hofmeister series is examined. Unfolding was initiated both thermally curate models, suggesting that it could be simulating the in vivo folding mech- and with chemical denaturants. Hofmeister ions were added singly and in pairs anism better. We are adapting the SAINT2 score to reflect the membrane to alter the stability of the native folded state, the unfolded state, and two environment in order to improve the accuracy of models produced and to partially folded intermediates. Protein stability was characterized by either more closely imitate in vivo folding. We are also exploring the effect of begin- the midpoint of the chemical denaturization curve or by the melting tempera- ning from a segment of the correct native structure and using SAINT2 to com- ture in the thermal studies. UV/VIS absorption spectroscopy and a basis spectra plete the structure. This is to simulate a folding mechanism where the first part fitting analysis were used to determine the populations of each protein confor- of the protein adopts part of its final fold even before the whole protein is mation along the folding pathway. These species can be differentiated by their synthesised.

BPJ 7716_7719 62a Sunday, February 12, 2017

Protein Dynamics and Allostery I amongst at least three conformational states and shows that ligands modulate the biological activity of the protease by differentially stabilizing a specific 312-Pos Board B77 protease conformation. Molecular Dynamics Simulations for Understanding IR Spectra and the 315-Pos Board B80 Distribution of Environments Around the Phosphopantetheine Arm of Allosteric Transmission Along a Loosely Structured Backbone Allows a Acyl Carrier Proteins Cardiac Troponin C Mutant to Function with only One Ca2D ion Michael R. Jordan, Louise K. Charkoudian, Casey H. Londergan. Mayra A. Marques1, Jose´ R. Pinto2, Adolfo H. Moraes3, Anwar Iqbal1, Chemistry, Haverford College, Haverford, PA, USA. Mariana T.Q. de Magalha˜es1, Jamila Monteiro1, Murilo M. Pedrote1, Natural product biosynthesis in polyketide synthases (PKSs) and fatty Martha M. Sorenson1, Jerson L. Silva1, Guilherme A.P. de Oliveira1. acid synthases (FASs) is heavily reliant on both specificity and selectivity 1Medical Biochemistry Institute, Rio de Janeiro, Brazil, 2Department of of protein-protein interactions. Interactions are mediated by the acyl 3 0 Biomedical Sciences, College of Medicine, Florida, FL, USA, Department carrier protein (ACP), which utilizes a 4 -phosphopantateine (Ppant) arm of Chemistry, Institute of Exact Sciences, Minas Gerais, Brazil. attached to a conserved serine residue to shuttle carbon intermediates be- Hypertrophic cardiomyopathy (HCM) is one of the most common cardiomyop- tween partner enzymes. Recent studies have shown that site-specific vibra- athies, and a major cause of sudden death in young athletes. The Ca2þ sensor of tional spectroscopy can identify the local environment of the arm through 0 the sarcomere, cardiac troponin C (cTnC), plays an important role in regulating cyanylation of the terminal sulfur of the 4 Ppant arm and introduction of muscle contraction. Although several cardiomyopathy-causing mutations have a unique SCN residue. We aim to use molecular dynamics simulations in been identified in cTnC, limited information about their structural defects has GROMACS to simulate IR spectra from atom-level physical interactions been mapped to the HCM phenotype. Here, we use high-resolution electron- and better elucidate the dynamics and structural distribution of the Ppant spray ionization mass spectrometry (ESI-MS), Carr-Purcell-Meiboom-Gill arm. A strong understanding of the behavior of the ACP’s Ppant arm will relaxation dispersion (CPMG-RD) and affinity measurements of cTnC for provide both a reliable and generalizable method for confirming experi- the thin filament in reconstituted papillary muscles to provide evidence of an mental IR data, and inform bioengineering efforts by further characterizing allosteric mechanism in mutant cTnC that may play a role to the HCM pheno- ACP’s interactions with PKSs and FASs that give rise to complex natural type. We show that the D145E mutation leads to altered dynamics on a ms-ms products. timescale, deactivates both divalent-cation binding sites of the cTnC C-domain and modifies cTnC binding to the troponin I inhibitory peptide (cTnI ). 313-Pos Board B78 128-147 CPMG-RD captured a low-populated conformation triggered by the Glu145 Caught in the Act: Trapping an Acyl Carrier Protein Interacting with a replacement of Asp. Paradoxically, although D145E C-domain is unable to Ketosynthase þ þ bind Ca2 or Mg2 , these changes along its backbone allowed it to attach Grace A. Thiele. more firmly to thin filaments than the wild-type isoform, providing evidence Chemistry, Haverford College, Haverford, PA, USA. þ for an allosteric response of the Ca2 -binding site II in the D145E N-domain. Polyketide synthases (PKSs) and fatty acid synthases (FASs) in microorgan- Our findings explain how the effects of an HCM mutation in the C-domain isms rely on selective and specific protein-protein interactions to build struc- þ reflect up into the N-domain to cause an increase of Ca2 affinity in site II, turally complex molecules. These synthases utilize acyl carrier proteins thus opening up new insights into the HCM phenotype. (ACPs) to carry reactive intermediates and substrates to different stops on the enzyme assembly line. ACPs collaborate with their partner enzymes 316-Pos Board B81 by tethering substrates and intermediates onto its highly conserved 40-phos- 0 Dynamics and Assembly of ASB-Containing E3 Ubiquitin Ligases phopantetheine (4 -Ppant) arm. Previously, we showed that the installation 1 2 1 1 0 Ryan Lumpkin , Alla Ahmad , Melinda Chan , Elizabeth Komives . of a site-specific thiocyanate vibrational spectroscopy probe onto the 4 -Ppant 1Chemistry and Biochemistry, University of California San Diego, La Jolla, arm allows us to visualize and evaluate the solvation environment of the 2 0 CA, USA, Chemistry and Biochemistry, University of California Irvine, 4 -Ppant arm using infrared spectroscopy. Herein, we extend these studies Irvine, CA, USA. to show that the thiocyanate probe can be used to probe ACP-protein interac- E3 Ubiquitin Ligases (UBLs) facilitate the highly-specific covalent attachment tions. We characterized a potential ACP-enzyme interaction using a range of activated ubiquitin to bound substrate proteins through an isopeptide bond on of biochemical and biophysical techniques including FTIR spectroscopy, an exposed lysine residue. Ubiquitin signaling regulates cellular protein degra- SDS PAGE, exclusion chromatography, and tandem proteolysis mass spec- dation which is essential for proper cell functioning. The multi-subunit Cullin- trometry. The ability to induce a covalent link between an ACP and its RING ligase (CRL) represents the most prominent form of E3 Ligases, and it is catalytic partner could represent a powerful tool for exploring the possibility responsible for a substantial percentage of ubiquitin-directed protein degrada- of synthesizing ‘‘unnatural’’ natural products through the bioengineering of tion through the K48 ubiquitin linkage. CRLs share a common structure, PKSs and FASs. composed (in order of arrangement) of a substrate receptor with a suppressor of cytokine signaling (SOCS) domain, one or two adapter proteins, a Cullin 314-Pos Board B79 protein, and a RING-box (RBX) protein that recruits E2 enzymes charged Allosteric Modulation of a Serine Protease by Conformationally Selective with activated ubiquitin. Among the substrate receptors belonging to this class Nanobodies of UBLs, all 18 proteins in the Ankyrin Repeat and SOCS-box (ASB) family of Tobias Kromann-Hansen. proteins associate with Cullin 5 and RBX2 through the Elongin B and C (EB/C) Department of Chemistry and Biochemistry, UC San Diego, San Diego, adapter proteins. ASB9 has previously been shown to tightly and specifically CA, USA. bind to Creatine Kinase (CK). I am building the entire seven-protein ASB9- Trypsin-like serine proteases regulate many important physiological pro- containing E3 ligase complex in order to characterize its dynamics. I have cesses including digestion, blood coagulation, tissue remodelling, comple- expressed and purified all components of the ASB9 UBL, along with the neces- ment activation and fibrinolysis. It is well known that ligand binding to sary Ubiquitin and Nedd8 conjugating enzymes. I have obtained crystals of allosteric sites modulates the function of the active site region through CK-ASB9-EB/C for structural determination, and I performed hydrogen- conformational and/or dynamic changes leading to a change in protease ac- deuterium exchange (HDX) on the same complex to characterize how protein tivity and altered substrate specificity. Furthermore, increasing biophysical complex formation influences the dynamics of each subunit in the complex. CK evidence supports the existence of multiple active and inactive conforma- binding to ASB9 reduced exchange across both of the first two Ankyrin repeats, tional states. However, molecular details about sparsely populated protease while the removal of the SOCS box increased deuterium exchange in the final conformations, and how ligands modulate the activity of a given protease re- Ankyrin repeats in ASB9. This demonstrates the complex relationship between mains largely elusive. Here we report the X-ray crystal structure of an unap- the components of the E3 ligase and the sophisticated substrate recognition by preciated inactive conformational state of the serine protease urokinase-type ASB proteins. plasminogen activator (uPA). Next, two distinct uPA conformations were stabilized by different single domain camelid antibodies – an active confor- 317-Pos Board B82 mation captured by an active site binding (orthosteric) nanobody (Nb22) and NFkB and IkBa Engage in an ‘I Fold You, You Fold Me’ Interaction an inactive conformation captured by an allosteric nanobody (Nb7). By Excluding DNA combining X-ray crystallography and hydrogen deuterium-exchange mass Kristen M. Ramsey, Holly E. Dembinski, Yi Chen, Elizabeth A. Komives. spectrometry, we identify the mechanism of allostery, which explains at Chemistry & Biochemistry, University of California San Diego, San Diego, the molecular level how ligand binding regulates uPA activity by changing CA, USA. the dynamics of several surface-exposed loops surrounding the active site re- Binding of IkBa to NFkB robustly causes cessation of NFkB signalling. This gion. Collectively, our data supports a model which assumes equilibrium phenomenon is facilitated by the sequestering of p50/p65 NFkB dimers in the

BPJ 7720_7725 Sunday, February 12, 2017 63a cytosol via IkBa’s masking of the p65 NLS. Interestingly, IkBa is one of the 320-Pos Board B85 genes upregulated by NFkB binding to its promoter upon translocation into The Effects of Protein Dynamics on Immune System Signaling Pathways the nucleus; IkBa then facilitates dissociation of NFkB from DNA, creating Helen T. Hobbs1, Susan Marqusee2, John Kuriyan2. a negative feedback loop to turn off the NFkB response. NFkB dimers consist 1Chemistry, University of California, Berkeley, CA, USA, 2Molecular and of two domains, a dimerization domain and an N-terminal domain (NTD), Cell Biology, University of California, Berkeley, CA, USA. which are responsible for dimerization/IkBa binding and DNA binding respec- Activation of signaling pathways in immune cells can be initiated by binding of tively. There are no direct contacts between IkBa and the NTDs and constructs membrane proteins, known as B-cell receptors (BCRs) or T-cell receptors lacking the NTDs bind IkBa with comparable KD values. I performed (TCRs), to fragments of an antigen. The proteins spleen tyrosine kinase hydrogen-deuterium exchange mass spectrometry to analyze changes in amide (Syk) and zeta-chain associated protein kinase 70 (ZAP-70) are among the first exchange in NFkB when in complex with IkBa and DNA. As expected, the re- proteins to be activated upon engagement of the BCR or TCR. Recruitment of sults show decreases in amide exchange at the binding interface for both part- these proteins to phosphorylated immunoreceptor tyrosine activation motifs ners. Surprisingly, we observe significant folding and stabilization in the NFkB (ITAMs) associated with the TCR or BCR activates the kinase whereupon it NTDs when IkBa is bound, particularly in the regions containing the DNA con- initiates downstream signaling pathways, eventually leading to an immune tacting residues in p65. Previous studies have shown that IkBa’s ankyrin re- response. Syk and ZAP-70 are paralogs, with Syk being present in B-cells peats (AR) 5 and 6 fold upon binding to NFkB; when NFkB binds a IkBa and ZAP-70 in T-cells. Additionally, ZAP-70 and Syk share the domain archi- mutant that is prefolded in ARs 5 and 6 we observe no folding in the NFkB tecture of tandem SH2 domains (tSH2) linked to a kinase domain. The tSH2 is NTDs, further supporting a synergistic ‘I fold you, you fold me’ interaction responsible for binding the phosphorylated ITAMs. Despite similarities in the wherein folding in the C-terminus of IkBa facilitates folding and stabilization structure and function of these two kinases, there are key differences in their of the NFkB NTDs which locks NFkB into a conformation that is unable to intrinsic activity and specificity. Using the technique of hydrogen exchange bind DNA. by mass spectrometry (HX/MS) we have identified the Inter-SH2 linker as be- ing significantly different in ZAP-70 compared to Syk. HX/MS experiments 318-Pos Board B83 indicate that the Inter-SH2 linker of the Syk tSH2 is very flexible and poten- HDXMS Reveals Dynamic Changes in the Anticoagulant W215A Mutant tially disordered unless bound to a phosphorylated ITAM. This is not the of Thrombin case for the ZAP-70 tSH2, which shows protection even in the apo state. These Riley Peacock, Jessie Davis, Sofia Zaragosa, Elizabeth Komives. findings suggest that the intrinsic flexibility of these proteins may be the cause University of California San Diego, San Diego, CA, USA. of some of the observed differences in auto-inhibition, activity, and specificity. The serine protease, thrombin, regulates the balance between the anticoagula- tion and coagulation in the blood clotting pathway. Thrombin ordinarily 321-Pos Board B86 cleaves fibrinogen generating fibrin, which forms the blood clot. Binding of Imatinib Binding to Human c-Src is Coupled to Inter-Domain Allostery thrombomodulin to thrombin switches its catalytic activity away from fibrin- and Suggest a Novel Kinase Inhibition Strategy ogen and towards activation of protein C, which initiates the anticoagulation Yuko Tsutsui1, Daniel Deredge2, Patrick L. Wintrode2, Franklin A. Hays1. pathway. Recently Trp 215 was shown to be a key residue in determining sub- 1Department of Biochemistry and Molecular Biology, University of strate specificity. The mutant W215A was reported to lose significant activity Oklahoma Health Sciences Center, Oklahoma City, OK, USA, 2Department towards fibrinogen without significant loss of activity towards protein C- shift- of Pharmaceutical Sciences, University of Maryland School of Pharmacy, ing thrombin’s activity toward anticoagulation, even in the absence of thromo- Baltimore, MD, USA. bomodulin. Our lab has obtained experimental and computational results One of non-receptor tyrosine kinases (nRTKs), c-Src, is a multi-modular pro- suggesting that enzyme dynamics are important for connecting the main allo- tein kinase. Because c-Src is involved in numerous oncogenic signaling cas- steric site on thrombin, where thrombomodulin binds, to the active site. In order cades, it is an important drug target to treat myeloid leukemia and to understand how mutation of Trp215, which is not on the pathway between gastrointestinal stromal tumors. Imatinib is a prototype nRTK inhibitor and the allosteric site and the active site may alter thrombin specificity, I carried binds to c-Src as well as another nRTK, c-Abl, with different affinities despite out hydrogen-deuterium exchange experiments (HDXMS) to compare the high sequence and structural conservations in their ATP/drug binding pockets. enzyme dynamics of the W215A mutant with those of wild type thrombin. Structural dynamics of c-Src has been extensively investigated to elucidate un- HDXMS results indicate that amide exchange throughout much of the thrombin derlying causes of the affinity difference between c-Src and c-Abl. One of such molecule remains unchanged in the W215A mutant. However, amide exchange studies suggests the presence of a conformer that plays an important role in se- in the 220s loop was strongly increased in the W215A mutant as compared to lective imatinib binding. However, because such a conformer accumulates wild-type, and amide exchange in the beta-sheet leading up to position 215 (res. slower than the timescale of conventional biophysical and computational ap- 209-214) was markedly decreased. We hypothesize that these changes are proaches, its structural identity cannot be studied using those techniques. We directly due to changes in solvent accessibility of the active site if thrombin, used hydrogen-deuterium exchange and mass spectrometry (HDX-MS) to which appears to be remodeled in the W215A mutant. It is likely that the mu- study structural dynamics of such a key c-Src conformer in the presence of im- tation directly alters substrate specificity rather than allosteric pathways within atinib. While most previous biochemical/biophysical studies used isolated thrombin. c-Src kinase domain, we used purified human c-Src spanning residue 83-536 containing SH3, SH2, and kinase domains. This allowed us to visualize changes 319-Pos Board B84 not only in the structural dynamics of the drug binding pocket in kinase domain Insights into Dynamics of Thrombin W215 Mutants using Synapt G2Si but also of functional regulatory sites in distant SH3 and SH2 domain upon im- with Ion Mobility Separation atinib binding, suggesting an allosteric communication among those sites. Our Jessie R. Davis, Riley Peacock, Elizabeth Komives. results also indicate similarities in structural changes between imatinib binding Chemistry/Biochemistry, University of California San Diego, San Diego, and c-Src activation steps. Such changes include loosening of the interface be- CA, USA. tween N- and C-lobes of kinase domain, loss of kinase domain anchoring to Thrombin is a serine protease central to regulation of the blood clotting SH2-kinase connecting loop, and disassembly of the hydrophobic spine. Based cascade. Active wild-type thrombin acts as a procoagulant and cleaves the on our findings, we propose a new cancer therapeutic strategy to improve bind- zymogen fibrinogen to produce fibrin, which forms the scaffold structure of ing of currently available nRTK inhibitors. a blood clot. Thrombin contains many intrinsically disordered loops near it’s active site and exosites that are believed to facilitate specificity of 322-Pos Board B87 thrombin towards it’s substrates through small time scale entropic movements A Phospho-Induced Theft of a Salt Bridge in RKIP Links Map Kinase and when cofactors are bound. To study the dynamic properties of these mutants, G Protein-Mediated Signaling we produced W215I and W215A mutants of human thrombin and carried Tobin R. Sosnick1, John J. Skinner1, Sheng Wang2, Jiyoung Lee3, out hydrogen-deuterium exchange (HDXMS) experiments using the Synapt Ruth Sommese4, Sivaraj Sivaramakrishnan4, Wolfgang Ko¨lmel5, G2Si instrument with ion mobility separation. This new instrument gave Maria Hirschbeck5, Hermann Schindelin5, Caroline Kisker5, much higher coverage of the thrombin sequence and we were able to Kristina Lorenz6, Marsha R. Rosner3. discover where the dynamics of the thrombin had changed with the mutations. 1Department of Biochemistry and Molecular Biology, University of Chicago, Amide exchange in the 220s loop was strongly increased in the W215A Chicago, IL, USA, 2Toyota Institute of Technology at Chicago, Chicago, IL, mutant as compared to wild-type, and amide exchange in the beta-sheet lead- USA, 3Ben May Department for Cancer Research, University of Chicago, ing up to position 215 (res. 209-214) was markedly decreased. These results Chicago, IL, USA, 4Department of Genetics, Cell Biology and Development, help to explain why mutants at Trp215 have markedly different substrate University of Minnesota, Twin Cities, MN, USA, 5Rudolf Virchow Center specificity. for Experimental Biomedicine, University of Wurzburg,€ Wurzburg,€

BPJ 7720_7725 64a Sunday, February 12, 2017

Germany, 6Institute of Pharmacology and Toxicology, University of additional host protein, Cul5. Understanding the dynamics and conformational Wurzburg,€ Wurzburg,€ Germany. ensemble of the Vif-host protein complex could reveal future directions for Phosphorylation is a ubiquitous post-translational modification that has been therapeutics. implicated in a myriad of biological functions but the underlying mechanism of action can be unclear. Here we study the phosphorylation-induced partial 325-Pos Board B90 unfolding reaction in Raf Kinase Inhibitory Protein (RKIP), a dual function Binding-Interface Dynamics between Calmodulin and its Targets Revealed protein that regulates key pathophysiological states including heart disease using Nonperturbative Infrared Probe Groups and cancer. RKIP transitions between inhibition of Raf/MAPK to activation Casey H. Londergan, Kristen L. Kelly. of Protein Kinase A via phosphorylation of a serine on RKIP. We show Chemistry, Haverford College, Haverford, PA, USA. by NMR and X-ray crystallography that switching is due to a ’theft’ by Calmodulin (CaM) binds to a wide array of natural targets, and most of these the phosphoserine of a lysine involved in a salt bridge with a pair of car- binding interactions have not been characterized structurally. The artificial boxylic acids. The helical region containing the phosphorylation site re- SCN functional group was placed at chosen sites on both a target peptide of mains intact whereas the region with the acidic groups unfolds, thereby CaM and on CaM itself to characterize site-specific details of the binding switching RKIP’s preferred binding partner. A database search finds candi- between these two species. The infrared CN stretching band of the probe dates that have the same structural motif underlying the theft mechanism. group reports mainly on solvent exposure, and reveals a dynamic distribution Three of them, Bax (1), troponin I & C (2), and Early endosome antigen of environments at each chosen site that report directly on the bound conforma- 1(3), had been more extensively characterized by mutations, and the results tional distribution of CaM and its partner. Dramatic site-specific changes are can be explained by a salt-bridge theft. These findings identify a facile and seen when CaM is free of calcium ions, saturated with calcium ions, or bound evolutionarily accessible mechanism for reorganizing a salt bridge network to the target peptide. Isothermal titration calorimetry experiments indicate with only a single mutation to trigger a functional switch. We anticipate that surprisingly that the SCN probe group does not substantially perturb binding the salt-bridge theft mechanism can be identified in other proteins and thermodynamics, even when placed at ‘‘key’’ hydrophobic sites. New insights complexes. into CaM-target binding will be discussed, as will the prospects for applica- 1. Arokium, H., et al., (2007) J Biol Chem 282, 35104. 2. Kooij, V et al., (2013) tion of this versatile and non-perturbative probe methodology to otherwise PLoS One. 8, e74847. 3. Ramanathan, H. N., Zhang, G., and Ye, Y. (2013) Cell uncharacterized CaM-target interactions and other protein-protein binding Biosci 3, 24. interfaces.

323-Pos Board B88 Enzyme Function, Cofactors, and Post- Characterization of Disease Causing Mutations Associated with FGF Receptor Tyrosine Kinases using NMR Spectroscopy translational Modifications William M. Marsiglia1, Huaibin Chen2, Min-kyu Cho1, 326-Pos Board B91 Moosa Mohammadi2, Nathaniel J. Traaseth1. 1New York University, New York, NY, USA, 2New York University School Nedd4-2 is a Functional Oligomer Exhibiting Cooperative Allosteric of Medicine, New York, NY, USA. Kinetics The kinase domain of receptor tyrosine kinases (RTKs) is a key factor in Dustin R. Todaro, Allison C. Augustus-Wallace, Jennifer M. Klein, the initiation of intracellular signaling cascades involved in development Arthur L. Haas. and metabolism. Pathogenic mutations in this domain are generally located Biochemistry and Molecular Biology, LSU Health Sciences Center, New within an allosteric network of residues involved in kinase activation, and Orleans, New Orleans, LA, USA. hijack this pathway to enhance the kinase domain’s intrinsic activity. This The Hect ligase Standard Model posits binding of the E2~ubiquitin co- leads to overstimulation of resulting signal cascades to cause many types of substrate to a single site within a monomeric Hect domain followed by trans- cancers and growth disorders. Although many crystal structures of kinase thiolation to form a Hect~ubiquitin thioester intermediate. Transfer of the domains harboring pathogenic mutations have been solved, little is known Hect-bound ubiquitin to lysyl residues on target proteins is then catalyzed about how these mutations affect the dynamics and stability of the kinase by the ligase, processesively assembling polyubiquitin chains by distal addi- domain. Here we assessed the dynamics and stability of pathogenic muta- tion. Recently, we demonstrated that the Hect ligase Nedd4-2 catalyzes pol- yubiquitin chain assembly by cooperative allosteric kinetics ([S]1/2=93520 tions within the kinase domain from the Fibroblast Growth Factor receptor 1 nM; kcat=0.06750.008 s ; nH=1.950.6) and exhibits substrate inhibition family using the Carr-Purcell-Meiboom-Gill (CPMG) experiment and differ- m 125 ential scanning calorimetry, respectively. Results from the CPMG experi- above 0.5 M Ubc5B~ I-ubiquitin, requiring the enzyme to function as ments show millisecond-to-microsecond motions for residues involved in an oligomer with two ordered E2~ubiquitin binding sites of different affin- the kinase allosteric activation network, and provide a metric to quantify ities. These data support an alternative model for Hect ligase conjugation the population of the active conformation imparted by the pathogenic muta- and are in agreement with our previous observations for E6AP. The current tions. Differential scanning calorimetry experiments provide evidence that studies employ kinetic and biophysical methods to further explore the mech- pathogenic mutations can lower kinase stability by increasing inter-lobal flex- anism of Nedd4-2. Gel filtration chromatography and dynamic light scat- ibility, and become stabilized in the presence of the ATP mimic AMP-PCP. tering are consistent with trimerization. Truncation of the N-terminus yielding GST-Nedd4-2HECT (residues 597-955) reveals cooperative ki- Together, these data provide detailed insight into the mechanisms of how 4 netics for polyubiquitin chain assembly (nH=2.651) with a ca. 10 -fold pathogenic mutations alter the FGFR kinase domain to overstimulate signal 5 6 1 cascade processes, and provide a direction for the development of future reduction in kcat (1.9 0.2x10 s ), demonstrating allostery intrinsic to drugs. the Hect domain and N-terminal contributions supporting oligomerization. Processing of GST yields monomeric Nedd4-2HECT resulting in monoubi- 6 quitination with hyperbolic kinetics (KM=3957nM;kcat=6.050.2x10 324-Pos Board B89 s 1) and loss of polyubiquitin chain assembly and substrate inhibition. Addi- Conformational Flexibility of HIV Vif in Complex with Host Proteins tion of Nedd4-2DHECT (residues 1-596) to wild type Nedd4-2 quantita- K. Aurelia Ball1, John D. Gross2, Matt P. Jacobson2. tively inhibits activity; however, addition of the dominant negative Nedd4- 1Chemistry, Skidmore College, Saratoga Springs, NY, USA, 2Pharmaceutical 2C922A mutant inhibits Nedd4-2 to 30% limiting activity, requiring the Chemistry, University of California, San Francisco, San Francisco, CA, USA. two sites to function in trans. These data support a model requiring trimeri- Like many viruses, HIV hijacks the host cell’s apparatus for normal protein zation and intersubunit interactions for polyubiquitin chain assembly consis- ubiquitination and degradation, using it to eliminate antiviral proteins. Under- tent with our model of Proximal Indexation. [Supported by GM34009 to standing how a virus recruits and targets the ubqiuitination complex is critical A.L.H.] for developing therapeutics to prevent it. One HIV protein responsible for this hijacking is Virion infectivity factor (Vif). Vif is intrinsically disordered but 327-Pos Board B92 loses flexibility as it binds more host proteins, a process that may be crucial Ubiquitination of Substrates by E6Ap/UBE3A Ligase for function. We are investigating the complex formed with Vif and the host Virginia Ronchi, Arthur Haas. proteins EloB, EloC, and CBF-beta. Using molecular dynamics simulations Biochemistry and Molecular Bilogy, Louisiana State University, New and NMR spectroscopy we have found that this Vif-host protein complex ex- Orleans, LA, USA. hibits global dynamics and occupies alternate conformational states. We Only a narrow range of E6AP ubiquitin ligase activity is allowed for normal have also characterized how these motions are affected by the binding of an neural development. Enzyme activation by Ube3A gene duplication is linked

BPJ 7720_7725 Sunday, February 12, 2017 65a with familial autism spectrum disorder while loss of function results in the 330-Pos Board B95 Angelman syndrome neurological disorder. BIOGRID recognizes 174 E6AP- A Computational Investigation into the Mechanism of the Histone Acetyl- interactors but only a few have been validated as substrates for ubiquitination. transferase, Gcn5 Recently, phosphorylation on E6APT485, an autism related residue, affected R. Hunter Wilson, Isaiah Sumner. RPN10/S5A substrate ubiquitination in cells. The current aim included kinetic Department of Chemistry and Biochemistry, James Madison University, analysis of E6AP-catalyzed conjugation of target proteins to provide new in- Harrisonburg, VA, USA. sights into the mechanism of substrate ubiquitination. E6AP-dependent Post-translational modifications (PTMs) can have a profound effect on protein Lys48-polyubiquitin chain assembly in the absence of substrate requires two structure and function. One such PTM is the acetylation of histone (a protein functionally distinct UbcH7~ubiquitin binding sites on the ligase surface and involved in DNA binding). In this reaction, an enzyme catalyzes the transfer oligomerization. Here, the E6AP ubiquitin-function was analyzed in the pres- of the acetyl group from acetyl CoA to a free lysine on the histone. This transfer ence of RNP10/S5A (regulatory subunit of proteasome 26S) and PRDX1 (anti- neutralizes the positively charged lysine, which ultimately allows the DNA to oxidant enzyme). Rates of substrate ubiquitin adduct formation were analyzed be exposed for transcription. In our study, we focus on the acetyltransferase, under E6AP rate-limiting conditions. Adducts of PRDX1-Ub1 or RPN10/S5A- Gcn5. Details regarding the reaction mechanism used by Gcn5 remain Ub1 showed Km values of 851 mM and 0.250.06 mM, respectively; while the obscured. However, current mechanistic hypotheses suggest that the reaction 1 1 kcat values were 0.3 s , comparable to 0.5 s observed for polyubiquitin chain occurs through a tetrahedral oxyanion intermediate, which is stabilized by a assembly without substrate and indicating that the ligase cannot distinguish the hydrogen bond to a nearby residue, i.e. an oxyanion hole. We utilize molecular lysine nucleophile in Lys48-ubiquitin and Lys-target protein ubiquitination. dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) cal- Analysis of pH-dependent E6AP ligase function inferred a pKa of ~8.4, either culations in order to further probe possible mechanistic schemes of this in the absence or presence of PRDX1. Removal of the first 250 N-terminal res- reaction. idues reduced ubiquitination of both substrates supporting the presence of pre- viously unrecognized substrate binding domains in this region. A T485D 331-Pos Board B96 mutation mimicking E6AP phosphorylation, or a D212A mutation, an Angel- Molecular Simulations of Bacterial Lipoprotein Biogenesis man syndrome mutation, abrogated substrate ubiquitination, although they re- Phillip J. Stansfeld. tained the polyubiquitin chain assembly function. The results provide new Biochemistry, University of Oxford, Oxford, United Kingdom. insights of the E6AP ubiquitination mechanism in the presence of target pro- Lipoproteins perform critical roles in bacterial physiology, pathogenicity, and teins that might explain the deleterious effect of some mutations associated antibiotic resistance. Their roles include modulation of the cell envelope struc- with Angelman syndrome. ture, signal transduction and transport. Lipoproteins are processed by a pathway of membrane proteins - Sec, Lgt, LspA, Lnt and Lol - which insert, cleave and 328-Pos Board B93 transport the protein substrate, while affixing lipid moieties to an absolutely Mechanistic Insights into Ubc13-Catalyzed Ubiquitination conserved cysteine and therefore permitting their tethering to the cell envelope. Isaiah Sumner1, R. Hunter Wilson1, Walker M. Jones1, Aaron G. Davis1, The recent determination of the three-dimensional protein structures of Lgt Serban Zamfir2. and LspA have enlivened lipoprotein research. Furthermore, the latest struc- 1Chemistry & Biochemistry, James Madison University, Harrisonburg, tures of the Sec translocon with associated signal peptide provide a means VA, USA, 2Chemistry, Virginia Commonwealth University, Richmond, to study the initiation point of this pathway and mechanism by which the lipo- VA, USA. proteins are inserted into the cell membrane. Structural studies of mature lipo- Ubc13 is an E2 enzyme that catalyzes lysine ubiquitination, a type of protein proteins have also recently come to the fore, including the BAM complex, of post-translation modification. Ubiquitinating a protein can signal for its which four of the five proteins are lipoproteins, and LptDE, an outer membrane degradation and affect its activity. Ubiquitination also plays a role in DNA barrel with a lipoprotein plug. Both complexes are essential to gram-negative repair and inflammatory response. Defects in this process are linked to bacteria; respectively, folding the protein-conduits that enable nutrient trans- different disorders including cancer, Parkinson’s and Alzheimer’s diseases. port through the outer membrane and assembling the outermost bacterial The accepted mechanism for Ubc13-catalyzed ubiquitination is a stepwise fortifications. pathway that proceeds through an oxyanion intermediate. This intermediate Here we have used a range of molecular simulation, modelling and bioinfor- is hypothesized to be stabilized by a nearby asparagine residue, which is matics methods to study this pathway. With initial focus on the first two en- known as the ‘‘oxyanion hole.’’ However, recent experimental results on zymes of the pathway, Lgt and LspA, our studies have elucidated the mode mutated Ubc13 have suggested an alternate role for the asparagine. In our of binding of signal peptides to both enzymes and interpreted from this the study, we use a combination of simulation techniques on the wild-type and molecular mechanisms involved in the enzymatic reactions. These studies mutated Ubc13 to examine its catalytic mechanism. Our calculations indicate highlight key roles for the most highly conserved residues, whilst also that several different intermediates are possible, that water may stabilize the providing a means to inhibit the enzymes, as illustrated by the antibiotic, glo- intermediate, and that the asparagine serves to stabilize a random coil near bomycin, bound to LspA. the active site. We have also developed molecular parameters for the cysteine lipid-moeities, and applied these post-translational modifications to simulate the dynamics of 329-Pos Board B94 both individual lipoproteins and the lipoprotein complexes of BAM and LptDE Characterization of the Essential Residues of Cyclooxygenase-1 and 2 within the bacterial outer membrane. Responsible for their Inter-Subunit Communications Upon their Binding to the Corresponding Substrates and Inhibitors 332-Pos Board B97 Inseok Song. Heme Trafficking by the Cytochrome C Biogenesis Pathways University of Seoul, Seoul, Korea, Republic of. Molly C. Sutherland1, Joel A. Rankin2, Robert G. Kranz1. Various conformational changes of a protein upon interaction with its endog- 1Washington University in St. Louis, St. Louis, MO, USA, 2Michigan State enous partner molecules or artificial synthetic compounds imply often an University, East Lansing, MI, USA. essential role structurally or functionally. Induction triggered by its binding Cytochromes function in electron transport chains to perform critical cellular to small ligands can contribute a long-range communication between intra- functions, such as respiration and photosynthesis. Cytochromes c are unique or inter-subunits, which has been exemplified in several model studies. due to their requirement for the covalent attachment of heme via two thioether Among these, cyclooxygenases (COX-1 and 2), also known as prostaglandin bonds at a conserved CXXCH motif. Three pathways have been identified for endoperoxide synthases, display a differential binding pattern between two cytochrome c maturation: System I (prokaryotes), System II (prokaryotes) and sequentially identical monomers. COXs catalyze the first committed step System III (eukaryotes). System I consists of 8 integral membrane proteins in the conversion of arachidonic acid into prostaglandins and thromboxanes. (CcmABCDEFGH), System II is comprised of 2 membrane proteins (CcsBA) Potential drug-like compounds against COXs have been enormously devel- and these pathways will be the focus of this presentation. Trafficking of heme oped and characterized up to date, which are exploited in this study with from the site of its synthesis (cytoplasm) to the site of attachment to apocyto- an aim to find core regions or residues responsible for their inter-subunit chrome c (periplasm) is critical for cytochrome c biogenesis, yet little direct and domain-domain communications. In addition to a careful examination evidence of heme trafficking exists. The study of heme trafficking has proved of the COX crystallographic data, extensive analysis of docking experi- elusive in this and most other systems due to tight cellular regulation, as well as ments with categorized NSAIDs and prediction of hot-spot(s) for protein- the cytotoxic and amphipathic nature of heme. Here, we use key putative heme protein interaction were performed, which suggest the theoretical basis transporters in the prokaryotic pathways as model systems to develop a novel for the functionally heterodimeric nature and half-of-the-sites behavior of technique to covalently ‘trap’ heme during the trafficking process. First, the COXs. conserved WWD domain, which is predicted to interact with heme in the

BPJ 7720_7725 66a Sunday, February 12, 2017 periplasmic space and is found in both System I and System II pathways, was coumarin derivatives. The coumarin derivatives were incorporated into pep- used to develop the heme trapping technique. Heme was covalently trapped in tides with various amino acid residues: WEHDA, WEHD, LEVD, LEHD as the WWD domains of CcmC (System I) and CcsA (System II), providing the either a side chain of a non-natural amino acid, or at the C-terminus. The fluo- first direct evidence of how heme is trafficked to the periplasm in cytochrome rescence quantum yields of these peptides were obtained, allowing the viability c biogenesis. Currently, this trapping approach is being used on other integral of these substrates to enhance the signal to noise ratio in caspase enzyme assays membrane proteins in the pathway to capture heme intermediates and delineate to be assessed. Biochemical assays were then carried out to determine if the the exact paths for trafficking in vivo. We envision that this approach will be signal to noise ratio indicated by the fluorescence quantum yield results corre- applicable to other heme transporters and trafficking pathways from prokary- lated with caspase activity and could be applied to inhibitor screening assays. otes through eukaryotes. 336-Pos Board B101 333-Pos Board B98 Single Molecule Enzymology with Outer Membrane Protein G Designed Enzymes and the Driving Forces Behind Interdomain Electron Bach G. Pham. Transfer University of Massachusetts - Amherst, Amherst, MA, USA. Mia C. Brown, Kelly Greenland, Lei Zhang, Ronald L. Koder. We have developed a nanopore sensor based on Outer membrane protein G City College of New York, New York, NY, USA. (OmpG) to study enzyme kinetics at the single molecule level. OmpG is a Synthetic biology and biodesign approaches to redox active enzymes will b -barrel porin with seven flexible loops that we had previously exploited in require the construction of artificial electron transport chains, particularly protein sensing. A recognition peptide sequence was inserted into one of chains which can interconvert between one- and two-electron chemistry. To OmpG’s loops allowing it to undergo enzymatic reaction. Caspase 7 both demonstrate this capability and explore the engineering parameters neces- (casp-7), a protease implicated in apoptosis, was chosen as the target enzyme sary for rapid and efficient electron transport through artificial electron trans- to interact and chemically alter the OmpG sensor. Currently, we can unambig- port chains, we have constructed a natural protein-designed protein chimera uously detect the enzyme-substrate complex of casp-7 and OmpG, as well as in which the diflavin domain of P-450 BM3 is connected to a de novo designed, the OmpG cleavage product in real time. We found that the key to the robust heme binding four helix bundle. This single chain protein contains one FMN, efficiency of casp-7 activity (and many other caspases) is that substrate is one FAD, and two heme cofactors. This chimera reacts with NADPH, taking in always cleaved once bound to casp-7. Casp-7 cleavage is essentially an irre- 1 its two electrons at the FAD cofactor, breaking them into single electrons at the versible reaction (k-1 =0;k2 = 8.3 s ). In addition, we manipulated the pH FMN cofactor, and then transferring them into the artificial heme domain. We and observe the effects on k2 that support the catalytic mechanism of cysteine have tested three different heme analogues with varying mid-point potentials to proteases. Our results allow us to probe more closely the catalytic mechanism examine the effect of driving forces on interdomain electron transfer rates. of an enzyme which cannot be probed using conventional ensemble assays. Finally, as our heme-binding domain is capable of binding oxygen in the Thus, we can use our single molecule OmpG enzymology platform to a variety reduced state, I will present some results using this construct as an artificial of other enzyme targets in a similar fashion. nitric oxide dioxygenase, which can perform NADPH-driven catalysis. 337-Pos Board B102 334-Pos Board B99 Protein Semi-Synthesis to Characterize Phospho-Regulation of Human Factors Governing Autooxidation of Human Hemoglobin UNG2 Andres S. Benitez Cardenas1, John S. Olson2. Brian P. Weiser, James T. Stivers, Philip A. Cole. 1Biosciences, Rice University, Houston, TX, USA, 2Biosciences, Rice Pharmacology & Molecular Sciences, Johns Hopkins University, Baltimore, University, HOUSTON, TX, USA. MD, USA. Determining mechanisms for the autooxidation of hemoglobin is required for The human nuclear Uracil DNA Glycosylase (hUNG2) initiates the base exci- understanding and treating unstable hemoglobinopathies and for developing sion repair pathway that removes uracil from genomic DNA. The catalytic more stable hemoglobin based O2 carriers. Previous studies suggested signifi- domain of hUNG2 is preceded by a disordered N-terminus that contains cant differences in autooxidation rates of a and b subunits. We used an azide numerous sites for post-translational modifications. We hypothesized that reaction assay to measure the concentrations of ferric a and b chains at different certain modifications might affect hUNG2 activity and/or its interactions time points during autooxidation. Our results showed no differences between with its protein binding partner, Proliferating Cell Nuclear Antigen (PCNA), the subunits. To obtain more accurate time courses for autooxidation, we de- which is thought to bind hUNG2 residues 4-11. Using protein semi- convoluted observed spectra into the decay of HbO2, metHb appearance, hemi- synthesis, we prepared full-length hUNG2 with a phosphorylation at either chrome generation, and increases in turbidity due to hemin loss and apoprotein Thr6 or Tyr8, and these constructs were purified to >95%. The uracil excision precipitation. The time courses for HbO2 decay at high concentrations activity of phospho-hUNG2 proteins was comparable to unmodified hUNG2 (R 100mM heme) accelerate implying cooperative autooxidation, where as when using a 19mer duplex DNA substrate. However, fluorescence anisotropy at low concentrations (% 10uM) the time courses are biphasic. These results measurements showed that the phospho-hUNG2s had >10-fold weaker affin- suggest that the biphasic time courses at low hemoglobin are due to differences ities for free PCNA. The PCNA affinity of a synthetic peptide that corresponds between tetramers and dimers. We have also measured autooxidation rates for a to UNG2 residues 1-19 was similar to that of full-length hUNG2. Our data in- recombinant hemoglobin, rHb0.1, that contains a genetically crosslinked di-a dicates that the N-terminal 11 residues of hUNG2 are necessary and sufficient subunit. This hemoglobin shows a monophasic time course for autooxidation for high-affinity binding to PCNA, and that phosphorylation within this motif at both high and low protein concentrations, and the azide binding assay disrupts binding. How phosphorylation affects other aspects of hUNG2 activity showed equal amounts of ferric a and b subunits. We have also examined re- is now being explored. combinant mutant hemoglobins to examine the structural factors that govern autooxidation. Increased rates of autooxidation were found for rHb Providence, 338-Pos Board B103 rHb Bethesda, rHb Presbyterian, and rHb Kirklareli. We have also confirmed Voltage Dependent Phosphatase Activity is Enhanced by Intracellular that the rate of autooxidation shows a bell-shaped dependence on oxygen con- Acidification centration and increases markedly as the pH is decreased. Angeliki Mavrantoni, Kirstin Hobiger, Dominik Oliver, Supported by NIH Grant P01 HL110900 and by Grant C-0612 from the Robert Christian R. Halaszovich. A. Welch Foundation. Neurophysiology, University Marburg, Marburg, Germany. Voltage sensitive phosphatases (VSPs) are PI(4,5)P2/PI(3,4,5)P3-5- and PI(3,4) 335-Pos Board B100 P2/PI(3,4,5)P3-3-phosphatases. For non-mammalian VSPs, this activity is regu- Assessing the Spectroscopic Properties and Enzyme Activity of Fluores- lated by membrane voltage via a voltage sensor domain (VSD). For mamma- cent Caspase Substrates lian VSPs the VSD seems insensitive to voltage changes yet still essential Gena Lenti, Nicholas Tassone, Srirajkumar Ranganathan, Caitlin Karver, for control of the phosphatase activity. Under physiological conditions, the Cathrine A. Southern. non-mammalian VSPs strongly deplete PI(4,5)P2 in a voltage dependent Chemistry, DePaul University, Chicago, IL, USA. manner. The physiological regulator of mammalian VSP activity remains Inflammatory caspases (caspase-1, 4 and 5 in humans and caspase-11 in elusive. VSPs are suggested to play a role in fertilization and development, mice) are cysteine-dependent, aspartate-specific proteases implicated in inflam- where changes in intracellular pH are known to occur. They are found to be ex- matory, autoimmune and autoinflammatory disorders. To date, assays seeking pressed in tissues like kidney, stomach, sperm, and ovary which are known to to test the activity of caspases-1, 5 and 11 have all used Ac-WEHD-AMC as undergo such pH changes. Therefore, we speculated that intracellular pH might their fluorogenic substrate. To explore the possibility that alternative fluoro- modulate VSP activity. genic peptides may exhibit enhanced assay properties, we have designed, To test this hypothesis we performed whole-cell patch-clamp experiments in synthesized, and characterized several novel fluorogenic peptides containing CHO cells expressing diverse VSPs and fluorescent PI(4,5)P2 reported

BPJ 7720_7725 Sunday, February 12, 2017 67a domains, the latter allowing for monitoring VSP activity by means of total in- structure-function studies on functionally important histidine residues in ternal reflection microscopy (TIRF-M). The whole-cell patch-clamp configura- enzymes. tion allowed for control not only over membrane voltage but also intracellular pH by dialysing the cell with solutions with the desired pH. 341-Pos Board B106 Origin of Chain Length Specificites of Starch Branching Enzyme We find that acidification of the cytoplasm results in increased PI(4,5)P2 deple- tion, accompanied by a shift of the apparent voltage dependence towards more Hadi Nayebi Gavgani, Remie Fawaz, Zahra Assar, Alireza Ghanbarpour, negative potentials. An increase in intracellular pH has the opposite effect. The David Walls, Sarah McGovern, James H. Geiger. voltage dependence of sensing currents was unaffected by the pH changes, sug- Chemistry, Michigan State University, East Lansing, MI, USA. gesting that alterations of the VSD are not causal for the observed changes in Starch Branching enzyme (BE) is one of the three enzymes involved in starch voltage dependent activity. Similar effects were observed in all tested VSPs We biosynthesis. It is responsible for synthesizing the alpha-1,6-glucan branches, conclude that the overall activity of the phosphatase is enhanced under acidic remodeling the linear alpha-1,4-glucan polymer to produce amylopectin. There and diminished under alkaline conditions. Kinetic modeling predicts a shift in are at least two isoforms of the enzyme in most plants, with each having distinct apparent voltage dependence under these circumstances that is in agreement reactivities and product chain-length specificities. Though the chemistry of the with the observed shift. active site of branching enzyme is relatively well studied, the origin of the chain In conclusion, we suggest that intracellular pH can play a role in the regulation length specificity is yet to be understood. Using protein crystallography and of the activity of VSPs. biochemical studies on rice branching enzyme, we aim to understand the fac- This work was supported by a research grant of the University Medical Center tors controlling the chain-length specificity of branching enzymes. The mech- Giessen and Marburg (UKGM32/2011MR) to C.R.H and by Deutsche For- anism of branching enzyme involves two steps: (1) An oligosaccharide (donor schungsgemeinschaft (SFB593 TP A12) to D.O. chain) binds to the enzyme and is cleaved by the action of a nucleophilic aspar- tate residue to form a covalently-linked enzyme-glucan intermediate. (2) A sec- 339-Pos Board B104 ond oligosaccharide (acceptor chain) then reacts, by nucleophilic attack of one Electric Field Effects in the Active Site of a Thermophilic Enzyme as of its alpha-1,6-hydroxyl groups, to form a new alpha-1,6-branch. We have Observed by FTIR and 2D IR Spectroscopy focused on discovering the surface glucan binding sites in branching enzyme Tayler D. Hill, Hannah H. Lepird, David A. Price, Sean D. Moran. because they are likely essential to understanding the specificity of the enzyme. Chemistry and Biochemistry, Southern Illinois University Carbondale, To this end we have obtained a crystal structure of an oligosaccharide (M12)- Carbondale, IL, USA. bound rice branching enzyme, which reveals oligosaccharide binding from the Our research aims to understand how changes in ultrafast dynamics compare outer surface of the enzyme almost to the active site. Mutations of the residues and correlate to thermophilic enzyme activity. We observe fluctuating electric interacting with the M12 can substantially compromise the enzyme’s activity, field effects in a promiscuous, hyperthermophilic ene-reductase from Pyrococ- though none have affected the branch chain specificity. On the other hand, cus horikoshii (PhENR) to address this. This enzyme catalyzes the reduction of comparison of an isoamylase maltoheptaose (M7)-bound structure with rice activated alkenes/alkynes to their respective alkanes/alkenes via proton and hy- branching enzyme suggested another potential glucan surface binding site. dride transfers from a flavin cofactor in the active site. We exploit the promis- Interestingly, mutations in this new site did effect the chain-length specificity cuity of PhENR in order to incorporate a variety of substrates and substrate of the enzyme. These results suggest that the bound M12 is part of the acceptor analogs into the active site for these studies. We have synthesized a set of chain and the newly identified binding site hosts the donor chain. Together, the covalently-attached substituted N-phenylmaleimide infrared labels, which data allow us to, for the first time propose a detailed mechanism for the enzyme, mimic the structures of the enzyme’s substrates, and contain unique vibrational explaining how disparate surface glucan binding sites far from the active site chromophores to probe the enzyme’s active site dynamics. Current studies create the enzyme’s activity and specificity against its polymeric substrate. focus on the vibrational frequencies and lineshapes of nitrile labels such as those of 4-cyano-N-phenylmaleimide, which sits proximal to the catalytic 342-Pos Board B107 flavin and can be attached in multiple orientations within the active site. Modifications of Alpha and Beta Carboxy-Terminal Tails Regulate Micro- When compared to the label in solution, the covalently attached label un- tubule Severing by Katanin dergoes significant inhomogeneous broadening in its FTIR spectrum reflecting Madison Tyler1, Corey Reed1, Dan Sackett2, Jennifer Ross1. 1 the distribution of active site microenvironments. Additionally, protein-based Physics, University of Massachusetts, Amherst, Amherst, MA, USA, 2 non-natural amino acid labels such as methionine to azidohomoalanine substi- National Institutes of Health, Bethesda, MD, USA. tutions are also being incorporated into the distal side of the flavin cofactor for Microtubules are part of a dynamic cytoskeletal network that is constantly be- similar studies in different location within the enzyme’s active site. Using 2D ing reorganized to control cell processes such as neuronal development and IR spectroscopy, we are examining the contributions of femtosecond to pico- maintenance, cell division, and cargo transport. Many stabilizing and destabi- second active site dynamics to the lineshapes of both the covalently attached lizing enzymes function to reorganize these networks for the specific needs of probes as well as the incorporated non-natural amino acid labels. Future the cell in a spatiotemporal manner. Katanin p60 is a microtubule destabilizing research aims to break the thermophilicity of the enzyme via specific mutations enzyme from the ATPases Associated with various Activities (AAAþ) family. in order to compare the active site dynamics to a corresponding mesophilic It recognizes the tubulin carboxy-terminal tails (CTTs) to sever microtubules. version of the protein. Our lab has previously shown free tubulin dimers and CTTs alone can inhibit katanin severing. We seek to determine the manner that tubulin CTTs sequence 340-Pos Board B105 can regulate katanin activity using polypeptide sequences of CTTs of different Infrared Structural Biology: How to Detect Protonation States of Histidine tubulin isoforms. We find that the sequence’s ionic, hydrophobic, and steric Side Chains in Proteins features play a role in determining katanin’s activity. Aihua Xie, Charle Liu, Matthew Cavener. Physics, Oklahoma State University, Stillwater, OK, USA. Ribosomes and Translation The imidazole group of histidine residues are found functionally important in a vast number of catalytic proteins. The remarkable catalytic power of histi- 343-Pos Board B108 dine side chains originates from its ionizable imidazole ring armed with a Protein Synthesis Times Scale with Gene Length because the Determinants pair of different tertiary amines and the ability of adopting three protonation of Translation Speed are Randomly Distributed Across Genes states near physiological pH environment. Knowledge on the protonation Edward P. O’Brien, Ajeet Sharma. states of key histidine side chains in enzymes at rest and during catalytic ac- Department of Chemistry, Penn State University, University Park, PA, USA. tions is indispensable to elucidation of the structure-function relationship un- Many of the molecular factors influencing codon translation speed have been derlying enzymatic catalysis. We report a rigorous method on how to detect identified, and their relative contributions estimated. These factors include the three protonation states of functionally important histidine imidazole tRNA concentration, the presence of charged nascent-chain residues in the rings in the static and dynamic states of enzymes using infrared structural ribosome exit tunnel, mRNA secondary structure, proline residues at the A biology. First principle computational methods based on density functional or P sites of the ribosome and steric interactions between ribosomes translating theory were employed to develop two vibrational structural markers a transcript. Here, we combine this information with genomic information from (VSM) of the imidazole group: VSMq for the charged states of the imidazole E. coli, yeast and humans in a simulation model of translation to estimate the group, while VSMt for distinguishing the D and E tautomers of charge synthesis time of cytopolasmic proteins. We find that regardless of the organ- neutral histidine. The accuracy of the VSMs is assessed by comparison of ism, the synthesis time of a protein scales linearly with the length of mRNA’s calculated VSMs with experimental FT-IR data of the 4-ethyl-imidazole coding sequence even though there is a large variation in the translation speed model compound. We will discuss how these VSMs may be employed in of individual codons. We demonstrate that this scaling arises because the

BPJ 7720_7725 68a Sunday, February 12, 2017 molecular determinants of translation speed are distributed randomly across tures that can form in repeating sequences of either guanine or cytosine, respec- the mRNA transcripts and that this distribution is generated from a Poisson tively. Both G4 and iM sequences may contain CpG sequences that can be point process. As a consequence, the Law of Large Numbers is followed methylated or hydroxymethylated. The effects of CpG islands on DNA second- and for any given transcript fast-translating segments are canceled out by a ary structures were determined by incorporating a single 5hmC at varying po- similar number of slow-translating segments resulting a constant average codon sitions in the Vascular Endothelial Growth Factor (VEGF) G4 and iM translation rate between different transcripts. This means that a protein’s sequences. An Olis DSM-20 spectropolarimeter and a Cary 100 UV-visible average synthesis time can be accurately predicted based solely on the corre- spectrometer were used to monitor the effect of 5hmC on G4 and iM thermal sponding gene length, provided the average codon translation speed is known. stability. Two of the three 5hmC-containing loops showed a notable decrease Thus, although evolution has biased codon usage between different genes in in stability for G4’s and increased intermolecular structure formation. Contrast- an organism, there is still a large degree of randomness associated with how ingly, the iM stability increased when 5hmC was incorporated into its individual codon translation speeds have been distributed. These results also sequence. Additionally, there was little change in the iM pka. In summary, provide an explanation for the observation from ribosome profiling that our results suggest the 5hmC has little effect on iM structures, but can destabi- different transcripts have the same average codon translation speed in lize the G4’s. mouse-stem cells. 347-Pos Board B112 344-Pos Board B109 Temperature Dependence of L-Proline RNA Duplex Destabilization The Role of L11 Stalk Fluctuations in aa-tRNA Accommodation Jeffrey J. Schwinefus, Kalpit Modi, Nadia Baka. Huan Yang, Paul Charles Whitford. Chemistry, St. Olaf College, Northfield, MN, USA. Physics, Northeastern University, Boston, MA, USA. L-Proline, an osmoprotectant and non-essential amino acid, destabilizes the The ribosome is a large ribonucleoprotein complex that is responsible for secondary and tertiary structures of nucleic acids. L-proline interactions with translation of proteins in all living organisms. Accommodation is a key the surface area exposed in unfolding RNA duplexes were quantified using conformational change during transfer RNA (tRNA) selection that allows m-values for ten RNA dodecamer duplexes that varied in GC content from for each molecule to fully associate with the ribosome. During tRNA accom- 17% to 83%. The RNA dodecamer unfolding transitions were facilitated by modation, there are large-scale fluctuations in the L11 stalk and the associ- thermal denaturation and isothermal titration with proline, both monitored us- ated protein. Experimental and theoretical work has shown that the L11 ing ultraviolet spectroscopy. The m-values from isothermal titration at 22 C protein and RNA exhibit dynamic behavior, where their precise position were independent of GC content. However, m-values from thermal denatur- can significantly affect the dynamics of elongation. To explore the impact ation were greater in magnitude and more negative for higher GC content of L11 stalk mobility on the kinetics of aa-tRNA accommodation, we used RNA indicating more favorable proline interactions with the accessible surface molecular dynamics simulations with a simplified model to evaluate the area exposed in unfolding high GC content duplexes. Since RNA duplex tran- free-energy as a function of aa-tRNA position. We find that as the flexibility sition temperatures increase with GC content, the more favorable L-proline in- of L11 is decreased, the position of the free-energy minima is robust. In teractions with the high GC content duplex surface area result from the contrast, the magnitude of the free-energy barrier for accommodation de- temperature dependence of L-proline interactions rather than the RNA surface pends on the precise scale of L11 fluctuations. We find that increasing the chemical composition. The enthalpy contribution to the m-value is positive and rigidity of the L11 stalk can destabilize the A/T ensemble. In addition to small (indicating a slight increase in duplex unfolding enthalpy with L-proline) the close relationship between L11 mobility and the free-energy landscape while the entropic contribution to the m-value is positive and increases with of accommodation, the direction of L11 stalk movement is also correlated temperature. Our results help facilitate using L-proline as a probe of solvent with aa-tRNA displacements. To probe the collective motion of the L11 accessible surface area changes during biochemical reactions at different reac- stalk, we performed Principal Component Analysis on the simulated trajec- tion temperatures. tories. When aa-tRNA is first delivered to the ribosome, the stalk moves away from the tRNA and adopts extended configurations. It then relaxes dur- 348-Pos Board B113 ing the accommodation process. Together, these calculations reveal a corre- Engineering DNA Looping in E. Coli lation between fluctuations in the L11 stalk and the dynamics of aa-tRNA Nicole A. Becker, Tanya L. Schwab, Karl J. Clark, L. James Maher III. during accommodation. This provides a quantitative foundation for interpret- Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA. ing experimental measures of accommodation and suggests how the dy- DNA looping plays an important role in many cellular functions including namics of the L11 stalk may contribute to aa-tRNA proof-reading and replication, transcription, DNA packaging, and recombination. DNA repres- accommodation. sion loop formation in prokaryotes acts to regulate transcription in response to a changing environment. One example of gene control by DNA loop for- 345-Pos Board B110 mation occurs in the well-studied lac operon. E. coli lac loop formation oc- Simulating Movement of the Ribosome Head during Translocation curs when LacI, a tetrameric protein, simultaneously binds two DNA Karissa Y. Sanbonmatsu1,2, Wataru Nishima2. operators within close proximity, resulting in a DNA loop. This structure re- 1Los Alamos National Laboratory, Los Alamos, NM, USA, 2New Mexico presses transcription of downstream operon genes. We are curious if repres- Consortium, Los Alamos, NM, USA. sion loop formation can be targeted in vivo. Our goal is an engineered protein Intrasubunit head movement has been identified as an essential motion fusion with both sequence specific DNA binding function and chemically required for translocation of mRNA through the ribosome. Recent single mole- induced dimerization (CID) function. The chemically dimerized protein cule FRET data has suggested that exaggerated motions of the head, beyond would have the ability to bind the DNA in a bidentate manner, analogous what are observed in structural studies, are required for the rate-limiting to LacI, resulting in DNA looping. Targeted DNA loop formation could step of translocation. We use molecular simulations of the 70S ribosome trans- act in gene repression and might be tunable by the concentration of CID location complexes to explore these exaggerated motions and identify regions agent. Using designed transcription activator-like effector (TALE) proteins of the ribosome that place constraints on the maximal displacements of the allows creation of DNA binding proteins with arbitrary DNA sequence spec- head. ificity. TALEs have been used for in vivo genome-engineering applications. FK506 binding protein (FKBP) provides a well-studied protein domain Nucleic Acid Structure and Dynamics I whose ability to undergo CID is exploited in our designs. We utilize our es- tablished lac promoter looping system to assess TALE-FKBP protein fusions 346-Pos Board B111 targeted to lac operators. Effects of 5-Hydroxymethylcytosine Epigenetic Modifications within the VEGF Promoter Region on G-Quadruplex and I-Motif DNA Structure 349-Pos Board B114 and Stability Investigating the Stability of DNA Duplex Hairpins using Optical Tweezers Michael M. Molnar1, Rhianna K. Morgan2, Tracy A. Brooks2, Leah Furman1, Micah McCauley2, Catherine A. Dietrich3, Randy M. Wadkins1. Mark C. Williams2, Megan E. Nunez1. 1Chemistry & Biochemistry, University of Mississippi, Oxford, MS, USA, 1Department of Chemistry, Wellesley College, Wellesley, MA, USA, 2Pharmacology, University of Mississippi, Oxford, MS, USA. 2Department of Physics, Northeastern University, Boston, MA, USA, Epigenetic modifications to DNA base sequences may regulate gene expres- 3Department of Chemistry, Mount Holyoke College, South Hadley, sion. CpG islands can contain methylated (5mC) or hydroxymethylated MA, USA. (5hmC) cytosine. Most CpG islands are found primarily in promoter regions Covalent modifications and replication errors lead to DNA damage and muta- that may also contain a high number of repeated cytosines and/or guanines. tion. Such errors occur frequently throughout the genome, disrupting the stabil- G-quadraplexes (G4) and i-motifs (iM) are two unique DNA secondary struc- ity of the double helix. The 8-oxoguanine (8-oxoG) lesion, one of the most

BPJ 7720_7725 Sunday, February 12, 2017 69a widespread forms of DNA damage, mimics guanine with respect to hydrogen 353-Pos Board B118 bonding, but perturbs base stacking, destabilizing the double helix. Other com- Live Cell Imaging of Genomic Loci using Fluorescent RNA Aptamers mon forms of damage include mismatches, such at G-T and C-T base pairs, that Adam Cawte1, Sunny Jeng2, Peter Unrau2, David Rueda1. are unable to form canonical hydrogen bonds. We have investigated the desta- 1MRC Clinical Science Centre, Imperial College London, London, United bilizing effects of three abnormal base pairs (8-oxoG-C, G-T, and C-T) on the Kingdom, 2Department of Molecular Biology and Biochemistry, Simon thermodynamic stability of DNA hairpins at the single molecule level. Each Fraser University, Burnaby, BC, Canada. damaged hairpin shows a distinct pattern of unfolding that allows us to quantify In recent years, there has been an explosion of SELEX-evolved fluorescent hairpin destabilization. These data are combined with an mFold-based analysis RNA aptamers, such as Spinach and Mango, with enhanced folding, fluores- to create energy landscapes of the unfolding pathway for perfectly matched, cence and a high affinity for their dyes. These aptamers have a drastically damaged, and mismatched hairpin duplexes. improved fluorescence contrast relative to EGFP and hold great promise for visualising vital cellular processes involving RNA molecules. However, the 350-Pos Board B115 use of aptamers in live-cell imaging has experienced limited resolution and Untwisting of Double-Stranded DNA and RNA Investigated by Molecular applicability due to the dynamic nature of RNA. We have recently developed Dynamics Simulations a new method that combines CRISPR/Cas9-based nuclear localization with Korbinian Liebl. fluorescent RNA aptamer-based imaging. A major difference with existing ap- Technical University Munich, Garching, Germany. proaches is that this system contains an engineered fluorescent RNA aptamer The response of dsDNA and dsRNA to torsional stress influences many of within the sgRNA scaffold in lieu of a fluorescent dCas9-EGFP fusion. This their biological functions, including binding by proteins, transcription initia- method enables the direct visualisation of genomic loci and their diffusion dy- tion and genome packaging. So far, the twist flexibility of DNA and RNA namics within live cells. We anticipate the development of this technology to has been studied comparatively with single-molecule experiments. However, improve our ability to target specific regions of the genome, as well as to while these studies capture the global deformability of duplexes with a length develop multi-colour imaging using different fluorescent sgRNA constructs. of several thousand base pairs (bp), detailed insight into conformational 354-Pos Board B119 changes on the base pair level remains elusive. We performed all-atom Mo- Elucidating the Role of Transcription in Shaping the 3D Structure of the lecular Dynamics (MD) simulations on 15 bp long DNA and RNA duplexes Bacterial Genome at femtosecond resolution. Employing an advanced sampling method based Hugo Brandao1, Xindan Wang2, David Rudner2, Leonid Mirny3. on a torsion-like restraining potential, we were able to untwist the molecules 1Harvard University, Cambridge, MA, USA, 2Harvard Medical School, to induce localized melting. The simulations allowed us to determine the Boston, MA, USA, 3Massachusetts Institute of Technology, Cambridge, relative free energy and structural changes as a function of the mean twist. MA, USA. Suppression of local bending has a strong influence on the onset of DNA Active transcription has been linked to several genome conformation changes melting. Significant differences in the response were also observed for in bacteria, including the recruitment of chromosomal DNA to the cell mem- DNA and RNA. The results can have important implications for understand- brane and formation of nucleoid clusters. Using genomic and imaging data ing the mechanism of replication and transcription of DNA the function of as input into mathematical models and polymer simulations, we sought to RNA molecules. explore the extent to which bacterial 3D genome structure could be explained by 1D transcription tracks. Using B. subtilis as a model organism, we investi- 351-Pos Board B116 gated via polymer simulations the role of loop extrusion and DNA super- Translocation of Structurally Defined Branched DNA through Nanopores coiling on the formation of interaction domains and other fine-scale features Philipp Karau, Kyle Briggs, Vincent Tabard-Cossa. that are visible in chromosome conformation capture (Hi-C) data. We then University of Ottawa, Ottawa, ON, Canada. explored the role of the condensin structural maintenance of chromosome com- We use solid-state nanopores to study the translocation characteristics of plex on the alignment of chromosomal arms. A parameter-free transcription different structurally defined DNA topologies. Site-specific modifications traffic model demonstrated that mean chromosomal arm alignment can be with non-natural nucleotides along the backbone of DNA fragments allow quantitatively explained, and the effects on arm alignment in genomically rear- grafting of side branches at specific locations by ‘‘click’’ chemistry. We pro- ranged strains of B. subtilis were accurately predicted. duce T- and pi-shaped DNA molecules with a 50bp double-stranded DNA backbone and either 25nt single-stranded or 25bp double stranded DNA 355-Pos Board B120 branches. Nanopores ranging in size from 3 to 10nm are fabricated by Investigation of the Melting Thermodynamics of a DNA 4-Way Junction: controlled breakdown (CBD) in ultra thin 10-nm SiN membranes and used One Base at a Time to electrophoretically translocate these short branched DNA molecules. We Rachel E. Savage1, Wujie Wang2, Francis W. Starr2, Ishita Mukerji1. can distinguish the topologies of these DNA molecules through analysis of 1Molecular Biology and Biochemistry Department, Wesleyan University, the ionic current blockages. Such structurally defined branched DNA mole- Middletown, CT, USA, 2Physics Department, Wesleyan University, cules can be used for the development of multiplexed nanopore-based Middletown, CT, USA. assays, and as position-controlled building blocks of much larger DNA DNA four-way junctions are branched structures that form between two homol- polymers, to further our understanding of the fundamentals of molecular ogous chromosomes. These junctions play key roles during several cellular pro- transport through nanopores by precisely measuring intra molecular velocity cesses, including meiosis and DNA double-stranded break repair, however their fluctuations. mechanism of formation and separation are not well understood, particularly with respect to branch migration. We have investigated the thermodynamic sta- 352-Pos Board B117 bility of the DNA four-way junction J3 using the fluorescent pteridine nucleo- Thermodynamic Linkage Analysis of pH-Induced Folding and Unfolding side analogues, 6-MAP and 6-MI, which provide site-specific information on Transitions of I-Motifs the melting process. We have incorporated these probes at different locations Byul Kim, Tigran Chalikian. throughout the junction to determine the influence of position on junction sta- Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada. bility. Preliminary fluorescence data suggest that the central region of the four- We describe the pH-induced folding/unfolding transitions of i-motifs by a link- way junction, a region under much torsional strain, is in fact more stable than age thermodynamics-based formalism in terms of three pKa’s of cytosines, previously hypothesized. We have also investigated the relative stability of the namely, an apparent pKa in the unfolded conformation, pKau, and two apparent different arms, by incorporating probes on each arm approximately the same 0 pKa’s in the folded state, pKaf1 and pKaf2. For the 5 -TTACCCACCCTACC distance from the junction center. These results are compared with those pre- CACCCTCA-3’ sequence from the human c-MYC oncogene promoter region, dicted from coarse-grained simulations of the junction using the 3 sites per the values of pKau,pKaf1, and pKaf2 are 4.8, 6.0, and 3.6, respectively. With nucleotide (3SPN.2) model. Already, we have demonstrated the ability of these pKa’s, we calculate the differential number of protons bound to the folded this model to reproduce many experimentally determined aspects of DNA junc- and unfolded states as a function of pH. Analysis along these lines offers an tion structure and stability, including the temperature dependence of melting on alternative interpretation to the experimentally observed shift in the pH- salt concentration, the bias between open and stacked conformations, the rela- induced unfolded-to-i-motif transitions to neutral pH in the presence of cosol- tive populations of conformers at high salt concentration, and the inter-duplex vents and crowders. Our simulations reveal that a significant increase in the angle between arms. We are now using a replica-exchange molecular dynamics transition midpoint pH can be achieved by an increase in the equilibrium con- approach to evaluate the fraction of bonded bases along each arm of the junc- stant between the folded and unfolded DNA conformations due to the excluded tion over the temperature range associated with melting. This approach allows volume effect. us to determine base-by-base the local melting temperature along the arm. We

BPJ 7720_7725 70a Sunday, February 12, 2017 specifically compare the patterns of melting observed experimentally with Through a combination of simulations and control experiments we demonstrate those obtained from the coarse-grained simulations. these limits, and present a collection of best-practices.

356-Pos Board B121 359-Pos Board B124 Role of Watson-Crick-Like Mismatches in DNA Replication Fidelity Small Molecule Aptamers for Biosensing Eric S. Szymanski1, Isaac J. Kimsey2, Hashim M. Al-Hashimi1. Gregory Wiedman, Yunan Zhao, David Perlin. 1Dept. of Biochemistry, Duke University, Durham, NC, USA, 2Nymirium, Public Health Research Institute, Rutgers New Jersey Medical School, Ann Arbor, MI, USA. Newark, NJ, USA. DNA replication, transcription, and translation rely on the strict Watson-Crick In this work, we used modified Synthetic Evolution of Ligands through Expo- base pairing rules to ensure faithful transmission of genetic information. The nential Enrichment (SELEX) to discover a DNA aptamer recognizing azole Watson-Crick pairing rules are determined by the predominant neutral tauto- class antifungal drugs. This aptamer undergoes a secondary structural change meric forms of the nucleic acid bases. Incorrect base pairing during replication, upon binding its target molecule as shown through fluorescence anisotropy if left unrepaired, leads to transition or transversion point mutations. Sponta- based binding measurements. Using circular dichroism spectroscopy, we found neous mutagenesis from replication errors is believed to be a prominent source a unique double G-quadruplex structure that was essential for binding to the of base substitution errors in tumor suppressor genes in multiple forms of target: azole antifungal drugs. This type of aptamer has the potential to be cancer. Rare tautomeric and ionized nucleotide bases can form mismatches used as a small molecule captor component of a device for therapeutic drug that conform to the Watson-Crick like geometry, subverting proof reading monitoring. mechanisms. These tautomeric and anionic mismatches have long been sus- pected to contribute to spontaneous replication errors; they have proved 360-Pos Board B125 difficult to visualize as the conformational changes are subtle and involve Improved Sampling in Molecular Dynamics Studies of DNA and the B To the rearmament of protons. Nuclear magnetic resonance relaxation dispersion Z[WC] To Z-DNA Transition techniques have allowed for the characterization of a highly sequence- Lam T. Nguyen, Ashutosh Rai, Micaela E. Bush, Alma Gracic, dependent kinetic network connecting the wobble dG$dT mismatch to multiple Ahsan A. Khoja, Jinhee Kim, Sunil Pun, Alexander K. Seewald, Watson-Crick-like tautomeric and anionic dG$dT mismatch ‘excited states’. Benjamin L. Yee, Michael G. Lerner. We have obtained evidence in support of a kinetic model for misincorporation Physics and Astronomy, Earlham College, Richmond, IN, USA. which introduces a rate-limiting on-pathway tautomerization or ionization Although DNA is most commonly found in the right-handed B-DNA structure, step that leads to Watson-Crick-like mismatches prior to incorporation it is known that biologically active systems also contain left-handed ZII- through the canonical synthesis pathway. This kinetic model can account for DNA. We used both steered and targeted molecular dynamics in combination i) the three orders of magnitude difference seen in vitro between rates of correct with umbrella sampling to produce potentials of mean force for the B to ZII and incorrect nucleotide incorporations, ii) nucleotide selectivity fidelity as low transition along both the direct B-ZII pathway as well as the B-Z[WC]-ZII as 10 6, and iii) the poorly understood sequence dependence of polymerization pathway. Full pathway sampling is not feasible on smaller computer clusters, errors. so we used Hamiltonian Replica Exchange to relax individual portions of the pathway. This technique is generalizable to larger systems and larger computer 357-Pos Board B122 clusters. Direct Observation of Single Biopolymer Folding and Unfolding Process by Solid-State Nanopore 361-Pos Board B126 Xin Shi, Rui Gao, Shao-Chuang Liu, Qiao Li, Yi-Tao Long. Energetic Contributions of Plectoneme Tips and Tails East China University of Science and Technology, Shanghai, China. Andrew Dittmore, Keir C. Neuman. Biomolecular conformation and their transition play a crucial role in various NIH, Bethesda, MD, USA. in vivo or in vitro system. The most of the practical techniques for resolving Global DNA topology is sensed locally by enzymes that act on plectonemes in the secondary structures of biomolecules could provide quite precise structural supercoiled DNA. Here we report that the formation and diffusion of plecto- information for their solid-state or steady state, even at atomic resolution. For nemes are determined by the energetic contributions of their tips and tails. First, example, Cryo-EM determines high-resolution structures for the frozen- to systematically vary the geometry and formation energy of plectoneme end- hydrated specimens of biomolecules. polymers, but it is still challenging to loops, we introduced base-pair defect regions of variable size (1-16 bp) using a resolve the dynamic process of multiple functional conformational states for cassette based single-strand nicking template generated by PCR. Direct manip- biomolecules at single-molecular scale. Here, we direct observed DNA folding ulation measurements with magnetic tweezers revealed that even a single and unfolding process in real-time by using sub-5 nm solid-state nanopores. In mismatch or abasic site is sufficient to nucleate formation of a plectoneme. Pre- our experiments, a single-stranded DNA adhered to single monovalent strepta- sentation of the defect precisely at an extruded plectoneme tip potentially vidin could be reversibly trapped in a solid-state nanopore. Then, the fluctua- serves as a damage-sensing mechanism and may facilitate the search process tions of the blockade current could be recorded, which reveals the dynamic of repair enzymes. Second, our measurements unexpectedly revealed that after structural transitions among DNA secondary structures. For example, after twisted DNA abruptly buckles into an initial plectoneme loop, further plecto- trapping the cytosine-rich DNA strains in slightly alkaline solution, the forma- neme extrusion occurs through a cascade of additional buckling steps in which tion of multiple unstable and semi-folded i-motif structures could be observed. the torque changes by roughly half of the initial overshoot value. These discrete More important, well time-resolved transitions between these structures could steps do not match any obvious scale of the system but are consistent with be obtained. When using slightly acidic solution, the stable structures with discontinuous feed-in of curving plectoneme tails. In light of these results, theo- stable blockade current could be found. With this new approach, we can retical models of plectonemes should include their overall structure, including directly observe the dynamic conformational change of biomolecules at the often neglected tips and tails. single-molecular scale, which would be of great help for resolving single mole- cule interactions, designing single-molecule machine and understanding the 362-Pos Board B127 working process of biomolecular in biological system. Multi-Scale Structure and Conformational Dynamics of Scaffolded DNA Origami Nanoparticles 358-Pos Board B123 William Bricker, Keyao Pan, Mark Bathe. Tuning Up Tethered Particle Motion Massachusetts Institute of Technology, Cambridge, MA, USA. Daniel T. Kovari, Eric Weeks, David Dunlap, Laura Finzi. Synthetic DNA can be programmed into self-assembled 3D nanoparticles us- Physics, Emory University, Atlanta, GA, USA. ing a DX design motif and the principle of scaffolded DNA origami. A top- Tethered Particle Motions assays are a simple but powerful tools for monitoring down design procedure (DAEDALUS) (Veneziano, Ratanalert, et al., Science, the effective length of individual DNA strands and other linear bio-polymers in 2016) facilitates the design of arbitrary DNA nanoparticle geometries on the 5 real-time. The technique has been employed in various capacities including to 100 nanometer scale, which we investigate in detail here using multi-scale characterization of DNA topology, transcription factor - DNA interactions, structural modeling. While coarse-grained modeling is useful for generating and monitoring progress of enzymes that translocate along DNA. At its core equilibrium structures of DNA nanoparticles (Pan et al., Nat. Comm., the technique is relatively simple to implement, only requiring a research- 2014), only all-atom models reveal fine structural details and mechanical grade microscope and a video camera; however, it is important to note that op- properties that contribute to overall structure and conformational dynamics. tical resolution, frame rate, exposure time, particle size, and solution viscosity Here, we first use all-atom molecular dynamics (MD) to simulate two 0.5 - all affect the ability to discriminate different tether lengths and detect changes. 1.0 MDa DNA polyhedral nanoparticles: a tetrahedron with 63 base pair

BPJ 7720_7725 Sunday, February 12, 2017 71a

(bp) edge lengths, and an octahedron with 52 bp edge lengths. Using 150 ns data, we give estimates for the base-pairing energies within the interference trajectories, we are able to elucidate subtle structural features seen in exper- complex for various CRISPR systems. imental cryo-EM maps, including right-handed twisting of the vertices in the octahedron and outward bowing of the edges in the tetrahedron. Next, 365-Pos Board B130 these results are compared with all-atom MD simulations of unconstrained Conformational Dynamics of Cas9 during DNA Binding vertex building blocks including the tetrahedron (3-arm vertex) and octahe- Yavuz S. Dagdas1, Janice S. Chen2, Samuel H. Sternberg3, dron (4-arm vertex). In these simulations, a notable feature is the significant Jennifer A. Doudna2,3, Ahmet Yildiz2,4. 1 out-of-plane bending angle away from the minor groove at the vertex due Biophysics Graduate Group, University of California, Berkeley, Berkeley, 2 to the chirality of dsDNA. Finally, equilibrium solution structures of 45 CA, USA, Department of Molecular and Cell Biology, University of 3 DX-based DNA origami nanoparticles are predicted by implementing an up- California, Berkeley, Berkeley, CA, USA, Department of Chemistry, 4 dated bulge stiffness parameter for our coarse-grained FE model CanDo (Kim University of California, Berkeley, Berkeley, CA, USA, Department of et al., Nucleic Acids Res., 2012). These multi-scale structural results show the Physics, University of California, Berkeley, Berkeley, CA, USA. interplay between coarse-grained and all-atom models in the ab initio predic- Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA tion and elucidation of complex features of DNA nanoparticles seen in exper- using two conserved nuclease domains, RuvC and HNH, as part of imental cryo-EM maps. CRISPR-Cas bacterial adaptive immune systems. Together with single- guide RNAs, Cas9 is also widely utilized as a programmable genome editing RNA Binding tool. DNA cleavage activity is controlled directly by the conformational state of the HNH nuclease domain, but Cas9 conformational dynamics during DNA 363-Pos Board B128 binding remain poorly characterized. Using single-molecule FRET assays, we Shedding Light on Cas9 Target Search identified a long-lived intermediate state of S. pyogenes Cas9. Upon DNA Viktorija Globyte1, Seung Hwan Lee1, Luuk Loeff1, Jin Soo Kim2, binding, the HNH nuclease reversibly transitions between RNA-bound, Chirlmin Joo1. DNA-bound and docked conformations, before it docks irreversibly into its 1TU Delft, Delft, Netherlands, 2Seoul National University, Seoul, Korea, catalytically active conformation for DNA cleavage. Docking of HNH to its Republic of. active state requires divalent cation, and HNH remains in the docked state af- The CRISPR/Cas adaptive immune system provides prokaryotes with a defen- ter cleavage of a complementary target. Increasing the number of mutations in sive mechanism against invading viruses and plasmids. The invading viral the target region distal to the protospacer adjacent motif (PAM) prevents tran- DNA fragments are incorporated into the CRISPR (Clustered Regularly Inter- sitions from intermediate to the docked conformation. The results provide a spaced Short Palindromic Repeats) locus in the bacterial genome and are later structural explanation for the lack of DNA cleavage activity when Cas9 binds used to recognize and destroy the invader when it returns. In the last stage of to off-target sites. CRISPR immunity, called the interference stage, Cas (CRISPR-associated) proteins assemble with short guide RNA molecules which are transcribed 366-Pos Board B131 from the CRISPR locus. These guide RNA molecules can be programmed to Repetitive Loop Formation by the CRISPR-Cas3 Helicase recognize any DNA sequence. In recent years the CRISPR/Cas adaptive im- Luuk Loeff1, Stan Brouns1,2, Chirlmin Joo1. 1 2 mune system has seen an immense growth in interest with the type II Technical University Delft, Delft, Netherlands, Wageningen University, CRISPR-Cas9 system being in the center of attention. In this system, the Wageningen, Netherlands. DNA of the invading virus is recognized and cleaved by a single protein E. coli maintain CRISPR-Cas adaptive immune systems to protect the cell Cas9 which is guided by an RNA duplex. Due to its simplicity, CRISPR- against invading genetic elements. Immunity relies on the RNA guided surveil- Cas9 system is a promising tool in gene engineering as its guide RNA can lance complex Cascade (CRISPR-associated complex for antiviral defense) and be programmed to recognize virtually any sequence in the genome. The the trans-acting Cas3 protein with helicase and nuclease activities. We recently CRISPR-Cas9 has been demonstrated to work in a variety of organisms, how- showed that Cas3 generates degradation products ranging from 30 to 150 nt that ever, despite the large interest in this system, the precise mechanism by which act as pre-cursors for primed spacer acquisition. However, it remains unclear Cas9 finds and cleaves its target remains ambiguous. We utilize biophysical which mechanism drives the generation of these fragments with a specific single-molecule techniques, namely total internal reflection fluorescence mi- size. Here we employed single-molecule FRET to probe the molecular dy- croscopy (TIRFM) together with Forster resonance energy transfer (FRET) namics of Cas3 in real-time. Our data shows that Cas3 repeatedly generates to investigate the mechanics of Cas9 target search with nanometer sensitivity. DNA loops in the target strand whilst remaining in tight contact with Cascade. We are probing one-dimensional diffusion of the protein along the DNA strand DNA loops are generated by breaking open the dsDNA helix in distinctive steps and investigating the effects different DNA sequences have on the mechanics of 3 bp, arising from the RecA like folds of the helicase domain. Repetitive of Cas9 target search. unwinding achieved by slipping of the helicase domain, which limits the average translocation distance to ~90 nt. Taken together, our data suggest 364-Pos Board B129 that the inherent helicase properties of Cas3 drive the generation precursors To Cleave or not to Cleave: Predicting the Target Specificity of CRISPR- of adequate size for primed spacer integration. Cas Systems through Theoretical Modeling Misha Klein, Martin Depken. 367-Pos Board B132 Kavli institute of Nanoscience, Departement of BioNanoScience, TU Delft, The Impact of DNA Topology on Target Selection by a Cytosine-Specific Delft, Netherlands. Cas9 Many prokaryotes employ the CRISPR-Cas (Clustered Regularly Interspaced Tsz Kin Martin Tsui, Travis H. Hand, Hong Li. Short Palindromic Repeat – CRISPR associated) system to fend off attacks Institute of Molecular Biophysics, Florida State University, Tallahassee, by hostile genetic elements. This adaptive immune system recognizes in- FL, USA. vaders based on their level of sequence complementarity with RNA tran- Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for scribed from a library of past invasions stored at the CRISPR locus. To genome editing and gene regulation. To be cleaved by Cas9, a double stranded avoid infection, the CRISPR interference complex must be able to single DNA, or the protospacer, must be 1) complementary to the Cas9-bound guide out a short target sequence (20 – 40 nt) among a total of 105-106 nt in the RNA and a short Cas9-specific sequence adjacent to protospacer, called Proto- cell, while at the same time recognizing targets that have evolved away spacer Adjacent Motif (PAM). Understanding the correct juxtaposition in time from the record stored at the CRISPR locus. Despite the tremendous interest and space of the protospacer- and PAM-interaction with Cas9 will enable the CRISPR-Cas9 system has gained as a novel genome editing tool, the development of versatile and safe Cas9-based technology. We report identifica- sequence preference of the interference complex remains poorly understood. tion and biochemical characterization of Cas9 from Acidothermus cellulolyti- 0 0 Experiments have shown that it is not just the amount of mutations, but also cus (AceCas9). AceCas9 depends strictly on a 5 -NNNCC-3 PAM and is their placement along the guide/target, that determine the level of more efficient in cleaving negative supercoils than relaxed DNA. We further interference. showed that mismatches to the guide RNA on a supercoiled protospacer are Through kinetic modeling we provide simple rules to assess sequence speci- tolerated by AceCas9, whereas the same mismatches on its relaxed form ficity based on mismatch patterns, and quantitatively explain the experimen- were not. The cytosine-specific and DNA topology-sensitive properties of the tally observed seed region over which no mismatches are permitted. Moving AceCas9 maybe explored for chromosome domain specific genome editing ap- beyond sequence complementarity, we also quantify how changes in the plications. Finally, our preliminary data showed that AceCas9 can disrupt conformation of the interference complex can serve to improve specificity dur- target DNA in in vivo assays, demonstrating its utility as a genome editing ing target recognition. Finally, by fitting our model to published experimental enzyme.

BPJ 7720_7725 72a Sunday, February 12, 2017

368-Pos Board B133 us to model biologically relevant mutations of these interactions and ultimately CRISPR-Cas9: Computational Insights Toward Improved Genome Editing design novel interactions. Despite recent advances in high throughput experi- Giulia Palermo1, Yinglong Miao1, Ross C. Walker1, Martin Jinek2, mental technologies that measure the energetics of these systems, quantitative J. Andrew McCammon1. computational prediction of relative RNA/protein binding affinities has re- 1Department of Pharmacology, University of California at San Diego/ mained a challenge. This is partly due to the observation that computational Howard Huges Medical Institute, La Jolla, CA, USA, 2Department of binding affinity prediction methods typically break down when the molecules Biochemistry, University of Zurich, Zurich, Switzerland. are highly flexible or undergo significant conformational changes, situations Life sciences are undergoing a transformative phase due to an emerging that often arise in RNA/protein binding. Here, we present a novel framework genome-editing technology based on the RNA-programmable CRISPR-Cas9 within Rosetta for predicting RNA/protein relative binding affinities that begins (clustered regularly interspaced short palindromic repeats-CRISPR associated to address this issue. Specifically, we show that the nearest neighbor energies, protein 9) system. In this system, the endonuclease Cas9 associates with a guide which are typically used for RNA secondary structure prediction, can be used to RNA to match and cleave complementary sequences in double stranded DNA, approximate the unbound free energy of the RNA, thus eliminating the need to forming an RNA:DNA hybrid and a displaced non-target DNA strand. explicitly account for the flexibility of the unbound RNA or conformational Although extensive structural studies are ongoing, the conformational dy- changes of the RNA upon binding. Using this method of calculating the un- namics of Cas9 and its interplay with the nucleic acids during association bound RNA free energy significantly improves the prediction accuracy over and DNA cleavage are largely unclear. This missing aspect hampers the precise a more typical 3D structure-based approach. We optimized this method using structure-based design of CRISPR-Cas9 genome-editing tools with improved a subset of published MS2 coat protein affinities and ultimately made predic- specificity. Here, we report the first biophysical study – based on extensive tions for the system with 1.11-1.28 kcal/mol root mean square (RMS) error. multi-microseconds molecular simulations integrated with structural data – Additionally, we show that this method is able to predict relative binding affin- revealing the conformational plasticity of Cas9 and identifying the key deter- ities for four diverse RNA/protein systems with 1.48 kcal/mol RMS error. minants that allow its large-scale conformational changes during nucleic acid Finally, to more rigorously assess this method, we independently measured binding and processing. We identify a remarkable conformational plasticity and made blind predictions for PUF3 and PUM2 binding affinities with RMS as an intrinsic property of the nuclease HNH domain, being a necessary factor errors of 1-2 kcal/mol, which is comparable to the accuracy achieved by predic- allowing for the HNH domain repositioning during catalysis. More impor- tion methods for other types of systems. tantly, we disclose a key role of the non-target DNA during the process of acti- vation of the HNH domain, showing how the non-target DNA positioning 371-Pos Board B136 triggers local conformational changes that favor the formation of a catalytically Biophysical Studies of Liposome Encapsulated Pokeweed Antiviral Protein competent Cas9. Our outcomes further suggest new and precise protein- and its use as a HIV Therapeutic O’Jay Stewart1, Artem Domashevskiy2. engineering modifications, which are of fundamental importance for the 1 2 rational design of more effective genome-editing tools. Overall, these novel CUNY - John Jay College, Manhattan, NY, USA, Department of Sciences, findings constitute a reference for future experimental studies aimed at a full CUNY - John Jay College, Manhattan, NY, USA. characterization of the dynamic features and at the improvement of biological Human Immunodeficiency Virus (HIV) is a virus that attacks the human im- applications of the CRISPR-Cas9 system. mune system, compromising its effect in regards to disease prevention. HIV re- sults in the destruction of CD4 cells, which are vital in the defense against 369-Pos Board B134 human immune responses. HIV can severely damage the immune system and Structural Insights into G-tract Recognition by the hnRNP H-RNA Recog- lead to Acquired Immunodeficiency Syndrome (AIDS). There is presently no nition Motif cure or effective HIV vaccine and as a result it is important to seek alternative Srinivasa R. Penumutchu. measures in the defense against HIV/AIDS. Pokeweed Antiviral Protein (PAP), Chemistry, Case Western reserve University, Cleveland, OH, USA. a protein isolated from pokeweed plant, Phytolacca americana, provides a new The heterogeneous nuclear ribonucleoprotein H (hnRNP H) family of proteins and profoundly promising direction in the field of HIV/AIDS research. PAP are involved in RNA splicing of cellular and viral mRNAs. These proteins plays a vital role in the immune system of pokeweed and is a ribosome inacti- function as both splicing activators and repressors. The hnRNP H family pro- vating protein (RIP), inhibiting viral protein production. PAP possesses anti- teins have previously been found to interact with poly-G sequences (G-tracts) viral properties, and reduces the infectivity of many plant and animal of cellular and viral mRNAs using quasi RNA recognition motifs (qRRMs) viruses, including HIV-1. My project focuses on mechanisms using liposomal including human immunodeficiency virus (HIV). hnRNP H proteins are encapsulated PAP, targeted to infected cells as a therapeutic for HIV infection. composed of three qRRMS, which are separated by linkers and two Glycine Steady state fluorescence was used to identify the thermodynamic parameters rich domains at C-terminal. The qRRM1 and qRRM2 domains are located at of interaction between PAP and HIV RNA. PAP binds to the m7 GTP cap the N-terminus and separated by 10-residue linker, whereas qRRM3 is located and the 50 UTR region of HIV RNA. Various isoforms of PAP were tested towards the C-terminus. To gain structural insights into hnRNP H protein, here and the optimal affinity was identified as a Km of 20.09 nM. Fluorescence we solved the solution structure of the HRRM12 domain of hnRNP H using observed V.S [m7 GTP] was plotted through nonlinear regression using the NMR spectroscopy. We used paramagnetic relaxation enhancement (PRE) Prism 6.0 program. Varying liposomes were prepared in order to identify the and Residual dipolar couplings (RDC) to obtain distance restraints and orien- optimal characteristics for PAP encapsulation. Liposomes were lyophilized tational restraints between the HRRM1 and HRRM2 domains. T1, T2 and through rotary evaporation and later extruded to ensure homogeneity. Fluores- NOE experimental data is consistent with calculated structure of HRRM12 cent titration enables the testing of liposomal PAP encapsulation efficiency. It domain and reveals that HRRM12 adopt the compact (closed) structure. To bet- was concluded that a 1/1 v% mixture of liposomes DOPE-DOTAP is most effi- ter understand principals of hnRNP H-RNA recognition, we systematically cient for PAP encapsulation. These accomplishments will enable further modi- screened G-tracts of RNA oligos against HRRM12 by using isothermal titration fication of the liposomes by covalently conjugating HIV-specific monoclonal calorimetry (ITC) and NMR spectroscopy. We examined the minimal G-tract antibodies (anti-pg120 and/or anti-pg41), thus facilitating the selectivity of RNA (–GGG–) sequence requirements for HRRM12 binding. The HRRM12 the liposomal PAP to the target tissues (HIV-infected CD4þ lymphocytes). domain of hnRNP H exhibited no substantial differences in binding affinities With the aforementioned completed the basis for PAP-liposome interactions for G-tracts of RNA (AGGGX) and HRRM12 residues involved in binding can be established and also the modulation of these interactions in order to G-tracts of RNA were detected by NMR chemical shift perturbation experi- target HIV-infected cells. ments and a data-driven model of the complex was determined using HADDOCK. Taken together, this study provides the molecular insights for bet- 372-Pos Board B137 ter understanding the role of the qRRM domains of hnRNP H in RNA splicing A Dead-Box Protein Acts through RNA to Promote HIV-1 Rev-RRE of cellular and viral mRNAs. Assembly Rajan Lamichhane, John A. Hammond, Raymond F. Pauszek, 370-Pos Board B135 Ingemar Pedron, Edwin van der Schans, James R. Williamson, Blind Predictions of RNA/Protein Relative Binding Affinities David P. Millar. Kalli Kappel, Inga Jarmoskaite, Pavan P. Vaidyanathan, Integrative Structural and Computational Biology, The Scripps Research William J. Greenleaf, Daniel Herschlag, Rhiju Das. Institute, La Jolla, CA, USA. Stanford University, Stanford, CA, USA. The HIV-1 Rev (Regulator of Expression of Virion) protein activates nuclear Interactions between RNA and proteins are pervasive in biology, shaping pro- export of unspliced and partially spliced viral mRNAs, which encode the viral cesses such as mRNA translation, localization, and alternative splicing. Devel- genome and the genes encoding viral structural proteins. Rev interacts with a oping a predictive understanding of the energetics of these systems would allow highly conserved region, the Rev Response Element (RRE), located within

BPJ 7720_7725 Sunday, February 12, 2017 73a the viral mRNA. In order to activate nuclear export, multiple Rev proteins must RNAs is regulated in vivo by interaction with both duplex and single stranded assemble on the RRE. The host DEAD box protein 1 (DDX1) enhances the regions. RNA export activity of Rev through an unknown mechanism. We used a single-molecule assembly assay utilizing immobilized full length RRE and Membrane Dynamics I fluorophore-labeled Rev to monitor each step of Rev-RRE assembly, in the presence or absence of DDX1. More Rev monomers were observed to bind 375-Pos Board B140 to the immobilized RRE in the presence of DDX1, indicating that DDX1 pro- Surfactant Micelle Self-Assembly with Coarse-Grained Martini Standard motes oligomeric Rev-RRE assembly. Further experiments using specific Water and Polarizable Water DDX1 mutants that are defective in either Rev binding or RNA binding indi- Eric Sefah, Blake Mertz. cate that DDX1 must be capable of associating with RNA in order to promote Chemistry, West Virginia University, Morgantown, WV, USA. assembly of the Rev-RRE complex. Single-molecule Fo¨rster resonance energy Molecular dynamics (MD) simulations of proteomicelle complexes are useful transfer (smFRET) experiments show that DDX1 transiently interacts with the for the investigation of membrane protein dynamics and function. However, RRE and that both DDX1 and Rev can occupy the same RRE molecule. Taken modeling assembly and dynamics of complex oligomeric systems require together, these results suggest that DDX1 acts as an RNA chaperone, folding length- and timescales that are traditionally inaccessible to all atom MD simu- the RRE into a conformation that is pre-organized to bind the first Rev mono- lations. Coarse grained (CG) force fields such as MARTINI decrease spatial mer, thereby promoting the overall Rev-RRE assembly process. Supported by resolution of a system, making biologically relevant time scales for phenomena NIH grant GM082545. such as membrane protein oligomeric assembly accessible [1]. In order to apply CG force fields to the study of large proteomicelle complexes it is important to 373-Pos Board B138 test their ability to reproduce experimentally observed properties. In this study Deciphering the Action Mechanism of DDX3: An RNA Helicase Implicated we have characterized the effects of the nonpolarizable and polarizable water in Cancer Propagation and Pathogenic Viral Infection models of the MARTINI force field [2] on assembly of surfactant micelles. Anthony F. Moore, Aliana Lopez, de Victoria, Eda Koculi. The two surfactant systems studied were a zwitterionic detergent (n-dodecyl- Chemistry, University of Central Florida, Orlando, FL, USA. phosphocholine (DPC)) and nonionic detergent (n-dodecyl-b-D-maltoside DDX3 is a human DEAD-box RNA helicase implicated in crucial cellular (DDM)). From a system of 50, 100, 150 and 200 random detergent molecules, processes including translation initiation, ribosome assembly, RNA transport, stable micelles formed with variable sizes for both models, in general agree- and microRNA processing. Consequently, DDX3 is implicated in many viral ment with experimental aggregation numbers. However, the polarizable water infections and cancer cell metabolism. Our goal is to obtain a detailed under- model formed larger micelles than the normal water model in a few cases. In standing of DDX3’s mechanism of action and employ this understanding to addition, the polarizable water model formed stable compact micelles in shorter discover DDX3 inhibitors that would serve as lead compounds for drugs times. that halt viral infections and cancer cell metabolism. While DDX3 is required [1] Marrink SJ, et al. J. Phys. Chem. B. 2007;111:7812. for many viral infections and cancer cell propagations, it is not essential for [2] Yesylevskyy SO, et al.PLoS Comp. Biol, 2010;6:e1000810. healthy cell metabolism, making DDX3 an ideal anticancer and antiviral drug target. Like all the members of the DEAD-box family of enzymes, 376-Pos Board B141 DDX3 uses ATP binding and hydrolysis to unwind short double-stranded Simulations of Glycerol and its Effect on the Phase and Behaviour of DPPC RNA helices. Our data show that different from many members of DEAD- Monolayers box family of enzymes, monomeric DDX3 is unable to perform RNA unwind- Jemma L. Trick1, Wachirun Terakosolphan2, Ben Forbes2, ing, and a multimeric DDX3 complex is required to support DDX3’s helicase Christian D. Lorenz1. activity. Furthermore, our data suggests that the single-stranded-double- 1Physics Department, Kings College London, London, United Kingdom, stranded RNA junction promotes the formation of the DDX3 multimer. We 2Institute of Pharmaceutical Science, Kings College London, London, United are in the process of performing mutagenesis studies combined with cross- Kingdom. linking and mass spectrometry to determine the DDX3 amino acids implicated Lung Surfactant (LS), a monolayer coating the alveolar surface undergoes in mulitmer formation and the amino acids that come in direct contact with changes during breathing. Glycerol, a known cryo-protectant is known to RNA during the DDX3 catalytic cycle. These experiments would produce in- induce folding in LS, stiffening in model LS monolayers, modulate area per formation both on DDX3’s mechanism of action, and elucidate why some lipid, and the transition temperature of such systems experimentally. Using DEAD-box proteins have evolved to act as mulitmers. Lastly, we have found atomistic molecular dynamics (MD) simulations, we model LS as a pure dipal- four natural compounds that are specific inhibitors of DDX3 ATPase activity. mitoylphosphatidylcholine (DPPC) monolayer, in which the concentration of These compounds will be used as probes to decipher DDX3’s action mecha- glycerol and simulation temperature is varied to investigate the molecular basis nism and could have translational potential as drugs that stop various viral in- of such variations in behaviour. Our Simulations suggest a possible dehydration fections and cancer progression. stage of DPPC head-groups under high concentrations of glycerol, which result in a change in DPPC transition temperature (6). This change could influence the 374-Pos Board B139 use of glycerol in possible aerosol devices and the permeability of the modu- Interaction of PKR with Single Stranded RNA lated monolayer. Christopher B. Mayo, James L. Cole. Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA. 377-Pos Board B142 Although the antiviral kinase PKR was originally believed to interact with Protocol and Validation of CHARMM-GUI Hex Phase Builder only duplex RNAs, evidence has accumulated that the enzyme can be acti- Andrew H. Beaven1, Alexander J. Sodt2, Richard W. Pastor3, Wonpil Im4. vated by a variety of structured RNAs. A potent PKR activating motif consists 1Chemistry, The University of Kansas, Lawrence, KS, USA, 2Unit on of a short stem loop containing single stranded tails (ss-dsRNA). The ssRNA Membrane Chemical Physics, National Institutes of Health, Bethesda, MD, tails contribute to binding and activation. However, PKR does not contain a USA, 3Laboratory of Computational Biology, National Institutes of Health, canonical ssRNA binding domain. Here, we demonstrate that isolated ssRNAs Bethesda, MD, USA, 4Department of Biological Sciences and interact with PKR. Both homopolymeric (rU)30 and heteropolymeric 30 nt Bioengineering Program, Lehigh University, Bethlehem, PA, USA. ssRNAs bind with micromolar dissociation constants. Addition of a 50-triphos- It is becoming increasingly apparent that protein dynamics, conformation, and phate slightly enhances binding affinity. A homopolymeric (dT)30 ssDNA therefore, function are dependent on the stresses within the bilayer – one of 0 binds more weakly than (rU)30, indicating a contribution of the 2 OH moiety. which being bending frustration. The amount of bending frustration is depen- –1 PKR contains a conserved region N-terminal to the kinase that is enriched in dent on the bending modulus (kc) and spontaneous radius of curvature (R0 ) basic residues. ssRNA binds to a construct containing the basic region and ki- for a given lipid composition. These quantities are difficult to obtain computa- nase and also binds to the isolated dsRNA binding domain. Both full length tionally, particularly by all-atom molecular dynamics (MD) simulation. For PKR and the basic region/kinase domain construct are weakly activated by many years, experimentalists (and much more recently, simulators) have ssRNA. However, the isolated kinase is not activated and does not bind used the lipid hexagonal phase to obtain these quantities. The inverse lipid hex- ssRNA. Photocrosslinking measurements were performed using a ss-dsRNA agonal phase exists in high temperature and low water domains of the certain containing 4-thiouridine and PKR constructs with a TEV protease cleavage lipid’s phase diagram. In this phase, lipids aggregate with their hydrophilic site at different positions between the kinase and dsRNA binding domain. heads oriented along hexagonally packed water pores. Although this phase Analysis of the products following crosslinking and TEV cleavage demon- spontaneously assembles, the equilibration time necessary for this type of pro- strates that that the basic region interacts with ss-dsRNA in the context of cess is unfeasible for typical all-atom MD simulations. Here, a simple and full length PKR. Our results support a model where PKR activation by reproducible methodology, Hex Phase Builder, is described and validated as

BPJ 7720_7725 74a Sunday, February 12, 2017 an integrated part of the CHARMM-GUI framework (http://www.charmm-gui. fatty acids (PUFA) by terminating a chain of chemical reactions that org/input/hexphase). By calculating the pressure within the water pore, kc and follow free radical attack. Due to the low global concentration of atoc in the –1 R0 can be obtained for any lipid composition. This sensitive technique pro- plasma membrane, we have proposed that atoc preferentially interacts with vides energetic insight into lipid systems and allows for further validation of the PUFA-containing lipids to optimize its local concentration. Here we tested the oft-used CHARMM C36 lipid force field. this hypothesis by calculating the binding energy between atoc and two different phospholipids. 1-stearoyl-2-docosahexaenoylphosphatidylcholine 378-Pos Board B143 (SDPC, 18:0-22:6PC) was used to represent a PUFA-containing lipid, while Reproduction of a Three-Component (DPPC/DOPC/Cholesterol) Phase 1-stearoyl-2-oleoylphosphatidylcholine (SOPC, 18:0-18:1PC) was used as a Diagram using Coarse Grained Molecular Dynamics monounsaturated control. Umbrella sampling molecular dynamics (USMD) 1 2 2 Cameron Montour , Timothy S. Carpenter , Felice C. Lightstone . simulations of atoc in bilayers composed of these lipids were performed 1Dept. of Biochemistry & Molecular & Cellular Biology, Georgetown ˚ 2 with 45 windows separated by 1 A and arranged from the center of bilayer University, Washington, DC, DC, USA, BBTD, Lawrence Livermore all the way to the bulk water to calculate the free energy as a function of atoc’s National Laboratory, Livermore, CA, USA. location. The results show slightly less binding energy between atoc and The plasma membrane consists primarily of a mixture of lipids and proteins, SDPC, which we ascribe to a more loosely packed arrangement of lipids in responsible for creating a homeostatic environment inside the cell. The plasma the PUFA-containing membrane. NMR experiments measuring the affinity membrane is also responsible for mediating cell-cell communication; specif- of atoc for SDPC and SOPC are currently ongoing in support of the ically, membrane proteins send and receive signals across the plasma mem- simulations. brane. However, in some cases the membrane proteins require lipids to create a locally optimized environment to create functional groupings. Lipids 381-Pos Board B146 may facilitate protein localization by forming phases of differing lateral orga- RBL-2H3 Proliferation is Modulated by Treatments that Shift Transition nization, which proteins are likely to segregate into based on overall lipid dy- Temperatures in Isolated Plasma Membrane Vesicles namics. These phases occur mainly as liquid-disordered (Ld) or liquid-ordered Rohan P. Desai, Sarah Veatch. (Lo); the Ld phase is composed of mainly unsaturated lipids, while the Lo Biophysics, University of Michigan, Ann Arbor, MI, USA. phase is composed of mainly saturated lipids and cholesterol. Membranes Giant plasma membrane vesicles (GPMVs) isolated from a variety of cell types can exist as a single fluid phase or coexistence of phases under the right com- appear homogeneous at physiological temperatures but separate into distinct positions and conditions. It is established that phase separation can occur in liquid-ordered and liquid-disordered phases below their transition temperature certain models but the ordering progression of the single fluid phase and (Tmix). Previous work in the lab has shown that Tmix is higher in GPMVs iso- whether or not a threshold event occurs between the Ld and Lo phases is still lated from sparsely plated cells than those plated at a higher density (Gray et al. largely unidentified. In this study we performed coarse-grained MARTINI mo- PLoS One 10(9):e0137741 (2015)). Since these cells are also contact inhibited, lecular dynamic simulations on a ternary system, consisting of a saturated lipid, we hypothesize that differences in T are related to their different growth dipalmitoylphosphatidylcholine (DPPC), an unsaturated lipid, dioleoylphos- mix rates. This previous study also found that Tmix is elevated in GPMVs isolated phatidylcholine (DOPC), and cholesterol (CHOL). A primary goal of the study from cells preparing to undergo cell division compared to cells in other phases was to observe how well the MARTINI forcefield could reproduce these of the cell cycle. Based on these past results, we hypothesized that compounds ternary phase diagrams. As such, no changes were made to the MARTINI which shift GPMV Tmix also affect the rate of cell growth when incubated forcefield, and no biases were imposed on the system. The MARTINI forcefield with intact cells. To test this hypothesis, we incubated intact RBL-2H3 cells performed remarkably well, replicating all of the single fluid phases and most in n-alcohols and combinations of n-alcohols which we have previously of the phase separated systems. This work were performed under the auspices shown to alter Tmix when added to isolated GPMVs (Machta et al. Biophys. of the U.S. Department of Energy by Lawrence Livermore National Laboratory J. 111(3):537-45 (2016)). We find that these treatments also alter cell growth under Contract DE-AC52-07NA27344, Release LLNL-ABS-704312. as measured by counting cells present after 24h of incubation with compounds 379-Pos Board B144 in complete media at 37 C. Specifically, fewer cells are counted in dishes con- Molecular Dynamics Simulations of 38 Types of Ganglioside in Homoegne- taining n-alcohols that lower Tmix in GPMVs than that of a control, whereas an ous Membrane Bilayers even greater number of cells are counted in dishes containing an n-alcohol that Steve Kim1, Wonpil Im2. raises Tmix in GPMVs. In addition, we find that cells adapt their membrane 1University of Kansas, Lawrence, KS, USA, 2Lehigh University, Bethlehem, composition to compensate for the effect of n-alcohols on GPMV Tmix when PA, USA. incubated with n-alcohols for 24h. Ongoing experiments are being conducted Ganglioside is a membrane lipid in which the hydrophilic head group is a to illuminate the biochemical pathways underlying the observed changes in complex oligosaccharide containing one or more sialic acids. The oligosac- cell proliferation. charides of the ganglioside extend out from the cell surface, interacting with various signaling molecules. Some of their functions include regulation 382-Pos Board B147 of protein activities, recognition of specific molecules, and communication be- Lipid Lateral Ordering Defined by High-Field EPR tween cells. Gangliosides are especially abundant in the brain, with three Zahra Hayati, Pavanjeet Kaur, Likai Song. times as much in grey as in white matter. It is believed that gangliosides National High Magnetic Field Laboratory, Florida State University, are involved in pathological states such as cancer, Tay-Sachs disease, Hun- tallahassee, FL, USA. tington’s disease, Alzheimer’s disease, et certera. To explore and understand EPR spectra at 94 GHz or higher are sensitive to lipid lateral ordering, which the orientation, structure, and dynamics of gangliosides, molecular dynamics provides key information on the structure and dynamics of biological simulations on 38 different types of gangliosides in homogenous membrane membranes as well as protein–lipid interactions. High-frequency high-field bilayer (POPC) were performed. CHARMM-GUI was utilized to build the EPR improves spectral resolution through increased g-factor resolution, membrane bilayers, and a ganglioside was inserted in both the upper leaflet enabling the determination of the motionally averaged gxx–gyy anisotropy, and the bottom leaflet, with three simulations for each ganglioside. Each sys- which reflects lipid lateral ordering. Lipid rafts are domains of plasma mem- tem was ran for up to 50 ns and multiple analyses were ran. Through compar- branes enriched with cholesterol and sphingolipids and are essential for ative analysis, the difference in properties and dynamics due to different cellular functions. Here, we investigated the lateral ordering of lipid rafts glycan sequence will be studied. Future work includes running the simulation using high-field EPR. The analyses reveal that lateral ordering is more longer up to 200~300 ns and assembling the information gathered through sensitive to raft-domain formation than the conventional order parameter. multiple analyses for greater understanding of gangliosides, which will aid Lateral ordering is a sensitive parameter for both lipid phase transition in glycome studies. and cholesterol/sphingomyelin content. Our data also show that raft and nonraft domains exhibit distinct lateral order profiles across the lipid bilayer. 380-Pos Board B145 In addition, we characterized the membrane interaction and peptide- Vitamin E does not Preferentially Bind to Polyunsaturated Lipids as induced lipid-lateral-ordering changes of an antibacterial g-AApeptide Revealed by Umbrella Sampling MD Simulations (AA1). The results illustrate that AA1 interacts strongly with bacterial mem- Xiaoling Leng1, Andres Cavazos1, Bruce Ray1, Mikel Ghelfi2, branes, induces significant lipid lateral ordering and membrane thinning, and Jeffrey Atkinson2, Fangqiang Zhu1, Stephen Wassall1. subsequently disrupts bacterial membranes. Our findings suggest that AA1 in- 1physics, IUPUI, Indianapolis, IN, USA, 2chemistry, Brock University, teracts with and disrupts bacterial membranes through a carpet-like mecha- St. Catharines, ON, Canada. nism. Taken together, these results highlight the sensitivity of high-field Alpha-tocopherol (atoc) is the active form of vitamin E that is retained in the EPR for elucidating membrane order and dynamics as well as protein–lipid human body. It is a lipid-soluble antioxidant, which protects polyunsaturated interactions.

BPJ 7720_7725 Sunday, February 12, 2017 75a

383-Pos Board B148 interfaces. Using single-molecule tracking, we study the diffusion of membrane Azobenzene-Cholesterol as a Photoactivator in Biomimetic Membranes: 1. components in the interfacial region and the effect of lipid composition and Lipid Dynamics environmental conditions on membrane behaviour. Chen Shen1, Jacques Ollivier2, Judith Peters3,Jo¨rg Pieper4, Beate Klo¨sgen1. 1Department of Physics, Chemistry and Pharmacy, University of Southern 387-Pos Board B152 Denmark, Odense, Denmark, 2Institut-Laue-Langevin, Grenoble, France, Influenza Binding Avidity Governed by Sterol-Dependent Ganglioside 3Universite´ Grenoble Alpes, Grenoble, France, 4Institute of Physics, Dynamics University of Tartu, Tartu, Estonia. Isabel Goronzy1, Robert Rawle2, Peter Kasson2, Steven Boxer1. Properties of biomembranes are modified in presence of additives such as 1Department of Chemistry, Stanford University, Stanford, CA, USA, cholesterol (chol) or proteins. Azobenzene-cholesterol (azo-chol) is a photoac- 2Department of Molecular Physiology and Biological Physics, University of tive variety of chol since its azo-headgroup exhibits a reversible change of the Virginia, Charlottesville, VA, USA. conformation upon illumination (365nm: trans to cis; 455nm: cis to trans). Influenza virions attach to host membranes by binding cell-surface glycans Quasielastic neutron scattering (QENS) was applied to resolve lipid dynamics containing sialic acid, initiating the viral life cycle and infecting cells. While within biomimetic membranes consisting of a 1-palmitoyl-2-oleoyl-sn-phos- the primary determinant of viral binding is thought to be the glycan chemical phatidyl-choline (POPC) matrix with embedded azo-chol in either of the two structure, single interactions between a viral hemagglutinin binding site and a conformational states. A pure POPC membrane and also a POPC membrane sialic acid are weak, so binding specificity is presumed to occur via multiva- with inactive chol were used as control systems. The results report on the lent interactions. Here, we show that target membrane composition can alter fast intramolecular motion of the protons in the POPC chains, a slow hop influenza binding while total receptor content is held constant. We hypothe- diffusion and an even slower mode both of the whole POPC molecule. The in- size that this effect occurs by changing the spatial distribution of target recep- tramolecular motion and the hop diffusion are modified by the presence of azo- tors. Viral binding was quantitated by monitoring individual viruses binding chol in the host membrane as compared with the control systems, but do not to synthetic supported lipid bilayers using fluorescence microscopy. Adding depend on the isomerization state. The slowest mode is though sensitive to cholesterol to target bilayers increased binding in a dose-dependent manner. the isomerization. The findings show the capacity of azo-chol for tuning the dy- Furthermore, membranes containing 5-cholesten-3-one, an oxidized variant namics and most probably also the structures of the host lipid membrane though of cholesterol, displayed significantly higher binding avidity compared to photoactivation. membranes supplemented with an equal amount of cholesterol. When sterol mole fractions were held constant, viral target receptor concentration and 384-Pos Board B149 binding exhibited a sigmoidal relationship, confirming the multivalent nature Anomalous Behavior in Lipid Bilayer Membranes of virus receptor binding. The cooperativity required for binding established Matthew R. Cheetham1, Helena L.E. Coker2, Mark I. Wallace1. the potential for local receptor concentration to regulate membrane binding 1Department of Chemistry, King’s College London, London, United affinity. To develop a physical model for sterol-induced changes in the lateral Kingdom, 2Physical and Theoretical Chemistry, University of Oxford, organization of GD1a receptors, we performed coarse grained molecular dy- Oxford, United Kingdom. namics simulations of target lipid bilayers. Irrespective of sterol content, In contrast to the diffusion of membrane components in artificial lipid bilayers, GD1a receptors showed strong tendencies to self-associate. The inclusion of diffusion in cells is both slower and non-ergodic. The physical origins of cholesterol in membranes increased pairwise receptor contacts, suggesting such anomalous diffusion are poorly understood, though cytoskeletal- that sterol addition stabilized target receptor clustering. This observation membrane interactions and nanoscopic heterogeneity in the membrane have supports the hypothesis that sterols can modulate membrane nanoscale orga- both been suggested to play an important role. There remains however a nization and thus alter viral binding avidity, illuminating a previously under- lack of controlled model systems on which to test these potential causes. appreciated mechanism by which target membrane context can influence Here we present a model system for reproducing this anomalous diffusion influenza infectivity. that utilizes supported lipid bilayers (SLBs) with varying degrees of excluded area fraction. By varying the concentration of PEG-lipids, we observe a dra- 388-Pos Board B153 matic and controllable switch in anomalous diffusion as we cross the estimated Secrets of the Enigmatic Lipid II Revealed by Molecular Dynamics Simu- percolation threshold. This provides a simple model system for investigating lations this phenomenon in lipid membranes. The behavior is well described by Syma Khalid1, Firdaus Samsudin1, Timothy S. Carpenter2, Sarah Witzke1. both theory and Monte-Carlo simulation of the anomalous crossover as a func- 1School of Chemistry, University of Southampton, UK, Southampton, United tion of obstacle density. Kingdom, 2Lawrence Livermore National Laboratory, Livermore, CA, USA. Lipid II is critical for the biosynthesis of peptidoglycan; the main component of 385-Pos Board B150 the bacterial cell wall. Lipid II is targeted by antibiotics such as the lantibiotics, All-Atom Molecular Dynamics Simulation of Stealth Liposomes which achieve their function by disrupting the biosynthesis of the cell wall. Seyed Hamid Tabari, Jeevapani Hettige, Mahmoud Moradi. Currently there is an the urgent need for development of novel antibiotics to University Of Arkansas, Fayetteville, AR, USA. counter the growing threat of pathogenic bacteria becoming resistant to Pegylation of liposomes has been widely used in the field of drug delivery to currently used antibiotics. To achieve this, it is imperative we gain a detailed increase the bloodstream circulation time of liposomes; however, simulation understanding of the molecules targeted by antibiotics. studies have focused on simple model of liposomes, including only one or Relatively little is known about the conformational dynamics of Lipid II, in two different kinds of lipids simulated by simplistic coarse-grained models particular about the unusually long tail. To this end, we present a molecular dy- that lack the atomic/chemical details. Implementing all-atom molecular dy- namics simulation study of the conformational dynamics of Lipid II within a namics (MD) simulations, stable structure of solvated stealth liposomes are detailed model of the Staphylococcus aureus cell membrane. We show that achieved. The liposomes are composed of different DOPC (dioleoylphosphati- Lipid II is able to adopt a range of conformations even within the packed lipidic dylcholine)/cholesterol/PEG2000-DSPE (polyethylene glycol 2000-distearoyl- environment of the membrane. Furthermore we present energetic analysis that phosphatidylethanolamine) molar ratio. Here we used coarse-graining/ reveals the free energy associated with removing Lipid II from the S. aureus backmapping method to speed up mixing of the stealth liposome molecules. membranes compared to other lipids. Thus, we provide unprecedented insights The MD simulations revealed that not only are geometric structure of lipo- into the conformational dynamics of Lipid II within a Gram-positive bacterial somes dominated by the cholesterol molecules, but also PEGylated lipids membrane. reduce leakage of contents from liposomes. 389-Pos Board B154 386-Pos Board B151 Evaluating Bilayer Mechanical Properties in Protein Reconstituted GUVs Lipid Diffusion in Membrane Junctions Measured by Single-Molecule Nestor Lopez Mora, Heather Findlay, Paula Booth. Tracking Chemistry, King’s College London, London, United Kingdom. Vivek Ramakrishna, Mark I. Wallace. Biological membranes are complex systems where membrane proteins are King’s College London, London, United Kingdom. surrounded by a bilayer composed of different types of lipids, with mem- The interface between lipid membranes is an important site for biochemical ac- branes from different organisms and organelles varying greatly in their tivity. Cellular signalling processes are often mediated by the diffusion of lipids composition and therefore physical properties. The mix of lipids in the mem- and other components in the membrane. We present a system comprising a sup- brane has additionally been shown to influence the activity, stability and ported lipid bilayer and a giant unilamellar vesicle as a model for membrane conformation of many integral membrane proteins. In order to investigate

BPJ 7720_7725 76a Sunday, February 12, 2017 the effect of incorporating protein on the mechanical properties of lipid 392-Pos Board B157 bilayers we have reconstituted the Escherichia coli transporter lactose Interdependence between Collective Thermal Fluctuations and Elastic and permease (LacY), a transmembrane protein representative of the major facil- Viscous Properties in Model Lipid Bilayers itator superfamily, into synthetic lipid vesicles. We observed directly the fluc- Michihiro Nagao1,2, Elizabeth G. Kelley1, Rana Ashkar3, tuation of the lipid bilayer in these protein reconstituted GUVs, with sizes in Robert Bradbury1,2, Paul D. Butler1,4. the order of micrometers, by phase contrast microscopy. This allowed the 1NIST Center for Neutron Research, National Institute of Standards and determination of the bending rigidity, which characterizes the ability of mem- Technology, Gaithersburg, MD, USA, 2Center for Exploration of Energy and branes to bend under low stress, by fluctuation analysis. Changes in the Matter, Indiana University, Bloomington, IN, USA, 3Biology and Soft Matter bending rigidity parameter allowed us to get better insights into the effect Division, Oak Ridge National Laboratory, Oak Ridge, TN, USA, of lipids and protein on the mechanical properties of GUVs in a quantitative 4Department of Chemical and Bimolecular Engineering, University of fashion. Delaware, Newark, DE, USA. Lipid membranes undergo an array of conformational and dynamic transitions, 390-Pos Board B155 ranging from individual lipid motions to undulations of micron-sized patches of Shear Stress Stimulated MSC Activities: Direct Changes of Membrane the membrane. However, the dynamics at intermediate length scales are largely Tension or Cytoskeletal Stress? unexplored due to experimental challenges in accessing the appropriate length Mohammad Mehdi Maneshi1, Frederick Sachs2, Susan Zonglu Hua1,2. and time scales. Over the past several years our group has used neutron spin 1 Mechanical and Aerospace Engineering, University at Buffalo, Buffalo, NY, echo spectroscopy (NSE) to provide unique insights into these elusive dy- 2 USA, Physiology & Biophysics, University at Buffalo, Buffalo, NY, USA. namics in model lipid bilayers, measuring collective bending and thickness 2þ Fluid shear stress induced membrane transport such as Ca influx and the fluctuations. These thermally induced collective membrane fluctuations are activation of ion channels has been widely reported. The shear stress can controlled by elastic and viscous properties of the membranes. It has long be mediated by a direct change in bilipid membrane tension and/or by a been known that the bending fluctuations are characterized by the bending change in cytoskeletal stress via binding proteins that link channels to actin, modulus, k, of the membranes and the motion is damped by the viscosity of but how the shear stress is coupled to ion channels is unclear. Using narrow solvent, h. By contrast, according to a recent theory proposed by Bingham, shear pulse stimuli generated by a pressure servo in a microfluidic chamber, Smye and Olmsted, the collective thickness fluctuations are characterized by we measured the changes of membrane tension and cytoskeletal protein stress the bilayer area compressibility modulus, KA, which is damped by the mem- simultaneously in astrocytes. The membrane tension was reported using mo- brane and solvent viscosities, m and h, respectively. Therefore, by measuring lecular motor probes (2-carboxy-2-cyanovinyl)-julolidine farnesyl ester these two collective membrane fluctuations the membrane’s elastic and viscous (FCVJ) and cytoskeletal tension reported by genetically encoded force probe parameters can be evaluated. Here we use this novel method to determine these actinin-cpst-FRET. Our results show that the changes of membrane tension characteristic parameters of lipid bilayers from neutron scattering data for a are highly localized and the gradient is relevant to the flow directions. A shear couple of simple saturated phosphatidylcholine bilayers. The estimated values 2 19 stress pulse (23 dyn/cm , 400 ms duration) caused a rapid increase in mem- are k ~10 J, KA ~ 0.3 to 0.4 N/m, and m ~ 10 nPa s m, which are all consis- brane tension at the front edge of the cell with respect to the flow and a tent with literature values. decrease in tension (compression) at the distal edge. The rise time was less than 30 ms, and the tension dropped to the initial state within ~30 ms post 393-Pos Board B158 stimulus, showing a typical elastic behavior. In contrast, the same shear pulse Hydration-Mediated Elastic Deformations in Biological Membranes generated profound and long-lasting tension in cytoskeletal cross-linking pro- Trivikram R. Molugu1, Soohyun K. Lee1, Xiaolin Xu2, Rami Musharrafieh1, teins a-actinin at the front edge of the cell, and the tension persisted for the K.J. Mallikarjunaiah1, Constantin Job1, Michael Brown1,2. entire experimental duration of 60 s. In situ Ca2þ imaging showed that the 1Department of Chemistry and Biochemistry, University of Arizona, Tucson, initial Ca2þ influx was strongly correlated with the region having high cyto- AZ, USA, 2Department of Physics, University of Arizona, Tucson, AZ, USA. skeletal tension, but weakly linked to the bilayer tension. The results suggest Lipid membranes are excellent examples of biological soft matter [1]. Many 2þ functions of biomembranes involve collective phenomena with motional time- the cytoskeletal tension plays primary role in shear stress activated Ca influx. This work was funded by NINDS. scales spanning several decades (10 12 s to s). For liquid-crystalline mem- branes atomistic interactions often explain bulk material properties in 391-Pos Board B156 relation to key biological functions. Solid-state 2H NMR spectroscopy provides Stress Propagation through Biological Lipid-Bilayers Revealed by Atom- such information by simultaneously probing structure and dynamics [2]. Here istic and Coarse-Grained Simulations we examine the effect of hydration on the liquid-crystalline properties of mem- Camilo Aponte-Santamaria1,2, Frauke Gr€ater1,2. branes using NMR relaxation methods. We performed 2H NMR longitudinal 1 QE Molecular Biomechanics, Heidelberg Institute for Theoretical Studies, (R1Z) and transverse quadrupolar-echo decay (R2 ) experiments on DMPC- 2 Heidelberg, Germany, Interdisciplinary Center for Scientific Computing, d54 bilayers, to study membrane-lipid dynamics. Plots of the R1Z rates versus 2 Heidelberg University, Heidelberg, Germany. squared segmental order parameters (SCD ) follow an empirical square-law Membrane tension has been shown to play various critical roles in cell showing the emergence of collective lipid dynamics [3]. Such a functional signaling. We here asked if and how pulses of local stress dynamically propa- behavior characterizes 3-D order-director fluctuations due to the onset of QE gate through membranes, and propose this mechanism as a novel way of membrane elasticity over mesoscopic dimensions [3]. The R2 rates also QE quickly propagating signals along the membrane. showed similar results. At high hydration there is an R2 enhancement of In both atomistic and coarse-grained MARTINI molecular dynamics simula- the functional square-law for the segments deeper in the bilayer. Additional tions of biological lipid-bilayers, we observed short stress pulses to very effi- contributions from slower dynamics involving water-mediated membrane ciently propagate laterally at a velocity of the order of km/s, in close deformation are evident over mesoscopic length scales on the order of bilayer agreement with the expected speed of sound. The temperature dependence of thickness. Such membrane deformations are also evident from bilayer struc- pulse propagation shows tendencies very comparable to analogous experiments tural parameters calculated using a statistical mean-torque model [4]. In addi- [1,2,3], with insightful differences between the atomistic and coarse-grained tion, the square-law confinement must be due to water penetration into the simulations. Remarkably, the propagation of the lateral stress was damped at hydrophobic interior of the bilayer. The slow dynamics at high hydration length scales in the ~100 nm range. Our data supports the notion of lateral stress must be a consequence of modulation of membrane elastic properties. The propagation through membranes as a potential ultrafast way of short-range QCPMG frequency dispersions provide quantitative viscoelastic properties of signal propagation in biology [4]. the liquid-crystalline membranes. Such studies on model membranes give in- [1] W. Schrader, et al. J. Phys. Chem. B. 106:6581-6586 (2002) sights into lipid rafts and membrane compositions relevant for biomembrane [2] S. Shrivastava S and MF Schneider. J. R. Soc. Interface 11:20140098 functions. [1] A. Leftin et al. (2014) BJ 107, 2274 (2014). [2] K.J. Mallikarjuniah et al. (2011) BJ 100, 98. [3] J. Kappler and R. Netz. EPL. 112: 19002 (2015) [3] T.R. Molugu et al. Chem. Phys.Lipids. (2016) [4] C. Aponte-Santamarı´a and F. Gr€ater. In preparation [4] A. Leftin et al. (2014) eMagRes 4, 199.

BPJ 7720_7725 Sunday, February 12, 2017 77a

Membrane Fusion and Non-Bilayer Structures pairs of small unilamellar vesicles have been shown using lipidated DNA [Chan et al., Biointerphases, 3:FA17, 2008] and peptide [P€ahler et al., Biophys- 394-Pos Board B159 ical Journal, 103:2295, 2012] ligand-receptor pairs. Here, we show strong ev- Interactions of Carbon Nanotubes Stabilized by Selected Gemini idence of the fusion of large unilamellar vesicles (LUVs) to a spatially confined Surfactants with Model Biomembranes region within liquid-liquid phase-separated giant unilamellar vesicles using the Michalina Skupin1, Justyna Izykowska_ 1, Weronika Andrzejewska1, abovementioned (i) hybridised DNA and (ii) coiled-coil peptide pairs as Maria Dobies1,2, Stefan Jurga1,2, Maciej Kozak1,3. SNARE mimetics. We quantified the (hemi-)fusion process using two different 1Department of Macromolecular Physics, Adam Mickiewicz University, FRET-based approaches which yielded similar results. Moreover, our system Poznan, Poland, 2NanoBioMedical Center, Adam Mickiewicz University, potentially offers to determine LUV docking and fusion efficiencies. Comple- Poznan, Poland, 3Joint Laboratory for SAXS studies, Adam Mickiewicz mentary experiments are currently performed using reconstituted SNARE pro- University, Poznan, Poland. teins (SNARE proteins were kindly provided by Claudia Steinem, Department Thanks to the extraordinary mechanical strength and high electrical conductiv- of Biomolecular Chemistry at Go¨ttingen University). This work is part of the ity multiwalled carbon nanotubes are currently used in electronics, medicine (as MaxSynBio consortium which is jointly funded by the Federal Ministry of biomedical sensors, transporters or drugs) as well as in the production of light- Education and Research of Germany and the Max Planck Society. weight and durable construction. The aim of this study was to determine the 397-Pos Board B162 possibility to use different cationic gemini surfactants with different spacer The Influence of Nanoparticles on SNARE-Mediated Membrane Fusion lengths or alkyl chain lengths in more efficient systems for dispersing nano- Michael J. Crowe, Jiajie Diao. structures in aqueous solutions. The most important advantages of these sys- Cancer Biology Department, University of Cincinnati, Cincinnati, OH, USA. tems are their non-immunogenic, biocompatible properties and generally low Due to their unique electrical, optical, and thermal properties, nanoparticles toxicity . Therefore nanotubes, surrounded by surfactants, have the potential have attracted significant research interest for biomedical applications [1-3]. to interact with biological membranes. For this purpose we studied the influ- A recent study showed that nanoparticles can affect synaptic transmission by ence of dispersed CN solution on the phase behavior of 1,2-dimyristoyl-sn- disrupting lipid composition and Ca2þ homeostasis [4]. To further elucidate glycero-3-phosphocholine (DMPC) - a phospholipid most often present in the effects of nanoparticles on the molecular mechanism of synaptic transmis- membranes of nerve cells. The microstructure of the stable suspension of sion, we utilized in vitro fusion assays based on proteoliposomes reconstituted carbon nanotubes was investigated using high-resolution Transmission Elec- with SNAREs. The v- and t-SNARE proteins are reconstituted in two indepen- tron Microscopy and Atomic Force Microscopy. Fourier transform infrared dent populations of liposomes that are labeled with acceptor (DiD) and donor (FTIR) spectroscopy and differential scanning calorimetry (DSC) were used (DiI) fluorophores. Upon fusion, lipid molecule mixing results in FRET be- to analyze the influence of surfactants studied, used for CN dispersion, on tween donor and acceptor molecules [5]. the phase behavior of DMPC bilayers. A series of measurements of toxicity 1. Diao, J.J. and Chen, G.D. J. Phys. D: Appl. Phys. 34, L79 (2001). of these systems were performed in HeLa and fibroblast cell cultures. This 2. Diao, J.J. and Chen, H. J. Chem. Phys. 124, 116103 (2006). work was supported by the Ministry of Science and Higher Education, within 3. Chen, Y. et al. J. Phys. Conf. Ser. 59, 548 (2007). the project ‘‘Najlepsi z najlepszych!’’ dec. DIR.5210.352016/1. 4. Bramini, M. et al. ACS Nano 10, 7154 (2016). 5. Brunger, A.T. et al. Crit. Rev. Biochem. Mol. Biol. 50, 231 (2015) 395-Pos Board B160 The Bilat: A Free Standing Lipid Bilayer Microarray Platform for 398-Pos Board B163 Membrane Fusion Calcium Sensitive Ring-Like Oligomers of Synaptotagmin 1: Implications Sathish K. Ramakrishnan1,2, Andrea Gohlke2, Paul Heo2, James Rothman1, for Neurotransmitter Release Frederic Pincet1,2. Shyam Krishnakumar. 1 Department of Cell Biology, Yale University, Stamford, CT, USA, Dept of Cell Biology, Yale University, West Haven, CT, USA. 2 Laboratoire de Physique Statistique, E´ cole normale supe´rieure, Paris, The synaptic vesicle protein, Synaptotagmin-1 (Syt1) is required to couple France. Ca2þ influx to the membrane fusion machinery. However, the structural mech- Planar lipid bilayer membranes are crucial for studying many physiological anism underlying this process is unclear. Using negative stain electron micro- process such as membrane fusion, transport, interactions and ion channels. scopy, we find that the Syt1 assembles into ring-like oligomeric structures on Despite the continuing technological progress in planar lipid membranes, there lipid monolayers under physiological ionic strength and lipid composition in is a lack of system with high membrane stability, protein mobility and free from the absence of free Ca2þ. These rings oligomers vary in diameter between support. Most in vitro membrane fusion studies are still carried out either by 19 - 42 nm (with an average size of 30 5 5 nm), corresponding to 12- 25 mol- bulk fusion assays or on supported membranes, where protein mobility is hin- ecules of Syt1. The ring-like oligomers are sensitive to Ca2þ and are disrupted dered. Here, we developed a planar bilayer microarray platform with integrated rapidly by the physiological concentrations of free Ca2þ. Analogous ring-like transmembrane proteins that mimics in vivo like environment by having supe- oligomers assemble from the C2AB domains of other Syt isoforms (Syt2, rior protein mobility and free from support. We will present our system and Syt7, Syt9) as well as related C2 domain containing protein, Doc2B and show the significance of freely diffusing transmembrane proteins in artificial extended Synaptotagmins (E-Syts). Evidently, the circular oligomerization is membranes. Both sides of the lipid bilayers can be modified and controlled a general and conserved structural aspect of many C2 domain proteins, separately allowing this setup to be adopted for a wide range of research. We including Synaptotagmins. Further, we find that both Syt1 ring formation and demonstrate the ability of the system by mimicking synaptic vesicle fusion dy- its disruption by Ca2þ principally involve well-established functional surfaces namics and regulation at a single vesicle level in a millisecond timescale. Each of the C2B domain which are important for neurotransmission. This includes membrane fusion states such as hemifusion and full fusion were monitored the Ca2þ -independent binding of the polybasic motif of C2B domain with through lipid mixing and content release assays. In addition, we report a proto- phosphatidylinositol 4,5-bisphosphate (PIP2) as a prerequisite for rings to type of an automated data analysis software, vesicle fusion analyzer, for faster assemble. This suggests that ring formation may be triggered at a very early membrane fusion data analysis. We suggest that the newly developed free step in synaptic vesicle docking, as C2B-PIP2 interaction is required for dock- standing bilayer system associated with this software can be used as a general ing in vivo. Ca2þ binding to the C2B domain and re-orientation/insertion into platform to study behavior of membrane-related molecules. the membrane, both required for triggering synaptic transmission, disrupts the ring oligomers. We advance a simple and novel mechanism wherein Syt1 ring 396-Pos Board B161 oligomer act as reversible washer/spacer to synchronize neurotransmitter Phase Specific Membrane Fusion with SNARE Mimetics release to Ca2þ influx. Supporting this hypothesis, we find mutations in Syt1 Bastian Kubsch1, Tom Robinson1, Torben-Tobias Kliesch2, that specifically disrupt the ring oligomer formation results in dysregulation Andreas Janshoff2, Reinhard Lipowsky1, Rumiana Dimova1. neuroexocytosis in PC12 cells. 1Theory and Bio-Systems, Max Planck Institute of Colloids and Interfaces, Potsdam, Germany, 2Go¨ttingen University, Go¨ttingen, Germany. 399-Pos Board B164 Biological membrane fusion is involved in a number of essential processes such Towards Understanding the Molecular Mechanism of Synchronous as neurotransmission, exocytosis, and viral infection. It is crucial for cells to Neurotransmitter Release spatially confine this process to specific organelles or sites in the plasma mem- Qiangjun Zhou, Thomas Christian Sudhof,€ Axel Thomas Brunger. brane. In nature, the SNARE complex induces fusion in eukaryotic cells. Its Howard Hughes Medical Institute, Molecular and Cellular Physiology, different constituents integrated into opposing membranes approximate the Stanford University, Stanford, CA, USA. two bilayers by interacting in a zipper-like fashion. Different studies examined Most communication between neurons is achieved at synapses by the membrane fusion in artificial systems using SNARE mimetics. Fusion between process of neurotransmitter release. Neurotransmitter release is initiated

BPJ 7726_7730 78a Sunday, February 12, 2017 by depolarization of a neuron, which in turn activates voltage-gated Ca2þ 402-Pos Board B167 channels. The resulting Ca2þ influx then triggers the fusion of the synaptic Structure of the Ebola Virus Envelope Protein MPER/TM Domain and its vesicles with the plasma membrane. Synaptic vesicle fusion is mediated by Interaction with the Fusion Loop Explains their Fusion Activity a core fusion machinery SNARE complex, a small regulatory factor com- Jinwoo Lee1,2, David A. Nyenhuis1,3, Elizabeth A. Nelson1,4, plexin (Cpx), and Ca2þ sensor synaptotagmin (Syt). However, it was un- David S. Cafiso1,3, Judith M. White1,4, Lukas K. Tamm1,2. known how they cooperate to trigger synaptic vesicle fusion. Combining 1Center for Membrane and Cell Physiology, University of Virginia, X-ray crystallography and electrophysiological recording techniques, we Charlottesville, VA, USA, 2Departments of Molecular Physiology and determined two atomic resolution crystal structures of the synaptic vesicle Biological Physics, University of Virginia, Charlottesville, VA, USA, fusion machinery at different states, revealing a large, specific, Ca2þ-inde- 3Department of Chemistry, University of Virginia, Charlottesville, VA, USA, pendent interface which is essential for synchronous neurotransmitter 4Department of Cell Biology, University of Virginia, Charlottesville, release in mouse neuronal synapses. We propose a working model and VA, USA. further reveal the molecular mechanism of synchronous neurotransmitter Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hem- release. orrhagic fever, enters cells by macropinocytosis and releases its genetic mate- rial into the cytoplasm after membrane fusion in a late endosomal 400-Pos Board B165 compartment. Membrane fusion is governed by the EBOV surface envelope a-Synuclein: A Functional Role as a Regulator of SNARE-Mediated glycoprotein (GP), which consists of subunits GP1 and GP2. GP1 binds to Fusion cellular receptors including Niemann-Pick C1 (NPC1) protein and GP2 is Siobhan Toal, Elizabeth Rhoades. responsible for membrane fusion at low pH. GP1 undergoes multiple steps of Chemistry, University of Pennsylvania, Philadelphia, PA, USA. proteolytic cleavage and binds to NPC1 at endosomal pH. GP2 is rearranged Fusion of vesicular and plasma membranes is mediated by SNARE in a fashion that exposes the hydrophobic fusion loop (FL) of GP2, which is proteins. In a vesicular fusion event, the t- and v-SNAREs assemble into then inserted into the cellular target membrane, ultimately forming a six- a four-helix bundle pulling the two membranes together to cause fusion. helix bundle structure and resulting in the formation of the fusion pore. A decrease in neurotransmitter release upon overexpression of the Although major portions of the GP2 structure that have been solved in pre- neuronal protein a-Synuclein (aS)hasbeenobservedinanimalmodels,sug- and post-fusion states and the current model places the transmembrane (TM) gesting that aS may act as a regulator of neurotransmission, altering and FL domains of GP2 in close proximity to each other at critical steps of SNAREdrivenfusionofsynapticvesicles.Recentworkinourlabhas membrane fusion, their structures in membrane environments and especially shown that aS is able to inhibit SNARE-mediated fusion in vitro, although interactions between TM and FL have not yet been characterized. Here we pre- the mechanism appears to be through binding to the lipid bilayer, not sent the structure of the membrane proximal external region (MPER) connected through direct interactions with SNARE proteins. Here, we investigate the to the TM domain, i.e. the missing parts of the EBOV GP2 structure. The possibility that aS may also modulate fusion through interactions structure, solved by solution NMR and EPR spectroscopy in membrane- with SNARE regulatory proteins, synaptotagmin and complexin, using an mimetic environments, consists of a helix-turn-helix architecture that is inde- in vitro fusion assay. We find that in the presence of synaptotagmin and pendent of pH. Moreover, the MPER region, not TM region, is shown to complexin, aS differentially alters SNARE-mediated vesicle fusion in a interact in the membrane interface with the previously determined structure concentration dependent manner. At low aS concentrations, fusion is signif- of the EBOV FL through several critical aromatic residues. Mutation of icantly enhanced in a concentration-dependent manner (i.e. increasing aromatic and neighboring residues in both binding partners decreases fusion fusion with increasing aS). However, once a threshold concentration is and viral entry highlighting the functional importance of the MPER/TM - FL exceeded, fusion is again inhibited, again in a concentration dependent interaction in EBOV entry and fusion. manner(i.e.decreasingfusionwithincreasingaS). Direct evidence of protein-protein interactions was monitored using fluorescence correlation 403-Pos Board B168 spectroscopy to measure the diffusion times of the protein components. Leakage Induced by the Influenza Virus Haemagglutinin Depends on SNARE complex formation can be observed as a function of time Target Membrane Spontaneous Curvature through an increase in the diffusion time of labeled t-SNARE protein. Sourav Haldar, Elena Mekhedov, Jane Farrington, Petr Chlanda, While aS does not appear to impact the rate of complex formation alone, Paul S. Blank, Joshua Zimmerberg. substantial increases in the diffusion time of the SNARE complex in the Section on Integrative Biophysics, NICHD/NIH, Bethesda, MD, USA. presence of aS and complexin were observed, suggesting an interaction be- A recent cryo-electron microscopy investigation(Chlanda et al.(2016)Nat. tween the two. Taken together, our results suggest that aS may have a dual Microbiol. 1:16050) of hemifusion structures mediated by the influenza vi- role in SNARE-mediated membrane fusion, as a chaperone of SNARE reg- rus haemagglutinin posited that there exist two pathways for hemi-fusion: ulatory components as well as, at high enough concentrations, a fusion hemifusion-stalk and rupture-insertion. Depending on target membrane inhibitor. material properties, such as spontaneous curvature, one pathway will be favored over the other. A prediction of this hypothesis is that leakage of sol- 401-Pos Board B166 uble content will be greater through the rupture-insertion pathway. To test Mitochondrial Fusion Proteins: A Tale of Two Membranes this prediction, we have developed a giant unilamellar vesicle (GUV)-based Andrew D. Kehr, Marisa A. Rubio, Jenny Hinshaw. dye influx assay that provides a direct measure of leakage. Our results show NIDDK, National Institutes of Health, Bethesda, MD, USA. that leakage (influx of soluble dye in GUV) induced by influenza virus Dynamins are a class of GTPase enzymes responsible for the fusion, changes from ~ 80 % to ~ 40 % as the spontaneous curvature is changed fission, and vesiculation of cellular lipid membranes throughout the from 0.02 nm 1 to 0.30 nm 1, supporting the hypothesis that leakage cell. The dynamin-like proteins Optic Atrophy 1 (Opa1) and Mitofusin is modulated by membrane spontaneous curvature. Surprisingly, with (Mfn) 1 and 2 are responsible for the fusion of the mitochondrial some lipid compositions, leakage was sub-maximal, i.e. there was a variable inner and outer membranes, respectively. Mutations in any of these degree of GUV filling. This result raised the possibility of a transient target proteins can lead to neuropathies including blindness and Charcot-Marie- membrane damage induced by the influenza virus. It was also possible Tooth, a disease characterized by progressive loss of distal muscle that the complete fusion of a leaky virus to a GUV was responsible for tissue. Currently, little is known structurally or biochemically about any the filling of the GUV. To control for this possibility, we compared leakage of these proteins. We have developed a protocol for expressing and induced by commercially prepared virus (containing significant damaged purifying biologically relevant and biochemically active shortened viral membrane, as evidenced by entry of a cell impermeant nucleotide- isoforms (OpaGG and Mfn1GG) in sufficient quantity to begin crystallo- binding dye) with lab-grown virus (with apparently minimal damaged viral graphic studies. Both have comparable GTPase activity compared to full- membranes). length, unstimulated Dynamin 1 when assayed at room temperature and interestingly OpaGG exists as a tetramerwhenassayedbysizeexclusion 404-Pos Board B169 chromatography. In addition, the long, membrane-bound isoforms of Opa1 Viral Fusion Efficacy of Influenza Virus H3N2 Reassortment Combination and Mfn1 have been expressed and purified in large quantities. To date, to the Suppoered Lipid Layer we have shown full length Mfn1 can be incorporated into proteoliposomes Hunglun Hsu1, Jean Millet2, Deirdre Costello1, Gary Whittaker2, and in the presence of GTP forms dense tethers as seen by cryo-EM. Susan Daniel1. This tethering is reversible as shown by confocal microscopy. Currently 1Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, we are developing tethering assays for Opa1 and fusion assays for both USA, 2Department of Microbiology and Immunology, Cornell University, Opa1 and Mfn1. Ithaca, NY, USA.

BPJ 7726_7730 Sunday, February 12, 2017 79a

Virus pseudotyping is a useful and safe technique for studying entry of 407-Pos Board B172 emerging strains of influenza virus. However, few studies have compared Probing Induced Structural Changes in Biomimetic Bacterial Cell different reassortant combinations in pseudoparticle systems, or compared Membrane Interactions with Divalent Cations entry kinetics of native viruses and their pseudotyped analogs. Here, vesic- Allison Whited, Alexander Johs, John Katsaras, Robert Standaert, ular stomatitis virus (VSV)-based pseudovirions displaying distinct influ- Aaron Jubb. enza virus envelope proteins were tested for fusion activity. We produced Oak Ridge National Lab, Knxoville, TN, USA. VSV pseudotypes containing the prototypical X-31 (H3) HA, either alone Biological membranes, formed primarily by the self-assembly of complex or with strain-matched or mismatched N2 NAs. We performed single- mixtures of phospholipids, provide a structured scaffold for compartmentali- particle fusion assays using total internal reflection fluorescence microscopy zation and structural processes in living cells. The specific physical properties to compare hemifusion kinetics among these pairings. Results illustrate that of phospholipid species present in a given membrane play a key role in medi- matching pseudoparticles behaved very similarly to native virus. Pseudopar- ating these processes. Phosphatidylethanolamine (PE), a zwitterionic lipid ticles harboring mismatched HA-NA pairings fuse at significantly slower present in bacterial, yeast, and mammalian cell membranes, is exceptional. rates than native virus, and NA-lacking pseudoparticles exhibiting the In addition to undergoing the standard lipid polymorphic transition between slowest fusion rates. Relative viral membrane HA density of matching the gel and liquid-crystalline phase, it can also assume an unusual polymor- pseudoparticles was higher than in mismatching or NA-lacking pseudopar- phic state, the inverse hexagonal phase (HII). Divalent cations are among ticles. An equivalent trend of HA expression level on cell membranes of the factors that drive the formation of the HII phase, wherein the lipid mole- HA/NA co-transfected cells was observed and intracellular trafficking of cules form stacked tubular structures by burying the hydrophilic head groups HA was affected by NA co-expression. Overall, we show that specific and exposing the hydrophobic tails to the bulk solvent. Most biological mem- influenza HA-NA combinations can profoundly affect the critical role branes contain a lipid species capable of forming the HII state suggesting that played by HA during entry, which may factor into viral fitness and the emer- such lipid polymorphic structural states play an important role in structural gence of new pandemic influenza viruses. biological processes such as membrane fusion. In this study, the interactions between Mg2þ and biomimetic bacterial cell membranes composed of PE and 405-Pos Board B170 phosphatidylglycerol (PG) were probed using differential scanning calorim- SERINC5 Inhibits HIV Fusion through Inactivation of Env Glycoproteins etry (DSC), small-angle x-ray scattering (SAXS), and fluorescence spectros- and Interference with Productive Refolding of Env copy. The lipid phase transitions were examined at varying ratios of PE to Chetan Sood1, Mariana Marin1, Ajit Chande2, Alexa L. Mattheyses3, PG and upon exposure to physiologically relevant concentrations of Mg2þ. Khalid Salaita4, Massimo Pizzato5, Gregory Melikian1. An understanding of these basic interactions enhances our understanding of 1Pediatrics, Emory University, Atlanta, GA, USA, 2University of Trento, 3 membrane dynamics and how membrane-mediated structural changes may Trento, Italy, Cell biology, Emory University, Atlanta, GA, USA, occur in vivo. 4Chemistry, Emory University, Atlanta, GA, USA, 5University, Trento, Italy. The multispan membrane proteins, SERINC3 and SERINC5, have been 408-Pos Board B173 recently shown to incorporate into HIV-1 particles and compromise their Role of trans to cis Transition in Viral Fusion Pore Dilation ability to fuse with target cells – an effect that is antagonized by the viral Brett E. Alcott1, Zhenyong Wu2,3, Josie Bircher4, Erdem Karatekin2,3, accessary protein Nef. Env glycoproteins from different HIV-1 strains Ben O’ Shaughnessy5. exhibit variable levels of sensitivity to SERINC-mediated restriction. The 1Biochemistry and Molecular Biophysics, Columbia University, New York, mechanism by which SERINCs interfere with HIV-1 fusion remains un- NY, USA, 2Cellular and Molecular Physiology, Yale University, New Haven, clear. Here, we show by real-time single particle imaging that incorporation CT, USA, 3Nanobiology Institute, Yale University, West Haven, CT, USA, of SERINC5 into virions in the absence of Nef inhibits the formation of 4Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, small fusion pores between viruses and cells. This effect was not USA, 5Chemical Engineering, Columbia University, New York, NY, USA. related to the SERINC5’s ability to oligomerize in the membrane or target Fusion of viral and host membranes is a key step during infection by the virus to degradation in lysosomes. Strikingly, we found that SERINC5 membrane-enclosed viruses. The fusion pore plays a critical role, and must promotes spontaneous inactivation of sensitive, but not resistant Env glyco- dilate to release the viral genome. Previous studies of fusion mediated by influ- proteins, and enhances the exposure of the conserved gp41 domains by de- enza A hemagglutinin (HA) revealed ~2-5 nm pores that flickered before laying the HIV-1 fusion reaction. Super-resolution imaging revealed that dilating to >10 nm. The mechanisms are unknown. SERINC5 also interferes with the formation of Env clusters on mature vi- Here we studied HA-mediated fusion pore dynamics using a novel single-pore rions, a step that is thought to be required for efficient HIV-1 fusion. These assay, combined with computational simulations accessing extraordinarily long results show that SERINC5 restricts HIV-1 fusion at a step prior to small ms-s timescales. We measured pores between HA-expressing fibroblasts and pore formation by selectively inactivating sensitive Env glycoproteins and bilayer nanodiscs. From pore currents we infer pore size with millisecond interfering with the function of the remaining active Env, likely by prevent- time resolution. Unlike previous in vitro studies, use of nanodiscs limited the ing the formation of large Env clusters and slowing down Env refolding. membrane contact areas and maximum pore sizes, better mimicking the initial This work was partially supported by the NIH R01 grant GM054787 to phases of virus-endosome fusion. With wild type (WT) HA, fusion pores G.B.M. flickered about a mean pore size ~1 nm. By contrast, fusion pores formed by 406-Pos Board B171 GPI-anchored HA nucleated at half the WT rate and were significantly larger. SERINC Inhibits HIV-1 Env Induced Membrane Fusion and Slows Fusion We developed radically coarse-grained, explicit lipid molecular dynamics sim- Pore Enlargement ulations of the fusion pore reconstituted with post-fusion, trans HA hairpins. Ruben M. Markosyan1, Shan-Lu Liu2, Fred S. Cohen1. With WT HA, fusion pores were small, similar to experiment. Over time hair- 1Rush University Medical Center, Chicago, IL, USA, 2Department of pins gradually converted from trans to cis, but contrary to a common view, cis Molecular Microbiology and Immunology, University of Missouri School of hairpins accumulated on the ‘‘viral’’ membrane, not the pore waist, due to the Medicine, Columbia, MO, USA. low mobility HA transmembrane domains. With GPI-HA the anchoring lipids The SERINC family of proteins are integral membrane proteins that regulate were far more mobile and the trans-cis transition much accelerated. Once most the incorporation of serine into phospholipids, to create PS, and into sphingo- hairpins had converted to cis, because apposing membranes were released the lipids. It has recently been shown that two members of the family, SERINC3 fusion pore dilated significantly. and SERINC5, inhibit HIV infectivity. Using a cell-cell fusion system to Our results suggest pore dilation requires the trans-cis transition. We hypothe- determine the extent to which inhibition of infectivity is due to reduced size that this transition is accelerated in GPI-HA by the more mobile lipid an- fusion, we found that the presence of SERINC3 or SERINC5 in either effector chor, explaining the larger observed pores. or target cells slows the kinetics and reduces the extent of fusion induced by HIV-1 Env. These two incorporators of serine greatly retard fusion pore 409-Pos Board B174 enlargement, as determined by the rate of aqueous dye transfer once a pore The Influence of Membrane Composition on the Kinetics of Influenza forms. Nef is an auxiliary protein of HIV that is well-known to enhance Virus Fusion Measured using a Single Particle Approach HIV infectivity. The presence of SERINC5 and Nef in effector cells leads Guus van der Borg1, Scarlett Braddock1, Jelle S. Blijleven1, to the same extent of fusion induced by expression of Env alone, showing Antoine M. van Ooien2, Wouter H. Roos1. that Nef eliminates the reduction of fusion caused by SERINC. (R01 GM 1Molecular Biophysics, Zernike Institute, Groningen, Netherlands, 2School 101 539). of Chemistry, Wollongong, Australia.

BPJ 7726_7730 80a Sunday, February 12, 2017

In order to infect a host cell the influenza virus fuses its envelope with the host largely unknown why virions would prefer nanoscopic ordered lipid domains cell membrane. This fusion of the viral and cell membranes is mediated by the over uniformly fluid membrane regions. Here, we show that HIV does not viral surface protein hemagglutinin (HA) which both docks the virus and fuses enter cells from within ordered membrane regions, but rather at the bound- the membranes. Using Total Internal Reflection Fluorescence (TIRF) micro- aries between raft-like and non-raft-like regions of the plasma membrane. scopy and a planar, fluid bilayer it is possible to study the kinetics of Using cell-derived giant plasma membrane vesicles (GPMVs), which are hemifusion in single virus particles. We have used this technique to study phase-separated into large-scale liquid-ordered (Lo) and liquid-disordered the yield and kinetics of the fusion of influenza with multiple different bilayer (Ld) membrane domains, we demonstrate that the HIV receptor CD4 is sub- compositions. This allows us to better understand the physics behind HA stantially sequestered into Lo domains while the coreceptor CCR5 localizes mediated membrane fusion, and thus to better understand the process of influ- preferentially at Lo/Ld domain boundaries. Lo/Ld phase coexistence is not enza infection. required for HIV attachment, but the recognition of Lo/Ld boundaries is a prerequisite for successful fusion of the viral envelope with the cell mem- 410-Pos Board B175 brane. We propose that virions localized to membrane domain boundaries Revisit the Correlation between the Elastic Mechanics and Fusion of Lipid utilize their interfacial energy as an additional driving force for fusion and Membranes cell entry. This study provides surprising answers to the long-standing ques- Zih-An Fan, Kuan-Yu Tsang, Si-Han Chen, Yi-Fan Chen. tion about the roles of lipid rafts in cell entry of HIV and perhaps other en- Department of Chemical and Materials Engineering, National Central veloped viruses. University, Taoyuan County, Taiwan. Membrane fusion is a vital process in key cellular events. The fusion capability 413-Pos Board B178 of a membrane depends on its elastic properties and varies with its lipid compo- Single-Virus Observation of pH-Triggered Zika Fusion in the Absence of a sition. It is believed that as the composition varies, the consequent change in C0 Cellular Receptor (monolayer spontaneous curvature) is the major factor dictating fusion, owing Robert J. Rawle1, Elizabeth Webster2, Isabel Goronzy2, Steven Boxer2, 1 to the associated variation in GEs (elastic energies) of the fusion intermediates Peter Kasson . 1 (e.g. stalk). By exploring the correlations among fusion, C0 and Kcp (monolayer Molecular Physiology and Biological Physics, University of Virginia, bending modulus), we revisit this long-held belief and re-examine the fuso- Charlottesville, VA, USA, 2Chemistry, Stanford University, Stanford, genic contributions of some relevant factors. We observe that not only C0 CA, USA. but also Kcp variations affect fusion, with depression in Kcp leading to suppres- Zika virus is a membrane-enveloped flavivirus which has garnered interna- sion in fusion. Variations in GE and inter-membrane interactions cannot tional attention as an emerging pathogen with causal links to birth defects account for the Kcp-fusion correlation; fusion is suppressed even as the GEs and neurological sequelae following infection. Because Zika virus has only decrease with Kcp, indicating the presence of factor(s) with fusogenic impor- recently been the subject of intense scientific study, little is known about tance overtaking that of GE. Furthermore, analyses find that the C0 influence the entry of Zika virus into host cells. From limited studies and by drawing on fusion is effected via modulating GE of the pre-fusion planar membrane, parallels to closely-related flaviviruses, Zika virus is presumed to first bind to rather than stalk. The results support a recent proposition calling for a paradigm a (as yet unknown) receptor on the cell surface, and then become internalized shift from the conventional view of fusion and may reshape our understanding by endocytosis. At some point during the endocytic pathway, the viral E pro- to the roles of fusogenic proteins in regulating cellular fusion machineries. tein is triggered by an unknown factor or factors, initiating membrane fusion with the endosomal membrane. To identify the factor(s) which trigger Zika 411-Pos Board B176 virus fusion, as well as to study the subsequent fusion kinetics, we used syn- Hemagglutinin Palmitoylation Contributes to Membrane Curvature in thetic DNA-lipid conjugates to tether Zika virus to target model lipid mem- Influenza a Virus Assembly and Membrane Fusion branes in the absence of receptor, a strategy demonstrated earlier for Petr Chlanda1, Elena Mekhedov1, Hang Waters1, Alexander Sodt2, influenza virus (Rawle et al., 2016, Biophysical Journal). This enabled us Paul S. Blank1, Joshua Zimmerberg1. to screen triggering conditions for Zika virus fusion and to monitor the result- 1SIB DBTB NICHD, NIH, Bethesda, MD, USA, 2UMCP DBTB NICHD, ing single virus fusion kinetics by quantitative fluorescence microscopy. NIH, Bethesda, MD, USA. We demonstrate that low pH, mimicking that inside the endosome, is suffi- Three cysteine residues in the cytoplasmic tail of influenza virus glycoprotein cient to trigger Zika virus fusion (lipid mixing). We also present the pH- hemagglutinin (HA) are covalently modified by three fatty acids and highly dependence of the Zika virus fusion kinetics, as well as implications for a conserved among HA subtypes. The importance of these S-acylation post- fusion mechanism. translational modifications is highlighted by a strain-dependence to their role in virus replication either in assembly or in fusion, but the mechanisms by which 414-Pos Board B179 the modifications exerts any effect are unknown. We studied the effects of HA Molecular Atlas Imaging and Osteoclast Formation: Multiscale Study of acylation on influenza virus-like particle (VLP) morphology, glycoprotein Cell-Cell Fusion Mechanisms spacing, protein incorporation, HA induced curvature, and membrane fusion us- Jesse L. Silverberg1, Pei Ying Ng2, Roland Baron2, Peng Yin1. ing cryo-electron tomography (cET), VLP-cell and cell-cell fusion assays, and 1Wyss Institute, Harvard University, Boston, MA, USA, 2Harvard School of molecular dynamics. Acylation has a significant effect on VLP envelope curva- Dental Medicine, Harvard University, Boston, MA, USA. ture but is not a determinant of either VLP morphology or HA lateral spacing. Advanced strategies for synthetic biomaterial design is a critical application De-acylated mutant HA is correlated with a flatter envelope curvature of the of fundamental research in tissue-scale biomechanics. Top-down experi- released particles in the absence of the M1 layer compared to wild type HA. ments seek to understand emergent material properties from the organization The de-acylated mutant HA failed to incorporate an M1 layer within spherical of individual constituents. While illuminating, bottom-up approaches are a VLP consistent with altered HA-M1 interactions. In cell-cell fusion assays, fusion necessary compliment since they provide information hidden by the com- pore enlargement was not observed, regardless of which strain of influenza was plex interactions in many-component multi-scale tissues. Thus, reducing de-acylated (H2 (A/Japan/305/57), H3 (A/Aichi/2/68), H3 (A/Udorn/72)), sug- complexity and studying minimal interactions allows us to better anticipate gesting that the role of acylation in membrane fusion is viral strain independent. and predict phenomenology in biomaterials engineered with living cells. Fusion without pore enlargement could be partially rescued by the expression of As a concrete example, fracture resistance in bone arises from mineral M1 and M2 proteins. The spontaneous curvature of palmitate was calculated by turnover driven by osteoclast and osteoblast cells. Osteoclasts are particu- molecular dynamic simulations, and was found to be comparable to curvature larly interesting since these large multi-nuclear cells resorb bone matrix. values derived from VLP size distributions. Our studies indicate that HA acyla- While it is widely accepted that efficient bone resorption requires osteo- tion is important for both influenza virus assembly and membrane fusion by con- clasts to become multi-nucleated, the exact biological mechanisms initiating trolling membrane curvature and modifying HA’s interactions with M1. and driving cell-to-cell fusion remains poorly understood. In this work, we are using Molecular Atlas Platform imaging technology to study the 412-Pos Board B177 formation of osteoclast cells from the fusion of progenitor bone marrow HIV Entry: Receptors Cooperate with Membrane Domain Boundaries to macrophages. These experiments utilize multiscale imaging to study the form Entry Sites in Host Cells cytoskeleton of progenitor cells fusing to create multi-nuclear osteoclasts. Sung-Tae Yang, Volker Kiessling, Lukas K. Tamm. At the smallest scales, super-resolution imaging enabled by DNA-PAINT University of Virginia, Charlottesville, VA, USA. allows us to observe the mechanisms when two progenitor cells come It has been proposed that lipid rafts of host cell membranes play pivotal roles into physical contact and their membranes fuse. At the largest scales, we for cell entry of many enveloped viruses including HIV. However, it remains study osteoclast development over fields of view spanning several mm. By

BPJ 7726_7730 Sunday, February 12, 2017 81a imaging across 5 orders of magnitude in length, we are able to both find disordered regions. The usefulness of the method is illustrated by analyzing these rare fusion events, and study a heterogeneous population of osteoclasts the concentration effects on the membrane order for the bilayers consisting for a contextualized understanding of this important cell type. The insights of 1) ganglioside GM1 and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho- gained in this work explore fundamental features of cell fusion required line (POPC), and of 2) cholesterol, 1,2-dimyristoyl-sn-glycero-3-phospho- for development of successful next generation biomaterials that cater to choline (DMPC), POPC, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamin basic osteoclast biology requirements and ensure better replication of the (DMPE), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine in vivo environment. (POPE). 417-Pos Board B182 415-Pos Board B180 Software for Direct Comparison of Membrane Scattering Experiments Optimizing Excitation Polarization to Probe Fusion Pore Dynamics using Data to Molecular Dynamics Simulations TIRF Microscopy Yevhen Cherniavskyi1, Svetlana Baoukina1, Bryan W. Holland1,2, Kasey Hancock1,2, Joerg Nikolaus3, Erdem Karatekin3, David Baddeley4. Norbert Kucerka3,4, Peter Tieleman1. 1Yale University, New Haven, CT, USA, 2Truman State University, 1Biological Sciences, University of Calgary, Calgary, AB, Canada, Kirksville, MO, USA, 3School of Medicine, Cellular and Molecular 2Simulations Plus, Inc., Lancaster, CA, USA, 3Frank Laboratory of Neutron Physiology, Molecular Biophysics and Biochemistry, Yale University, West Physics, Joint Institute for Nuclear Research, Dubna, Russian Federation, Haven, CT, USA, 4Department of Cell Biology, School of Medicine, Yale 4Department of Physical Chemistry of Drugs, Comenius University, University, New Haven, CT, USA. Bratislava, Slovakia. During hormone or neurotransmitter release via exocytosis, fusion pores may Detailed information on lipid bilayers structure is very important for under- open and close (flicker) repeatedly before resealing (‘‘kiss & run’’ fusion) or standing the processes in cellular membranes. Nowadays X-ray and neutron dilating (full fusion) irreversibly. Pore dynamics regulate the amount and ki- scattering experiments are widely used as a source of lipid bilayers structural netics of cargo release, and determine the mode of recycling, but mechanisms parameters (area per lipid, thickness, etc.), but in contrast to scattering pat- that govern pore dynamics are not understood. This is in large part due to a terns from crystal structures, lipid membranes produce very broad peaks in lack of reconstituted assays with single-pore sensitivity and millisecond scattering intensity profiles due to their fluid nature. This makes the process time resolution. We recently described a polarized total internal reflection of retrieving structural details indirect and introduces approximations to (pTIRF) microscopy assay to monitor fusion of proteoliposomes to planar convert experimental structure factors to electron or neutron scattering pro- lipid bilayers supported on a soft polymer cushion with single molecule sensi- files. On the other hand, molecular dynamics simulations are able to provide tivity and ~15 ms temporal resolution. Fluorescently labeled small unilamellar direct sub-atomic level information on lipid bilayers structure, but can pro- vesicles, reconstituted with exocytotic/neuronal v-SNAREs (v-SUVs), fuse duce unreliable data because of inaccuracy of the force fields. Simulation to with a supported bilayer containing the cognate t-SNAREs (t-SBL). Each Experiment (SIMtoEXP) software is designed to facilitate direct, model free, fusion event is accompanied by changes in the total fluorescence intensity sur- comparison of MD simulations to experimentally measured structure factors rounding the fusion site, as the lipid-linked labels diffuse from the liposome for lipid bilayers. We present SIMtoEXP version 2.0 - an updated version of into the supported bilayer through the fusion pore. Analysis of the intensity the original software. The new version of this program is written in Cþþ changes, combined with a mathematical model, provides information on using the Qt GUI library, instead of the original C/Tcl implementation. pore dynamics (Stratton et al. Biophys. J. 2016). In principle, three factors New features, including a MD trajectory reading module, were added to can contribute to intensity changes upon fusion: 1) dequenching of fluoro- simplify the workflow for users. In future releases SIMtoEXP software phores, 2) evanescent field decay and 3) a change in the average orientation will support structure factor calculations from simulations with the coarse- of the fluorophore dipole moments with respect to the excitation polarization grained Martini force field. The Martini force field provides significant field, as the fluorophores move from the round liposome into the flat bilayer. computational speedup while retaining significant chemical detail of the Here, we systematically varied the polarization of the excitation field and lipids. This allows simulating large lipid-protein aggregates on longer time- quantified its contribution to intensity changes for different lipid-linked fluo- scales and investigate the structure and lateral organization of cell mem- rophores. Large increases facilitate detection of fusion events and quantifica- branes. Incorporation of Martini support in SIMtoEXP will allow direct tion of lipid release kinetics. comparison of coarse-grained simulations to experiments, which will benefit both Martini force-field parametrization and the interpretation of experi- Membrane Structure I mental results.

416-Pos Board B181 418-Pos Board B183 Analysis of Lipid Domains in Bilayer Simulations using Observables for Modeling Ethers with Molecular Dynamics: Updated CHARMM Force Lipid Packing Field Parameters for Ethers in Model Compounds and Lipid Membranes Soohyung Park, Wonpil Im. Alison M. Leonard. Biological Sciences, Lehigh University, Bethlehem, PA, USA. UMCP, Crofton, MD, USA. Lipid rafts are micro- or nano-sized dynamic domains of membranes en- Small ether molecules such as polyethylene glycol (PEG) have extensive riched in cholesterols, glycosphingolipids (and other saturated lipids), and industrial and medical applications. Additionally, phospholipids containing specific membrane proteins, which are involved in many important biolog- ether linkages make up 30% of the glycerophospholipids in the human ical processes such as membrane trafficking, signaling, protein sequestra- brain. Preliminary research suggests that the CHARMM All-Atom Additive tion, and so on. Though the existence of lipid rafts has been widely Force Field does not accurately recreate experimental results for bilayers accepted, the study of their dynamics (size and lifetime) is still challenging composed of ether lipids such as 1,2-di-O-hexadecyl-sn-glycero-3-phospho- due to the limitations in experimental techniques. In this context, computa- choline (DHPC). 100-ns simulations of pure DHPC bilayers do not repro- tional studies can be a useful approach that complements experiments and duce essential target data such as surface area per lipid and electron provides insight into the properties and dynamics of lipid rafts by moni- density profile, which describe the structural properties of the membrane. toring and analyzing membrane structures at the atomic resolution. So far, Additionally, the initial parameterization of ethers tested a limited popula- the analysis of lipid domains in computational studies has been mostly based tion of linear ethers and did not accurately reproduce potential energy scans on the order parameter and neighbor composition of lipids, which could be about the O-C-C-O dihedral (J. Chem. Theory Comput., 3(3):1120 - 1133). complicated in the analysis of multicomponent bilayers. To address this We have used the MP2 density and Dunning diffuse aug-cc-pVQZ basis issue, we propose a simple yet robust method for the analysis of lipid do- sets to compute the charge distributions of model ethers, including various mains in bilayer simulations using physically transparent observables for lengths of PEG. We have also used Hybrid Methods for Interaction Energies lipid packing, area and thickness. In our method, the ordered states of lipids (HM-IE) to compute potential energy scans about the O-C-C-O angle effec- are inferred by a hidden Markov model analysis. Then, the ordered state tively at the CCSD(T)/aug-cc-pVQZ level (J. Phys. Chem.A,108: 107 - map onto the Voronoi tessellation of lipids is analyzed using the Getis- 112). We found that the charge on ether oxygens is substantially more Ord local spatial autocorrelation statistics to obtain clusters of ordered lipids negative than the current CHARMM force field suggests. Adjusting this (lipid domains). The method is general and can be applied to various situa- charge allows more water to penetrate a DHPC bilayer, increasing the sur- tions in that 1) it does not require information other than the mechanical face area per lipid and improving the bilayer’s structural representation. properties of lipids to assign their ordered states, and 2) the spatial autocor- Additionally, our new dihedral parameters for the central O-C-C-O angle relation statistics allows a robust assignment of the clusters of ordered and more accurately reproduce the quantum potential energy scan and bring

BPJ 7726_7730 82a Sunday, February 12, 2017 the free energies of solvation of model compounds closer to experimental 4INIFTA (CONICET-UNLP), La Plata, Argentina, 5CIPROVE values. Further research will involve evaluating the radius of gyration of (CONICET-UNLP), La Plata, Argentina. PEG in salt solutions to improve the interaction of ether oxygens with Sphingomyelins (SM) and phosphatidylcholines (PC) are major lipid classes ions in simulation (Colloid Jour., 72(2): 279 - 281). in the external plasma membrane leaflet of mammalian cells. A preferential interaction between SM and cholesterol (Cho) in both cell and model mem- 419-Pos Board B184 branes has been proposed as central for the formation of Cho- and SM-rich Simulation of Linoleoyl-Containing Pure Lipid Bilayer and Soybean domains in membranes. In this context, the relevance of the SM hydro- Plasma Membranes phobic moiety on its interaction with Cho for domain stabilization has Xiaohong Zhuang, Anna Ou, Jeffery B. Klauda. been investigated by our group (1-2). We report here on the effects of Chemical and Biomolecular Engineering, University of Maryland, College sphingomyelin structure on the orientational and conformational properties Park, MD, USA. of monolayers of pure lipids and of two ternary lipid mixtures (DOPC/ Molecular dynamics (MD) simulations have been extensively used to study 16:0SM/Cho and DOPC/24:1SM/Cho), which are relevant as mammalian lipid membranes in addition to experimental studies as they help better un- cell membrane models. We investigated interchain interactions, hydrogen derstand membranes in the atomic level. Computational models of bacterial bonding, conformational and structural properties using in situ polarization- (E. coli) membranes have been developed and applied to study the antimi- modulated infrared reflection absorption spectroscopy (PM-IRRAS). Our crobial peptide proteins. Plant membranes are less frequently studied results indicate that the particular properties conferred on sphingolipids compared to the bacterial membrane. In this work, we will present the soy- by unsaturation have profound implications on membrane organization. bean plasma membranes models. The compositions of cell plasma mem- Finally, we also explored the orientational and conformational changes in branes of soybean vary depending on the species, stage of development, lipid monolayers of DOPC/16:0SM/Cho 2:1:1 after the adsorption/insertion and the part of the plant. The two parts of the plant that we study are the of the active toxin HlyA and its unacylated nonhemolytic precursor ProHlyA, hypocotyl and the root. Each model consists of 100 lipids per leaflet, with so as to complement our knowledge on the action mechanism of both the composition based on the weighted and averaged values from past exper- proteins. imental studies. Specifically, the hypocotyl membrane contains 7 types of (1) Sabina Mate´, Jon V. Busto, Aritz B. Garcı´a-Arribas, Jesu´s Sot, Romina unsaturated phospholipids and two types of sterols, while the root membrane Vazquez, Vanesa Herlax, Claude Wolf, Laura Baka´s and Fe´lix M. Gon˜i contains 8 types of phospholipids and two types of sterols. All types of phos- (2014). ‘‘N-nervonoylsphingomyelin (C24:1) prevents lateral heterogeneity pholipids in soybean contains the 18:2 (cis D9, 12) linoleoyl tail which was in cholesterol-containing membranes’’. Biophys J. Jun 17;106(12):2606-16. not well studied before, therefore, the simulations on the pure 18:0/18:2 and (2) Sabina M. Mate´, Romina F. Va´zquez, Vanesa S. Herlax, Marı´a A. Daza 18:2/18:2 phosphocholine (PC) lipid bilayers are also performed. The struc- Millone, Marı´a L. Fanani, Bruno Maggio, Marı´a E. Vela, Laura S. Baka´s tural properties such as surface area per lipid, bilayer thicknesses, order pa- (2014). Boundary region between coexisting lipid phases as initial binding sites rameters, and spin-lattice relaxation time are analyzed for all membranes. for Escherichia coli alpha-hemolysin: A real-time study. Biochimica et Bio- Moreover, the analyses of the sterols tilt angle distributions, hydrogen physica Acta 1838 (2014) 1832-1841. bonding, and clustering are also conducted for the soybean membranes. The structural properties of pure bilayers agree well with NMR experimental 422-Pos Board B187 data validate the accuracy of 18:2 linoleoyl-containing lipids, based on Investigating Lipid Domain Formation in Asymmetric Large Unilamellar which the soybean membrane models also result in reasonable structural Vesicles using Fo¨rster Resonance Energy Transfer (FRET) properties. These results imply that the two soybean membrane models are Johnna R. St Clair, Qing Wang, Erwin London. realistic, and can facilitate the further study of soybean and other plant Biochemistry and Cell Biology, Stony Brook University, Stony Brook, membranes. NY, USA. Many living cell membranes display lipid asymmetry, with a distinct dif- 420-Pos Board B185 ference between the phospholipid and sphingolipid compositions of the Lateral Heterogeneity of Cholesterol on Binary Lipid Mixtures of inner and outer leaflets of the lipid bilayer. We recently developed improved POPC/Chol Imaged with AFM methods to prepare asymmetric lipid vesicles using using alpha- Arturo Galva´n-Herna´ndez1, Fernando Favela-Rosales2, cyclodextrins. These hexasaccharide rings can promote the exchange of Jorge Herna´ndez-Cobos1, Iva´n Ortega-Blake1. lipids between vesicles, but do not transport sterols. This allows the efficient 1Universidad Nacional Auto´noma de Me´xico, Cuernavaca, Mexico, 2Instituto and selective replacement of the outer leaflet lipids of lipid vesicles without Tecnolo´gico Superior Zacatecas Occidente, Sombrerete, Mexico. perturbing sterol content. Here we show, using several types of asymmetric Phase diagrams on ternary and quaternary lipid mixtures showing the vesicles, it is possible to investigate ordered domain formation in both the existence of liquid-ordered (lo), liquid-disordered (ld) and a mixed phase inner and outer leaflets of large unilamellar vesicles using FRET. To do (loþld) are now well established. However the existence of such a phase this, large unilamellar vesicles are prepared with FRET acceptor probes in diagram on binary mixtures is still in debate despite reports of a phase either only the inner or only the outer leaflet. FRET was used to study diagram on the mixtures POPC/Chol and POPC/Erg and nystatin activity cholesterol-containing vesicles in which sphingomyelin was the predominant co-related to this purported phase diagram; with a maximal activity ap- lipid in either the inner leaflet or the outer leaflet, and the opposite leaflet pearing in the mixed phase. Recent Molecular Dynamics simulations of was composed of unsaturated phosphatidylethanolamine and phosphatidyl- POPC/Chol at different temperatures and cholesterol concentrations along serine, or of unsaturated phosphatidylcholine. Preliminary results show this phase diagram, suggest the existence of cholesterol enriched nano- that in at least some of the lipid compositions studied ordered lipid domains sites in this mixed phase, indicating the presence of cholesterol lateral in one leaflet can induce ordered lipid domains in leaflets composed of lipids heterogeneity. Here we present atomic force microscopy images of sup- which do not normally form ordered domains by themselves. These results ported lipid bilayers made up of POPC and different cholesterol concentra- support the findings of our previous studies of domain formation and inter- tions along the phase diagram. We observe structures that are consistent leaflet coupling using microscopy, and show that the FRET approach can with the Molecular Dynamics predictions of order parameters and mem- be extended to study interleaflet coupling in membranes containing sub- brane thickness, supporting the idea of cholesterol lateral heterogeneity in microscopic domains. binary mixtures, which can enrich the discussion on the existence of lipid rafts and domains in biological membranes. Funding: DGAPA- 423-Pos Board B188 PAPIIT-RG100416. Lipid Mixing in Model Membranes Ruo-Xu Gu, Svetlana Baoukina, D. Peter Tieleman. 421-Pos Board B186 Centre for Molecular Simulation and Department of Biological Sciences, Orientational Properties of DOPC/SM/Cholesterol Mixtures: A University of Calgary, Calgary, AB, Canada. PM-IRRAS Study The plasma membrane constitutes the boundary between the cell and its Sabina M. Mate´1,2, Romina Vazquez1, Felippe J. Pavinatto3, environment. It is believed to have a highly organized lateral structure M. Antonieta Daza-Millone4, Vanesa Herlax1, Laura Bakas5, (the raft hypothesis). Due to the complex composition of plasma membranes, Osvaldo N. Oliveira Jr3, Marı´a E. Vela4. model lipid bilayers of simple composition are often used in experiments 1INIBIOLP-CONICET-UNLP, La Plata, Argentina, 2Comisio´n de and simulations to study the lateral organization. Here we conducted all- Investigaciones Cientı´ficas de la Pcia. de Buenos Aires, La Plata, Argentina, atom (AA) and coarse-grained (CG) simulations of bilayers contain- 3Instituto de Fı´sica de Sa˜o Carlos, Sa˜o Paulo, Sa˜o Carlos, SP, Brazil., Brazil, ing POPC, POPC:cholesterol, DPPC:DOPC, DPPC:DOPC:cholesterol and

BPJ 7726_7730 Sunday, February 12, 2017 83a

POPE-POPG at different temperatures to investigate bilayer properties and 426-Pos Board B191 mixing behaviour. We compare the AA and CG results. The POPC:choles- Functional and Structural Characterization of Pulmonary Surfactant terol bilayer formed a random mixture, with small ordered cholesterol Fractions Obtained from Bronchoalveolar Lavages sub-structures. DPPC:DOPC formed a homogeneous liquid phase at Jose´ Carlos Castillo-Sa´nchez, Alejandro Cerrada, Mikel Conde, 310 K, and showed notable lipid segregation at 290 K and 280 K. Choles- Je´sus Pe´rez-Gil, Antonio Cruz. terol enhanced lipid segregation in the DPPC:DOPC:cholesterol bilayers Dept. of Biochemistry, Faculty of Biology, and Research Institute Hospital 12 by displacing DOPC lipids from the local environment of DPPC. The de Octubre, Universidad Complutense, Madrid, Spain. cholesterol-cholesterol and cholesterol-phospholipid interactions in AA The alveolar surface of the lung is protected by a lipophilic material, the pulmo- and CG models were found to differ significantly. Lipid segregation in nary surfactant, mainly composed by lipids and a few specific proteins: SP-A, the CG simulations showed weak temperature dependence, due to partial SP-B, SP-C and SP-D. The main function of pulmonary surfactant is to reduce substitution of entropic components by enthalpic components intrinsic in the surface tension of the thin aqueous layer covering the alveolar epithelium, coarse-graining. In the bilayers containing charged lipids, repulsion be- avoiding the collapse of the smaller alveoli during the expiration. To achieve tween POPG lipids in the CG simulations was stronger than in AA simula- this function, surfactant lipids form an interfacial film at the air-liquid interface tions, and also differed between the standard and polarizable Martini water of this aqueous lining layer. Nevertheless, pulmonary surfactant is synthetized, models. packed and secreted by type II alveolar pneumocytes as tightly packed mem- branes assembled in specialized organelles, the lamellar bodies. Currently, the 424-Pos Board B189 molecular mechanisms that let pulmonary surfactant be transformed from the bi- Glycolipid Crosslinking is Required for Cholera Toxin to Partition into layers of lamellar bodies into the functional interfacial film remain unclear. This and Stabilize Ordered Domains is in part because most of the previous studies were made using surfactants ob- Krishnan Raghunathan1, Tiffany Wong1, Daniel J. Chinnapen2, tained from bronchoalveolar lavages, a complex mixture that likely combines Wayne I. Lencer3, Michael G. Jobling4, Anne K. Kenworthy1. freshly secreted surfactant and surfactant that has been already exposed to respi- 1Dept of Molecular Physiology and Biophysics, Vanderbilt University, ratory dynamics and can be at least partially deactivated. In this work, we have Nashville, TN, USA, 2Harvard Medical School and the Harvard Digestive characterized different fractions obtained by density gradient ultracentrifugation Diseases Center, Cambridge, MA, USA, 3Harvard Medical School and the of pulmonary surfactant purified from bronchoalveolar lavages of porcine lungs. Harvard Digestive Diseases Center, Cambridge, MA, USA, 4University of The analysis of these fractions by transmission electron microscopy showed a Colorado Anschutz Medical Campus, Aurora, CO, USA. heterogeneous mixture of membranous structures. With regard to composition, Current models of lipid rafts propose that lipid domains normally exist each fraction showed important differences in cholesterol and the content of as nanoscale compositional fluctuations at steady state in cells, but can surfactant proteins. All fractions presented a relatively good surface activity be stabilized to form functional entities. However, the mechanism when analyzed in the captive bubble surfactometer, except for some minority behind how stabilized rafts assemble and function remains unclear. fraction. These results indicate that surfactant from bronchoalveolar lavage is Here, we test the role of glycolipid crosslinking as a raft targeting and composed by a heterogeneous mixture of membranes differing in composition ordering mechanism using the well-studied raft marker cholera toxin and structure. We propose that some of these structures could be associated B pentamer (CTxB) as a model. We show that when bound to cell- with different stages of surfactant dynamics, including freshly secreted material, derived Giant Plasma Membrane Vesicles, a variant of CTxB containing interfacial structures and recycled membranes. only a single functional GM1 binding site exhibits significantly reduced 427-Pos Board B192 partitioning in the ordered phase compared to wild type CTxB with five Simulations Provide Insight into Improving the Tolerance of the E. coli binding sites. Moreover, monovalent CTxB does not increase membrane Membrane heterogeneity, unlike wild type CTxB. These results support the long-held Pouyan Khakbaz1, Jeffery Klauda2. hypothesis that CTxB stabilizes raft domains via a crosslinking mechanism 1Chemical and Biomolecular engineering, University of Maryland College and uncover an unexpected role for crosslinking in the targeting of CTxB to Park, College Park, MD, USA, 2Chemical and Biomolecular engineering, ordered domains. University of maryland college park, College Park, MD, USA. Damage of the cell membrane is identified as the principal mechanism for 425-Pos Board B190 toxicity. Partitioning of chemicals like ethanol in the membrane could change Model for Lipid Droplets within Endoplasmic Reticulum membrane properties and eventually destabilize lipid bilayers. Lipid composi- 1 2 Gonen Golani , Michael M. Kozlov . tion, specifically head group populations, affect membrane properties such as 1School Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel, 2 integrity, electrochemical potential, and intracellular pH. Here, a set of molecu- Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. lar dynamics (MD) simulations were carried out to investigate the effect of Endoplasmic Reticulum (ER) is a continuous network of membrane tubules ethanol on native and PssA mutant models for the inner membrane of E. coli. and sheets that plays a central role in the biogenesis of Lipid Droplets (LD). Properties such as surface area per lipid, order parameters, rigidity, and thick- The bulk of a LD is filled by hydrophobic molecules while its surface is nesses were calculated. The main differences between PssA and native models covered by a phospholipid monolayer. It has been suggested that LDs were the ratio of phosphoethanolamine (PE)/phosphatidylglycerol(PG) head form between two leaflets of an ER membrane as a result of accumulation groups and the concentration of the cyclic moiety in fatty acid chains. The of enzymatically synthesized triacylglycerols and sterol esters. Our work PssA model had more PE head groups and more unsaturated lipids, while native aims at analyzing the shape and stability of a tubular ER membrane contain- model had more cyclic moiety in lipid tails. Equilibrium bulk molar concentra- ing LDs and determination of conditions of LD detachment from the tions of ethanol with the PssA model were 0%, 0.62%, 1.84%, and 4.56% relative membrane. We consider the ER membrane as a thin elastic sheet, whose to water in the bulk. The molar concentration of ethanol in native system was bending energy is determined by Helfrich model. The tubular membrane 4.64%. For the pure water simulations, the PssA model resulted in a thicker is assumed to be subjected to a vanishing or small lateral tension, vanishing membrane, which may reduce ethanol permeation to cytoplasm of E. coli. For trans-membrane pressure difference and periodic boundary conditions. The simulations with ethanol, higher surface area and less order parameters were shape of an ER tubule is determined by a homogeneous spontaneous curva- observed as ethanol concentration increased in PssA and native models. MD re- ture of the membrane. The LD is modeled as a liquid drop with relatively sults also proved higher ethanol concentration will result in a reduction in rigidity high surface tension. The transition area between the LD and the ER of membranes with the native being less rigid at higher ethanol concentrations. membranes is modeled as a line with constant line tension. We used Less hydrophobic core thickness of native model compared to the PssA model the finite element numerical simulations (using Surface Evolver program) indicated likely increased permeation of ethanol into the cytoplasm. In overall, to compute the minimal energy configurations of the system. We found our MD simulation results support the hypothesis that PssA is more protective of the conditions under which the LD embedding into the membrane the cell with increased thickness and rigidity. is energetically more favorable than LD release in the cytosol depending on the LD size, the lateral tension of the tubular membrane, the line tension 428-Pos Board B193 of the tubule-LD contact line, the membrane bending and saddle splay Intermembrane Crosstalk in E. coli moduli. We showed that the membrane lateral tension and negative saddle Patrice Rassam, Colin Kleanthous. splay modulus favor, whereas the line tension disfavors the LD embedding Biochemistry, University of Oxford, Oxford, United Kingdom. into the membrane. We found that LD growth results in the droplet detach- We have previously described how promiscuous protein-protein interactions ment from the membrane. between outer membrane proteins (OMPs) cause them to self-associate into

BPJ 7726_7730 84a Sunday, February 12, 2017 large platforms, which we call OMP islands, and that these supramolecular GUV, along with Bodipy-PC fluorescence intensity added to the GUVs assemblies are the basis by which OMPs are turned over in bacterial popu- from the MLV donors. lations (Rassam et al., Nature, 2015; Kleanthous et al., Current Opinion in Structural Biology, 2015). Combining advanced microscopy methods with 431-Pos Board B196 new analytical tools, we have discovered that the organisation inherent in The L-Gamma Phase of Pulmonary Surfactant 1 1 1 1 the bacterial outer membrane is reflected in the inner membrane when the Kamlesh Kumar , Mariya Chavarha , Ryan W. Loney , Maayan P. Dagan , 2 3 1 two become connected by a protein bridge. Periplasmic protein bridges Thomas M. Weiss , Shankar B. Rananavare , Stephen B. Hall . 1Oregon Health & Science University, Portland, OR, USA, 2SLAC/SSRL, serve many important functions in Gram-negative bacteria, including 3 secreting lipopolysaccharides and virulence factors to the cell surface and Menlo Park, CA, USA, Portland State University, Portland, OR, USA. importing iron siderophores across the outer membrane. Current work is When compressed, alveolar films of pulmonary surfactant resist collapse from aimed at determing the biological consequences of this outer-inner mem- the air/water interface. This physiologically essential characteristic remains brane cross-talk. unexplained. Recent studies show that self-assembled films of pulmonary surfactant formed by adsorption are more resistant to collapse than spread 429-Pos Board B194 monolayers containing the same constituents. One candidate for the self- Low-Enthalpy Phase Transitions Yield Entropy-Driven Lateral Reorgani- assembled stable structure is the Lg phase. The unit cell of this structure zation and Phase Separation in Synthetic and Natural Multi-Component contains two lamellar bilayers. Half of the monolayers are ordered, and half DIB Membranes are disordered. Because ordered monolayers resist collapse, formation of the Graham Taylor1, Frederick A. Heberle2, Jason Seinfeld3, John Katsaras2, Lg phase with the correct orientation at the air/water interface could explain C. Patrick Collier4, Stephen A. Sarles3. the stability of the adsorbed film. Preparations related to pulmonary surfactant 1Joint Institute of Biological Sciences (JIBS), UTK-ORNL, Knoxville, TN, form the Lg phase over a poorly defined range of hydrations. The studies USA, 2Shull Wollan Center, Oak Ridge National Laboratory, Knoxville, TN, reported here used small angle and wide angle X-ray scattering to determine USA, 3Mechanical, Aerospace, and Biomedical Engineering, UTK, factors that affect formation of the Lg phase. Variables considered include tem- Knoxville, TN, USA, 4Center for Nanophase Materials Sciences, Oak Ridge perature, hydration, the influence of an interface, and important constituents of National Laboratory, Knoxville, TN, USA. pulmonary surfactant. The complete set of surfactant phospholipids formed the Temperature is an important parameter affecting biomembrane behavior, Lg phase on solid supports at relative humidities from 70-99%. The electron however, model membrane studies combining electrophysiology and active density profiles confirmed prior evidence suggesting that the two bilayers in temperature control are relatively scarce. Here, we report on the recently the Lg phase are equal and asymmetric, with each containing one ordered discovered ability to use controlled heating to form droplet interface bila- and one disordered leaflet. The anionic phospholipids were essential for these yers (DIBs) composed of lipids that exhibit phase transitions above ambient structures, which otherwise formed standard lamellar structures. Physiological temperatures. Using heating-assisted monolayer formation, we formed DIBs levels of cholesterol had minimal effect. In dispersed samples, Lg structures from single lipids or multi-component lipid mixtures, as well as complex were evident up to a water-content of ~35%. The structures continued to swell natural total lipid extracts (TLE) from E. coli and porcine brain tissues. with additional water. Because the lattice-constants for single bilayers at these DIB capacitance measurements detected thermotropic phase transitions in hydrations depends on their electrostatic composition, whether our samples TLEs and some phase-separating ternary systems that were not observed represented the Lg phase was uncertain. These results show that pulmonary with differential scanning calorimetry, pressure-perturbation calorimetry, surfactant can form the Lg phase approaching physiological conditions, or densitometry measurements. We also performed electrical measurements particularly at the solid/liquid interface. Whether this behavior extends to the of membrane protein insertion using the model pore-forming peptide air/water interface remains unknown. Alamethicin, to directly show how bilayer phase transitions are coupled to dramatic transitions in membrane protein behavior. Our results provide a General Protein-Lipid Interactions I new picture of low-enthalpy phase transitions in complex membranes where the ensemble average lipid area, length, and volume do not undergo abrupt 432-Pos Board B197 changes, but where lateral reorganization of lipids is driven largely by Lipid-Protein Interactions are Unique Fingerprints for Membrane entropic forces. We discuss how a DIB, which is sensitive to lipid order Proteins and packing as well as membrane tension, is especially well-suited to the Valentina Corradi1, Eduardo Mendez-Villuendas1, Helgi Ingolfsson2, study of such low-enthalpy transitions in complex synthetic and natural Siewert-Jan Marrink2, D. Peter Tieleman1. 1 model membranes. Biological Sciences, University of Calgary, Calgary, AB, Canada, 2University of Groningen, Groningen, Netherlands. Cell membranes function as physical barriers for the cell and control the ex- 430-Pos Board B195 change of ions, peptides, and small molecules between the interior and the exte- Utilizing Asymmetric GUVs to Inspect Plasma Membrane Phase Behavior rior of the cell. The main constituents of cell membranes are lipid molecules, and Binding of Polybasic Proteins whose hydrocarbon tails provide the barrier-like properties, and membrane pro- 1 2 1 Josephine Gonzales , Milka Doktorova , Gerald Feigenson . teins, which carry out specific functions. Complex lipid-protein interactions 1Molecular Cellular Biology and Genetics, Cornell University, Ithaca, NY, 2 take place in the membrane, where proteins and lipids affect each other, strictly USA, Physiology, Biophysics and Systems Biology, Weill Cornell Medical regulating a wide range of cellular tasks. Here, we use coarse grained (CG) mo- College and Field of Biophysics, New York City, NY, USA. lecular dynamics (MD) simulations to characterize the lipid environment of ten The lipid distribution is asymmetric across the plasma membrane (PM) membrane proteins, which include examples of receptors, transporters, chan- with cytosolic leaflet enriched in anionic lipids, and the outer leaflet nels, and enzymes. To provide a realistic lipid environment, the proteins are enriched in sphingomyelin and phosphatidylcholine and perhaps embedded in a model plasma membrane, where more than 60 lipid species containing ordered, functional domains, or lipid rafts. Our lab has previ- are represented, asymmetrically distributed between upper and lower leaflet ously analyzed cellular events by using symmetric giant unilamellar vesicles (JACS, 2014, 136, 14554-59). The simulations show in detail how each protein (GUVs) to model peripheral membrane protein binding to the modulates its local lipid environment in a unique way through local lipid cytosolic leaflet of the PM. As a way to inspect the dynamics and composition, thickness, curvature, lipid dynamics and other properties. domain behavior of an asymmetric PM, we make GUVs with asymmetric membranes. The goal of this project is to prepare asymmetric GUVs as a 433-Pos Board B198 model system to study plasma membrane phase behavior and binding of Lipid-Dependence of the Membrane Interactions of the Tim23 Channel polybasic proteins. For convenience in study of protein binding the GUV Subunit of the Mitochondrial Protein Import Machinery outer leaflet will have plasma membrane cytosolic composition of PE/PS/ Melissa K. Skoryk, Kevin J. Boyd, Eric R. May, Nathan N. Alder. cholesterol; the GUV inner leaflet will have plasma membrane exoplas- Molecular and Cell Biology, University of Connecticut, Storrs, CT, USA. mic composition SM/PC/cholesterol. We are using a-cyclodextrin to ex- The TIM23 complex of the mitochondrial inner membrane mediates the trans- change the initially symmetric GUV of SM/PC/cholesterol with location and integration of most nuclear-encoded polypeptides that reside multilammeller (MLVs) donor vesicles. Initial confocal microscopy imaging within the mitochondrion. The central subunit of this complex is Tim23, results show good exchange of the GUV outer leaflet, and almost complete a voltage-gated channel-forming protein that constitutes part of the removal of MLV donor vesicles. Currently, leaflet exchange is polypeptide-conducting channel. Tim23 has a bipartite domain organization estimated from the LR-DOPE fluorescence intensity remaining in each with a carboxy-terminal integral membrane domain and an amino-terminal

BPJ 7726_7730 Sunday, February 12, 2017 85a intrinsically disordered region that resides in the intermembrane space (IMS). endoplasmic reticulum. Therefore, coarse-grained molecular dynamics Work from our group and others has shown that cardiolipin, the signature phos- (CGMD) simulations were applied to study the interactions of 15LOX-2 pholipid of the mitochondrion, is required for the activity and subunit interac- with an endoplasmic reticulum membrane mimic. Our modeling data re- tions of the TIM23 complex, as well as for specific membrane interactions of vealed that phosphatidylethanolamine and phosphatidylinositol are the the Tim23 subunit. In this work we further address the role of cardiolipin in most likely membrane substrates for 15LOX-2. CGMD simulations (2 ms modulating membrane interactions of Tim23 from Saccharomyces cerevisiae. runs) confirmed that the amino-terminal b-barreldomain(calledPLAT)of Using a site-specific fluorescence mapping approach, we show that in organ- 15LOX-2 is responsible for membrane binding. This domain entered deeply ello, key sites in the Tim23 IMS domain partition into a nonpolar environment, into the membrane. To establish the positioning of the catalytic site’s consistent with past NMR experiments. Using a reductionist approach with opening and its orientation with respect to the membrane surface, we model membranes, we measure the cardiolipin-dependence of this interaction reconstructed an atomistic model based on the CGMD data. The opening as well as the depth of membrane penetration and how variations in cardiolipin of the J-shaped catalytic site invariably oriented itself toward the physiochemical properties affect Tim23 bilayer interactions. These results are membrane surface facilitating phospholipid hydroperoxidation. Accord- verified and extended using computational approaches. Based on molecular dy- ingly, we propose a model indicating that the PLAT domain strongly namics simulations, membrane binding is initiated by interaction of the Tim23 entangles 15-LOX in the membrane providing accessibility of the active amino terminus with the bilayer that is stabilized by electrostatic interactions site to esterified lipids embedded in the membrane. These data between lipid phosphate groups and the protein amino group. The presence have been supported by grants P01HL114453, U19AI068021 and HFSP- of cardiolipin promotes the insertion of key hydrophobic residues into the RGP0013/2014. nonpolar core, stabilizing the membrane bound state of Tim23. We attribute this phenomenon to packing defects in cardiolipin-containing bilayers that 436-Pos Board B201 expose acyl chains to the solvent. These results are discussed in light of heri- Investigation of acyl Protein Thioesterase Activity at the Membrane table disorders in cardiolipin biogenesis known to disrupt protein-lipid Kathrin Estel, Patricia Stege, Ingrid Vetter. interactions. Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Phyisiology, Dortmund, Germany, Dortmund, Germany. 434-Pos Board B199 As a member of the family of GTPases, Ras proteins officiate as Deciphering Membrane Protein Energetics using Deep Sequencing; molecular switches (i.e. cycling between a GDP-bound OFF and a Towards Robust Design and Structure Prediction of Membrane Proteins GTP-bound ON state) in many signal transduction cascades. Oncogenic mu- Assaf Elazar, Jonathan Weinstein, Sarel Fleishman. tations in Ras genes can lead to constitutively active proteins, which are Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel. found in 20-30% of all human tumors. Interfering with Ras localization at The energetics of membrane-protein interactions dictates protein topology the plasma membrane would disturb the oncogenic proliferation signal, and structure, which in turn determines the function and expression levels making Ras a potential anti cancer target. There are three human Ras of all plasma membrane proteins. However, systematic and reliable quanti- isoforms (H/N/K), which are all posttranslationally prenylated. H- and fication of membrane-protein energetics has been challenging. I recently N-Ras are additionally S-palmitoylated. Both modifications increase the developed a deep mutational scanning method, dsTbL(Elazar et al., hydrophobicity and therefore the membrane affinity of Ras. The reversibility 2016a) (deep-sequencing TOXCAT-b-lactamase), to monitor the effects of of palmitoylation allows the spatial regulation of Ras via a reaction- hundreds of point mutations on insertion and association within the bacterial diffusion mechanism called ‘‘acylation cycle’’. This is achieved by an ubiq- inner membrane. The assay quantifies insertion-energy profiles for each uitous depalmitoylation of Ras followed by a rapid diffusion to the golgi amino acid residue across the membrane, revealing that the hydrophobicity apparatus where it gets palmitoylated and transferred back to the plasma of biological membranes is significantly higher than appreciated. In addi- membrane via a vesicular transport. This cycle ensures the enrichment of tion, a key feature of the dsTbL profiles is that they show asymmetries Ras on the plasma membrane and prevents its mislocalization on other for Arg, Lys, and His, in agreement with the ‘positive-inside’ rule. The three cellular membranes. profiles, however, are not identical: whereas Lys and Arg are favored by 2 The depalmitoylation of Ras is catalyzed by the Acyl Protein Thioesterase kcal/mol near the cytoplasm compared to near the periplasm, the titratable (APT). Its function is to prevent mislocalization of H- and N-Ras to the endo- amino acid His shows a more modest asymmetry of 1 kcal/mol; membranes and it allows the acylation cycle to reestablish the physiological moreover, only Arg stabilizes the segment near the cytosol, whereas Lys plasma membrane localization. Inhibiton of APT inhibits this cycle and Ras and His are nearly neutral at the cytosol- membrane interface. Based on remains distributed on the endomembranes. The absence of Ras from the these findings, together with Jonathan Weinstein, we developed a novel plasma membrane leads to a weaker Ras mediated proliferation signal. APT graphical topology-prediction algorithm named TopGraph(Elazar et al., itself undergoes a dynamic palmitoylation for steady state membrane 2016b), based on a sequence search for minimum energy of insertion using localization. the dsTbL experimental insertion scale rather than statistics derived from The effect of membranes on APT and their targets remains still unclear. known structures. Unlike many existing predictors, TopGraph exhibits In the presented work, several biochemical methods were used to high accuracy even on large transporters with no structural homologues. investigate how the physiological membrane environment influences the Furthermore, results suggest that the ‘positive-inside’ rule, which is known APT activity. to orient segments with respect to the membrane, can also drive insertion of marginally hydrophobic segments in large membrane domains. These in- 437-Pos Board B202 sights may aid structure prediction, engineering, and design of membrane MG56, A Membrane Bound O-Acyltransferase Protein, Regulates Lipid proteins. Composition and Membrane Vesicle Size in Skeletal Muscle Matthew Sermersheim1, Arpad Somogyi2, Jordi Torrelles3, Miyuki Nishi4, 435-Pos Board B200 Hiroshi Takeshima4, Pei-Hui Lin1, Jianjie Ma1. Governing Mechanism of Phospholipids Peroxidation via 15-Lipoxyge- 1Surgery, The Ohio State University, Columbus, OH, USA, 2The Ohio State nase, A Key Player in Ferroptosis Cell Death Pathway University, Columbus, OH, USA, 3Microbial Infection and Immunology, The Dariush Mohammadyani1, Judith Klein-Seetharaman2, Valerian E. Kagan3. Ohio State University, Columbus, OH, USA, 4Kyoto University, Kyoto, 1Biophysics, Johns Hopkins University, Baltimore, MD, USA, 2University of Japan. Warwick, Coventry, United Kingdom, 3University of Pittsburgh, Pittsburgh, The human genome encodes 11 genes belonging to the membrane bound PA, USA. O-acyltransferase (MBOAT) family of proteins. MBOAT family proteins Ferroptosis is a newly discovered cell death pathway that is characterized are known for containing a large number of transmembrane domains and by the production of lipid hydroperoxides. Although the exact lipid peroxi- exhibiting acyltransferase enzymatic activity. While collectively these dation pathways have not been identified, recent findings indicate the proteins cover a broad-spectrum of acylation, substrates ranging from involvement of lipoxygenases (LOXs), particularly, 15LOX acting phospholipids to peptides, each member possesses specificity with regards directly —independently from phospholipases— on the membrane. Here, to the target of acylation and the lipid species utilized. Mitsugumin 56 a combination of computational approaches was used to study interactions (MG56), also known as hedgehog acyltransferase-like protein, is an of human 15LOX-2 with a membrane, and to identify preferred phospho- MBOAT protein predominantly expressed in cardiac and skeletal muscle. lipid substrates for this enzyme. Using molecular docking modeling, the MG56 resides along the terminal cisternea of the Sarcoplasmic Reticulum binding poses and the binding affinities of various esterified lipids and (SR) in striated muscle. Ablation of MG56 in mice results in seemingly arachidonic acid —a known substrate of LOXs—were compared. It has normal pups at birth, however severe growth retardation is observed around been shown that ferroptosis-triggering events occur predominantly in the day 7, shortly followed by death under starvation conditions. Skeletal

BPJ 7726_7730 86a Sunday, February 12, 2017 muscle from knockout mice displays swollen SR and dilated vacuoles within biotinylated protein to the binding of monovalent streptavidin, is advantageous sarcomeres, however this phenomenon is not seen in cardiac tissue. Lipid due to its ability to measure protein stability directly under native conditions analysis of skeletal muscle via matrix-assisted laser desorption/ionization without using denaturants. Interestingly, in mild detergents, the direct hydrogen fourier transform ion cyclotron resonance mass spectrometry (MALDI bond between His254 and Ser201 was moderately unfavorable, with the o FT-ICR) shows a larger abundance of phospholipids in knockout muscle interaction energy DDG Interzþ1.0 kcal/mol, while the water-mediated compared to wild type. Interestingly, knockout muscle also shows an hydrogen bond between Ser201 and Asn154 was moderately strong o increased abundance of high molecular mass phospholipids, indicative of (DDG Interz1.5 kcal/mol). His254 and Asn154, which are distantly located, o tri-acyl phospholipids. Tri-acylated phospholipids, such as N-acyl phospha- were weakly coupled (DDG Interz0 kcal/mol). Our result suggests that the tidylethanolamine (NAPE), have an additional acyl chain attached to the strength of polar interactions within the membrane are strongly context- phosphate group, and have been shown to alter membrane size and serve dependent and their burial in the membrane have been stringently selected as precursors to N-acyl ethanolamine (NAE), a hormone shown to be an for function, not stability, during evolution. appetite suppressant in rodents. It is our hypothesis that MG56 functions to negatively regulate NAPE production, thereby fostering normal muscle 440-Pos Board B205 development. We are working to identify the high molecular mass lipids Reconstitution of FtsH-Mediated Membrane Protein Degradation in seen in knockout skeletal muscle in hopes of determining the substrate of Bicelles 1 1 1 2 MG56. Yiqing Yang , Miyeon Kim , Ruiqiong Guo , Kristen Gaffney , Heedeok Hong1. 438-Pos Board B203 1Chemistry, Michigan State University, East Lansing, MI, USA, The Role of Packing Defects in the Stability and Function of the Intramem- 2Biochemistry, Michigan State University, East Lansing, MI, USA. brane Protease GlpG Controlled degradation of misassembled and dispensable proteins is a crucial Ruiqiong Guo1, Zixuan Cang2, Deans Erin3, Guowei Wei2,3, cellular process for maintaining the quality control of proteomes. While a ma- Heedeok Hong1,3. jority of studies have focused on degradation of water soluble proteins, it is 1Department of Chemistry, Michigan State University, East Lansing, MI, poorly understood how membrane proteins are degraded in cells. This discrep- USA, 2Department of Mathematics, Michigan State University, East Lansing, ancy is largely due to the difficulties in reconstituting the degradation process in MI, USA, 3Department of Biochemistry and Molecular Biology, Michigan vitro, which is necessary for biochemical and biophysical analysis of the mech- State University, East Lansing, MI, USA. anism. In this study, we successfully reconstituted ATP-dependent proteolysis Although proteins are efficiently packed, structural analysis has revealed of membrane proteins mediated by the membrane-integrated AAAþ protease that packing defects (i.e., voids, tunnels and pockets) are prevalent inside FtsH of E. coli. proteins despite their unfavorable contribution to protein stability. It has To achieve this, we established robust overexpression and purification proto- been speculated that those packing defects may be necessary for ligand cols of E. coli FtsH, and reconstituted its ATPase and protease activities in binding, transport or conformational changes required for function. Despite Triton X-100 micelles and DMPC/CHAPS bicelles. We successfully trans- the potential importance, the role of packing defects in the stability and formed a stable E. coli membrane protein, the intramembrane protease function of proteins is not clearly understood especially for membrane pro- GlpG, into an efficiently degraded substrate of FtsH by fusing the N-terminal teins. Here, using the rhomboid intramembrane protease GlpG as a model, or C-terminal degradation markers. This construct was specifically recog- we test the hypotheses that 1) improving the packing through cavity- nized by FtsH both in vivo and in vitro. We also developed a fluorescence- filling substitution can be a general way to stabilize a membrane protein, based high-throughput assay to monitor FtsH-mediated degradation of and 2) if packing defects are critical for function, it would be possible to membrane proteins in real time. These newly developed tools enabled the lock the protein conformation into either inactive or constitutively active Michaelis-Menten analysis of membrane protein turnover by FtsH. Our state through engineering of the cavities. We first identified packing defects experimental system may serve as a versatile platform to study the molecular in several rhomboid proteases from different origins by homology modeling, mechanisms of FtsH-mediated membrane protein degradation and elucidate and mapped conserved cavities onto the structure of GlpG. Then we de- the quantitative relationship between folding and degradation of membrane signed cavity-filling substitutions, and measured the stability and activity proteins. of those variants. Among the 18 ‘‘cavity-filling’’ substitutions tested, three o 441-Pos Board B206 were significantly destabilizing (DDG U > 1 kcal/mol), three were moder- o Impact of Plasma Protein Binding on Cargo Release by Thermosensitive ately stabilizing (DDG U =0.6~1.0 kcal/mol), and the rest retained the sta- bility to the level similar to wild-type. Among the three stabilizing Liposomes Studied by Fluorescence Correlation Spectroscopy 1 2,3 1 2 substitutions, one was fully active while the other two were inactive. Those Judith J. Mittag , Barbara Kneidl , Tobias Preiß , Martin Hossann , 3 4 4 € 1 two inactivating substitutions did not involve the active-site residues but Gerhard Winter , Stefan Wuttke , Hanna Engelke , Joachim O. Radler . 1Department of Physics and Center for Nanoscience, Ludwig Maximilian targeted the sites that belonged to the previously identified more flexible re- 2 gions in GlpG. This result suggests that the packing defects may be required University of Munich, Munich, Germany, Department of Internal Medicine III, University Hospital Munich, Ludwig Maximilian University of Munich, for the movement of structural elements of proteins, and implies that protein 3 packing may have evolved for function through the delicate balance be- Munich, Germany, Department of Pharmacy, Pharmaceutical Technology tween stability and flexibility. and Biopharmaceutics, Ludwig Maximilian University of Munich, Munich, Germany, 4Department of Chemistry and Center for NanoScience, Ludwig 439-Pos Board B204 Maximilian University of Munich, Munich, Germany. Dissecting the Side Chain Interaction Energies of the Active Site Hydrogen Thermosensitive liposomes (TSLs) whose phase-transition temperature (Tm) Bond Network in a Rhomboid Protease GlpG lies slightly above body temperature are promising candidates for induced Kristen A. Gaffney1, Jeff Cho1, Heedeok Hong2. drug release via local hyperthermia. Recent studies, however, have revealed 1Biochemistry & Molecular Biology, Michigan State University, East disruptive shifts in the transition temperature in mouse plasma, which are Lansing, MI, USA, 2Chemistry and Biochemistry & Molecular Biology, attributed to unknown interactions with blood proteins. Here, we study the Michigan State University, East Lansing, MI, USA. effects of four major plasma proteins – serum albumin, transferrin, apolipo- The intramembrane rhomboid proteases are of particular interest due to their protein A1 and fibrinogen – on the temperature-dependent release of fluores- ability to hydrolyze a substrate peptide bond at the active site which is buried cein di-b-D-galactopyranoside (FDG) from TSLs. The amount of fluorescein within the membrane. The high-resolution structure of the E. coli rhomboid released was quantified by fluorescence correlation spectroscopy (FCS) after protease GlpG revealed the catalytic dyad Ser201/His254, which is surrounded hydrolysis of FDG with b-galactosidase. This approach is more sensitive by a protein matrix and connected to bulk water via a narrow water channel. and thus superior to established release assays, as it is non-sensitive to Although multiple crystal structures have been solved for GlpG, its catalytic the confounding effects of Triton on conventional fluorescence measure- mechanism is not clearly understood. Because it is a serine protease, hydrogen ments. The assay determines the molar release ratio, i.e. the number of bonding interactions among active site residues are thought to play a critical molecules released per TSL. We show that shifts in the Tm of release do role in the catalytic cycle. In this study we dissected the interaction energies not correlate with protein affinities for the liposomes derived from adsorp- among the active site residues His254, Ser201 and Asn154, that form a tion isotherms. A remarkable shift in induced release towards lower temper- hydrogen bonding network in the active site of GlpG. To do this, we utilized atures in the presence of mouse plasma is confirmed. In contrast, exposure to the double-mutant cycle analysis combined with stability measurement by rat or human plasma, orfetal bovine serum (FBS), has no effect on the steric trapping. Steric trapping, which couples the unfolding of a doubly- release profile.

BPJ 7726_7730 Sunday, February 12, 2017 87a

442-Pos Board B207 isoforms E3, E2, E4, the latter two have been associated with increased A Mass-Spectrometry Based Methodology to Unravel the Molecular risk of cardiovascular and Alzheimer’s disease respectively, yet all Mechanisms of Sugar Transport three contain an identical C-terminal (CT) domain. The CT domain (resi- Chloe Martens, Antoni Borysik, Paula Booth, Argyris Politis. dues 201-299) is responsible for tetramerization and has been found to Chemistry, King’s College London, London, United Kingdom. initiate lipid binding that influences critical functional features of apoE. Our work aims at providing a molecular description of the transport mech- The N-terminal (NT) domain of apoE has four a-helices arranged in a anism(s) of sugar transporters from the Major Facilitator Superfamily (MFS) bundle similar to apolipophorin III (apoLp-III), a model insect apolipopro- using a novel combination of mass-spectrometry based methods and model- tein containing five a-helices. To better understand the role apolipoprotein ling. According to the widely acknowledged alternating-access model, MFS domains play in structure and function, a novel chimeric apolipoprotein transporters have to adopt at least two conformations open to opposite sides was designed by attaching apoE-CT to apoLp-III. Recombinant apoLp-III/ of the membrane to allow vectorial translocation of their substrate. How- apoE-CT, apoE3, apoE-CT, and apoLp-III were expressed in bacterial ever, a molecular description of the mechanism underlying such transition cells, purified by affinity chromatography, and purity verified by is not well established and the actual models fail to integrate the role of SDS-PAGE. Western blot analysis using monoclonal apoE-CT specific anti- the lipid environment. By combining reconstitution in different lipid envi- body confirmed the presence of apoE-CT in the chimera. Crosslinking ronments, mutagenesis and modelling, we plan to derive a detailed confor- studies using dimethylsuberimidate revealed that the apoLp-III/apoE-CT mational landscape of the transporters XylE and LacY in lipid bilayers. We chimera formed oligomers similar to apoE, while apoLp-III was monomeric. use native mass spectrometry to identify with high-resolution the binding of The chimera was highly a-helical, and incubation with 1-anilinona- lipid species, ion-mobility mass spectrometry to assess the global conforma- phthalene-8-sulfonic acid showed increased fluorescence intensity when tional changes and the variation in stability of the protein, and hydrogen- apoE-CT was added to apoLp-III, consistent with the presence of water- deuterium exchange mass spectrometry to follow the structural dynamics. shielded dye binding sites upon addition of a structured apoE-CT segment. Preliminary results show that the transporters have a signature pattern of Our results suggest that the chimeric protein has structural features similar bound lipids and that this pattern is affected by changes in the conforma- to the parent proteins, and that self-association properties of apoE can be tional equilibrium transferred to apoLp-III. Future studies will include measuring the ability of the chimera to solubilize phospholipid bilayers and binding to low density 443-Pos Board B208 lipoproteins. The chimeric approach offers the potential to obtain insight Vectorial Cholesterol Transport by STARD4 is Mediated by Specific PIP2 into domain interactions and the structure-function relationships in Membrane Composition apolipoproteins. Derek M. Shore1, David B. Iaea2, Radda Rusinova1, George Khelashvili1, Michel A. Cuendet1, Olaf S. Andersen1, Frederick R. Maxfield2, Membrane Receptors and Signal Transduction I Harel Weinstein1. 1 Department of Physiology and Biophysics, Weill Cornell Medicine, New 445-Pos Board B210 2 York, NY, USA, Department of Biochemistry, Weill Cornell Medicine, New Computational Model of Integrin Clustering in Response to Actin Turn- York, NY, USA. over STARD4 (steroidogenic acute regulatory protein-related lipid-transfer Tamara C. Bidone1, Aravind R. Rammohan2, Matt McKenzie2, domain containing 4) is known to transport cholesterol from the plasma Gregory A. Voth1. membrane (PM) to intracellular organelles, such as the endoplasmic reticu- 1University of Chicago, Chicago, IL, USA, 2Corning Inc, Elmira, NY, USA. lum and endocytic recycling compartment, but it has not been known if the Cell adhesion is a physical process that requires coordinated changes in cell transport is selective. Identification of a surface-exposed basic region en- morphology and interactions with the extracellular matrix (ECM). Coupling compassing the C-terminal helix in the crystal structure of STARD4 sug- between reorganization in cell morphology and contacts with the ECM is gested the possibility that anionic lipids confer membrane selectivity. To provided by dynamic actin filaments and integrin-mediated adhesions. How- test this hypothesis we carried out combined biochemical and computational ever, owing to the complexity and potential regulators of both actin fila- studies. Results of fluorescence-based sterol transport assays show that ments and adhesions, the interplay between actin dynamics and adhesions phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2) selectively increased assembly is not fully understood. Here, we developed a coarse-grained the rate of STARD4-mediated sterol removal from PM-like membranes, Brownian Dynamics computational model, which includes explicit semiflex- whereas phosphatidylinositol-(3,5)-bisphosphate (PI(3,5)P2)increasedthe ible actin filaments, coupled to particles diffusing on a quasi-2D rate of cholesterol release into membranes. From the analysis of extensive surface, representing integrins on the ventral surface of cells. In the model, unbiased atomistic molecular dynamic simulations (20 ms), we identified integrins can establish interactions with actin filaments and regulate their distinct STARD4/membrane binding modes regulated by associations with assembly and disassembly dynamics. Using this model, we determined different PIP2 lipids, and identified primary and secondary binding sites of the effect of tuning ligand affinity, patterns of actin (de)polymerization, STARD4 to the membranes. Evaluation of STARD4/membrane interaction and actin flow on integrin clustering and ligand binding. Our results provide trajectories with a Molecular Mechanics-Generalized Born surface area insights into the interplay between these factors in regulating adhesion initi- approach revealed differences in the binding geometry of STARD4 to mem- ation as well as coupled changes in the architecture of a dynamic actin branes containing PI(4,5)P2 compared to membranes containing PI(3,5)P2. network. Thus, the C-terminal helix adopts a smaller angle with the membrane normal in binding to membranes containing PI(4,5)P2, reflecting a differen- 446-Pos Board B211 tial presentation of the exposed basic region to the two membrane types. The M2 Muscarinic Receptor Signaling Complex Resolved by Single Both PI(4,5)P2 and PI(3,5)P2 strongly bind a region of basic residues on Molecule Tracking in Live Cells 1 2 3 3 the b1/b2sheetsthatservesasaprimaryPIP2 anchoring site, but PI(3,5) Yuchong Li , Rabindra V. Shivnaraine , Huiqiao Ji , Fei Huang , P lipids also interact strongly with the distal end of the C-terminal helix. Kevin Braeckmans4, James W. Wells3, Claudiu C. Gradinaru1. 2 1 Altogether, the results have identified the basis of selectivity in the compo- Department of Chemical and Physical Sciences, University of Toronto 2 sition of the two types of membranes, and the computational analysis has Mississauga, Mississauga, ON, Canada, Department of Molecular and revealed a specific structural context for STARD4’s experimentally Cellular Physiology and Medicine, Stanford University, Stanford, CA, 3 measured functional selectivity at membranes containing different PIP USA, Department of Pharmaceutical Sciences, University of Toronto, 2 4 subtypes. Toronto, ON, Canada, Department of Pharmacy, Ghent University, Ghent, Belgium. 444-Pos Board B209 Many aspects of the cellular signaling pathways via G protein-coupled recep- Chimera of Apolipophorin III and C-terminal Domain of Apolipoprotein E tors (GPCRs) are not completely understood. In particular, three questions to Study Apolipoprotein Structure Function have been the focus of much attention and debate: the oligomeric status of Paul M. Weers, Leesa M. Kakutani, James V. Horn, the receptor, the coupling strength between the receptor and the G protein, Vasanthy Narayanaswami. and the regulation of the receptors in response to external stimuli. Here we Chemistry and Biochemistry, California State University Long Beach, Long examine those questions by 2D tracking the M2 muscarinic receptors with To- Beach, CA, USA. tal Internal Reflection Fluorescence Microscopy (TIRFM) in the membrane of Human apolipoprotein E (apoE) is a two-domain anti-atherogenic lipid live cells. transport protein that mediates plasma cholesterol homeostasis by serving M2 Receptors and G proteins were genetically fused with fluorescent pro- as a ligand for the low density lipoprotein (LDL) receptor. Of its three teins (GFP and mCherry) and expressed in Chinese Hamster Ovary

BPJ 7726_7730 88a Sunday, February 12, 2017

(CHO) cells. The distributions of oligomeric sizes and diffusion coefficients Cannabinoid CB1 receptors are one of the most abundantly expressed G-pro- for both the receptors and the G proteins were extracted from single-particle tein coupled receptors (GPCRs) in the central nervous system and play critical tracking data obtained with TIRFM. Both of the receptors and G proteins roles in various neurophysiological processes such as reward, emotion and mo- were found to be localized in membrane micro-domains, showing multi- tor activity. Growing evidence supports that GPCRs form heteromers that cause step photobleaching and exhibited a wide range of diffusion behaviors on significant changes in their signaling. Utilizing electron microscope immuno- a slower time scale than single protein controls. Corroborated with dual- cytochemistry, CB1 receptors were found to co-localize with dopamine D2 re- color fluorescence correlation spectroscopy on the same samples, we pro- ceptors in the somata and dendrites of GABAergic neurons. Furthermore, pose that multiple oligomeric receptors and oligomeric G proteins sizes fluorescence resonance energy transfer and co-immunoprecipitation studies co-exist in close proximity inside a spatially confined signaling scaffold have provided evidence for heteromerization of CB1 and D2 receptors in het- in the membrane of living cells. In addition, two-color simultaneous erologous expression systems. CB1 receptors primarily signal through Pertussis tracking of co-expressed receptors and G proteins shows preliminary evi- toxin (PTX) sensitive Gi/o, leading to a decrease in cAMP levels. Interestingly, dence for receptor-G protein decoupling and receptor internalization upon stimulating the CB1 receptor in a cell line co-expressing CB1 and D2 receptors induction with excessive agonist. was shown to increase cAMP levels, suggesting a possible Gs signaling switch through the CB1-D2 heteromer. Our preliminary data using GIRK channels as 447-Pos Board B212 reporters of GPCR signaling in the Xenopus oocyte heterologous expression Quantitative Fluorescence Microscopy Reveals Higher Order Oligomeri- system corroborated the Gs switching behavior of CB1 receptors upon hetero- zation of FGFR5 merization with D2 receptors. To increase the signal to noise ratio of the CB1 1,2 1,2 1 Romario Regeenes , Pamuditha Silva , Dawn M. Kilkenny , receptor Gs signaling switch, we have turned to a calcium mobilization Jonathan V. Rocheleau1,2. 1 2 assay. Naturally, the promiscuous G alpha 16 belongs to the Gq superfamily IBBME, University of Toronto, Toronto, ON, Canada, Advanced and its activation leads to phospholipase C activation and calcium release. Diagnostics, Toronto General Research Institute, UHN, Toronto, ON, G alpha 16 chimeras in which 11 or 30 amino acids from the C-terminus Canada. were replaced by the corresponding sequence of Go and Gs were constructed. The fibroblast growth factor receptor (FGFR) family of tyrosine kinase re- Calcium mobilization was monitored by GCaMP 6s in a FlexStation. We found ceptors transactivate in response to ligand binding (e.g. FGF2) to initiate that activation of Gi-coupled D2 receptor causes increases in intracellular classical intracellular signaling cascades. Despite not having an intracellular calcium levels with G16/o chimeras but not with G16wt. Gs-coupled Glucagon kinase domain like canonical FGFR1, FGFR5 retains the ability to activate receptor signal to both G16/s chimeras and G16wt but G16/s chimeras signif- the MAPK signaling pathway, and particularly in the absence of ligand- icantly left-shift the dose-response curve of Glucagon, indicating G16/s receptor binding. Both receptors exhibit high affinity for FGF2, and are chimeras as good Gs signaling reporter. We are in the process of testing therefore likely to compete for ligand binding. To examine the extent of signaling of homomeric and heteromeric CB1 receptors through G16 chimeras competition between FGFR1 and FGFR5 for ligand in living cells, we aimed in this system. to use the complementary techniques of fluorescence-anisotropy based homo-Fo¨rster resonance energy transfer (homoFRET) and Number and 450-Pos Board B215 Brightness analysis (N&B). In this study, we expressed varied combinations Examining the Effects of Neurotrophin 3 and Nerve Growth Factor on the of dark, Venus-tagged, and Cerulean-tagged FGFR5 and/or FGFR1. Homo- Interaction of Tropomyosin Receptor Kinase A and C FRET imaging using the smaller Fo¨rster distance of Cerulean-tagged recep- Fozia Ahmed, Kalina Hristova. tor revealed that FGFR5 existed in pre-formed homodimers. However, the Materials Science and Engineering, Johns Hopkins University, Baltimore, larger Fo¨rster distance of Venus-tagged FGFR5 revealed anisotropy lower MD, USA. than the tandem Venus-Dimer control, suggesting it more loosely formed Tropomyosin receptor kinases (Trks) are known to initiate signaling cascades higher order oligomers. Furthermore, N&B imaging revealed that removal D in response to their ligands, the neurotrophins. Trks are involved in multiple of the receptor’s intracellular domain (R5 C) resulted in higher order ag- important key events responsible for the development of the mammalian ner- gregation, suggesting FGFR5 associates through an interaction in either vous system. They are known to control synaptic strength, plasticity, neuronal the transmembrane or extracellular domains. Co-expression of Cerulean- D survival, proliferation, migration, axonal growth/guidance and patterning, tagged FGFR5 and R5 C confirmed this potential alternate interaction injury protection, and neuronal apoptosis. Trks belong to the second largest domain. Both techniques suggest FGFR5 forms pre-formed heterodimers class of membrane proteins, known as receptor tyrosine kinases (RTKs). There with canonical FGFR1. Interestingly, the addition of FGF2 ligand resulted are three known members of the Trk family: Tropomyosin receptor kinase A, B in no change in FGFR5 aggregation state. These data suggest that FGFR5 and C. The signaling mechanism of Trks in response to neurotrophins is com- natively exists as a higher order oligomer with the ability to complex plex and the exact mechanism of their activation is still under study. We study with FGFR1. Collectively, these findings shed new light on the signaling the mechanism of activation of the Trk receptors in the presence and absence of mechanisms of FGFRs that can be used to develop novel therapeutics to different neurotrophins, and the effects of different neurotrophins on the dimer- treat a number of diseases. ization energetics of each Trk receptor, using quantitative Forster Resonance 448-Pos Board B213 Energy Transfer and Western Blotting techniques. Investigating the Hetero-Interations of Receptor Tyrosine Kinases in Live Cells 451-Pos Board B216 Michael Paul, Fozia Ahmed, Kalina Hristova. High Content Analysis of Intracellular Heterogeneity to Study GPCR Johns Hopkins University, Baltimore, MD, USA. Oligomerization Receptor tyrosine kinases (RTKs) are the second largest class of membrane re- Artu’ Breuer, Samuel Mcewen Walsh, Anna Mantsiou, Dimitrios Stamou. ceptors, and are one of the main players in transducing signals across the mem- University of Copenhagen, Copenhagen, Denmark. brane. They have an important role in development and regulating cell growth Heterogeneities are a fundamental feature of biology that enable organism and migration, and mutations of these receptors are associated with many can- resilience through flexible responses. We quantified diffraction limited intra- cers. Although in vitro studies have suggested that receptors from different sub- cellular heterogeneities of recombinant receptors in an in vitro cell culture families of RTKs (Ephs, VEGFRs, FGFRs, etc.) might interact with each other, using conventional fluorescence microscopy. We exploited intracellular little is known about whether this actually occurs in cells. We use a quantitative heterogeneities to quantitate the oligomerization of two prototypical G-pro- FRET spectroscopy method known as fully quantified spectral imaging (FSI) to tein coupled receptors (GPCRs): b1 adrenergic receptor (b1AR) and the Y2 identify association partners between subfamilies in live cells. receptor (Y2R). Our results suggest that intracellular heterogeneities can severely skew single cell, and many cell, average properties. 449-Pos Board B214 Decoding the Signaling through Homomeric and Heteromeric Cannabi- 452-Pos Board B217 noid CB1 Receptors Interactions between G Proteins and Non-Stimulated GPCRs Revealed by Guoqing Xiang1, Takeharu Kawano2, Apostolia Baki2, Two-Photon Polarization Microscopy Diomedes Logothetis2. Alexey Bondar1, Josef Lazar2. 1Physiology and Biophysics, Virginia Commonwealth University, 1Center for Nanobiology and Structural Biology, Institute of Microbiology, Richmond, VA, USA, 2Pharmaceutical Sciences, Northeastern University, Nove Hrady, Czech Republic, 2Institute of Organic Chemistry and Boston, MA, USA. Biochemistry, Prague, Czech Republic.

BPJ 7726_7730 Sunday, February 12, 2017 89a

G protein-coupled receptors (GPCRs) and G proteins are key players of 455-Pos Board B220 cellular signal transduction. They transduce signals from a multitude of Investigating Initial Events of IgE Receptor Signaling with Super- extracellular stimuli into the cells. Despite a number of studies, many as- Resolution Microscopy and Monte Carlo Simulations pects of molecular mechanisms and spatiotemporal dynamics of G protein Eshan Mitra1, James P. Sethna2, David Holowka1, Barbara Baird1. interactions with GPCRs remain unclear. In particular, it is uncertain 1Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA, whether G proteins can form complexes with inactive GPCRs, and what 2Physics, Cornell University, Ithaca, NY, USA. is the stability and functional significance of such complexes. The high affinity IgE receptor (FcεRI) in mast cells plays a central role in initi- In order to address these issues, we have investigated the interactions ating allergic responses, and also serves as a model system for immune receptor between the G proteins and various GPCRs using the technique of two- signaling. FcεRI binds IgE antibody, which confers specificity for antigen. The photon polarization microscopy (2PPM). 2PPM, developed in our labora- cross-linking of IgE-FcεRI by antigen stimulates a transmembrane signal that þ tory, allows sensitive monitoring of protein conformational changes and leads to downstream events including Ca2 mobilization and degranulation. protein-protein interactions, in living cells, in real time, using a single fluo- Our work seeks to understand the molecular basis of these initial signaling rescent protein tag. Our results demonstrate that 2PPM indeed allows events. observing interactions between G proteins and representative GPCRs, using To examine signal initiation experimentally, we performed PALM/STORM a single fluorescent protein tag. Our experiments reveal an important role of super-resolution imaging. We employed structurally defined ligands to gain receptor basal activity for GPCR - G protein interactions, yielding insights molecular-level control over the structure of the IgE cluster formed. Two triva- into receptor - G protein precoupling and other aspects of GPCR - G protein lent ligands studied, dsDNA based Y16 and Y46, differ in the spacing of recep- signal transduction. tor binding sites. With one-color STORM on live cells, we imaged the dynamic clustering of receptors upon stimulation. We found that Y16 and Y46 differ in 453-Pos Board B218 the density of receptors in clusters formed, as quantified by autocorrelation Investigating MUC1 Transmembrane Dimer Structure using Replica functions. With two-color PALM/STORM, we quantified the extent to which Exchange Molecular Dynamics the differing structures of these IgE clusters affect their capacity to recruit other Christina M. Freeman, Alexander J. Sodt. membrane components, including signaling partner Lyn kinase and markers for NICHD, National Institutes of Health, Bethesda, MD, USA. liquid ordered (Lo) membrane phase. The epithelial single-pass transmembrane glycoprotein Mucin 1 (MUC1) is a We used theoretical modeling to further address the potential role of membrane main constituent of mucus and has roles in cell signaling and differentiation. lipids in promoting the recruitment of a kinase to a cluster of receptors. We In addition, overexpression and subsequent homodimerization of the C-termi- modeled the membrane with a 2D Ising model, and calculated the change in nal subunit of MUC1 has been implicated in the progression of many cancers, free energy associated with recruiting a Lo-preferring kinase into a Lo- particularly breast cancer. preferring receptor cluster. Using this framework, we asked what structural fea- Recent experiments have shown that strong dimerization is dependent on the tures of a receptor cluster are most important for effective lipid-mediated formation of disulfide bonds between two cysteine residues in the juxta- signaling. Ongoing work uses an extension of the Ising model in order to membrane region, but weak dimers can also be formed without them. address other models of membrane phase behavior, including microemulsions. Certain mutations in the TMD can also partially disrupt the strength of the dimer. This suggests that protein-protein or protein-lipid interactions 456-Pos Board B221 are also mediating dimer formation. However, the transmembrane domain Fluorescence Fluctuation Spectroscopy of Dopaminergic Signaling in of MUC1-C doesn’t appear to contain common oligomerization motifs Pancreatic Beta Cells such as the Sm-X3-Sm sequence found in glycophorin A and many receptor Daniel J.P. Foust, Alessandro Ustione, David W. Piston. tyrosine kinases. Cell Biology and Physiology, Washington University School of Medicine, Understanding the physical mechanisms by which MUC1-C dimerizes within St. Louis, MO, USA. the surrounding plasma membrane environment will help in creating drug Dopamine inhibits insulin secretion from pancreatic beta cells via D2-like targets, as well as generating a more comprehensive model of protein-lipid in- dopamine receptors and we seek to understand the molecular details of this in- teractions. To investigate this, atomistic replica exchange molecular dynamics hibition using the tools of fluorescence fluctuation spectroscopy. Normal insu- simulations are being used to find stable conformations of wild-type and mutant lin secretion is critical to maintaining blood glucose homeostasis and the MUC1 TMDs. prevention of type II diabetes. Although glucose stimulated insulin secretion is well understood, there are hundreds of receptors expressed in beta cells, 454-Pos Board B219 including G-protein coupled dopamine receptors, which may provide addi- Electrostatics Facilitates the Trimer-of-Dimers Formation of the Chemo- tional mechanisms for regulating insulin secretion. Previously, we have shown receptor Signaling Domain that D2-like (D2, D3, D4) dopamine receptors confer beta cell sensitivity to Marharyta Petukh1, Davi Ortega2, Igor B. Zhulin1. 1 2 dopamine. Furthermore, we demonstrated that the D3 homolog is the receptor UTK/ORNL, Oak Ridge, TN, USA, The California Institute of Technology, primarily responsible for dopamine sensitivity, although the other receptors Pasadena, CA, USA. may compensate in a D3-knockout scenario. Electrophysiology has revealed Chemoreceptors are crucial components of the bacterial sensory system an inwardly-rectified current in dopamine-stimulated beta cells, implicating that modulates cellular motility. They detect changes in the environment ion channels as possible downstream signaling targets. G-protein coupled and transmit information to CheA histidine kinase, which ultimately inwardly rectifying Kþ (GIRK) channels are known effectors of D2-like recep- controls cellular flagellar motors. The prototypical Tsr chemoreceptor in tors, although transactivation of GIRK channels by dopamine receptors has not E. coli is a homodimer containing two principle functional modules: (i) a been demonstrated explicitly in beta cells. GIRK channels are activated by periplasmic ligand-binding domain and (ii) a cytoplasmic signaling domain tetrameric binding of Gbg subunits dissociated from the trimeric G-protein comprising an antiparallel, four-helix coiled-coil bundle. Receptor dimers complex. To study dopamine receptor interactions with downstream effectors are arranged into a trimer-of-dimers, which is a minimal physical unit we express fluorescently labeled proteins (receptors, G-proteins, channels) in essential for enhancing the CheA activity several hundredfold. Recent MIN6 cells. Cells are imaged with single- or two-photon excitation and these advances in cryo-electron tomography showed that trimers-of-dimers are images are analyzed by considering the intensity distribution (photon counting arranged into highly ordered hexagon arrays at the cell pole; however, the histogram) and correlation (image correlation spectroscopy). Using these ana- mechanism underlying the trimer-of-dimer and higher order array lyses we show that dopamine receptors co-cluster with G-proteins at discrete formation remains unknown. Current evidence from structural and bio- sites on the plasma membrane. At these cluster sites, the apparent brightness chemical studies suggest that trimers-of-dimers are maintained exclusively of labeled Gbg subunits increases with dopamine stimulus. Brightness in- by contacts at the chemoreceptor cytoplasmic tip. Here, using all-atom, creases indicate oligomerization, therefore these results support our hypothesis microsecond-range MD simulation of the Tsr trimer-of-dimers crystal struc- that dopamine receptor activation promotes the opening of GIRK channels via ture, we show that dimers within the trimer may interact throughout the tetrameric binding of dissociated Gbg subunits to channels. entire length of the signaling domain. While inter-dimer contacts at the chemoreceptor tip occur via hydrophobic interactions, complete 457-Pos Board B222 dimer ‘‘zipping’’ is facilitated by electrostatics, especially by the polar Dynamics of Various Phospholipase C-B Complexes solvation component. We also show that many of the residues involved in Ashima Singla, Suzanne Scarlata. establishing hydrogen bonds and salt bridges between dimers are evolu- Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, tionary conserved. MA, USA.

BPJ 7726_7730 90a Sunday, February 12, 2017

Phospholipase C-b (PLCb) is activated by G protein signals from different hor- Cellular Organization and Signalling, National Centre for Biological mones and neurotransmitters on the plasma membrane, to mediate increases in Sciences, Bengaluru, India. intracellular calcium. Surprisingly, we find that PLCb also localizes to the cyto- Cells regulate their membrane area and tension by finely tuned mechanisms. plasm where it plays an important role in RNA-induced gene silencing by in- Exo-endocytic processes, by addition or removal of membrane respectively, teracting with the component 3 promoter of the RNA-induced silencing play a major role in controlling physical properties of the membrane. complex (C3PO) as well as one of the key components of the RNA-induced However, the cellular processes and molecular mechanisms behind silencing machinery, Argonaute 2 (Ago2). We also find that PLCb distributes this regulation are poorly understood. Here we have explored the role of between its two binding partners, Gaq and C3PO/Ago2, to promote calcium multiple endocytic pathways in the maintenance of membrane homeostasis. signaling or RNA-induced silencing. For example, Using bimolecular fluores- We have used either a custom made cell stretching device, osmotic cence complementation, FLIM and FCS we find that the interaction of PLCb shocks or deadhering to modulate membrane tension and optical or with Ago2 and C3PO change with neuronal differentiation. We propose that magnetic tweezers to measure membrane tension. We find that a specific en- the ability of PLCb to shuttle between its two binding partners to carry out these docytic pathway, the dynamin-independent CLIC/GEEC (CG) endocytic two different functions plays a key role in the normal physiological processes pathway is transiently up regulated during change of membrane tension of cells. by stretch-induced relaxation or by multiple other means. By contrast, the extensively studied clathrin-mediated or caveolar endocytic pathways are 458-Pos Board B223 not modulated. The CG pathway is a high capacity pathway that rapidly Optical Control of cGMP Signaling endocytoses and recycles a major fraction of endocytosed contents within 1 1 2 3 Ulrike Scheib , Katja Stehfest , Christine E. Gee , Heinz G. Ko¨rschen , minutes of internalization. Modulating the CG pathway via perturbation Shatanik Mukherjee1, Thomas G. Oertner2, Peter Hegemann1. 1 of key regulatory genes correspondingly modifies membrane tension Experimental Biophysics, Humboldt-Universitaet zu Berlin, Berlin, and membrane retrieval after strain-induced relaxation. Using knock out Germany, 2Institute for Synaptic Physiology, University Medical Center 3 cell lines we further find that focal adhesion related molecules help in Hamburg-Eppendorf, Hamburg, Germany, Molecular Sensory Systems, sensing and transducing membrane tension to regulate CG pathway. These Center of Advanced European Studies and Research (caesar) Bonn, Bonn, features indicate that the CG pathway could be a vital component of the Germany. cellular machinery that senses and responds to regulate plasma membrane Zoospores of the aquatic fungi Blastocladiella emersonii are phototactic and a tension. rhodopsin-guanylyl cyclase (RhGC) was identified as the putative phototaxis receptor. Due to the direct linkage between the rhodopsin and the cyclase 461-Pos Board B226 domain, RhGC represents the first member of a novel class of enzyme linked Munc13 And Munc18 Cooperate to Properly Assemble SNAREs for Fast rhodopsins. In this work, we characterized RhGC biophysically. We show Neurotransmitter Release that green light maximally activates RhGC and cyclic GMP is produced. After Ying Lai. a short green laser flash, RhGC (D525) converts in 8 ms into a blue-shifted Molecular and Cellular Physiology, Stanford University, palo alto, CA, USA. signaling state P380 and recovers within 100 ms. RhGC expresses well in Neuronal SNAREs (synaptobrevin, syntaxin, SNAP-25), synaptotagmin, com- Xenopus oocytes, yeast, CHO cells, mammalian neurons and cyclic GMP plexin, Munc13, and Munc18 are essential for fast Ca2þ-triggered synaptic production was light dose-dependent, rapid and reproducible. Thus, RhGC is vesicle fusion. Syntaxin forms a closed complex with Munc18 that prevents a versatile tool for optogenetic analysis of cGMP-dependent signaling SNARE complex formation, and Munc13 catalyzes the opening and transit processes in cell biology and the neurosciences. of syntaxin into the SNARE complex. However, Munc13 knockout in mice cannot be fully rescued with a constitutively open mutant of syntaxin. We 459-Pos Board B224 report a new function of Munc13, independent of Munc18: it promotes the Photo Regulation of Small G-Protein Ras using Photochromic Peptide proper syntaxin/synaptobrevin subconfiguration during assembly of the ternary Masahiro Kuboyama, Kaori Masuhara, Shinsaku Maruta, Kazunori Kondo, SNARE complex. In cooperation with Munc18, Munc13 additionally ensures Kazuo Fujiwara. the proper syntaxin/SNAP-25 subconfiguration. In a reconstituted fusion assay Soka University, Hachioji, Japan. with SNAREs, complexin, and synaptotagmin, inclusion of both Munc13 and The small guanine nucleotide binding protein (G-protein), which are known as Munc18 quadruples the Ca2þ-triggered amplitude, and improves the Ca2þ- a molecular switch is central regulator of cellular signaling pathway. The sensitivity by an order of magnitude, reaching physiological sensitivity. If regulation mechanism of the protein is well studied at molecular level. Ras Munc13 or Munc18 is deleted, improperly assembled SNARE complexes is one of the G-protein which have essential role in signaling cascade reaction may occur, explaining the observed abrogation of neurotransmitter release in in cell is regulated by guanine nucleotide exchange factor (GEF) and GTPase neurons. activating protein (GAP). GTP bout Ras induced by GEF is active state and GDP bound Ras induced by GAP is inactive state. The conformational change 462-Pos Board B227 induced by GTP binding enable Ras to transduce a signal into downstream Effects of Opsonin Density and type on the Phagocytosis of Beads through direct interaction with its effectors. Also, mutant Ras known as con- Sebastian Hendrickx-Rodriguez1, Michael R. Falvo2, stant active state cause excess signaling transduction to downstream effectors, E. Timothy O’ Brien III,2, Richard Superfine2. which is pathogenesis of canceration, In this study, we focused on the inter- 1Physics, New Mexico Institute of Mining and Technology, Socorro, NM, action between Ras and its GEF, Son of sevenless (SOS) in order to control USA, 2Physics, UNC - Chapel Hill, Chapel Hill, NC, USA. Ras function using photochromic compounds. Previously it is shown that When a pathogen enters the body, the immune system releases small proteins the peptide mimicking the aH-helix, Ras binding region of SOS effectively that opsonize the foreign entity. This serves as a signal to macrophages to inhibits Ras function competing with native SOS. Therefore, it is expected engulf the pathogen, a process known as phagocytosis. Depending on the that photo-induced structural change of the peptide conjugate with photo- type of opsonin covering the invasive species, phagocytosis can occur in switch enable us to control Ras activity. We designed and synthesized SOS structurally different ways. My research aimed to examine how different aH-helix peptide which contains two cysteine residues in order to cross- opsonin types and densities affect phagocytosis both qualitatively and quan- link with bi-functional photochromic molecule of azobenzene derivative, titatively. Microspheres were used to replicate pathogens, and IgG antibody ABDM which change its structure reversibly by UV-VIS irradiation. The pep- alongside iC3b complement were the opsonins studied. First, a Dose- tide modified with ABDM exhibited secondary structural change reversibly Response curve was made to show it was possible to cover the bead with upon UV and visible light irradiation. Binding of ABDM-peptide to Ras different amounts of opsonin, i.e. change the density of antibody or comple- and SOS dependent nucleotide exchange reaction were also altered photo- ment on the bead surface. Then, the distinctly opsonized beads were fed reversibly. We also tried to examine the photo-reversible effect of ABDM- to cells and the effectiveness of phagocytosis was analyzed using two peptide for HeLa cell. different techniques: time-lapse movies and 3D image reconstruction. Through the former method, it was quantitatively found that more densely Exocytosis and Endocytosis I opsonized beads were more effectively phagocytosed, and that cells transi- tioned through phagocytosis quicker with IgG covered beads. This may sug- 460-Pos Board B225 gest that the actin dynamics and cytoskeleton remodeling occur faster Dynamin Independent Endocytic Pathway Operates in a Negative Feed- through the IgG signaling pathway than through that of iC3b. Progress back Loop to Sense and Regulate the Resting Membrane Tension was made through the 3D image reconstruction to better observe the phago- Joseph J. Thottacherry. cytic cup structure formed by the cell and to more efficiently note a

BPJ 7726_7730 Sunday, February 12, 2017 91a difference between iC3b and IgG consumption in a qualitative sense. Phago- such as PIP2, we show that the effective binding strength is enhanced by or- cytic studies can supply useful information for drug treatments involving ders of magnitude and becomes, surprisingly, independent of the protein- passage of the drug through the cell membrane to reach its activation site. protein binding strength. We quantify how this effect varies for proteins My research showed that opsonin density and type affect phagocytosis involved in later stages of vesicle trafficking and cell division in yeast. tremendously. Coupled with detailed spatially and structurally resolved simulations, we have further measured the effect of membrane recruitment on controlling 463-Pos Board B228 the speed of assembly, and influences of crowding and diffusion on this A Supported Tubulated Bilayer System Shows Effects of Synaptotagmin-7 process. on Membrane Curvature Peter Dahl1, Joseph Vasquez2, Hai Tran2, Jeff Knight2, Arun Anantharam1. 466-Pos Board B231 1University of Michigan, Ann Arbor, MI, USA, 2University of Colorado, Mechanoregulation of Clathrin-Mediated Endocytosis in Isolated Cells Denver, CO, USA. and Developing Tissues Fusion and fission of cellular membranes involve dramatic, precisely Comert Kural. regulated changes in membrane curvature mediated by a number The Ohio State University, Columbus, OH, USA. of proteins. The mechanisms by which proteins influence membrane curva- Clathrin-coated assemblies bear the largest fraction of the endocytic load ture are not well understood, and current methods for investigating curvature from the plasma membrane of eukaryotic cells. However, dynamics of changes using well-controlled systems are limited. We have developed a clathrin-mediated endocytosis (CME) have not been established within system based on supported lipid bilayers in which lipid tubules spontane- tissues of multicellular organisms due to experimental and analytical ously form in a manner that can be tuned by varying the ionic character bottlenecks in determining the lifespan of clathrin-coated structures. We and strength used during bilayer deposition and imaging. Using this found that clathrin coat growth rates obtained from fluorescence microscopy supported tubulated bilayer system, which we term ‘‘STuBS,’’ we have acquisitions can be utilized as reporters of CME dynamics. Growth rates investigated membrane-targeting C2 domains from synaptotagmin-7, a canbeassembledwithintimewindowsshorter than the average clathrin member of the synaptotagmin protein family that triggers exocytosis in coat lifetime and, thereby, allow probing the changes in CME neurons and neuroendocrine cells. We find that addition of dynamics in real time. Furthermore, this novel approach is applicable to purified synaptotagmin-7 C2AB domains, but not synaptotagmin-1 C2A, tissues as it is not prone to particle detection and tracking errors, which leads to a Ca2þ-dependent disappearance of tubules with concomitant result in underestimation of the clathrin coat lifetimes. Exploiting these ad- formation of vesicles. These studies demonstrate that synaptotagmin-7 can vantages, we detected spatial and temporal changes in CME dynamics alter membrane morphology by driving changes in membrane cur- within Drosophila amnioserosa tissues at different stages of embryo devel- vature. STuBS is a novel experimental system useful for monitoring solute- opment. We also found that increased membrane tension impedes CME and protein-mediated effects on membrane topology in aqueous media and through inhibition of formation and dissolution of clathrin-coated structures. in real time. Therefore, the parameters defining clathrin coat dynamics (i.e., lifetime, formation density and growth rates) can be utilized to monitor the spatio- 464-Pos Board B229 temporal gradients of the plasma membrane tension during cell migration Exosomes Fractionation by Biophysical Properties and spreading. Soheyl Tadjiki1, Robert Reed1, Samer Al-Hakami2, Mikhail Skliar3. 1Postnova Analytics, Salt Lake City, UT, USA, 2University of Utal, Salt Lake 467-Pos Board B232 City, UT, USA, 3University of Utah, Salt Lake City, UT, USA. Regulation of Lysosomal Exocytosis by Oxidative Stress and Calcium Ions Exosomes are small extracellular vesicles containing nucleic acid and Sreeram Ravi, Andrew P. VanDemark, Kirill Kiselyov. protein, which have shown a great potential for cancer diagnostics and University of Pittsburgh, Pittsburgh, PA, USA. therapeutic applications. Characterization of exosomes is challenging due Lysosomal exocytosis has emerged as an important mechanism of cellular to their inherent heterogeneity and complexity. A fractionation step is repair and clearance. Cellular reactions supported by lysosomal exocytosis necessary to provide narrow-sized fractions to enable a more accurate sub- include membrane repair and expulsion of various toxins. Lysosomal exocy- sequent size analysis. Asymmetrical Flow Field-Flow Fractionation tosis involves delivery of lysosomes to the plasma membrane followed by (AsFlFFF) is a high resolution elution technique for fractionation of macro- SNARE-dependent fusion. The processes of lysosomal delivery and fusion molecules and biological nanoparticles based on their hydrodynamic sizes. require calcium ions. How these processes are regulated is not completely In this study the AsFlFFF system was interfaced with Multi Angle Static understood, especially how they are modulated by pathological conditions. Light Scattering (MALS) detector to characterize the MCF-7 tumor exo- Our data show that lysosomal exocytosis is biphasically regulated by some sample. The analysis revealed a size distribution between 30-120 reactive oxygen species. Low levels of reactive oxygen species increase nm in diameter. Several narrow fractions were collected along the size dis- lysosomal exocytosis, while high levels of oxidative stress suppresses it. tribution and were analyzed by PCR. The study showed that the population Activation of lysosomal exocytosis by reactive oxygen species requires of tumor exosomes in circulation are heterogeneous in their cancer both the lysosomal ion channels TRPML1 and calcium entry across biomarker miR21. the plasma membrane. High levels of oxidative stress suppressed lysosomal acidification and significantly the ability of calcium to stimulate 465-Pos Board B230 lysosomal exocytosis. We propose that stimulation of lysosomal exocytosis Membrane Recruitment Enables Weak Binding Endocytic Proteins to by oxidative stress is a cytoprotective mechanism that limits lysosomal Form Stable Complexes permeabilization aos cells death under oxidative stress condition. Osman Yogurtcu, Margaret E. Johnson. Suppression of lysosomal exocytosis by oxidative stress is a pathologic Biophysics, Johns Hopkins University, Baltimore, MD, USA. mechanism probably driven by inhibition of lysosomal delivery to the Membrane targeting and assembly of proteins is required for vesicle traf- plasma membrane. ficking and receptor mediated signaling, but it is not known to what extent the proteins recruited to these events may have evolved to exploit the 2D 468-Pos Board B233 surface for assembly, versus pre-assembling in solution. We show that the Local Turgor Pressure Reduction via Channel Clustering phospholipid targeting proteins of clathrin-mediated endocytosis dramati- Jonah K. Scher-Zagier. cally enhance their effective binding strength and subsequent complex for- Physics, Washington University in St. Louis, St. Louis, MO, USA. mation to one another after surface recruitment in yeast and metazoans. For The primary drivers of yeast endocytosis are actin polymerization and proteins such as clathrin that do not directly bind lipids, the enhancement is curvature-generating proteins, such as clathrin and BAR domain proteins. still achieved by using three distinct binding sites to stabilize the clathrin to Previous work has indicated that these factors may not be capable of gener- peripheral membrane proteins on the surface. We derive simple formulas ating the forces necessary to overcome turgor pressure. Thus local reduction that quantify the degree of binding enhancement as a function of the protein of the turgor pressure, via localized accumulation or activation of solute and lipid concentrations, binding constants, and critically, the ratio of vol- channels, might facilitate endocytosis. The possible reduction in turgor pres- ume to membrane surface area. Our results thus apply to any cell type or sure is calculated numerically, by solving the diffusion equation through a geometries, including in vitro systems and the targeting of internal organ- Legendre polynomial expansion. We find that for a region of increased elles from the cytoplasm. With a sufficient concentration of lipid recruiters, permeability having radius 45 nm, as few as 60 channels with a spacing

BPJ 7726_7730 92a Sunday, February 12, 2017 of ten nanometers could locally decrease the turgor pressure by 50%. We Clathrin-mediated endocytosis is one of the major endocytosis pathways. identify a key dimensionless parameter, P1 a/D ,whereP1 is the increased Formation of clathrin coated vesicles (CCV) begins from relatively flat permeability, a is the radius of the permeable region, and D is the plasma membrane regions. Clathrin molecules bind to the CCV budding solute diffusion coefficient. When p > 0.44, the turgor pressure is locally sites and assemble into a lattice covering growing vesicle. Initial flat clathrin reduced by more than 50%. An approximate analytic theory is used to lattice displays a hexagonal honeycomb architecture whereas the final CCV generate explicit formulas for the turgor pressure reduction in terms of displays a mixture of hexagons and pentagons. The relation between clathrin key parameters. These findings may also be relevant to plants, where the lattice conversion and curvature generation in that process remains un- mechanisms that allow endocytosis to proceed despite high turgor pressure known. No dynamic structural analysis of the detailed mechanisms during are largely unknown. CCV budding was achieved so far. High-speed atomic force microscopy (HS-AFM) permits observation of structural dynamics in biological 469-Pos Board B234 processes because it allows performing imaging with nanometer spatial Effects of Sterol Substitution in Plasma Membrane of Host Cell upon and subsecond temporal resolution. To study the CCV transformation by Yersinia Pseudotuberculosis Internalization of HS-AFM, we use a cell-free endocytosis assay based on plasma membrane 1 2 2 1 JiHyun Kim , Hana S. Fukuto , James B. Bliska , Erwin London . patches. Combination of HS-AFM together with cell-free assay should 1Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, 2 permit direct, real-time observation of clathrin lattice rearrangement during USA, Microbiology, Stony Brook University, Stony Brook, NY, USA. CCV budding. Cholesterol is a major component of mammalian plasma membrane, and can participate in the formation of ordered membrane domains (lipid rafts). 472-Pos Board B237 To investigate its functional role, it can be substituted with various Dilation of Fusion Pores by SNARE Protein Crowding sterols having different chemical structures and varying abilities to support Zhenyong Wu1, Oscar D. Bello2, Sathish Thiyagarajan3, Sarah M. Auclair2, or inhibit ordered domains formation by incubating cells with sterol-loaded Wensi Vennekate1, Shyam S. Krishnakumar2, Ben O’shaughnessy4, methyl b cyclodextrins. We recently used this technique to investigate Erdem Karatekin1. the role of sterol properties upon endocytosis of proteins. Several sterols 1Department of Cellular and Molecular Physiology, Yale University, New could support endocytosis. It was found that a sterol having both raft- Haven, CT, USA, 2Department of Cell Biology, Yale University, New promoting abilities and a 3b-OH group in its structure is necessary and suf- Haven, CT, USA, 3Department of Physics, Columbia University, New York, ficient for that sterol to support endocytosis. These studies have been NY, USA, 4Department of Chemical Engineering, Columbia University, extended by investigating the effect of sterol substitution upon entry of a New York, NY, USA. bacterial pathogen into host cells. Entry of Yersinia pseudotuberculosis Hormones and neurotransmitters are released through exocytotic fusion into the human breast cancer cell line MDA-MB-231 was measured. Thir- pores that can fluctuate in size and flicker open and shut multiple times. teen different sterols/steroids were substituted for cholesterol. In all cases, The kinetics and the amount of cargo released, and the mode of vesicle a similar level of Y. pseudotuberculosis attachment to cells was obtained. recycling depend on the fate of the pore, which may reseal or dilate irrevers- However, other than cholesterol, only desmosterol and 7-dehydrocholes- ibly. Pore nucleation requires zippering between vesicle-associated v- and terol could support Y. pseudotuberculosis internalization. The sterols are target membrane t-SNAREs, but the mechanisms governing the the immediate precursors of cholesterol, and thus are very similar to subsequent pore dilation are not known. Past approaches either monitored cholesterol in structure. Thus, it appears that Y. pseudotuberculosis inter- single exocytotic pores in live cells with unknown biochemistry, or used nalization into host cells needs more specific property of the sterol than reconstitutions that lacked single pore sensitivity. Here, we probed dilation the endocytosis. Whether this sterol specificity reflects a requirement for of single fusion pores using v-SNARE-reconstituted ~23 nm diameter active bacterial invasion or passive endocytosis by the host cells is being discoidal nanolipoprotein particles (vNLPs) as fusion partners with cells studied with Y. pseudotuberculosis mutants lacking one or more surface ectopically expressing cognate, ‘‘flipped’’ t-SNAREs. A flipped t-SNARE adhesins. cell is patch-clamped in the cell-attached configuration with the vNLPs included in the pipette solution. Fusion of a vNLP with the cell surface 470-Pos Board B235 produces a pore connecting the cytosol with the pipette solution, through Recovery of ESCRT-III Filaments Subjected to Force: An ‘Invasive Mode’ which direct-currents are measured under voltage clamp. The magnitude HS-AFM Study of the current reports pore size with sub-millisecond time resolution 1,2 1,2 3 Nebojsa Jukic , Lorena Redondo-Morata , Aure´lien Roux , (Wu, Z. et al. Sci. Rep. 2016). We found that pore nucleation required a 1,2 Simon Scheuring . minimum of 2, and reached a maximum above ~4 copies of v-SNAREs 1Weill Cornell Medicine, New York, NY, USA, 2U1006, INSERM / Aix- 3 per NLP face. In contrast, the mean conductance of single pores Marseille Universite´, Marseille, France, Department of Biochemistry, increased as copy number was increased and was far from saturating at University of Geneva, Geneva, Switzerland. 15 copies, the NLP capacity. Thus, very different numbers of SNARE Endosomal sorting complex required for transport III (ESCRT-III) proteins complexes cooperate at the distinct stages of fusion pore nucleation are crucial to membrane sculpting processes, including cytokinesis and and pore dilation. We calculated pore size distributions and free energy biogenesis of multivesicular bodies. How ESCRT-III polymerization gener- profiles versus pore size. Combined with a mathematical model, these ates membrane curvature remains debated. Using High-Speed Atomic Force results suggest crowding of SNARE complexes at the pore waist drive Microscopy (HS-AFM), a versatile technique with unprecedented spatial pore expansion. and temporal resolution, we acquired insights into how Snf7 assemblies, the major component of the ESCRT-III system, changed architecture in 473-Pos Board B238 the presence of divalent cations and how they recover after being dissected Imaging Regulatory Lipids and Protein Kinase C at Sites of Exocytosis by applying increased forces to well-defined delimited areas of the sample Adam J. Trexler, Justin W. Taraska. surface. The dissected assemblies present free ends of broken filaments National Heart Lung and Blood Institute, National Institutes of Health, onto which new monomers from the imaging solution can polymerize. After Bethesda, MD, USA. initial perturbation, the recovering assemblies show a tendency toward The control of exocytosis is physiologically essential. In vitro SNARE pro- maximization of interfilament contacts, manifesting as nascent filaments teins are sufficient to drive model membrane fusion, but in cells there are or elongation of broken filaments along pre-existing filaments that act as over twenty additional proteins and lipids that work together to drive effi- scaffolds, as well as reparation of broken filaments. Based on these results, cient, fast, and timely release of vesicular cargo. Many of these important we hypothesize about a novel mechanism by which lateral interactions be- accessory proteins are controlled via phosphorylation, and protein kinase tween ESCRT-III filaments drive constriction of the assemblies in order C (PKC) has previously been linked to the modulation of exocytosis. We to induce membrane deformation. use total internal reflection fluorescence microscopy to observe the spatio- temporal dynamics of dozens of proteins and lipids relative to single sites 471-Pos Board B236 of exocytosis in living endocrine INS-1 cells. INS-1 cells are a model Structural Dynamics of Endocytosis by High-Speed Atomic Force system for insulin secretion. We use a vesicle cargo marker NPY-GFP to Microscopy identify where exocytosis occurs and tag proteins-of-interest with a red Grigory Tagiltsev1, Frederic Eghiaian2, Simon Scheuring1. fluorescent protein. Lipid species are visualized using lipid sensors: fluores- 1Weill Cornell Medicine, New York, NY, USA, 2JPK Instruments, Berlin, cent proteins fused to protein domains with known specificity for single Germany. lipids. After stimulating exocytosis using ionomycin, we observe a transient

BPJ 7726_7730 Sunday, February 12, 2017 93a accumulation of PIP2- and DAG-sensors at exocytic sites centered on the We have developed a fluorescence-based turn-on molecular sensor (Neuro- time of membrane fusion and only lasting a few seconds. Intriguingly, we Sensor 521) based on a coumarin aldehyde scaffold that allows for the se- observe a concomitant recruitment of conventional PKC isoforms with lective recognition and sensing of norepinephrine in live and fixed kinetics broadly similar to PIP2 and DAG. Novel and atypical PKCs do chromaffin cells. Based on NeuroSensor 521, we have now developed not appear to be recruited. PIP2, DAG, and PKC recruitment only occur Miz-2: a promising member of a new series of catecholamine-selective sen- at sites of exocytosis and not at control docked-vesicles nearby to sites of sors, which has both a functional aldehyde group to bind to primary amines, exocytosis. PKC is known to target several critical exocytic proteins, and other recognition elements to improve selectivity for binding catechol- including munc18 and dynamin-1, and we are currently investigating amine over other amines. The excitation maximum of Miz-2 is near 488 nm. whether these targets are phosphorylated in response to the recruitment of Fluorescence is quenched when the sensor interacts with epinephrine and PKC that we observe. These data suggest that a regulatory lipid cascade dopamine whereas the fluorescence increases upon binding with norepineph- may recruit and activate PKC to sites of exocytosis to regulate membrane rine. We are using adrenal chromaffin cells as a model cell to test fluores- fusion or cargo release. cent catecholamine sensors and sub-populations of cells enriched in epinephrine versus norepinephrine can be isolated upon gradient centrifuga- 474-Pos Board B239 tion. Norepinephrine-enriched chromaffin cells exhibit distinct fluorescence A CMOS based Sensor Array Platform for Analysis of Exocytosis Events punctae by total internal reflection fluorescence (TIRF) microscopy when 1,2 3 2 loaded with Miz-2 that are consistent with labeling of granules. Upon stim- Meng Huang , John C. Ruelas , Shailendra S. Rathore , þ Joannalyn B. Delacruz4, Manfred Lindau2. ulation with a high-K solution, punctae abruptly disappear during TIRF 1Materials Science and Engineering, Cornell University, Ithaca, NY, USA, imaging, consistent with granule fusion and loss of dye via exocytosis. 2Applied Engineering Physics, Cornell University, Ithaca, NY, USA, Using underlying transparent electrodes, we recorded amperometric spikes 3Electrical and Computer Engineering, Cornell University, Ithaca, NY, USA, consistent with quantal exocytosis of catecholamine in cells loaded with 4Field of Pharmacology, Cornell University, Ithaca, NY, USA. the sensor. Experiments are in progress to combine TIRF imaging with The release of neurotransmitters into the extracellular space is an important amperometric measurements in underlying transparent electrodes to resolve drug target and crucial for molecular manipulation. Amperometric measure- features of norepinephrine release from individual granule. Supported by ments provide details about the amount and kinetics of transmitter release in NIH R01EB020415. single quantal events. However, amperometric spikes vary from cell to cell even under the same condition; therefore, a large number of release events 476-Pos Board B241 must be analyzed to achieve the statistical significance. We present CMOS SNARE Protein Structure Altered in Response to pH chips with 100 or 1,024 active electrodes with surface modifications for Kara L. Woodbury, Sam K. Zenger, Peter Weitzel, Curtis D. Nelson, amperometry measurements. To enable amperometry measurements, Sterling M. Jones, Trey S. Winter, Wade J. Whitt, Ani C. Henriksen, polarizable electrodes such as platinum are patterned on the original Dixon J. Woodbury. aluminum metal contact on the chip. To prevent potential current leakage, Brigham Young University, Provo, UT, USA. a new shifted electrode design was used to enhance the reliability of The SNARE proteins SNAP-25, syntaxin, and VAMP play critical roles in post-fabrication. Platinum electrode material is deposited on the aluminum neuronal exocytosis by providing the four helical regions (SNARE domains) contact extending onto the periphery area and further insulated to redefine that form a coiled-coil complex required for neurotransmitter release. This the openings of the active electrodes by lithography and sputtering. The complex is continually formed and unwound as exocytotic vesicles fuse and insulation of the surface is performed by lithography using photoresist. are recycled. Using multiple techniques, we observe clear structural changes The 100 sensor array was validated by simultaneous recording from all of these proteins in response to changes in pH, ionic strength, and oxidation electrodes at 1MS/s using dopamine injection and recordings from live state. Here we focus on pH changes. chromaffin cells cultured on-chip. A new chip with 1024 working electrodes Using Circular Dichroism to measure the secondary structure of SNAP-25B capable of bi-directional current recordings was developed for fast scan in low ionic strength solutions, we observe an increase in alpha-helical struc- cyclic voltammetry. Positive as well as negative currents are measured in ture when pH is lowered from 7 to 4.5, similar to the helical shift observed the same working manner, and a current mirror and a biased cascode tran- when SNAP-25 forms a complex with syntaxin and VAMP. This shift is sistor ensure a gain error <1%. A sign detection function is implemented to not observed in 150 mM NaF solutions. This conformation change correlates report the polarity of the current input. Simulations of the circuit revealed with our observation that SNAP-25 precipitates when the pH is dropped from reliable capacitive current recording and the linearity of the gain over a 7to4.5. wide range of input with the correct sign-bit output. We anticipate that Using the fluorescent signal from the single tryptophan in SNAP-25B, we the chip will serve as a universal platform for characterizing various phys- observe a reversible shift in emission wavelength as the pH is increased from iological events. 7 to 10. The shift has a pK of ~8.8, similar to the expected pKa for the sulfhy- dryl of cysteine. Replacing all four cysteines with serines prevents this shift. 475-Pos Board B240 The conformational changes observed above may lead to a change in accessi- Miz-2, A New Catecholamine-Selective Fluorescent Sensor for Visualizing bility of the cysteines in these SNARE proteins. Consistent with this hypothe- Norepinephrine sis, we observe that decreasing the pH from 7 to 5.5 of solutions containing Xin A. Liu1, Le Zhang2, Kevin D. Gillis3,4, Timothy E. Glass2. syntaxin and SNAP-25B varies the number of cysteines accessible for labeling 1Dalton Cardiovascular Research Center, University of Missouri-Columbia, with a Biotin-maleimide tag technique developed in our lab (Woodbury et al., Columbia, MO, USA, 2Department of Chemistry, University of 2011. Anal. Biochem. 417:165-173). This correlates with the pKa of Histidine, Missouri-Columbia, Columbia, MO, USA, 3Dalton Cardiovascular Research present in both proteins. Center; Department of Medical Pharmacology & Physiology, University of These data support the hypothesis that the structures of SNAP-25B and syn- Missouri-Columbia, Columbia, MO, USA, 4Department of Bioengineering, taxin are sensitive to changes in pH, which may contribute to modulation of University of Missouri-Columbia, Columbia, MO, USA. exocytosis in vivo.

BPJ 7726_7730 94a Sunday, February 12, 2017

Calcium Signaling I 1Memorial University, St John’s, NL, Canada, 2Electrophysiology and Heart Modeling Institute, Bordeaux, France, 3Columbia University, New York, 4 477-Pos Board B242 NY, USA, Libin Cardiovascular Institute of Alberta, Calgary, AB, Canada. Optogenetic Dissection of STIM1 Conformational Switch and Oligomeri- BACKGROUND. The trigger of ventricular tachyarrhythmias after myocar- zation dial infarction (MI) has been attributed to increased incidence of electrogenic 2þ 2þ Guolin Ma, Lian He, Qian Zhang, Yubin Zhou. Ca waves in cardiac Purkinje cells (Pcells). A change in Ca release chan- 2þ Center for Translational Cancer Research, Institute of Biosciences & nel (CRC) gating properties and overload of intracellular Ca store are 2 Technology, Texas A&M University, Houston, TX, USA. potential causes of this increase. We analysed through a model of intracellular 2þ 2þ Store-operated calcium release-activated calcium (CRAC) channel constitutes Ca dynamics the spontaneous Ca transients of Pcells after MI. Regional a major route for calcium influx in many cell types. To study the impact of protein expression was assessed for potential remodelling associated with calcium signals on specific cellular functions, several approaches based on ischemia. pharmacological tools or photoactivatable ‘caged’ compounds have been METHODS. Pcells were dispersed enzymatically from dog, pig, sheep, and developed to manipulate intracellular calcium concentrations, but they often human Purkinje fibers dissected from normal hearts and from hearts with 2þ lack rapid reversibility and spatial precision. The recently developed optoge- MI. Spontaneous local Ca transients were studied by high resolution netic tools provide exciting opportunities to remotely and reversibly modulate confocal fluorescence microscopy techniques. Specific antibodies were 2þ cell signaling at superior temporal and spatial resolution. Here we report two used to map proteins involved in Ca release/uptake functions (ERCs, major optogenetic engineering strategies to confer light sensitivities to CRAC SERCAs). 2þ channel (termed ‘‘Opto-CRAC’’) to achieve light-operated calcium entry in RESULTS. In Pcells of MI heart, the density and firing rate of Ca -release mammalian cells: i) fusion of light-inducible dimerization/oligomerization sites were increased by 20% [Normal: 3.0 5 0.3 sites /25mm (14 scans; 12 domain with the cytosolic domain of STIM1 or ii) fusion of a photoswitch cells) vs MI: 3.6 5 0.2 sites /25mm (9 cells); P<0.05] and 120% LOV2 with SOAR/CAD to mimic CC1-SOAR/CAD intramolecular trapping. [Normal:0.77 5 0.10 sparks/s (42 sites, 12 cells) vs MI: 1.74 5 0.23 sparks 2þ In the first strategy, light induced dimerization or oligomerization overcomes /s (40 sites, 9 cells); P<0.05), respectively. A model fit of Ca -waves in MI the autoinhibition of STIM1, much like calcium-depletion induced oligomer- Pcells revealed 50% increase in amplitude, 25% faster decay, and 35% eleva- 2þ ization of the STIM1 luminal domain, thereby exposing the SOAR/CAD tion of Ca -uptake rate. No change was observed in CRC expression. How- domain to gate ORAI1 channels on the plasma membrane. In the second ever SERCAs redistribution to the cell periphery was detected in all animal approach, STIM1 cytosolic fragments containing SOAR/CAD domain were MI models and in human heart with large MI. Peripheral protein expression fused with LOV2. In the dark, LOV2 sterically masks function of the effector and gene expression of SERCA2 showed 4.5 and 2.3 times increases post MI, domain. Upon blue light illumination, LOV2 underwent conformational respectively. Interestingly, no change was observed in regulatory protein changes to unleash the minimal ORAI-activating domain SOAR/CAD to phospholamban. induce calcium flux through ORA1 channels. The Opto-CRAC system can CONCLUSION. An unknown factor associated with myocardial ischemia be conveniently used to generated user-defined calcium signals to control mediates a dramatic increase in SERCA expression at the Pcell periphery, calcium-dependent activities in cells of the immune system. The similar opto- i.e. wherein the CRCs IP3R and RyR3 are specifically located. We propose a 2þ genetic engineering approach can be further extended to study conformational mechanistic model of intracellular Ca cycling demonstrating that augmenta- switch and oligomerization of other signaling proteins. [This work was sup- tion of peripheral SERCA activity is sufficient to promote pro-arrhythmic spon- 2þ ported by grants from the NIH (R01GM112003 to Y.Z.) and the American taneous Ca release in Pcells of post-MI human heart. Cancer Society (RSG 129848 to Y.Z.)] 480-Pos Board B245 R420Q 478-Pos Board B243 Functional Effects of the RyR2 Catecholaminergic Ventricular Structural Differences between Cardiac and Skeletal Ryanodine Polymorphic Tachycardia in Mouse Cardiomyocytes Receptors Riccardo Rizzetto1, Miguel Fernandez-Tenorio2, Sonali Dhindwal, Joshua Lobo, Montserrat Samso. Alexandra Zahradnikova Jr1, Simona Boncompani3, Elena Marque´s-Sule´1, Physiology and Biophysics, VCU School of Medicine, Richmond, VA, USA. Yue Yi Wang1, Jean-Pierre Benitah1, Esther Zorio4, Feliciano Protasi3, Cardiac and skeletal muscles have different mechanisms of excitation- Ernst Niggli2, Ana M. Gomez1. 1 2 contraction coupling that depend on a set of tissue-specific protein isoforms. UMR-S 1180, Inserm, Chaˆtenay-Malabry, France, University of Bern, 3 4 In heart, ryanodine receptor isoform 2 (RyR2), cardiac dihydropyridine recep- Bern, Switzerland, University G. d’Annunzio, Chieti, Italy, Hospital La Fe, tor (DHPR) and FKBP12.6 together with other proteins couple the plasmalem- Valencia, Spain. mal depolarization to the release of Ca2þ from the sarcoplasmic reticulum (SR), Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a genetic via a calcium-induced calcium release mechanism. In skeletal muscle, RyR1, disease characterized by stress-induced syncope and/or sudden death. CPVT1 skeletal DHPR and FKBP12 together with other proteins couple these two mutations are clustered in three regions: the N-terminal, the central, and the 2þ events via an entirely different mechanism that requires a more direct RyR- C-terminal domains of the cardiac Ca release channel (type-2 ryanodine re- DHPR coupling, and different long-range allosterism pathways. ceptor, RyR2). We have created a knock-in (KI) mouse model bearing the R420Q While the 3D structure of RyR1 has been solved at near-atomic resolution, the RyR2 mutation, which is located in the N-terminal region, found in three 3D structure of RyR2 lags significantly in resolution (currently ~30 A˚ ). To start families diagnosed with CPVT. Electrocardiographic measurements of wake uncovering the different molecular mechanisms of these two main RyR iso- animals showed bidirectional ventricular tachycardia after epinephrine/caffeine R420Q forms, we have determined the 3D structure of RyR2 in complex with injection in all the KI (heterozygous for RyR2 ) but in none of the WT FKBP12.6 in the closed state at nanometer resolution. This has enabled build- (wild type) animals, validating the model. Consistent with the in-vivo pheno- ing a pseudo-atomic model for RyR2. Comparative analysis between the RyR2 type, during action potential (AP) measurement by patch-clamp in the presence and RyR1 isoforms shows significant structural differences localized to specific of 100 nM isoproterenol all KI myocytes displayed delayed afterdepolariza- domains, but not all structural differences can be explained by primary tions (DADs) and triggered activity (TA), while the incidence of pro- sequence divergence. These differences are interpreted in the context of the arrhythmic events was significantly lower in WT myocytes. AP characteristics two modes of excitation-contraction coupling. The analysis of heterogeneity and potassium currents showed only minor differences between KI and WT. 2þ 2þ in the dataset allowed us to further define two conformations representing We measured ICa, [Ca ]i transients and intra-SR [Ca ] by patch-clamp and two functional states of RyR2. confocal microscopy. ICa density was similar between WT and KI cardiomyo- 2þ This work was supported by American Heart Association grant No. cytes, but the associated [Ca ]i transient amplitude was unchanged or 14GRNT19660003 and Muscular Dystrophy Association grant No. enhanced depending on the protocol, as well as its response to beta- 2þ MDA352845 (to MS). adrenergic stimulation. Intact cardiac myocytes showed more Ca sparks occurrence with a lowered SR Ca2þ threshold to initiate Ca2þ waves. Electron þ 479-Pos Board B244 microscopy analyses indicate that Ca2 release sites (or estimates indicate that Increased Density of SERCA Pumps at the Periphery of Cardiac Purkinje couplons) in KI cardiomyocytes were fewer in number, significantly smaller Cells after Myocardial Infarction and, hence, may contain a significantly lower number of RyR2. This morpho- þ Bruno Stuyvers1,2, Penelope Boyden3, Henk EDJ ter Keurs4, Yunbo Guo2, logical changes in couplons could participate to the differences in Ca2 sparks Wen Dun3, Kazi Haq1, Meleze Hocini2, Michel Haissaguerre2, characteristics recorded in KI cells compared to WT. Taken together, we have 2 2 2 2 Olivier Bernus , Sebastien Chaigne , Sabine Charron , Caroline Cros , found a unique cellular alteration in this N terminal RyR2 mutation at structural Fabien Brette2. and functional levels.

BPJ 7731_7735 Sunday, February 12, 2017 95a

481-Pos Board B246 483-Pos Board B248 Suppression of Arrhythmia by Enhancing Mitochondrial Calcium Uptake The Interplay between Length-Dependent Calcium Affinity of Troponin in Experimental Models of Catecholaminergic Ventricular Tachycardia and X-ROS Signaling on Myoplasmic Calcium Levels in Heart Maria K. Schweitzer1, Fabiola Wilting1, Simon Sedej2, Lisa Dreizehnter3, Sarita Limbu1, Benjamin L. Prosser2, W. Jonathan Lederer3, M. Saleet Jafri1. Nathan J. Dupper4, Alessandra Moretti3, Ohyun Kwon4, Silvia G. Priori5, 1School of Systems Biology and Krasnow Institute for Advanced Studies, Karl-Ludwig Laugwitz3, Michael Mederos y Schnitzler1, George Mason University, Fairfax, VA, USA, 2Department of Physiology Thomas Gudermann1, Johann Schredelseker1. and Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, 1LMU Munchen,€ Munich, Germany, 2Medial University of Graz, Graz, PA, USA, 3Department of Physiology and Center for Biomedical Austria, 3TU Munchen,€ Munich, Germany, 4UCLA, Los Angeles, CA, USA, Engineering and Technology, University of Maryland School of Medicine, 5IRCCS Salvatore Maugeri Foundation, Pavia, Italy. Baltimore, MD, USA. We have recently identified a critical role of mitochondria to shape intracel- The stretching of a cardiomyocyte leads to increased production of reactive lular Ca2þ signals and to regulate cardiac rhythmicity. Activation of mito- oxygen species and an elevation in ryanodine receptor open probability chondrial Ca2þ uptake by efsevin, an agonist of the voltage-dependent through a process termed X-ROS signaling. Stretching also increases the anion channel 2 in the outer mitochondrial membrane, restored rhythmic calcium binding to the myofilaments by elevating troponin C calcium cardiac contractions in a zebrafish cardiac arrhythmia model. Here we inves- affinity. Here an integrative experimental and modeling study is pursued tigated the potential of pharmacological activation of mitochondrial Ca2þ that explains the interplay of length-dependent changes in troponin’s uptake as a novel pharmacological strategy for human cardiac arrhythmia calcium affinity and stretch activated X-ROS calcium signaling. The in a translational approach. To this aim we first used a murine model of decreased myoplasmic calcium concentration caused by increased calcium ryanodine receptor 2 (RyR2)-mediated catecholaminergic polymorphic ven- affinity of troponin during myocyte stretching is offset by increased calcium tricular tachycardia (CPVT). In freshly isolated cardiomyocytes of release from the sarcoplasmic reticulum caused by X-ROS signaling. The RyR2R4496C/WT mice, harboring the human RyR2R4496C mutation associated combination of modeling and experiment also help with proper interpreta- with CPVT, efsevin restricted diastolic Ca2þ sparks and prevented the tion of the length-dependent changes to the calcium binding affinity of formation of propagating Ca2þ waves and spontaneous, diastolic action troponin in the presence of blebbistatin. The model suggests that in order potentials. This anti-arrhythmic effect was abolished in the presence of to explain all the experimental data, the decrease in calcium affinity of mitochondrial Ca2þ uniporter (MCU) blocker Ru360 , but could be repro- troponin with increasing blebbistatin is accompanied by a reduction and duced with the MCU activator kaempferol, demonstrating an immediate eventual elimination in length dependent differences in calcium affinity. role of mitochondrial Ca2þ uptake for the anti-arrhythmic effect of Therefore, this work suggests that X-ROS signaling may serve to maintain efsevin. In RyR2R4496C/WT mice both mitochondrial Ca2þ uptake enhancers free myoplasmic calcium concentrations during a change in myocyte length. (MiCUps), efsevin and kaempferol, significantly reduced episodes of ven- Furthermore it defines the relative contributions of X-ROS signaling and the tricular tachycardia after catecholaminergic stimulation by a bolus injection length-dependent changes in calcium binding by troponin on myoplasmic of epinephrine and caffeine in vivo while baseline ECG was unaffected. calcium levels. Finally, we used stem cell-derived cardiomyocytes from a CPVT patient 484-Pos Board B249 to show efficacy of MiCUps in a human model. Both MiCUps abolished 2D arrhythmogenic events in human CPVT cardiomyocytes. Our results Mitochondrial Ca Uptake and Superoxide Generation Regulates demonstrate that enhancement of mitochondrial Ca2þ uptake by MiCUps Angiotensin II-Induced Proliferation in Neonatal Cardiac Fibroblasts 1 2 2 1 is a promising pharmacological strategy for treatment and prevention of Jin O-Uchi , Deming Fu , Jyotsna Mishra , Bong Sook Jhun , 2 Ca2þ-triggered arrhythmias, such as CPVT. Shey-Shing Sheu . 1Cardiovascular Research Center, Medicine, Rhode Island Hospital, Brown 482-Pos Board B247 University, Providence, RI, USA, 2Center for Translational Medicine, Antidepressant Drugs Citalopram and Escitalopram but not Paroxetine Medicine, Thomas Jefferson University, Philadelphia, PA, USA. Induce Arrythmogenic Sarcoplasmic Reticulum Calcium Release Cardiac fibroblasts (CFs) are one o the most abundant cell types in the Daniel Blackwell, Bjo¨rn Knollmann. heart and play key roles in regulating myocardial physiological function Vanderbilt University, Nashville, TN, USA. and pathophysiological remodeling especially for the cardiac fibrosis. The The antidepressant citalopram, a selective serotonin reuptake inhibitor levels of Angiotensin II (AT-II) are increased in the remodeling heart and (SSRI), has been associated with increased risk of sudden cardiac death. Angiotensin signaling participates in pathological CF proliferation. It has Epidemiological data from the Danish population suggest that in been shown that CF proliferation may occur via the increased levels of patients taking citalopram, co-administration of carvedilol reduced sudden cellular reactive oxygen species (ROS), but the detailed signal transduction death risk compared to two other beta-blockers, metoprolol and bisoprolol. remains unclear. We previously reported that the enhancement of mito- Since carvedilol is the only beta blocker that suppresses store overload- chondrial Ca2þ uptake by mitochondrial Ca2þ uniporter (MCU) induces induced calcium release, we hypothesized that citalopram promotes calcium mitochondrial superoxide (mtSO) generation in cardiac myofibroblast cell release from sarcoplasmic reticulum (SR) calcium stores. Left ventricular line H9C2 cells. Therefore, we hypothesize that Ang-II stimulation enhances myocytes were isolated from black 6 mice, permeabilized, and loaded mitochondrial Ca2þ-induced mtSO generation in primary CFs, which can with the calcium indicator, Fluo-4. Incubation of citalopram or escitalopram activate ROS-dependent proliferation signaling in primary CFs. First, we (S-enantiomer) for 20 minutes significantly increased calcium wave confirmed that AT-II (R1 mM) stimulation induces significant mitochon- frequency and decreased calcium wave amplitude in a dose-dependent drial Ca2þ uptake assess by mitochondria-targeted Ca2þ biosensor in manner. This response was more sensitive to escitalopram, indicating response to the Ca2þ release from the endoplasmic reticulum in selectivity for the S-enantiomer in the high nanomolar range. At 30 mM, neonatal rat CFs (NCF). In addition, AT-II stimulation increases the calcium waves were no longer evident, however cytosolic calcium mtSO levels detected by a mtSO indicator MitoSOX Red. We also was elevated approximately two-fold, indicating possible constitutive confirmed that AT-II application activates proliferative pathway, including calcium release from the SR stores. Incubation with a different SSRI, ERK1/2, p38 and JNK1/2 in time-dependent manner, which was abolished paroxetine, did not significantly alter calcium wave frequency or by losartan pretreatment. Lastly, pretreatment of a mitochondria-targeted amplitude at concentrations as high as 30 mM. Calcium wave activation antioxidant, Mito-tempo significantly inhibited AT-II-mediated activation by citalopram and escitalopram resembledthoseseeninacalsequestrin of the mtSO production as well as proliferative pathway without knockout mouse model of catecholaminergic polymorphic ventricular changing the AT-II-induced the mitochondrial Ca2þ uptake profile. Our tachycardia (CPVT), suggesting a possible similar mechanism for results indicate that mtSO generation induced by mitochondrial Ca2þ accu- citalopram. Pre-incubation of isolated myocytes with 1 mM carvedilol, mulation via MCU serves as an important regulator for the Ang II-induced but not metoprolol or bisoprolol, for 30 minutes significantly reduced proliferation in CFs. calcium wave frequency and increased SR calcium stores in myocytes treated with citalopram or escitalopram. Taken together, increased calcium 485-Pos Board B250 leak from SR stores through ryanodine receptor may contribute to the TRPV4 Enhances Cardiomyocyte Calcium Transients and Cardiac increased risk of sudden death associated with citalopram. Hence, our in Contractility Following Hypoosmotic Stress and Ischemia-Reperfusion vitro findings support the existence of drug-induced CPVT caused by cita- John L. Jones, Deborah Peana, Michelle D. Lambert, Timothy L. Domeier. lopram or escitalopram, which can be prevented by co-administration of Medical Pharmacology and Physiology, University of Missouri, Columbia, carvedilol. MO, USA.

BPJ 7731_7735 96a Sunday, February 12, 2017

The Transient Receptor Potential (TRP) ion channel family plays an impor- 487-Pos Board B252 tant role in cardiomyocyte calcium homeostasis, particularly in diseased Genetic Ablation of Fibroblast Mitochondrial Calcium Uptake Increases states. Here, we test the hypothesis that the osmotically-activated TRP Myofibroblast Transdifferentiation and Exacerbates Fibrosis in Myocar- Vanilloid 4 (TRPV4) channel enhances cardiomyocyte calcium transients dial Infarction in the aged heart following hypoosmotic stress. Expression of TRPV4 was Alyssa A. Lombardi, Ehtesham Arif, Timothy S. Luongo, John W. Elrod. low in cardiomyocytes of Young (3-6 month) mice, but significantly Temple University School of Medicine, Philadelphia, PA, USA. increased in cardiomyocytes of Aged (24-26 month) mice. In cardiomyo- When the heart is injured, fibroblasts transition from a structural role into con- cytes of Aged, hypoosmotic stress (250 mOsm) induced an increase in tractile, synthetic myofibroblasts. This is crucial for scar formation after calcium transient amplitude (F/F0:3.650.1 hypoosmotic versus 2.950.2 myocardial infarction to prevent ventricular rupture, but excessive fibrosis is 2þ isosmotic, P<0.05). This effect was prevented by the TRPV4 inhibitor maladaptive and leads to heart failure. While intracellular calcium (iCa ) HC067047 (1 mM, F/F0:2.950.1 hypoosmotic) and was absent in cardio- elevation has been shown to be necessary for myofibroblast transdifferentia- 2þ myocytes of Young mice (F/F0:3.250.1 hypoosmotic versus 2.950.2 isos- tion, the mitochondrial calcium (mCa ) domain has not been explored. Mcu motic). However, cardiomyocytes of Young mice with cardiac-specific encodes the channel-forming portion of the mitochondrial calcium uniporter 2þ TRPV4 Overexpression exhibited enhanced calcium transient amplitude complex and is required for acute mCa uptake. We generated a conditional, fl/fl following hypoosmotic stress (F/F0:3.550.2 hypoosmotic versus 2.450.1 fibroblast-restricted Mcu knockout mouse by crossing Mcu mice with a isosmotic, P<0.05) that was prevented by HC067047 (F/F0:2.650.1 hypo- mouse expressing a tamoxifen-inducible Cre recombinase under control of osmotic). Ischemia-reperfusion (I-R) injury is a pathological scenario asso- the collagen1a2 promoter (fibroblast Mcu-cKO). Fibroblast Mcu-cKO mice ciated with pronounced osmotic stress on cardiomyocytes. We therefore and controls were subjected to ligation of the left coronary artery and cardiac monitored left-ventricular pressure development in Langendorff-perfused function was examined weekly by echocardiography. Deletion of fibroblast hearts of Young, Aged, Young TRPV4 Overexpressor, and Aged TRPV4 Mcu worsened left ventricular dysfunction and increased fibrosis. To examine knock-out mice subjected to 45 minute global ischemia followed by reper- cellular mechanisms responsible we isolated mouse embryonic fibroblasts fl/fl fusion. Prior to ischemia, all hearts exhibited similar contractility (dP/dtMax (MEFs) from Mcu mice and deleted Mcu using Cre-adenovirus. When -/- 2þ range: 19345206 to 2226562 mmHg/s). Following I-R, hearts of Aged challenged with pro-fibrotic ligands, Mcu MEFs exhibited decreased mCa 2þ -/- mice exhibited enhanced contractile performance (dP/dtMax:27705180 uptake and enhanced iCa transient amplitude. Mcu MEFs displayed mmHg/s) versus hearts of Aged TRPV4 knock-out mice (dP/dtMax: enhanced myofibroblast transdifferentiation as evidenced by decreased mi- 14005300 mmHg/s, P<0.05). Similarly, hearts of Young TRPV4 Overex- gration and proliferation as well as increased a-SMA expression and gel -/- pressor mice exhibited enhanced contractile performance (dP/dtMax: contraction. Metabolically, Mcu MEFs display a shift towards more glycol- 27505180 mmHg/s) versus hearts of Young mice (dP/dtMax:16505260 ysis with less oxidative phosphorylation, correlating with increased phosphor- mmHg/s, P<0.05). In conclusion, TRPV4 enhances calcium transients ylation (inactivation) of the pyruvate dehydrogenase subunit E3. These results 2þ following hypoosmotic stress and contributes to hypercontractility following suggest that alterations in mitochondrial buffering of pro-fibrotic iCa I-R. Our findings have potential clinical implications in the treatment of signaling and changes in energetic pathways may be mechanisms driving my- elderly populations at increased risk of myocardial infarction and reperfu- ofibroblast transdifferentiation and fibrosis. sion injury. 488-Pos Board B253 486-Pos Board B251 Gain-of-Function Mutation in Ryanodine Receptor Type 1 Modulates Mu- Characterization of Intracellular Sodium Homeostasis in Murine Atrial rine Thymocyte Calcium Signaling and Autoimmune Response in Mice Myocytes Lukun Yang, Athena Soulika, Lillian Cruz-Orengo, Paul D. Allen, Libet Garber1, W. Jonathan Lederer2, Maura Greiser2. Alla F. Fomina. 1Bioengineering, University of Maryland College Park, College Park, MD, Univ. California, Davis, Davis, CA, USA. USA, 2Center for Biomedical Engineering and Technology and Department Gain-of-function mutations in gene encoding ryanodine receptor type 1 of Physiology, University of Maryland School of Medicine, Baltimore, (RyR1), an endo/sarcoplasmic reticulum Ca2þ release channel, are linked to MD, USA. a variety of skeletal muscular disorders. Other tissues, including cells of the þ Intracellular sodium concentration ([Na ]i) is an important regulator of immune system, have been shown to express RyR1. A previous study demon- intracellular Ca2þ and provides insight into the activation of the sarco- strated enhanced immune responses in mice with gain-of-function RyR1 muta- lemmal sodium calcium exchanger (NCX) and the behavior of both Naþ tion (Vukcevic et al., 2013, J Cell Sci, 126: 3485-92). Here we report that after channels and the Naþ,Kþ-ATPase. In atrial diseases such as atrial fibrilla- induction of an experimental autoimmune encephalomyelitis (EAE), a T cell- tion (AF), intracellular Ca2þ signaling is profoundly altered. While the mediated autoimmune disease, the mice carrying gain-of-function RyR1 mechanisms underlying altered intracellular Ca2þ homeostasis are well R163C mutation (R163C mice) displayed an earlier disease onset and augmen- þ characterized, the role that [Na ]i may play and its dysregulation in disease tation of neurological symptoms compared with wild-type (WT) mice. In vitro þ in atrial myocytes is less well understood. For example, we have previously studies revealed that splenocytes from the R163C mice have altered Ca2 shown that high rate (10 Hz) atrial activation for 5 days in a rabbit model signaling compared with those from WT mice, which was revealed by chal- þ 2þ leads to an expected but significant reduction in [Na ]i (Greiser et al, JCI, lenging the cells with different concentrations of extracellular Ca and þ 2014; 124: 4759-72). Ca2 mobilizing agent thapsigargin. No differences were found in the relative Here we characterize intracellular Naþ homeostasis in murine atrial myo- abundances of immune cell subtypes in spleens and lymph nodes from R163C cytes using the Naþ indicator SBFI (Molecular Probes) in quiescent cells. and WT mice prior to the onset of the EAE. Nevertheless, immunostaining of Dual UV excitation (340 nm and 380 nm) was provided by a rapid switching the lumbar spinal cord sections after the onset of the clinical symptoms, re- Sutter DG5-plus illuminator using a 300 W xenon light source with wide- vealed the larger pro-inflammatory infiltrate and axonal damage in the field imaging by an EMCCD camera (Princeton Instruments). Freshly iso- R163C mice compared with the WT mice, indicating that R163C mutation lated atrial myocytes were loaded with 10 mmol/l SBFI-AM for 90 min in may affect immune cell trafficking to the CNS. These data indicate that altered þ the presence of the non-ionic surfactant Pluronic F-127 (0.05 %w/v). After Ca2 handling in R163C mice immune cells may enhance these cells autoim- washout of the extra cellular dye, SBFI-AM de-esterification was allowed mune responses and that gain-of-function mutations in RYR1 may represent a þ for 20 minutes before proceeding with [Na ]i measurements. Fluorescence previously unknown risk factor for autoimmune diseases. images (F340 and F380) were collected at 510 5 40 nm. The calibration þ of [Na ]i was performed in each cell separately after the measurements of 489-Pos Board B254 þ quiescent [Na ]i by exposing the SBFI-loaded myocytes to various extracel- Regulation of Cardiac Pacemaker Activity by PDE4 Isoforms þ þ 1 1 2 lular [Na ]([Na]o) in the presence of 10 mmol/l gramicidin D and Delphine Mika , Ana Maria Gomez , Marco Conti , þ 1 1 100 mmol/l-strophanthidin. Resting [Na ]i in mouse atrial myocytes was Rodolphe Fischmeister , Gre´goire Vandecasteele . 8.2 5 0.6 mM. External field stimulation (4 ms, 20 V, 1 Hz) increased 1INSERM UMR-S 1180, Chatenay-Malabry, France, 2ObGyn department þ þ [Na ]i by 3.1 5 0.2 mM. These data show that [Na ]i in murine atrial my- University of California San Francisco, San Francisco, ocytes is lower than in murine ventricular myocytes (12 5 1mM,Correll CA, USA. et al, Circ Res, 2014;114:249-56). These data and more will provide a broad Background Numerous epidemiological and clinical studies have revealed a understanding of atrial Naþ homeostasis and its implication for atrial myo- positive correlation between heart rate (HR) and cardiovascular morbimor- cyte Ca2þ signaling. tality. The autonomic nervous system is the major extracardiac determinant

BPJ 7731_7735 Sunday, February 12, 2017 97a of HR. During sympathetic stimulation, the activation of b-adrenergic re- lation of the N-terminal domain (NTD) which is not only the best resolved ceptors (bAR) induces an increase in cAMP levels, leading to a positive structural component of RyRs, but also a hotspot of disease mutations. chronotropic effect. Among the 5 cAMP-PDE families expressed in the First, we simulated the tetrameric NTD of wild-type RyR1 and three disease heart, PDE4 is critical for controlling excitation-contraction coupling mutants (K155E, R157Q, and R164Q) that perturb the inter-subunit interfaces. (ECC) during bAR stimulation in atrial and ventricular cells. PDE4 may Our simulations identified a dynamic network of salt-bridges involving also be important for automaticity. 3 genes encode for cardiac PDE4s: charged residues at inter-subunit/subdomain interfaces and disease-mutation pde4a, pde4b and pde4d. Their respective contribution to the regulation of sites. By perturbing this key network, the above three mutations result in pacemaker activity remains ill-defined. Methods The total enzymatic PDE greater flexibility and unstable inter-subunit interfaces that favour channel activity was determined in mouse sinoatrial node (SAN) tissue as the opening. cAMP hydrolytic activity measured in the absence of PDE inhibitor and Next, we simulated the monomeric NTD of RyR2 in the presence or absence of the fraction corresponding to PDE4 activity was assessed by including the a central Cl anion which is known to stabilize the interfaces between the three PDE4 inhibitor Ro-20-1724 (10 mM). The in vitro pacemaker activity was NTD subdomains (A, B, and C). We found that the loss of Cl restructures the assessed by measuring the spontaneous Ca2þ transients in Fluo4-loaded- salt-bridge network near the Cl-binding site, leading to rotations of subdomain SAN intact tissue. Images were obtained using confocal microscopy. A/B relative to subdomain C and enhanced mobility between the subdomains. Results Ro-20-1724 increased the beating rate of intact mouse SAN and This finding supports a mechanism for some disease mutations in the NTD of increased PKA-phosphorylation levels of key ECC actors (ryanodine recep- RyR2 via perturbation of the Cl binding. tor, phospholamban). PDE4 enzymatic activity was found to account for The structural and dynamic insights gained from this study will guide future 60% of the total cAMP-PDE activity in SAN. The 3 isoforms PDE4A, 4B functional/mutational studies of the NTD of RyRs. and 4D were found to be expressed in mouse SAN. In PDE4D-, but not in PDE4B-deficient mice, Ca2þ homeostasis was altered in control condi- 492-Pos Board B257 tions and after bAR stimulation. Indeed, ablation of PDE4D induced a Genotype-Phenotype Correlations of the Central Core Disease Mutations decreased beating rate and an increased Ca2þ spark frequency in control in the C-Terminal Region of the RyR1 Channel 1 1 2 and bAR-stimulated conditions. Conclusion Our preliminary results reveal Takashi Murayama , Nagomi Kurebayashi , Haruo Ogawa , Toshiko Yamazawa3, Takashi Sakurai1. that PDE4 controls pacemaker function in mice and that PDE4D ablation 1 strongly perturbs normal SAN activity. Department of Pharmacology, Juntendo University School of Medicine, Tokyo, Japan, 2Institute of Molecular and Cellular Biosciences, The 490-Pos Board B255 University of Tokyo, Tokyo, Japan, 3Department of Molecular Physiology, Functional Role of TRPC1 Channels in Neonatal Cardiomyocytes Jikei University School of Medicine, Tokyo, Japan. Ahmad A. Azmi, Chris Hunter, Hu Qinghua, Frank B. Sachse. Central core disease (CCD) is a congenital myopathy and has been linked to CVRTI, University of Utah, Salt Lake City, UT, USA. mutations in the type 1 ryanodine receptor (RYR1), a Ca2þ release channel Transient receptor potential cation (TRPC) 1 channels are thought to play a of the sarcoplasmic reticulum. CCD has been classified into two groups in role in store-operated and receptor-operated calcium entry in various terms of susceptibility to malignant hyperthermia (MH), i.e., MH/CCD mammalian cell types. However, our understanding of the functional role and CCD alone. MH/CCD is frequently found in the cytoplasmic regions, of these channels in cardiomyocytes remains to be ill-defined. Here, we and CCD alone mutations mostly reside in the C-terminal region. Functional test the hypothesis that the channels are involved in calcium leak from analyses have proposed that MH/CCD is caused by gain-of-function of the the sarcoplasmic reticulum (SR) and modulate the cytosolic calcium channel, whereas loss-of-function of the channel leads to the CCD alone concentration in cardiomyocytes. Our studies were performed on neonatal phenotype. However, how specific mutations cause different phenotypes re- rodent ventricular myocytes. After cell isolations, cells were plated on glass mains largely unknown. In this study, we investigated the channel activity of slides. Two days after isolation cells were infected with adenoviral vectors 16 mutations (14 CCD alone and 2 MH/CCD) in the C-terminal region using of TRPC1 tagged with eGFP. Measurements were performed 4-5 days after a heterologous expression system in HEK293 and myogenic C2C12 cells. infection. Immunolabeling, three-dimensional scanning confocal micro- Ca2þ-induced Ca2þ release (CICR) activity was evaluated with HEK293 2þ 2þ 2þ scopy and quantitative colocalization analysis revealed an abundant intracel- cells by caffeine-induced Ca release, cytoplasmic Ca ([Ca ]i)and 2þ 2þ 3 lular density of native TRPC1 and TRPC1 expressed via adenoviral vectors. ER luminal Ca ([Ca ]ER) measurements, and [ H]ryanodine binding TRPC1 was not associated with the sarcolemma, but the SR. We measured assay. MH/CCD mutants consistently exhibited a gain-of-function 2þ the rest decay and caffeine induced peak calcium release using rapid effect; an enhanced sensitivity to caffeine, a reduced [Ca ]ER,an 2þ 3 scanning confocal microscopy on infected cells loaded with the calcium increased [Ca ]i and enhanced [ H]ryanodine binding. In contrast, CCD sensitive dye Rhod-3. We found an increased SR calcium content in the alone mutations showed divergent CICR phenotypes: gain-of-function, presence of the TRPC channel blocker SKF-96365. SR calcium content loss-of-function and no appreciable change. Effect of these mutations on exhibited a decreasing relationship with TRPC1 expression. In a computa- the excitation-contraction coupling is now in progress using C2C12 myo- tional model, activated SR TRPC1 channels increased the systolic and tubes. Plausible mechanisms of how these mutations alter the channel gating diastolic cytosolic calcium concentration with only minor effects on will be discussed based on recent near-atomic structures of the RYR1 action potential and SR calcium content. Our studies indicate that TRPC1 channel. channels are not involved in sarcolemmal electrophysiology of rodent ventricular myocytes, but localized in the SR. The studies support our 493-Pos Board B258 2D hypothesis that the channels play a role as SR calcium leak channels. The The Role of TRIC Channels in SR Countercurrent during SR Ca findings could guide us to an understanding of TRPC channels as physiolog- Release and SERCA Re-Uptake ical modulators of intracellular calcium and contractility in cardiac Vilmos Zsolnay, Claudio Berti, Michael Fill, Dirk Gillespie. myocytes. Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, IL, USA. Excitation-Contraction Coupling In heart, the release of Ca2þ from the SR that generates sparks or AP-driven transients requires countercurrent to prevent large SR membrane potential þ 491-Pos Board B256 changes and maintain normal excitation/contraction coupling. SR K Probing the Inter-Subunit/Subdomain Interactions Relevant to Disease channels (TRIC channels) have been proposed to be an important counter- þ Mutations in the N-Terminal Domain of Ryanodine Receptors by Molecu- current pathway during SR Ca2 release. It has separately been proposed lar Dynamics Simulation that TRIC channels conduct countercurrent during diastole for SERCA re- þ Wenjun Zheng. uptake of Ca2 . To probe the dependence of countercurrent on the number SUNY at Buffalo, Buffalo, NY, USA. and location of TRIC channels in the SR, we used an equivalent circuit The ryanodine receptors (RyR) are essential to calcium signaling in skeletal model of a compartmentalized SR and the surrounding cytosol to simulate and cardiac muscles, and numerous disease mutations have been found in the cycling of ions into and out of the SR during a heartbeat. We altered two RyR isoforms (RyR1 and RyR2). A deep understanding of the activa- the total number of TRIC channels at heart rates ranging between 60 and tion/regulation mechanisms of RyRs has been hampered by the shortage of 600 bpm. Our results agree with experiments that show removal of half of high-resolution structural and dynamic information for this giant tetrameric the SR TRIC channels is not fatal in mice, whereas total removal is. In complex in different functional states. Toward elucidating the molecular mech- our simulations, there is no physiological difference between having anisms of disease mutations in RyRs, we performed molecular dynamics simu- 100% or 50% the normal number of TRIC channels, but if there were

BPJ 7731_7735 98a Sunday, February 12, 2017

0 TRIC channels, the model SR generated non-physiological a diastolic contractile force (relative force after 10 tetani in adult vs. aged: membrane potential (~20 mV). We also explored the dependence of 41.053.1%, and 53.451.5%). Our results indicate that TAs a) accumulate countercurrent on the location and distribution of TRIC channels on the STIM1, which is likely visible as electron-dense strands between tubes; junctional SR (JSR)-sublemmal, JSR-cytosol, and nonjunctional SR b) while contain STIM1, do not functionally contribute to Ca2þ entry during (NSR)-cytosol membranes. The only qualitative difference occurred when repetitive stimulation. the TRIC complement of the NSR-cytosol membrane (SERCA pump 496-Pos Board B261 location) was varied. Overall, we found that TRIC channels provide coun- 2D tercurrent both during SR Ca2þ release and SERCA uptake. Mitsugumin 53 Regulates Extracellular Ca Entry and Intracellular CA2D Release via Orai1 and RyR1 in Skeletal Muscle 494-Pos Board B259 Mi Kyoung Ahn1, Keon Jin Lee1, Chuanxi Cai2, Mei Huang1, A Method for Validating Mutations Associated with Malignant Hyper- Chung-Hyun Cho3, Jianjie Ma4, Eun Hui Lee1. thermia using CRISPR/Cas9 and Dual Integrase Cassette Exchange 1Dept. of Physiology, College of Medicine, The Catholic Univ. of Korea, Kevin J. De Leon1, Shane Antrobus2, Paul Denney Allen2, Isabelle Marty1, Seoul, Korea, Republic of, 2Center for Cardiovascular Sciences, Department David Segal1. of Molecular and Cellular Physiology, Albany Medical College, Albany, NY, 1Genome Center, University of California, Davis, Davis, CA, USA, 2VM USA, 3Dept. of Pharmacology, College of Medicine, Seoul National Molecular Biosciences, University of California, Davis, Davis, CA, USA. University, Seoul, Korea, Republic of, 4Dept. of Surgery, Davis Heart Malignant Hyperthermia (MH) is a pharmacogenetic disorder characterized and Lung Research Institute, The Ohio State University, Columbus, by a hypermetabolic skeletal muscle response to volatile anesthetics. While OH, USA. there are hundreds of mutations in the Ryanodine Receptor 1 gene (RyR1) Mitsugumin 53 (MG53) participates in the membrane repair of various associated with MH, the current diagnostic protocol has only confirmed 35 cells, and skeletal muscle is the major tissue that expresses MG53. Except mutations to be causative. This study aims to develop a more efficient for the regulatory effects of MG53 on SERCA1a, the role(s) of MG53 in method for validating mutations. The CRISPR/Cas9 system has emerged the unique functions of skeletal muscle such as muscle contraction have as an efficient and targetable gene-editing tool used to assist in not been well examined. Here, a new MG53-interacting protein, Orai1, is homology-driven repair (HDR) for the insertion of donor templates in vitro. identified in skeletal muscle. To examine the functional relevance of the Serine integrase site-specific recombination offers a more efficient method MG53-Orai1 interaction, MG53 was over-expressed in mouse primary or of gene editing but requires specific recognition sites. In this study, we C2C12 skeletal myotubes and the functional properties of the myotubes use CRISPR/Cas9 and Dual Integrase Cassette Exchange (DICE) to create were examined using cell physiological and biochemical approaches. The RyR1-mutant cell lines. Using calcium imaging, myotubes consisting of PRY-SPRY region of MG53 binds to Orai1, and MG53 and Orai1 are co- differentiated myoblasts are tested for their sensitivity to the calcium- localized in the plasma membrane of skeletal myotubes. MG53-Orai1 releasing triggers KCl, caffeine, and 4-chloro-m-cresol. The sensitivities interaction enhances extracellular Ca2þ entry via a store-operated Ca2þ en- of RyR1-mutant myotubes are compared to wild-type myotubes. An in- try (SOCE) mechanism in skeletal myotubes. Interestingly, skeletal myo- crease in sensitivity is indicative of MH and is used to classify the mutations tubes over-expressing MG53 or PRY-SPRY display a reduced intracellular as likely causative. The study is performed on primary murine myoblasts Ca2þ release in response to Kþ-membrane depolarization or caffeine stimu- that have been immortalized via lentiviral transduction of cyclin- lation, suggesting a reduction in RyR1 channel activity. Expressions of dependent kinase 4 (CDK4). We have designed and tested guide RNAs TRPC3, TRPC4, and calmodulin 1 are increased in the myotubes, and (gRNAs) used in the CRISPR/Cas9 system, identifying three gRNAs with MG53 directly binds to TRPC3, which suggests a possibility that TRPC3 DNA-cleavage efficiencies of 25-30%. The combined use of CRISPR/ also participates in the enhanced extracellular Ca2þ entry. Thus, MG53 Cas9 and DICE allows for the efficient and rapid creation of RyR1- could participate in regulating extracellular Ca2þ entry via Orai1 during mutant cell lines in which the mutations can subsequently be functionally SOCE and also intracellular Ca2þ release via RyR1 during skeletal muscle validated. In doing so, we hope to expedite the validation of contraction. MH-associated mutations for the purpose of assisting the development of a genetic screen. In addition, our methods can be applied to other regions 497-Pos Board B262 of the genome to create an improved, high-throughput method of genomic Localization of Junctophilin-1 at the Junctional Sarcoplasmic Reticulum engineering. Requires a Sequence in the Transmembrane Domain Daniela Rossi1, Angela Maria Scarcella1, Stefania Lorenzini1, Enea Liguori1, 495-Pos Board B260 Mirko Messa2, Pietro De Camilli2, Vincenzo Sorrentino1. Role of STIM1 And Orai1 in the Formation of Tubular Aggregates in 1Molecular and Developmental Medicine, University, Siena, Italy, Ageing Skeletal Muscle Fibers 2Departments of Neuroscience and Cell Biology, Yale University School of Claudia Pecorai, Antonio Michelucci, Laura Pietrangelo, Feliciano Protasi, Medicine, New Haven, CT, USA. Simona Boncompagni. The endoplasmic reticulum of striated muscle cells, called sarcoplasmic re- University G. D’Annunzio, Chieti, Italy. ticulum (SR), is mainly dedicated to Ca2þ homeostasis and regulation of Tubular aggregates (TAs), ordered arrays of sarcoplasmic reticulum (SR) muscle contraction. The SR is organized in longitudinal (l-SR) and junc- tubes, form in ageing fast-twitch fibers of mice, preferentially in males. tional domains (j-SR). In skeletal muscle, this latter domain is closely asso- TAs are also found in biopsies from patients affected by TA Myopathy ciated with sarcolemmal invaginations, the T-tubules, to form specific (TAM), a muscle disorder linked to mutations in STIM1 and Orai1 (proteins junctions called triads, where proteins regulating the excitation- involved in store-operated Ca2þ entry, SOCE) We have previously shown contraction coupling mechanism assemble. Junctophilin-1 (JPH1) protein using Electron Microscopy (EM) that tubes of TAs appear linked by small tethers the membrane of the T-tubules with those of the j-SR. This interac- bridges, resembling aggregated STIM1 molecules. Here, we compared tion is mediated by eight phospholipid-binding modules (MORN) in the extensor digitorum longus (EDL) muscles from 2 groups of mice N-terminus and by a trans-membrane domain (TMD) in the C-terminus of (~4 months, adults and R24 months, aged) to determine: a) presence of JPH1. The mechanisms that regulate targeting of JPH1 at triads are STIM1 (and Orai1) in TAs; b) the relative contribution of Ca2þ entry to currently unknown. When expressed in muscle cells, GFP-JPH1 protein muscle function during repetitive stimulation in ageing muscle. Immunoflu- was localized selectively only to triads. Deletion of the TMD resulted in orescence indicates that ageing causes STIM1 (a SR protein), but not de-localization of this GFP-JPH1 deletion mutant on the surface sarcolemma Orai (that resides in TTs) to accumulate in TAs. This finding is consistent and on T-tubules. Phospholipase C-mediated PI(4,5)P2 hydrolysis decreased with: a) EM data showing that TTs are rarely seen in TAs; and the association of the TMD-deleted JPH1 with the surface sarcolemma and b) western-bot analysis showing increased expression levels of both T-tubules, indicating that MORN motifs bind PI(4,5)P2, but this interaction STIM1 splicing variants (arbitrary units: STIM1-short=0.8750.09 vs. is not sufficient to selectively direct localization of JPH1 at triads. In 1.1850.04; STIM1-long=0.8150.1 vs. 1.1150.05, respectively adult vs. contrast, progressive deletion of the cytosolic sequence of JPH1 restricted aged). During a repetitive stimulation protocol (30 x 1s-60Hz pulses every the region for triadic localization to the TMD of JPH1. Fusion of the 5 seconds): i) in 2.5 mM Ca2þ external solution, EDL muscles from aged TMD of JPH1 to SEC61ß relocated this Sec61ß-JPH1TMD fusion protein mice exhibit a decreased capability to maintain contractile (relative force to triads. Finally, site-directed mutagenesis of selected residues of JPH1 after 10 tetani in adult vs. aged: 61.053.2%, and 49.952.4%); ii) in TMD identified a short amino acid sequence required for triadic localization Ca2þ-free external solution, aged EDL muscles display a lower decay in of JPH1.

BPJ 7731_7735 Sunday, February 12, 2017 99a

498-Pos Board B263 increase in Ca2þ sensitivity is the main underlying cause of functional up- Interaction of Junctophilins and the C-Terminus of Cav1.1 Subunits regulation of SKs in diseased hearts. We developed the protocol to assess Regulates Localization and Function of L-Type Calcium Channels in Ca2þ sensitivity using simultaneous recordings of currents with intracellular Skeletal Muscle Ca2þ in voltage clamped cardiomyocytes. By converting Rhod-2 fluores- Tsutomu Nakada, Toshihide Kashihara, Mitsuhiko Yamada. cence into [Ca2þ]i we estimated Ca2þ sensitivity of rat SK2 channels over- Shinshu University School of Medicine, Matsumoto, Nagano, Japan. expressed in cells using Adenovirus gene transfer. We obtained apparent Junctophilins (JPs) are known to contribute to the stabilization of the junc- affinity of rSK2 ~ 500 nM which is in consistence with published values. tional membrane (JM) complex by bridging the plasma membrane and To test whether SK channels from diseased hearts are more sensitive to sarcoplasmic reticulum; however, the role of JPs on the JM-targeting and Ca2þ we used rat model of cardiac hypertrophy induced by thoracic aortic function of L-type calcium channels is still unclear. Thus, we explored banding (TAB). In contrast to Shams, myocytes from TAB hearts exhibited structural and functional consequences of JP knockdown (KD) in significant prolongation of APD upon application of SK inhibitor UCL-1684 C2C12 and GLT myotubes. JP1 or JP2 KD significantly inhibited the (1 mM). Voltage clamp experiments showed SK inhibitor-sensitive current JM-targeting of L-type calcium channels whereas JP2 but not JP1 KD with amplitude ~1 pA/pF. Simultaneous measurements of caffeine-induced significantly decreased the current density of L-type calcium channels. Cal- ISK (10 mM) and [Ca]i at 30 mV before and after application of UCL- cium imaging assay showed that JP1 or JP2 KD significantly decreased the 1684 demonstrated that sensitivity of SKs in TAB myocytes is low (Kd number of myotubes exhibiting calcium transient in response to electrical ~900 nM). This result suggests that sensitivity to [Ca2þ]i in hypertrophy stimulation. Co-immunoprecipitation study showed that CaV1.1 interacted is rather reduced than increased. To explore potential mechanisms respon- with JP1 and JP2 in mouse skeletal muscle. Pull down assay with sible for changes in SK sensitivity to [Ca2þ]i we treated cultured rat myo- GST-fusion proteins bearing cytosolic regions of CaV1.1 indicated that the cytes overexpressing SK2 with Phenylephrine (100 mM) and Propranolol 2þ proximal C-terminus of CaV1.1 is necessary for the binding with JPs. This (1 mM). We found that SK2 Ca dependence in Phe/Pro was shifted to JP binding domain (JPD) was well conserved between CaV1.1 and cardiac the right (Kd ~850 nM), which was preventable by coexpressing CRNK, 2þ CaV1.2, which also targeted into JM when expressed in GLT myotubes. an inhibitory peptide for ROS and Ca -dependent tyrosine kinase Pyk2. However, JPD was not conserved in neuronal CaV2.1, which did not Western blot analysis demonstrated that Pyk2 levels were upregulated in localize into JM when expressed in GLT myotubes. Reduced binding of TABs. These data implicate Pyk2 as a novel negative regulator of SK the GST fusion proteins to JPs were observed by alanine substitutions Ca2þ sensitivity in diseased hearts. of several residues in JPD. We introduced same alanine substitutions 501-Pos Board B266 in JPD of CaV1.1, and transiently expressed it in GLT myotubes. Immunocytochemical analysis revealed that the JM-targeting rate of the Modified Calcium Homeostasis in Aged Mouse Skeletal Muscle La´szlo´ Csernoch,Ja´nos Fodor, Da´na Al-Gaadi, Tama´s Czirja´k, Tama´s Ola´h, JPD-mutated CaV1.1 was significantly reduced compared to the wild Beatrix Dienes, Pe´ter Szentesi. type. These results suggested that interaction of CaV1.1 with JPs via JPD is important for the proper localization and function of L-type calcium Department of Physiology, University of Debrecen, Debrecen, Hungary. channels. In aging decreased physical activity and reduced muscle mass (sarcopenia) leads to impaired muscle force and increased fatigability accompanied by a 499-Pos Board B264 decline in sarcoplasmic reticulum (SR) calcium release. As an essential trace Dynamics of Triad Organization element selenium plays a significant role in muscle functions, as in selenium Muriel Se´bastien, Perrine Teyssier, Julie Brocard, Eric Denarier, deficiency skeletal muscle disorders manifesting in muscle pain, fatigue, Isabelle Marty, Julien Faure´. proximal weakness, and serum creatine kinase elevation could develop. Grenoble Insitut des Neurosciences, Inserm U1216, Grenoble, France. Here in vivo physical activity of control and myostatin deficient (Cmpt) Skeletal muscle contraction results of massive intracellular calcium release af- mice during 22 months were examined. Their performance in grip and volun- ter stimulation. This calcium release is mediated by the calcium release com- tary wheel tests reached its maximum 3 month after birth and declined after plex (CRC) and occurs in a very specific sub-compartment of muscle cells, the 10 month. This decrease was faster in Cmpt mice as the average daily running triads. Triads are formed by the close apposition of two sarcoplasmic reticu- distance decreased to 37% in control and to 25% in Cmpt mice in old age. lum (SR) terminal cisternae (TC) on both sides of an invagination of the In vitro force was measured on soleus and extensor digitorum longus plasma membrane, the transverse-tubule. All CRC proteins are exclusively (EDL) muscles from 20-month-old control, Cmpt, and selenium supple- localized at the triad, but the molecular mechanisms leading to their traffic mented mice. Albeit the absolute force was higher in Cmpt than in control and localization at the triads are so far unknown. Among CRC proteins, tria- mice, after normalization to cross section EDL was significantly stronger in din was proposed to act as a triad anchor for the other reticulum proteins. To the latter (4.7050.88 vs 7.6150.96 mN/mm2, respectively). Selenium investigate the mechanisms leading to triad protein targeting and to the orga- supplementation significantly increased the maximal force of EDL nization of triad membranes, we have recorded movements of fluorescent chi- (10.8950.60 mN/mm2). Changes in intracellular calcium concentration meras of triadin expressed in primary myotubes from triadin KO mice. The were measured on enzymatically isolated intact flexor digitorum brevis mus- mobility of GFP-triadin was recorded during cell differentiation. Although cle fibers using Fura-2. The rate of KCl depolarization-evoked SR calcium immobile at late differentiation stages, GFP-triadin clusters can move at release was greater in selenium supplementedthanincontrolanimals earlier stages. These movements require intact microtubule cytoskeleton and (6915100 vs 481533 mM/s, respectively). Western-blot analysis revealed may be necessary for triad organization at the A-I junctions along cell differ- no change in the expression of the dihydropyridine receptor in the three entiation. What these moving structures precisely are is still under investiga- animal groups while that of the ryanodine receptor declined with aging which tions. A photoactivatable version of triadin was used to study the dynamics of was reversed by long-term training. Our results support the positive effects of limited pools of activated molecules. At both early and late myotube differen- selenium and training on SR calcium release in old age associated muscle tiation stages, a continuous diffusion of triadin molecules in the whole SR was weakness. On the other hand, the increased muscle mass of Cmpt mice revealed. The progressive accumulation of triadin in the TC was also evi- during their lifespan doesn’t improve their physical performance in old denced, and we have shown that it depends on its C-terminal luminal domain. age. Supported by: NKFIH-K-115461 Overall, triadin clusters move along microtubules during early differentiation 502-Pos Board B267 stage probably to reach their final localization along the sarcomeres. During 2D all observed differentiation stages, a continuous diffusion of triadin allows Shear Stress Induces Transverse Ca Waves via Autocrine Activation of its traffic to the TC. Yet its retention is mediated by triadin C-terminal P2X Purinoceptors in Rat Atrial Myocytes domain. Joon-Chul Kim, Sun-Hee Woo. College of Pharmacy, Chungnam National University, Daejeon, Korea, 500-Pos Board B265 Republic of. D Assessment of Ca2 Sensitivity of SK Channels in Rat Ventricular Atrial myocytes are exposed to high shear stress during blood regurgitation D Cardiomyocytes using Intrinsic CA2 Cycling Machinery and high intra-atrial pressure due to valve diseases and heart failure, since Iuliia Polina, Radmila Terentyeva, Karim Roder, Gideon Koren, Jin O-Uchi, such disturbances disrupt endocardium. We have previously reported that Dmitry Terentyev. shear stress induces two types of global Ca2þ waves in atrial myocytes, lon- Cardiovascular Research Center, Rhode Island Hospital, Providence, RI, gitudinal and transverse Ca2þ waves (T-waves) (Biophys J 2012;102 USA. (3, Suppl 1);227a), and that the longitudinal wave is triggered by Ca2þ 2þ þ Small conductance Ca activated K (SK) channels are thought to play release via P2Y1 purinoceptor-inositol 1,4,5-trisphosphate receptor signaling significant roles in ventricular arrhythmias. It has been hypothesized that (J Physiol 2015;593:5091-5109). Here, we investigated cellular mechanism

BPJ 7731_7735 100a Sunday, February 12, 2017 for the generation of T-wave in atrial cells under shear stress. Shear stress of including the L-type calcium channel, phospholamban and RyR2. In addi- ~16 dyn/cm2 was applied onto single myocytes using micro fluid-jet, and tion, cAMP activates ‘exchange protein directly activated by cAMP’ two-dimensional confocal Ca2þ imaging was performed. Shear stress- (Epac): a cAMP-activated guanine nucleotide exchange factor (GEF). How- induced T-waves were observed repetitively under 3-4 min intervals be- ever, little is known about Epac signalling via its role as a GEF and effects tween the stimuli, and occurred at ~1 event per 10 s. They were eliminated mediated via downstream effectors such as the small GTPase Rap1. In the by inhibition of the voltage-gated Naþ or Ca2þ channels, or ryanodine re- present study on adult rat ventricular myocytes (ARVMs), the Epac2 inhib- 2þ ceptors, suggesting that the T-wave is mediated by action potential- itor ESI-05 induced prolongation of the [Ca ]i transient, followed by devel- 2þ 2þ triggered Ca release. Blockades of key stretch signaling molecules, opment of a plateau phase with [Ca ]i oscillations, characteristic of early stretch-activated channel, Naþ-Ca2þ exchange, and NADPH oxidase, did after depolarisations (EADs). This effect was blocked by pre-incubation þ not suppress T-wave generation by shear. However, shear-induced T-wave with (i) the late Na current (INalate) inhibitor ranolazine (ii) the mitochon- generation was abolished by pre-incubation of cells with external ATP- drial ROS scavenger mitoTEMPO or (iii) the CaMKII inhibitor KN93. All metabolizing enzyme apyrase, the gap junction blocker carbenoxolone, or effects of ESI-05 were mimicked by downstream inhibition of Rap1GTP with P2X purinoceptor antagonist iso-PPADS. Inhibition of P2Y1 purinergic using GGTI-298. Active Rap1 (Rap1GTP) was detectable under basal signaling that mediates the longitudinal Ca2þ wave under shear did not conditions in ARVMs and was reduced ~50% by ESI-05. These data suggest attenuate the occurrence of T-waves. Our data suggest that shear stress in- (i) that Epac2 regulates the level of active Rap1GTP in the heart and (ii) duces activation of P2X purinoceptors via gap junction-mediated ATP that active Rap1GTP may supress mitochondrial ROS production and release, thereby triggering action potential with subsequent T-wave in atrial susceptibility to EAD arrhythmias, which involve activation of CaMKII myocytes. and INalate. 503-Pos Board B268 505-Pos Board B270 Optical Mapping in Rat Models of Atrial Dilation Generation and Characterization of a Human iPSC Cardiomyocyte Model Samantha Cannazzaro1, Claudia Crocini1, Marina Scardigli1, of Troponin T I79N Linked Hypertrophic Cardiomyopathy Raffaele Coppini2, Ping Yan3, Leslie M. Loew3, Chiara Tesi2, Lili Wang1, Dmytro Oleksandrovych Kryshtal1, Kyungsoo Kim1, Elisabetta Cerbai2, Francesco S. Pavone1, Corrado Poggesi2, Shan Parikh1, Kevin Richard Bersell1, Jose R. Pinto2, Huan He3, Leonardo Sacconi1, Cecilia Ferrantini2. Bjorn Christian Knollmann1. 1LENS, Sesto Fiorentino, Italy, 2University of Florence, Florence, Italy, 1Vanderbilt University Medical Center, Nashville, TN, USA, 2Department of 3University of Connecticut Health Center, Farmington, CT, USA. Biomedical Sciences, Florida State University College of Medicine, Atrial fibrillation (AF) is commonly associated with atrial dilatation caused Tallahassee, FL, USA, 3Institute of Molecular Biophysics, Florida State by pressure or volume overload. Acute atrial stretch may create a myocar- University, Tallahassee, FL, USA. dial substrate to promote AF via stretch-activated channels and mechano- Objective: The aim of this study was to examine the functional conse- electrical feedback mechanisms. However, enhanced AF vulnerability in quences of a troponin T mutation (I79N) associated with hypertrophic car- chronic dilated atria occurs as a result of complex remodeling that includes diomyopathy (HCM) in cardiomyocytes derived from human induced changes of myocyte membrane currents, altered intracellular Ca2þ homeo- pluripotent stem cells (hiPSC). Method and Results: We generated a stasis and alterations of the extracellular matrix (e.g. interstitial fibrosis). TnT-I79N hiPSC HCM model by inserting the 236T>A mutation into the Here, we dissect the role of acute stretch-related changes versus those occur- TNNT2 gene of one of the alleles of hiPSC derived from a healthy donor ring as part of the long-term remodeling process. Spontaneous hypertensive using CRISPR/Cas9. There was no off-target cleavage produced by rats (SHR) with chronic atrial dilation were compared to normotensive CRISPR/Cas9. Nano-liquid chromatography mass spectrometry showed Wistar-Kyoto rats (WKY) and to WKY rats with acute biatrial dilation that 43% of TnT protein was mutant in cardiomyocytes (CM) derived (dil-WKY) obtained with atrial balloons (inserted into both atria and inflated from TnT-I79N hiPSCs. In 2D monolayers, the impedance amplitude with controlled pressure after baseline recording). Briefly, the hearts were (a measure of contractility) was increased and the Ca-contractility relation- perfused on a Langendorff’s apparatus and then the ventricles were excised ship leftward shifted in TnT-I79N hiPSC-CMs compared to isogenic control just below the atrio-ventricular junction. To maximize atrial perfusion and hiPSC-CM. Results were confirmed using single hiPSC-CM cultured on a staining main coronary ventricular branches were cauterized. Atria were matrigel matress: The TnT-I79N mutation altered cytosolic Ca buffering stained with a bolus injection of a red-shifted voltage-sensitive dye (di-4- properties by lowering the apparent dissociation constant Kd (0.4050.04 ANBDQPQ). An ultrafast wide-field macroscope operating at 2 KHz mM in TnT-I79N, n=12 vs. 0.7250.10 mM in control, n=16, P<0.05) (100 x 100 pixel) was developed to optically map action potential propaga- without changing the maximal binding capacity Bmax, indicating that tion of Langendorff’s perfused-atria and to test AF vulnerability. AF was TnT-I79N mutation enhanced the Ca-binding affinity of myofilaments. induced through burst pacing and occurred in 5.8% of WKY, 10.6% of Likely as a result of the increased cytosolic Ca-binding affinity, I79N dil-WKY and 17.4% of SHR atria. Action potential duration at 90% repolar- hiPSC-CM paced at 0.2 Hz exhibited significantly reduced intracellular ization (APD90) was prolonged in SHR as compared to WKY and dil-WKY. Ca-transients (DF/Fratio 0.8550.03 in I79N vs. 1.0150.06 in control, APD90 variability between contiguous regions was increased in both dil- P<0.05) and reduced caffeine transient (DF/Fratio 0.9250.06 in I79N vs. WKY and SHR compared to WKY, potentially contributing to the increased 1.1050.06 in control, P<0.05), whereas Ca-decay kinetics where un- AF vulnerability. Next, we analyzed activation maps during AF episodes. changed. Despite the reduced Ca-transient amplitude, TnT-I79N mutation Occasionally, they exhibited a stable reentry pattern characterized by similar enhanced contractility but delayed the relaxation in single hiPSC-CMs, origin and propagation on a beat-to-beat basis. Alternatevely, the origins of indicating an increased sensitivity to Ca. Conclusion: The I79N hiPSC- successive reentrant waves varied randomly and the arrhythmia was perpet- CM model reproduces key findings from I79N transgenic mice: increased uated by coexisting reentrant circuits, maintained through the continuous myofilament Ca-sensitivity, enhances contractility, impaired relaxation annihilation and creation of multiple wavelets. Voltage oscillations during and enhanced cytosolic Ca-binding affinity. Human iPSC-CM could be a AF were analyzed in time and frequency domains, and the spectrogram promising tool to model HCM in vitro. (Fourier transform) revealed coexisting reentrant circuits at different fre- quencies. Analysis of the dispersion of the action potential durations and 506-Pos Board B271 conduction velocity maps will clarify the mechanism of AF in chronic Cardiac Electromechanical Coupling Model of Myocardial Contractile and acute models of atrial dilation. Function under Ischemic Conditions Yasser Aboelkassem, Natalia Trayanova. 504-Pos Board B269 Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA. Epac2-Rap1 Signaling Influences Reactive Oxygen Species Production Hyperkalemia, acidosis, and anoxia are major pathophysiological conditions and Susceptibility to Cardiac Arrhythmias of myocardial ischemia. Cardiac arrhythmias induced by these conditions Zhaokang Yang1, Hannah M. Kirton1, Moza Al-Owais2, Chris Peers2, are widely investigated. However, the myocardium contractile function Derek S. Steele1. during acute ischemic perturbations is not fully explored. A fully coupled 1School of Biomedical Sciences, University of Leeds, Leeds, United electromechanical computational model is used to investigate cardiac Kingdom, 2Division of Cardiovascular Medicine, University of Leeds, Leeds, inotropic response under various ischemic conditions. The coupling is United Kingdom. accomplished with a detailed myofilament model (MF) that allows for a In the heart, the inotropic and lusitropic effects of b1-adrenergic stimulation dynamic feedback between both electrophysiology and contractile model are known to involve the second messenger cAMP acting via protein components. Additionally, an ATP-sensitive potassium channel (KATP) kinase A (PKA) to increase phosphorylation of intracellular targets kinetics is incorporated in the model to account particularly for anoxia

BPJ 7731_7735 Sunday, February 12, 2017 101a effects. Simulations are performed on a highly resolved cellular scale where We recently documented that phospholamban (PLB), a sarcoplasmic reticu- Ca2þ sensitivity to ischemic perturbations is well predicted and fed to the lum (SR) resident protein regulator of cardiac SR Ca-ATPase (SERCA2a), MF model, thus inotropic alterations due to ischemia can be calculated. is localized to the nuclear envelope (NE) of cardiomyocytes (CMs). It is Both Ca2þ transient and twitch dynamics are calculated to establish an well-known that heart failure (HF) remodeling involves altered protein ischemic-mechanics relationship. When compared with non-ischemic expression of PLB and SERCA2a; however, with the demonstrated compart- control case, Ca2þ levels have shown to be attenuated as a result of both mentalization of PLB accumulation, we asked whether HF alters trafficking anoxia and acidosis but not with hyperkalemia perturbations. Mechanical re- between ER/SR subcompartments affecting steady-state intracellular distri- sponses represented by twitch dynamics as triggered by these changes in bution of PLB and SERCA. In this study, we examined heart tissue from Ca2þ behavior are computed. Simulations suggest that, the activation ki- non-failing and pacing-induced HF dogs with the monoclonal PLB (2D12 netics of the KATP – channel (% f-ATP) is the main ischemic parameter or 1F1) and SERCA (2A7-A1) antibodies. Confocal immunofluorescence that significantly alters the cellular excitation-contraction coupling process microscopy was used to compare subcellular concentration of PLB and in the form of; action potential shortening, reduced excitability, delayed re- SERCA.Atleast3regionsfromNEandSRin10CMswereanalyzed covery, Ca2þ attenuation, significant decrease in the myocardium twitch for each dog, n = 4 dogs). Both PLB and SERCA antibodies stained SR production. and NE in these CMs. Fluorescence intensity ratios between NE and SR for PLB was 2.0550.82 (mean 5 SD) in control dog CMs, consistent 507-Pos Board B272 with results just reported. In HF CMs we found a highly decreased ratio Cardiomyocyte Functional Kinetic Reserve is Lost in an Ossabaw Swine of PLB between NE and SR to 1.26 5 0.34 (n = 8 dogs). In contrast to Model of Heart Failure with Preserved Ejection Fraction re-distribution of PLB, intensity ratios for SERCA in NE and SR remained 1 1 1 2 Adam B. Veteto , John L. Jones , Joel C. Robinett , T. Dylan Olver , unchanged (1.0150.14 in Control, and 0.9850.26 in HF). Furthermore, 2 1 2 Jenna C. Edwards , Michelle D. Lambert , Pamela K. Thorne , while there was only minor levels of phosphoPLB in Control CMs, we found 1 1 3 4 Maike Krenz , Kerry S. McDonald , Jaume Padilla , David A. Ford , that phosphoPLB (S16) were very significantly higher in NE of failed CMs. 2 3 2 Christopher Baines , R. Scott Rector , Craig A. Emter , The decreased PLB relative to SERCA in the NE of CMs suggests that 1 Timothy L. Domeier . retention and accumulation of PLB in the NE is decreased in HF, leading 1Medical Pharmacology and Physiology, University of Missouri Columbia, 2 to a smaller, possibly hyper-phosphorylated steady-state of PLB concentra- Columbia, MO, USA, Biomedical Sciences, University of Missouri tion in the NE. Columbia, Columbia, MO, USA, 3Nutrition and Exercise Physiology, University of Missouri Columbia, Columbia, MO, USA, 4Biochemistry and 509-Pos Board B274 Molecular Biology, Saint Louis University School of Medicine, St. Louis, The Effect of Ovariectomy on Calcium (Ca2D) Handling in Guinea Pig MO, USA. Cardiomyocytes Heart failure with preserved ejection fraction (HFpEF) comprises half of HF Hsiang-Yu Yang1,2, Anita Alvarez-Laviada1, Jahn M. Firth1, diagnoses and is clinically characterized by comorbidities including aging, Alice J. Francis1, Kenneth T. MacLeod1. obesity, hypertension, and type 2 diabetes (T2D), with increased prevalence 1National Heart and Lung institute, Imperial College London, London, in females. Animal models accurately representing clinical HFpEF are lack- United Kingdom, 2Tri-Service General Hospital, National Defense Medical ing, thus limited data exists describing functional properties of myocardium Center, Taipei, Taiwan. in HFpEF. We therefore examined cardiomyocyte function in an Ossabaw This study addressed the hypothesis that long term absence of ovarian hor- swine model of HFpEF induced by combined T2D (Western-diet; WD) mones alters calcium handling in ventricular myocytes. Female guinea pigs and pressure-overload (aortic-banding; AB). Female WD-AB animals ex- were randomly assigned to have either a bilateral ovariectomy (Ovx) or a hibited T2D, including increased body weight, HOMA-IR, hyperlipidemia, sham operation. Pellets containing 17b-estradiol (E) (1mg, 60-day release), and elevated liver enzymes (P<0.05) compared to control lean Ossabaw were placed subcutaneously in selected Ovx animals. Cardiac myocytes swine (CTL). Pressure-overload increased LV diastolic wall thickness were enzymatically isolated and action potential durations and L-type 2þ without changing internal diastolic dimension (i.e. concentric hypertrophy) Ca currents (ICa,L) were measured under current- or voltage-clamp respec- in WD-AB (P<0.05 versus CTL) with preserved ejection fraction. Enzymat- tively with a switch clamp system. Action potential morphology was inves- 2þ ically isolated cardiomyocytes were utilized to monitor intracellular Ca tigated and, while action potential duration at 90% repolarisation (APD90) homeostasis (fura-2/AM) and contractile function in response to action- remained unchanged, APD10 was 1.46 times longer and the rate of repolar- potential stimulation (0.5 Hz). Ca2þ transient and shortening amplitude isation was slower in the Ovx group. In Fluo-4 loaded cells, Ca2þ transients were similar between CTL and WD-AB, yet Ca2þ transient upstroke and were 1.34 fold larger in Ovx group compared with sham along with a greater contraction kinetics were faster (P<0.05) in WD-AB compared to CTL fractional release. Sarcoplasmic reticulum Ca2þ stores were greater by 20% with a shorter time-to-peak Ca2þ and time-to-minimum sarcomere length. in the Ovx group and these cells also showed higher frequency of Ca2þ Spontaneous Ryanodine Receptor-mediated Ca2þ spark (fluo-4/AM) fre- sparks and waves during a quiescent period following 2 Hz field- quency and amplitude were also elevated (P<0.05) in WD-AB versus stimulation. The changes occurring in the Ovx group did not take place in CTL. Following treatment with the beta-adrenergic agonist dobutamine (1 the OvxþE group. Cardiac myocytes isolated from the Ovx group showed 2þ uM), Ca transient and shortening amplitude each increased (P<0.05) increased peak ICa,L with the voltage dependence of activation and inactiva- versus baseline conditions yet remained similar between CTL and tion shifting to more positive voltages which will increase the likelihood of WD-AB groups. However, dobutamine-induced changes in kinetic parame- channel opening. The protein kinase A inhibitor H-89, resulted in less 2þ 2þ ters (time-to-peak Ca , time-to-minimum sarcomere length, Ca transient reductioninpeakICa,L in the Ovx group compared with sham. These find- recovery tau, relaxation rate) were all blunted (P<0.05) in WD-AB versus ings suggest long term absence of ovarian hormones lead to potentially CTL. In conclusion, WD-AB cardiomyocytes exhibited enhanced Ca2þ detrimental changes in Ca2þ handling mechanisms that may cause the for- and contractile kinetics under basal conditions, yet lost functional kinetic mation of a more proarrhythmic substrate. Estradiol replacement prevented reserve following beta-adrenergic challenge. Our findings suggest b-adren- these adverse effects. ergic cardiomyocyte functional reserve is impaired in a novel translational model of HFpEF. 510-Pos Board B275 Biochemical and Mechanical Properties of Murine Extraocular Muscles Jan Eckhardt1, Marijana Sekulic-Jablanovic1, Susan Treves1,2, 508-Pos Board B273 Francesco Zorzato1,2. Heart Failure Re-Distributes Phospholamban between Nuclear 1Department of Anesthesiology, University Hospital, Basel, Switzerland, Membranes and Sarcoplasmic Reticulum in Cardiomyocytes 2University of Ferrara, Ferrara, Italy. Zhipeng Tian1,2, Yan Li1,3, Peng-Sheng Chen1, Steven Cala4, Extraocular muscles (EOMs) are among the fastest and most fatigue resis- Zhenhui Chen1. tant skeletal muscles. The distinct origin and innervation of EOMs are prob- 1Medicine, Krannert Institute of Cardiology, Indiana University, ably responsible for their different gene and protein expression. Indeed, Indianapolis, IN, USA, 2Cardiology, Central Hospital Affiliated to Shenyang western blot analysis revealed that the expression of the protein isoforms Medical College, Shenyang, China, 3Geriatrics, Shengjing Hospital of China forming the Excitation-Contraction Coupling Macromolecular Complex Medical University, Shenyang, China, 4Physiology, Wayne State University, (ECC-MC) of human EOM is substantially different from human quadriceps Detroit, MI, USA. muscles. Human EOM muscles express, in addition to the skeletal muscle

BPJ 7731_7735 102a Sunday, February 12, 2017

isoforms of proteins involved in ECC, Cav1.2, CASQ2 and RYR3. Because dantrolene present, we observed that dantrolene reduced RyR activity by of the use of genetically modified mouse models to study human diseases we altering its affinity for Mg2þ. thought it important to investigate the biochemical and mechanical proper- ties of murine EOM. We found that the expression level of the ECC-MC 513-Pos Board B278 proteins in mouse EOM is different from that of mouse hind limb muscles MG53 Interacts with Cardiolipin to Protect Mitochondria from Ischemia- and from that of human EOM. We also studied the mechanical properties of Reperfusion Induced Oxidative Stress Hanley Ma1, Xinyu Zhou1, Xinxin Wang1, Junwei Wu1, Kristyn Gumpper1, mouse EOM. In the presence of 1.8 mM CaCl2 in the extracellular medium 1 1 1 1 the force induced by a single action potential and by a train of action poten- Tao Tan , Timothy Ayodele Adesanya , Chunlin Yang , Yongqiu Zheng , 5 m 5 m Heather Chandler1, Jingsong Zhou2, Jianjie Ma1, Hua Zhu1. tials delivered at 100 Hz was 40,4 35,7 N(n=8),359 213 N (n=8 ), 1 2 respectively . The half time to peak, the time to peak and the half relaxation The Ohio State University, Columbus, OH, USA, Kansas City University of times of force development evoked by a single action potential were Medicine and Bioscience, Kansas City, MO, USA. 2.5350.69 ms (n=8), 10.150.3 ms (n=8), 42.3527.8 ms (n= 8), MG53 is a tripartite motif protein that is essential to plasma membrane respectively. Addition of 100 mMLa3þ to the extrcellular medium caused regeneration. It binds to phosphatidylserine and facilitates the formation a mean decrease of 75 % of the peak tetanic tension. The effect of of repair patches at sites of plasma membrane disruption. Our previous La3þ was reversed by washing the muscle with Tyrode’s solution contain- studies have reported MG53’s efficacy in ameliorating ischemia- reperfusion (I/R) injury to multiple organs including the heart, lungs, kid- ing 1.8 mM CaCl2 This study defines the biochemical and mechanical properties of preparations of mouse EOM under normal conditions and ney, and brain. In I/R injury, mitochondria, however, are the primary victim will be used as reference to investigate the phenotype of EOM from mice of damage and the exposure of mitochondrial cardiolipin (CL) in response to carrying modifications of genes encoding key proteins encompassing the I/R-induced oxidative stress procures cell death. Thus far, the role of MG53 ECC-MC. in the preservation of mitochondrial function has not been studied. Here, we test the hypothesis that MG53 interacts with CL to protect mitochondria 511-Pos Board B276 from oxidative stress. We find that mitochondria in the heart of MG53 The Maintenance Ability and Ca2D Availability of Skeletal Muscle are knockout mouse are more susceptible to I/R injury than those of wild Enhanced by Sildenafil type mice. We also observe that MG53 targets mitochondria under chronic Mei Huang1, Keon Jin Lee1, Kyung-Jin Kim2, Mi Kyoung Ahn1, oxidative stress conditions, such as high-fat diet induced metabolic syn- Chung-Hyun Cho2, Do Han Kim3, Eun Hui Lee1. drome and amyotrophic lateral sclerosis. We demonstrate that MG53 binds 1Dept. of Physiology, College of Medicine, The Catholic Univ. of Korea, to CL through a lipid dot-blot and a quantitative ELISA lipid-protein bind- Seoul, Korea, Republic of, 2Dept. of Pharmacology, College of Medicine, ing assay. Additionally, we show that exogenous recombinant human MG53 Seoul National University, Seoul, Korea, Republic of, 3School of Life (rhMG53) protein can be up-taken by a variety of cells, including mesen- Sciences and Systems Biology Research Center, Gwangju Institute of chymal stem cells, human corneal epithelial cells, and valvular interstitial Science and Technology, Seoul, Korea, Republic of. cells. Once internalized, rhMG53 translocates to mitochondria in Sildenafil relaxes vascular smooth muscle cells, and is used to treat pulmo- response to oxidative stress induced by anoxia-reoxygenation treatment as nary artery hypertension as well as erectile dysfunction. However, the effec- revealed by live-cell imaging and 3D reconstruction of confocal micro- tiveness of sildenafil on skeletal muscle and the benefit of its clinical use scopy. These data suggest that MG53 can target damaged mitochondria have been controversial, and most studies have focused primarily on tissues through a potential CL signaling mechanism and may implicate MG53 in and organs from disease models, without cellular examinations. Here, the mitochondrial membrane protection. To our knowledge, this study is the effects of sildenafil on skeletal muscle at the cellular level were examined first to explore the interaction of MG53 with subcellular structures and pro- using mouse primary skeletal myoblasts (the proliferative forms of skeletal vides a novel mechanism for MG53-mediated amelioration of I/R-induced muscle stem cells) and myotubes along with single-cell Ca2þ imaging ex- tissue injury. periments and cellular and biochemical examinations. The proliferation of the skeletal myoblasts was enhanced by sildenafil, without dose- dependency. In the skeletal myotubes, sildenafil enhances the activity of Voltage-gated Na Channels I ryanodine receptor 1, an internal Ca2þ channel, and Ca2þ movements that promote skeletal muscle contraction, possibly due to an increase in 514-Pos Board B279 the resting cytosolic Ca2þ level and a unique microscopic shape in the Domain Specific Role of S4 for Stepping into and Recovering from the myotube membranes. Therefore, these results suggest that the maintenance Inactivated State as Obtained from Omega- and R4H Mutants in Nav1.2 ability of skeletal muscle mass and the contractility of skeletal muscle Nikolaus Guenter Greeff, Hansjakob Heldstab, Claudia Lehmann. could be improved by sildenafil via enhancing the proliferation of skeletal Biophysics Institute Greeff, Uetikon am See, Switzerland. myoblasts and increasing the Ca2þ availability of skeletal myotubes, The macroscopic time courses of activation, inactivation and recovery are respectively. voltage dependent. Control of these processes on the molecular level by the voltage sensors S4 of each domain together with the DIII-IV loop (the 512-Pos Board B277 inactivation particle) is not completely understood. With omega current D Dantrolene Shifts the Affinity of the Ryanodine Receptor for MG2 mutations (RR//QQ) along S4 we explore the position or state of S4 in Rocky H. Choi, F. Xaver Konig, Tanya R. Cully, Bradley S. Launikonis. the gating-pore and also disturb the normal gating kinetics in a domain School of Biomedical Sciences, University of Queensland, St Lucia, and state specific manner. In addition, we used the mutation S4/R4H in Australia. either DIII or DIV which slowed recovery from inactivation about 10 times; Dantrolene is the only approved drug for the treatment of life-threatening the return of the loop into the recovered position releasing the a-pore par- malignant hyperthermia episodes. It has been shown by several studies allels the appearance of the omega current reflecting the arrival of S4 in þ that the voltage-dependent Ca2 transient or force response is reduced the resting state. This confirms that both domains DIII and DIV control by dantrolene; and indeed a binding domain for dantrolene on the recovery. ryanodine receptor (RyR) has been identified. However, studies of isolated Stepping from resting into inactivated state appears more complex. Previous RyRs have mostly failed to observe alterations in the conductance of data for squid showed that the inactivation time constant tau-h has a kink at ions through the channel in the presence of clinically relevant concentra- about 10 mV. Combination with high-resolution gating currents showed tions of dantrolene. This may be the case because the ionic conditions in that below this voltage tau-h obtained its steeper voltage dependence by bilayer studies typically do not match those occurring in the muscle coupling to activation; the smaller voltage dependence of tau-h above fibre. To examine the mechanism of dantrolene action on the RyR we 10 mV appeared as result of a single inactivation voltage sensor (Greeff used mechanically skinned fibres where the imposed ionic conditions and Forster, 1991). In Nav1.2, we now see a similar kink at about mimic those occurring in the body. The skinned fibre allowed us to 10 mV. Gating currents are too small to be resolved. Instead, we use þ track the effect of dantrolene on Ca2 transients in the cytoplasm omega mutations in DI to IV to study changes of voltage dependence of elicited by action potentials; and a recently developed technique inactivation. Modifying DIV/S4 with RR//QQ along 3 positions shifts the allowed the detection of the activity of the RyR under defined ionic kink to the left and seems to reduce the voltage dependence indicating the conditions, including basal levels of activity, with a high degree of sensi- role of DIV/S4 as voltage sensor of inactivation. In contrast, in domain I þ þ tivity. By varying the [Ca2 ]and[Mg2 ] of the cytoplasm, with and without to III the resting state RR//QQ mutation does not change tau-h. However,

BPJ 7731_7735 Sunday, February 12, 2017 103a omega mutations in the inner region of DI, II and III, especially DII34QQ, 517-Pos Board B282 increased tau-h substantially combined with a strong right shift of the kink. Effects of Cysteine Substitutions In D1-S6 on Fast and Slow Inactivation in Conclusion for Nav1.2: Recovery from inactivation clearly depends on S4 in Nav1.4 DIII and DIV. Going into the inactivated state, i.e. closing of the a-pore by John O’Reilly, Penny Shockett. the DIII-IV loop, needs as prerequisite the activation of DI and DII at lower Southeastern Louisiana University, Hammond, LA, USA. voltages, while at higher voltages the slower DIV is rate limiting. The relation Previous studies in voltage-gated Na channels (Navs) have suggested a role of DIII to a specific function when stepping up is less clear from these for segment 6 of domain 1 (D1-S6) in slow inactivation of Navs. In the experiments. current studies, we used scanning site-directed mutagenesis to substitute cysteine residues for the native amino acids in D1-S6 of the human skeletal 515-Pos Board B280 muscle Nav isoform (hNav1.4) spanning from the putative ‘‘gating hinge’’ Divergence in Domain IV of an Electric Fish NaV Channel Tunes its Fast to the ‘‘bundle crossing’’ of S6 segments. Presently, we have obtained Inactivation to Support Rapid Firing Rates by Electro-Motorneurons data from mutants I439C, N440C, L441C, I442C, and L443C. Compared Daniel Thomas Infield1, Ammon Thompson2, Troy Smith3, with wild-type hNav1.4, the activation curve (G-V) was only affected Harold H. Zakon2, Christopher A. Ahern1. 1 (increased) in the slope factor (k) of L443C. The V1/2 of the steady-state Department of Physiology and Molecular Biophysics, University of Iowa, fast inactivation curve (hN) in N440C was hyperpolarized and the slope Iowa City, IA, USA, 2Department of Neuroscience, University of Texas, 3 was increased. The slope factor for hN was also increased in I442C and Austin, TX, USA, Department of Biology, University of Indiana, L443C. In slow inactivation, all mutants differed from hNav1.4. Enhanced Bloomington, IN, USA. development of slow inactivation (faster entry) was observed in N440C In some neuronal cell types, persistent or resurgent current through voltage- and I442C, while I439C, L441C, and L443C all showed resistance to devel- gated sodium channels enables the regular firing of ‘‘spontaneous’’ opment of slow inactivation. Steady-state slow inactivation (SN)wasaltered action potentials from a few to a hundred Hz. However, the neurons in I439C (depolarized V1/2 and increased k) and N440C (hyperpolarized V1/2 with the fastest spontaneous firing rates known are found in the nocturnal and decreased k). We conclude that cysteine substitutions in D1-S6 of ghost knifefish (family apteronotidae), which accomplish active electroloca- hNav1.4 disrupt the normal molecular gating kinetics of slow inactivation tion via a unique neuronal electrical organ that spontaneously fires action in hNav1.4. Ongoing and future experiments include analysis of additional potentials at rates exceeding 1000 Hz. Voltage-clamping of electro- cysteine mutants in D1-S6 and application of methanethiosulfonate (MTS) motorneurons from apteronotidae revealed voltage-gated sodium currents reagents to examine potential molecular rearrangements of D1-S6 during with incomplete fast inactivation (i.e., the existence of a persistent sodium slow inactivation gating. current). Unexpectedly, RT-PCR revealed that Nav 1.4b is the dominant Nav isoform in the apteronotid spinal cord, and it is expressed here nearly 518-Pos Board B283 D exclusively. We found that this gene contains five apteronotid-specific Molecular Mechanisms of Cardiacvoltage-Gated Na Channel Regulation substitutions in the S4-S5 cytoplasmic linker of Domain IV, a region that by Acidic pH has been implicated in the process of fast inactivation of mammalian voltage Bicong Li, Wandi Zhu, Jon Silva. gated sodium channels. Using two electrode voltage clamp electrophysi- Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, ology, we assayed the effects of making combinations of these substitutions USA. in the human cardiac sodium channel Nav 1.5 (R1644W, L1647W, M1651R, Background: During ischemic heart disease, pH drops from 7.4 to 6.0 within I1660F, G1661S). Interestingly, when all five apteronotid substitutions are 10 minutes of onset, severely affecting ion channel gating. The cardiac so- incorporated into DIV S4-5 of Nav 1.5, a persistent sodium current is dium channel (Nav1.5) is particularly susceptible to this abrupt pH change, observed that is qualitatively similar to that of the apterotontid Nav 1.4b and its altered gating is thought to predispose patients suffering ischemia to in native cells. In addition, the effects observed in some partial substitutions arrhythmia and sudden cardiac death. We observed the voltage-sensing do- (for example, loss of voltage dependence of inactivation in R1644W/ mains (VSDs) of NaV1.5 to discover molecular mechanisms of its regulation M1651R-Nav 1.5), were apparently lost in the context of the quintuple- by pH. mutant. Taken together, these results suggest that the evolution of rapid Methods: A cysteine mutation was made in each of the four VSDs (DI-DIV) of electrical organ discharge in knifefish was driven by the tissue-specific Nav1.5. Synthesized RNA from these constructs was injected into Xenopus expression of and sequence divergence within the voltage gated sodium oocytes. Once channels were expressing, a fluorophore was tethered to the channel Nav 1.4b. Moreover, they help define the structural requirements cysteine via a disulfide bond. By measuring the kinetics and change in magni- within the DIV S4-S5 linker for normal inactivation in mammalian sodium tude of the fluorescence, we were able to track VSD conformational changes channels. along with the current-voltage relationship. Results:Reducing the pH of the extracellular solution from 7.4 to 6.0 causes INa 516-Pos Board B281 to decrease in magnitude by 50%, and shifts in both activation and fast inacti- Role for Fast Inactivation in Domain I of Voltage Gated Sodium Channels vation rightward, consistent with previous results. At a pH of 6.0, time to peak James R. Groome, Ryann Camp. was reduced by 300% while inactivation was only 10% slower. Observation of Biological Sciences, Idaho State University, Pocatello, ID, USA. the VSDs showed that the DII-VSD is not affected by pH, and the DIII-VSD In sodium channels, the voltage sensing S4 segments move outward in showed a small depolarizing activation shift ~6.65 mV. The DIV-VSD response to membrane depolarization to promote activation and fast inactiva- displayed a complex phenotype, not shifting after short pulses, but shifting tion of the channel. Domain-specific roles for S4 segments include activation prominently (23.27 mV) after prolonged pulses. Its kinetics were also slowed and fast inactivation. In the present work we investigated the role of domain I by a factor of 2 at a pH of 6.0. in activation, deactivation and fast inactivation of the skeletal muscle sodium Conclusions:These results suggest an important role for the DIV-VSD in channel hNaV1.4. Charge reversal at R1 (R219D) produced a significant determining regulation of NaV1.5 by pH. hyperpolarizing shift of the activation curve, an effect confirmed in an inac- tivation deficient background (R219D/QQQ) and with gating currents. Charge 519-Pos Board B284 reversal at residues lower in the S4 segment (R225D, K228D) produced a de- Omega Mutations along S4 in Nav1.2 Channels Give Insight into Domain polarizing shift of the activation curve. R219D selectively enhanced fast inac- Specific Contribution to Activation and Steady State Inactivation tivation from closed states (hyperpolarizing shift of the steady-state fast Claudia Lehmann, Hansjakob Heldstab, Nikolaus Guenter Greeff. inactivation curve, and accelerated kinetics of closed-state fast inactivation. Biophysics Institute Greeff, Uetikon am See, Switzerland. Fast inactivation from the open state was not affected by R219D. Lesser We previously identified the resting state positions of the voltage sensor S4 effects on closed-state fast inactivation were observed for charge reversal at for each domain of Nav1.2 by means of omega mutations. We found that a DIS4 residues lower in that voltage sensor. Charge reversal at inner negative double gap is needed to open the omega pore (narrow part of the countercharges (E171R, S2; D197R, S3) also produced a hyperpolarizing shift gating pore) resulting in detectable omega current, also known as gating of the steady-state fast inactivation curve and accelerated kinetics of closed- pore current. At hyperpolarizing conditions, the resting state of S4 was state fast inactivation. These results suggest that in the wild type NaV1.4 found for double gap RR1,2QQ in domain I, II and IV and for double gap channel, interactions of inner negative charges with the outer charge in RR2,3QQ in domain III. In this work we evaluated additional conforma- DIS4 restrict outward movement of that voltage sensor in response to weak tional states of the voltage sensor S4 moving through the gating pore depolarization, and limit fast inactivation elicited from closed states. This by further double gap mutations along S4 (second double gap 2,3QQ work was supported by NIH 1R15NS093579-01A1 to JRG and NIH and third double gap 3,4QQ). Two electrode voltage clamping on X. laevis P20GM103408 to ISU. oocytes expressing rat brain sodium channels Nav1.2 was used to measure

BPJ 7731_7735 104a Sunday, February 12, 2017 macroscopic ionic current through the alpha pore and, if present, omega WT on a separate construct suggesting gating cooperation between current through the omega pore. In DI and DII we detected clear channels. Therefore, our objective was to investigate whether trafficking- outward omega current for S4 mutants RK3,4QQ at depolarizing conditions. efficient but gating-deficient channels could also lead to a dominant- Furthermore, we found that activation of sodium currents was right shifted negative effect by impairing the gating of the WT. Methods: Cell surface by about 30 mV towards higher potentials compared to the first and second biotinylation was used to assess trafficking efficiency of the channel and double gap mutants or wild-type sodium channel. These findings its presence at the cell surface. Current density was measured using the suggest two sequential gating steps of S4 between resting and activated state whole-cell configuration of the patch-clamp technique. Open probability in both domains DI and DII. In DIII and DIV no clear outward omega of sodium channels was measured using single-channel recordings in the current could be detected at depolarized potentials either for the second or cell-attached configuration. Results: Co-expression of WT with the for the third double gap mutant. However steady state inactivation was dominant-negative mutant Nav1.5-L325R led to a dominant-negative effect. strongly left shifted by about 50 to 100 mV to more hyperpolarized poten- However, the presence of difopein, a 14-3-3 protein inhibitor, was able to tials for the second and third double gap mutant in both domains, consistent abolish this effect leading to restored current density. Surprisingly, cell with involvement in recovery from inactivation and immobilization, surface biotinylation results showed that the channel density at the cell sur- respectively. face was not altered when WT and L325R mutant were coexpressed together, even though we observed a dominant-negative effect when cur- 520-Pos Board B285 rents were measured. Interestingly, our single channel recordings showed Characterization of a NaV1.5 Gain-of-Function Mutation (G213D) causing that Nav1.5 alpha-subunits display coupled gating properties. Importantly, Multifocal Atrial and Ventricular Premature Ectopies and an Increased this effect was abolished in presence of difopein, the 14-3-3 protein inhib- Risk of Dilated Cardiomyopathy itor. Hence, this uncoupling of the WT and mutant channels seen at the 1 2 3 Kirstine Calloe , Anders K. Broendberg , Alex H. Christensen , single-channel level could explain the suppression of the dominant- 4 5 1 6 Lisbeth N. Pedersen , Morten S. Olesen , Maria A. Tejada , Soren Friis , negative effect observed at the whole-cell level. Conclusions: Our data 7 3 2 Morten B. Thomsen , Henning Bundgaard , Henrik K. Jensen . strongly support that dominant-negative suppression exerted by dominant 1Department of Veterinary Clinical and Animal Science, University of 2 negative mutant is not only due to trafficking deficiency but could also be Copenhagen, Copenhagen, Denmark, Department of Cardiology, Aarhus due to impairment of the WT gating probability. University Hospital, Aarhus University Hospital, Aarhus, Denmark, 3Unit for Inherited Cardiovascular Diseases, the Heart Centre, National University 522-Pos Board B287 4 Hospital, University of Copenhagen, Copenhagen, Denmark, Department of Open and Closed States of the NaVAb Activation Gate Molecular Medicine, Aarhus University Hospital, Aarhus, Denmark, Michael Lenaeus, Tamer M. Gamal El-Din, Karthik Ramanadane, 5Laboratory for Molecular Cardiology, the Heart Centre, National University Ning Zheng, William A. Catterall. Hospital, University of Copenhagen, Copenhagen, Denmark, 6Nanion University of Washington, Seattle, WA, USA. 7 Technologies, Munich, Germany, Department of Biomedical Sciences, The bacterial voltage-gated sodium channel NavAb is a structural model for University of Copenhagen, Copenhagen, Denmark. eukaryotic voltage-gated sodium channels, and it has been crystallized in Background: Mutations in SCN5A have been linked to different cardiac both the pre-open and inactivated states (Payanadeh et al 2011, 2012). diseases including multifocal ectopic Purkinje-related premature contrac- These structures revealed the general architecture of bacterial voltage- tions (MEPPC) characterized by ventricular ectopy and dilated cardiomyop- gated sodium channels and provided insight into mechanisms of voltage- athy (DCM). Here we characterize the Nav1.5 G213D variant found in a sensing, electromechanical coupling, and the selective conductance of large Danish family with frequent premature ventricular and atrial contrac- sodium ions. Comparison with structures of other bacterial sodium channels tions, frequent nonsustained ventricular tachycardia and DCM. Methods (Zhang et al 2012, Bagneris et al 2013) has also informed hypotheses of and Results: The family was clinically assessed and an MEPPC-like activation gating, an opening transition now thought to involve twisting phenotype including complex atrial and ventricular arrhythmias and dilated movements of the S6 helix that ultimately result in an iris-like dilation of cardiomyopathy was found. Genetic screening revealed a G213D Nav1.5 the intracellular ends of the four S6 segments. We sought to capture closed missensemutationinthelinkofsegment3and4indomain1ofthe and open states in the same channel protein through x-ray crystallography Nav1.5 protein. The MEPPC-like phenotype co-segregated with G213D. and electrophysiological studies of mutations in the S6 of NavAb. Our Electrophysiological studies of wild type (WT) hNav1.5 and hNav1.5_ studies show that paired mutations of hydrophobic residues in the S6 helix G213D expressed in CHO-K cells was performed using conventional can uncouple voltage-sensor movement from pore opening and thereby lock patch clamp and a SynchoPatch 384PE (Nanion Technologies) high the channel in a permanently closed state in which the intracellular ends of throughput automatic patch clamp. The half-maximal inactivation (V½) the four S6 helices interact tightly with each other. On the other hand, trun- was significantly more negative for hNav1.5_G213D compared to WT and cation of the intracellular extensions of the S6 segments results in an open the V½ of steady-state inactivation was shifted towards more positive conformation of the activation gate. These analyses allow for comparison of values for hNav1.5_G213D, resulting in increased window-currents. This the closed, open, and inactivated states of NavAB’s activation gate, a first suggests that hNav1.5_G213D activates at more negative potentials and a for a voltage-gated sodium channel. Our results help confirm and sharpen larger fraction of channels will recover from inactivation during the diastole the previously hypotheses for the pore-opening transition of sodium chan- resulting in a gain-of-function phenotype. A combination of wild type and nels and reveal key conformational changes that connect the closed, open, G213D mimicking the heterozygote state exhibited an intermediate pheno- and inactivated states of NavAb. type. Flecainide or amiodarone resulted in a markedly reduced number of premature atrial and ventricular contractions in patients. Conclusions: 523-Pos Board B288 In Vitro Single-Molecule Study of Nachbac using Planar Lipid Bilayer The G213D Nav1.5 variant is associated with a gain-of-function and is associated with multifocal atrial and ventricular ectopy and dilated Device 1,2 1,2 2,3 1,2 cardiomyopathy. Hiofan Hoi , Andrew Jo , Manisha Gupta , Carlo D. Montemagno . 1Chemical and Materials Engineering, University of Alberta, Edmonton, AB, 521-Pos Board B286 Canada, 2Ingenuity Lab, Edmonton, AB, Canada, 3Electrical and Computer Voltage-Gated Sodium Channel Mutations can Exert Dominant-Negative Engineering, University of Alberta, Edmonton, AB, Canada. Suppression by Coupled Gating Voltage-gated sodium channels (Navs) are essential component for the gen- Jerome Clatot1, Aurore Girardeau2, Celine Marionneau2, eration and propagation of electric signals in excitable cells. The successes Isabelle Deschenes1. in the biochemical, biophysical and crystallographic studies on prokaryotic 1Case Western Reserve University, Cleveland, OH, USA, 2Universite de Nav in recent years has greatly promote the understanding of the molecular Nantes, Nantes, France. mechanism underlie these proteins and their eukaryotic counterparts. In this Introduction: Mutations in voltage-gated sodium channels have been linked paper, we aim to investigate the conductance and ionic selectivity of the to channelopathies such as cardiac arrhythmias, epilepsy and myotonia. prokaryotic Nav NaChBac at single-molecule level. Purified NaChBac pro- We previously demonstrated that trafficking-deficient mutant channels could tein was first reconstituted into lipid vesicles to form proteoliposome, which lead to a dominant-negative effect by impairing trafficking of the wild-type is subsequently incorporated into lipid bilayer with known lipid composition cardiac sodium channel (WT). We and others have also reported that sodium by proteoliposome fusion and studied using a planar bilayer device. We channel polymorphisms present on one construct can affect the gating of the were able to insert a single NaChBac into the bilayer by using a high

BPJ 7731_7735 Sunday, February 12, 2017 105a lipid-to-protein ratio for the proteoliposome preparation. At single-molecule with competitive binding of Naþ and Kþ, shows how modifying the struc- level, we observed three distinct conductance sub-states, 26 5 6pS ture and fluctuations of the selectivity filter, either directly or indirectly, al- (mean 5 SEM, n=4), 92 5 18 pS (n=3), and 268 5 45 pS (n=2), respec- ters both the ion conduction mechanism and the ionic selectivity of the tively. The first two values are comparable to previous reported ones that channel. These findings have implications for the study of cation permeation were obtained by single-channel patch-clamp and multi-channel recording and selectivity in a large class of tetrameric ion channels containing acidic with planar bilayer device, respectively. But the third conductance sub- side chains in the conduction pore. state is reported for the first time. In addition, our data at single-molecule recording shows similar permeability among Naþ,Kþ and Ca2þ, indicating 526-Pos Board B291 that the reconstituted NaChBac is non-selective in the artificial membrane An Open State Model of the Navab Channel Explored by Rosetta and environment. Study of NaChBac at single-molecule level in an artificial Molecular Dynamics Simulation 1 1 2 environment reveals new properties that were not observable in in vivo Phuong T. Nguyen , Kevin R. DeMarco , Igor Vorobyov , 2 3,4 5 studies. Our results provide novel insight to understanding the biophysical Coleen E. Clancy , Toby W. Allen , Vladimir Yarov-Yarovoy . 1Biophysics Graduate Group, UC Davis, Davis, CA, USA, 2Department of properties of Navs. Pharmacology, UC Davis, Davis, CA, USA, 3School of Applied Sciences, 524-Pos Board B289 RMIT, Melbourne, Australia, 4Department of Chemistry, UC Davis, Davis, 5 Control of Slow, Use Dependent Inactivation of NaVAb by its C Terminal CA, USA, Department of Physiology and Membrane Biology, UC Davis, Tail Davis, CA, USA. Tamer M. Gamal El-Din, Michael J. Lenaeus, Karthik Ramanadane, Voltage-gated sodium (Nav) channels play a pivotal role in propagating Ning Zheng, William A. Catterall. electrical signals in excitable cells and key targets for development of novel Pharmacology, University of Washington, Seattle, WA, USA. therapeutics. Despite recent progress in determining x-ray structures of bac- Bacterial voltage-gated sodium channels are composed of four identical terial Nav channels in closed, inactivated, and partially open states, we are subunits. They share major biophysical features with eukaryotic counter- still missing a stable open state structure of a Nav channel that will be useful parts. Crystallization of full-length bacterial sodium channels (Payandeh to study ion conduction, channel gating and drug - channel interactions. We et al. 2011, 2012; Zhang et al. 2012) makes them invaluable models for used Rosetta molecular modeling software to build the full-length open state studying the structural basis of ion conductance, activation, inactivation, model of the bacterial NavAb channel. The fully activated state of the and drug interaction. Mammalian sodium channels have two main types NavAb voltage sensor domain (VSD) was modeled using the crystal struc- of inactivation: fast inactivation on the order of milliseconds, which is ture of NavRh channel VSD as a template. The initial open state of NavAb mediated by the IFM motif in the intracellular loop between domains III pore was modeled using the crystal structure of NavMs with partially open and IV; and slow inactivation on the order of hundreds of milliseconds to pore. Rebuilding the interacting regions of NavAb (S3, S4, S5, S6, and S4- seconds, which is believed to occur through pore collapse. The bacterial so- S5 linker) using Rosetta loop modeling and relax approaches was essential dium channel NavAb activates at very negative membrane potentials, and it to generate stable NavAb open state model. Output models were evaluated has a late use-dependent phase of slow inactivation that reverses very using RosettaMembrane energy function and Rosetta clustering approach. slowly (Gamal El-Din et al. 2013). We studied the effect of the NavAb The top Rosetta models represented different alternative open states of the C-terminal cytoplasmic domain on use-dependent slow inactivation. Dele- NavAb channel. We have conducted a set of molecular dynamics (MD) sim- tion of 40 residues of the cytoplasmic tail (a40) abolished late use- ulations on the NavAb channel open state model using the Anton supercom- dependent inactivation. Progressively smaller deletions to yield a28, a10, puter to examine its stability. The simulations revealed that the NavAb a7, and a3causedgradedeffects.However, deletion of only 10 residues channel open state model reproducibly sustained a fully hydrated open was sufficient to abolish most of the late, use-dependent inactivation of pore that conducted sodium ions under an applied membrane potential for NavAb. In addition to modulating the extent of use-dependent inactivation, periods of hundreds of nanoseconds. These results suggest that our structural the progressively truncated constructs showed decremental slowing of the modeling approach could be useful for modeling multiple states of other ion decay of sodium current during depolarizations. NaVAb-a40 has voltage- channels and for rational design of novel therapeutics targeting ion independent kinetics of current decay, while other constructs have differen- channels. tial profiles of decay kinetics that are correlated with C-terminal length. Our experiments reveal a surprisingly crucial role for the C-terminal 527-Pos Board B292 domain, which forms a coiled-coil structure unique to bacterial sodium Simulating the Access and Binding of Subtype Selective Sodium Channel channels, in both early and late phases of use-dependent slow inactivation Inhibitors Ben Corry. of NaVAb. Research School of Biology, Australian National University, Acton, ACT, Australia. 525-Pos Board B290 Sodium channel blockers are commonly used as local anaesthetics, anti- Role of Channel Fluctuations in Ion Transport and Selectivity in Bacterial arrhythmics and anti-epileptics, however they generally cannot distinguish Sodium Channel NavAb between the different sodium channel subtypes expressed in humans. The 1,2 1 3,4 Christopher Ing , Nilmadhab Chakrabarti , Ning Zheng , development of highly selective channel inhibitors will allow for a range of William A. Catterall3,Re´gis Pome`s1,2. 1 new clinical applications and a decrease in side effects. To help achieve this Molecular Structure and Function, Hospital for Sick Children, Toronto, ON, aim we here examine how two recently discovered subtype selective voltage Canada, 2Biochemistry, University of Toronto, Toronto, ON, Canada, 3 4 sensor inhibitors interact with a bacterial sodium channel (NavAb) and with Pharmacology, University of Washington, Seattle, WA, USA, Howard the eukaryotic channel Nav1.7 (as part of a Nav1.7/NavAb chimera). Using a Hughes Medical Institute, Seattle, WA, USA. range of advanced sampling techniques and tens of ms of molecular dynamics The elucidation of high-resolution structures of voltage-gated sodium chan- simulations we show both where these compounds bind and how they access nels has opened the way to elucidating the mechanism of sodium permeation this site. Our simulations help unravel how subtype selectivity is achieved and selectivity. Molecular simulation studies of bacterial sodium channel and are able to accurately predict the binding affinity in each channel. This NavAb (Chakrabarti et al., PNAS 110, 11331-11336, 2013) suggested that highlights that for these compounds binding is much weaker for bacterial þ Na binding and permeation through the selectivity filter are coupled to channels than for Nav1.7. The simulations also answer the puzzle as to why the conformational isomerization of the Glu177 side chains of the EEEE the channels have to be held in the inactivated state for prolonged periods in ring from an outfacing conformation to a lumen-facing conformation, result- þ order to measure inhibition, as the compounds face large barriers to access ing in a high rate of Na diffusion through the selectivity filter. To clarify the binding site due to specific interactions with the protein. These results the role of channel dynamics on ion permeation and selectivity, we examine will assist in developing strategies to speed up the kinetics of inhibition and the mechanism of ion permeation in various systems in which either the na- to improve selectivity of the inhibitors for specific sodium channel subtypes. ture of the EEEE ring or the extent of channel fluctuations have been modi- þ þ fied. Specifically, we study how the molecular mechanism of Na and K 528-Pos Board B293 permeation in NavAb is affected by protonating a single Glu177 side chain, Propofol is a Potent Gating Modifier of Voltage-Gated Sodium Channels by replacing all four Glu177 side chains by Asp, by preventing conforma- Elaine Yang1, Daniele Granata2, Roderic Eckenhoff3, Vincenzo Carnevale2, tional isomerization of Glu177 side chains, by removing the voltage- Manuel Covarrubias1. sensing domains, and by introducing artificial structural restraints on the 1Vickie and Jack Farber Institute and Department of Neuroscience, transmembrane helices of the pore domain. The analysis of unbiased equi- Thomas Jefferson University, Philadelphia, PA, USA, 2Institute for librium simulations totalling over 200 microseconds, including simulations Computational Molecular Science, Temple University, Philadelphia, PA, USA,

BPJ 7731_7735 106a Sunday, February 12, 2017

3 Department of Anesthesiology and Critical Care, University of Pennsylvania The availability of recent high-resolution structures for bacterial Nav chan- School of Medicine, Philadelphia, PA, USA. nels makes them ideal tools for exploring functional mechanisms. Bacterial þ þ Voltage-gated Naþ channels (Navs), which play a pivotal role in the electri- and mammalian channels have been proposed to select for Na over K via cal excitability of the central nervous system, are inhibited by clinically different mechanisms, owing to their distinct selectivity signature se- relevant concentrations of many general anesthetics and are thus highly rele- quences, with bacterial channels making use of a 4-fold ring of Glu side vant anesthetic targets. The molecular mechanisms of this inhibition, how- chains, in place of Asp, Glu, Lys and Ala (DEKA) in mammalian channels. þ ever, remain unclear. Here, we investigated the electrophysiological We have investigated the underlying causes of Na selectivity by carrying response of NaChBac and NaVMs, two anesthetic-sensitive bacterial homo- out multi-ms fully atomistic simulations that capture long time-scale protein logs of eukaryotic Navs, to the intravenous anesthetic propofol at 2, 5, and movements essential for ion permeation. Our previous bacterial channel 10 mM. In both NaChBac and NaVMs, propofol induced hyperpolarizing simulations demonstrated that the ability of the high-field strength Glu þ shifts of the pre-pulse inactivation and conductance-voltage (G-V) relation- ring to accommodate 2 Na ions provides more efficient conduction for þ þ ships, reduced the time constant of inactivation, and increased the time con- Na than for K (for which only 1 ion binds to this ring). To probe stant of deactivation in a dose dependent manner, without significant effects conduction mechanisms in mammalian channels, a model of Nav1.2 was on recovery from inactivation. Previous investigations suggested that gen- constructed by grafting residues of its selectivity filter and external eral anesthetics might inhibit NaChBac by open channel block, much like vestibular regions into bacterial NavAb and NavRh channels. We have local anesthetics. Contrary to predictions based on a mechanism of pore observed similarities in the multiple ion mechanisms, enabled by blockade, however, propofol induced hyperpolarizing shifts in the G-V carboxylate-ion complexes involving DEKA and carboxylates of the vestib- curve with minimal effects on peak current and current decay in a non- ular region, previously thought to play passive roles in modulating conduc- inactivating NaChBac mutant at both 2 and 5 mM. Propofol may bind to tion. Our simulations also demonstrate that the DEKA Lys is an active the channel to stabilize the open and inactivated-open states, a mechanism participant in permeation, with its protonation state influencing the conduc- supported by kinetic modeling. Guided by molecular dynamics simulations, tion mechanism. Our findings suggest a possible common mechanism for þ we are evaluating putative propofol binding sites in the pore and voltage- Na -selective conduction across bacterial and mammalian Nav channels, þ sensing domains with electrophysiology and mutational analysis to identify and provide insight into the origins of Na -selective binding across the structural determinants of Nav gating involved in modulation by propofol. proteome. Furthermore, using the propofol analog ‘‘fropofol’’, which contains a fluo- rine substitution that selectively weakens its hydrogen bonding capability, 531-Pos Board B296 we are also evaluating the contribution of the propofol 1-hydroxyl to molec- How C-Terminal Domain Stabilize the Gate of Voltage-Gated Sodium ular recognition. Channels Song Ke1, Bonnie Ann Wallace2, Jakob Ulmschneider1, 529-Pos Board B294 Martin Ulmschneider3. The Sodium Ion Binding Region at the Focus of P1 Helices Attracts both 1Institute of Natural Sciences, Shanghai Jiaotong University, Shanghai, Charged and Electroneutral Ligands of Sodium Channels China, 2Department of Biological Sciences, Birkbeck College, University of Denis B. Tikhonov1, Boris S. Zhorov1,2. London, London, United Kingdom, 3Department of Materials Science and 1Sechenov Institute, RAS, St. Petersburg, Russian Federation, 2Biochemistry, Engineering, Johns Hopkins University, Baltimore, MD, USA. McMaster University, Hamilton, ON, Canada. Sodium ion conduction plays pivotal role in action potential of excitable cells; The inner pore of eukaryotic voltage-gated sodium channels is targeted this selective, rapid and transient process is regulated by the opening, inactivat- by ligands of dramatically different chemical structures. These include ing and closing of voltage-gated sodium channels (Navs). organic cations such as a local anesthetic lidocaine and electroneutral drugs A wide variety of life-threatening diseases such as epilepsy, cardiac such as an anticonvulsant carbamazepine. Mutations of the critical phenyl- arrhythmia, and chronic pain syndrome were attributed to pathogenic muta- alanine residue in helix IVS6 and some other inner pore-facing residues are tions of human sodium channels; hence these channels are subject to extensive known to affect action of both charged and neutral ligands. The structural structural and functional studies and enlisted as key targets for drug cause of this ligand-binding promiscuity of sodium channels is unclear. development. Here we used the X-ray structure of a prokaryotic sodium channel NavMs Several crystal structures of bacterial sodium channels (BacNavs) have been to model the pore domain of the Nav1.x channels. We further employed published recently (J. Payandeh et al. 2011; Jian Payandeh et al. 2012; Zhang the Monte Carlo energy-minimization method to perform intensive docking et al. 2012; McCusker et al. 2012; Bagne´ris et al. 2013; Bagne´ris et al. 2014; of lidocaine and carbamazepine from thousands starting points in the Shaya et al. 2013), which serve good starting points in understanding structural inner-pore region. The sodium ion NaIII, which is located between the and mechanistic details of Navs. Although the proteins are present in different four backbone carbonyls at the C-ends of P1 helices and does not make functional states, these studies render a consensus transmembrane architecture direct contacts with the channel protein, attracted carbamazepine, but for BacNavs. Transmembrane (TM) helices 1 to 4 form the voltage sensor repelled lidocaine. Therefore we further docked electroneutral ligands domain (VSD) to sense the voltage changes a cross the cell membrane. In addi- (lamotrigine, carbamazepine, phenytoin, lacosamide and bisphenol A) and tion, TM5 and TM6 form the pore domain (PD) to selectively allow Naþ ion cationic ligands (lidocaine, QX-314, cocaine, quinidine, and sipatrigine) passage across membrane. in the channel models, respectively, with and without NaIII. In our models Besides that, the cytoplasmic C-terminal domain (CTD) that follows the all the ligands interacted with the phenylalanine residue in IVS6 and most pore-lining TM6 transmembrane helices has two parts: a membrane proximal of the ligands also interacted with the tyrosine residue in IVS6. Some charged region termed the ‘‘neck’’ and a unique ‘‘coiled-coil’’ region for ligands extended their moieties in the III/IV sidewalk (fenestration). The BacNaVs. Structural and electrophysiological studies suggest this CTD has electroneutral ligands bound the sodium ion with their electronegative fundamental role in maintaining channel activity and recovery from channel groups and lacosamide chelated this ion. The ligand-bound ion remained inactivation (Tsai et al. 2013; Bagne´ris et al. 2013; Shaya et al. 2013) close to the NaIII position due to attraction to the pore-facing backbone Here, molecular dynamics (MD) simulations elucidate key interplays between carbonyls. The same region attracted the charged group of the cationic li- CTD and other domains such as, VSD and PD, and shed lights on the mecha- gands. In the predicted binding modes even small-size ligands would block nistic roles of this domain during channel gating. the ion permeation by the electrostatic and steric mechanisms. Our study proposes a common pharmacophore for the diverse ligands. It includes a 532-Pos Board B297 cation (the ligand’s ammonium group or the ligand-bound sodium ion) Molecular Modeling of Mammalian Nav1.4 Channel Ali O. Acar1, Esra Korpe1, Murat Cavus2, Serdar Kuyucak3, Turgut Bastug4. and an aromatic moiety, which are usually linked by four bonds. Supported 1 by NSERC and RFBR. Micro and Nanotechnology, TOBB University of Economics and Technology, Ankara, Turkey, 2Faculty of Education, Bozok University, 530-Pos Board B295 Yozgat, Turkey, 3School of Physics, Sydney University, Sydney, NSW, Comparison of Ion Selectivity Mechanisms in Bacterial and Mammalian Australia, 4Material Science and Nanotechnology Engineering, TOBB Sodium Channels University of Economics and Technology, Ankara, Turkey. Emelie Flood,Ce´line Boiteux, Toby W. Allen. Cell membranes consist of two layers of lipid molecules and are impermeable RMIT University, Melbourne, Australia. to ions. Ion transportation across the membrane is mediated via membrane pro- Voltage-gated sodium (Nav) channels play essential roles in electrical sig- teins which are pumps, transporters and ion channels. Voltage gated sodium nalling in the body and are associated with many physiological disorders. (NaV) channels are essential elements in ion channels family that responsible

BPJ 7731_7735 Sunday, February 12, 2017 107a for the rapid upstroke of the action potential. The crystal structures of bacterial the RyR during VA exposure, as observed experimentally. The integrated NaV channels that have been determined recently, have made way for studies computational model predicts the combined actions of the LCC and RyR and 2þ of mammalian NaV channels through homology modeling. However, homol- their modulations by VA on the dyadic space Ca dynamics and how the ogy modeling is not straightforward because of the differences between two factors collectively regulate the LCC and RyR kinetics during mammalian and bacterial NaV channels. The Selectivity Filter (SF), which excitation-contraction (EC) coupling in cardiac cells during VA exposure. plays a key role in ion permeation, has EEEE residues in bacterial channel This mechanistic computational model provides a strong foundation for a but has DEKA residues in mammalian channels. We have constructed a homol- whole-cell level model of cardiomyocytes Ca2þ dynamics and electrophysi- ogy model for the NaV1.4 channel from the crystal structure of NaVMs (PDB ology during EC coupling and VA exposure during cardioprotection against ID: 4CBC) bacterial channel. A recent study indicate that there are four stable IR injury. inter-domain links between SF and neighboring domains. We additionally real- ized that there are extra residues making inter-domain links around the SF in 535-Pos Board B300 Electrophysiological Characterization of T-Type Calcium Channels in NaVMs channel which are also conserved in the alignment between NaV1.4 Central Medial Nucleus of the Rat Thalamus and NaVMs channel. The distances between the linked residues were used as restraints in our homology model. The time series of N-O distances have Tamara Timic Stamenic, Slobodan M. Todorovic. been obtained between linked residues from a 100 ns MD simulation which Anesthesiology, CU Anschutz Medical Campus, Aurora, CO, USA. is performed using NAMD with the CHARMM36 force field. The N-O dis- Centralmedialnucleus(CeM)isapart of intralaminar thalamus that is tances of extra links were found to be stable. We also studied channel stability, involved in the control of arousal but mechanisms that regulate its activity sodium ion hydration and coordination as well as the permeation mechanism. are not well studied. It is well known that low-voltage-activated T-type Comparison of the our simulation results with experiments show that our ho- calcium channels (T-channels) are abundantly expressed in the thalamus where they regulate neuronal excitability but their role in CeM was not pre- mology model provides a realistic representation of the mammalian NaV1.4 structure. viously investigated. Here, we investigated properties of T-channels in CeM using patch-clamp technique in acute coronal brain slices of adolescent rats. 533-Pos Board B298 We used voltage-clamp recording with three different internal solutions: tet- Mechanism and Energetics of Ion and Tetrodotoxin Binding to NavMs ramethylammonium hydroxide (TMAOH) without ATP, cesium hydroxide Channel (CsOH) with ATP and CsOH ATP-free solution. TMAOH internal solution Esra Korpe1, Ali Osman Acar1, Murat Cavus2, Serdar Kuyucak3, induced a profound hyperpolarizing shift in steady-state inactivation curves Turgut Bastug4. of about 20 mV when compared to CsOH with ATP (p<0.001, one-way 1Micro and Nanotechnology, TOBB University of Economics and ANOVA). Smaller but significant shift of about 8 mV was achieved with Technology, Ankara, Turkey, 2Faculty of Education, Bozok University, CsOH ATP-free internal solution (p<0.001). In contrast, voltage- Yozgat, Turkey, 3School of Physics, Sydney University, Sydney, NSW, dependent steady-state activation kinetics of T-currents were not different Australia, 4Material Science and Nanotechnology Engineering, TOBB under identical recording conditions. Additionally, we found that pan- University of Economics and Technology, Ankara, Turkey. selective T-channel blocker TTA-P2 at 5 mM decreased T-current density Voltage-gated sodium channels (Nav) are important targets for treating by about 70% (p<0.001, two-way RM ANOVA) and induced hyperpolariz- various diseases. Crystal structure of the bacterial voltage-gated sodium chan- ingshiftof4mV(p<0.001, t-test) in steady-state inactivation curves after nel NavMs in the open conformation has been obtained by Ulmschneider et al 10 minutes of application. Moreover, we used current-clamp recordings to recently. We used this structure in our simulation work in order to study chan- investigate the effects of TTA-P2 on firing patterns and passive membrane nel stability, sodium ion coordination as well as the ion permeation mecha- properties of CeM neurons. We found that TTA-P2 reduced tonic action nism. We have employed free energy techniques to calculate the potential potential frequency by 23% (p<0.05, two-way RM ANOVA), completely of mean force (PMF) for ion movement through the NavMs channel. The abolished rebound burst firing, reduced low-threshold spike (LTS) ampli- PMF calculations revealed the ion-binding sites in the channel and the mech- tude by 78% (p<0.001), slightly increased input resistance by 15% anism of ion conductance. We also studied tetrodotoxin binding to the NavMs (p<0.001) and latency to LTS by 18% (p<0.05). Our results strongly channel. Using docking and molecular dynamic simulations, we have con- suggest that T-channels are important regulators of neuronal excitability structed a model for the NavMs-tetrodotoxin complex. The toxin binds to in CeM, which may be finely tuned by voltage-dependent phosphorylation. various parts of the channel and occludes the ion-conducting pore. Our results Supported by GM102525 to SMT. help to explain experimental data and provide insights into the Nav inhibition 536-Pos Board B301 process. The complex structures we have found provide templates for devel- 2D oping new sodium channel blockers with improved affinity and selectivity Unmasking the Molecular Determinants Important for Ca -Dependent properties, which will be useful in the design of novel drugs targeting sodium Regulation of CaV2.2 ion channels. Jessica R. Thomas, Jussara Hagen, Amy Lee. University of Iowa, Iowa City, IA, USA. (CDI) and facilitation (CDF), respectively, which contribute to short-term syn- Voltage-gated Ca Channels I aptic plasticity. Both CDI and CDF are mediated by calmodulin (CaM) binding to sites in the C-terminal domain (CT) of the Cav2.1 a1 subunit, including a 534-Pos Board B299 consensus CaM-binding IQ-domain. Cav2.2 (N-type) channels display CDI Modeling the Effects of Volatile Anesthetics on L-Type Ca2D Channels but not CDF but the underlying mechanism that blocks CDF in Cav2.2 is un- and Ca2D Induced Ca2D Release in Cardiomyocytes known. Here, we tested the hypothesis that Cav2.2 does not undergo CDF since Neeraj Manhas. it lacks essential molecular determinants for CDF that are present in Cav2.1. Physiology, MCW, Milwaukee, WI, USA. We find that alternative splicing of exons in the proximal and distal CT, which Studies have shown that volatile anesthetics (VA; e.g. isoflurane) exert negative regulates CDF of Cav2.1, has no effect on CDF of Cav2.2. However, replace- inotropic effects on cardiac cells, which could be one way in mediating cardi- ment of the entire CT of Cav2.2 with that of Cav2.1 produces robust CDF of oprotection against ischemia-reperfusion (IR) injury. Specifically, VA is shown 2þ 2þ the chimeric channel. Further analyses reveal that transfer of the Cav2.1 to decrease the rate of Ca entry into the dyadic space via the L-type Ca EF-hand, Pre-IQ- IQ domains, and a downstream CaM-binding domain channels (LCC), while also activating the ryanodine receptors (RyR) and 2þ 2þ (CBD) are sufficient to support CDF in chimeric Cav2.2 channels. Our results Ca induced Ca release (CICR) process. However, the kinetic mechanisms highlight the importance of the CT in distinguishing Ca2þ feedback regulation of VA actions in these two processes and the consequent effects on the dyadic 2þ of Cav2.2 and Cav2.1, and underscore how molecular distinctions may underlie space Ca dynamics, which also regulate these two processes, are not the unique contributions of these channels in regulating neurotransmitter well-understood. We developed here a computational model to investigate release. the effects of VA on Ca2þ dynamics in the dyadic space, by extending and integrating contemporary kinetic models of the LCC and RyR, incorporating 537-Pos Board B302 their regulations by VA. The extended kinetic model of the LCC systematically C-Terminal Splice Variation Reveals New Insights into Calmodulin reproduces the various aspects of the LCC and its regulations by VA, observed Regulation of CaV1.4 Channels experimentally; specifically, the VA inhibition of the LCC open probability and Brittany Williams, Vasily Kerov, Daniel Soh, Amy Lee. VA modulation of the LCC activation and inactivation gates that are mechanis- University of Iowa, iowa city, IA, USA. 2þ tically inferred through additional VA bound states of the LCC. Also, the In synaptic terminals of retinal photoreceptors, Cav1.4 (L-type) Ca chan- extended kinetic model of the RyR is able to simulate the increased activity nels mediate Ca2þ influx that promotes neurotransmitter release. Mutations

BPJ 7731_7735 108a Sunday, February 12, 2017

2þ in Cav1.4 are associated with multiple vision disorders including congenital water around Sr suggests a stable 8-fold geometry in the local hydration 2þ 2þ stationary night blindness (CSNB2). Cav1.4 does not undergo Ca -depen- environment, similar to Ba . While the predicted hydration free energy of dent inactivation (CDI) – a negative feedback mechanism seen for other 331.8 kcal/mol is comparable with the experimental result of 334 kcal/mol, L-type channels mediated by calmodulin (CaM) binding to a consensus the value is significantly more favorable than the 305 kcal/mol hydration IQ-domain in proximal C-terminal domain (pCT) of the pore-forming sub- free energy of Ba2þ. When placed in the innermost Kþ channel blocking 2þ unit. The lack of CDI in Cav1.4 is due to a C-terminal automodulatory site, the solvation free energies and lowest energy structures of both Sr domain (CTM), located in the distal CT. The CTM is thought to suppress and Ba2þ are nearly unchanged compared with their respective hydration prop- CDI of Cav1.4 channels by competing with CaM-binding to sites in the erties. That result suggests that differences in blocking behavior may arise due pCT. A CSNB2-causing mutation (K1591X) in Cav1.4 that deletes the to kinetic properties associated with exchange of water ligands for channel li- CTM promotes CaM-binding and CDI, but also causes channel activation gands instead of equilibrium thermodynamic properties. at more negative potentials than full-length channels (Cav1.4FL). Here, we demonstrate that similar properties are exhibited by a naturally 540-Pos Board B305 The Calcium Channel A2D Subunit Increases the Gating Charges of occurring human Cav1.4 splice variant lacking exon 47 (Cav1.4Dex47), and characterized an unexpected role for CaM in the regulation of this Cav1.2 Channels Gustavo F. Contreras1, Nicoletta Savalli2, Antonios Pantazis2, channel. By qPCR, we found that Cav1.4Dex47, which lacks the initial 43 1 2 1 amino acids of the CTM, is expressed in primate but not mouse retina. In Carlos Gonzalez , Riccardo Olcese , Alan Neely . 1Centro de Neurociencias de Valparaiso, Valparaiso, Chile, 2Division of electrophysiological recordings of transfected HEK293T cells, Cav1.4Dex47 Ca2þ currents activate at more negative voltages and display stronger CDI Molecular Medicine, Department of Anesthesiology, David Geffen School of Medicine at UCLA, LA, CA, USA. than Cav1.4FL, similar to K1591X. These effects were blunted by IQ- Optically tracking the movement of individual voltage sensors (VSD) of human domain mutations known to disrupt CaM-binding to Cav1.4. Mutations þ a d a that prevent Ca2 -binding to either N- or C-terminal CaM lobes suppress CaV1.2 revealed that association of 2 -1 with 1C resulted in a substantial CDI of Ca 1.4Dex47. However, mutations in the N-terminal but not the change in the intrinsic voltage-sensing properties of VSDs I-III (Savalli et al v a d C-terminal lobe of CaM abolish the effect of exon 47 deletion on channel 2016, JGP 142, 147-159). Co-expression of 2 -1 increased the sensitivity of activation. We conclude that exon 47 contains key molecular determinants activation for several VSDs indicating that this mostly extracellular subunit within the CTM for regulating CDI and activation, and that CaM plays increased the electrical distances traversed by voltage-sensing charges. In distinct roles in these processes. this study we measured the slope of the voltage-dependence of the channel’s probability of being open (Po) at extremely low probability using marcro- 538-Pos Board B303 patches of Xenopus oocytes expressing CaV1.2/CaVb3 by themselves or in Structural Characterization of Calmodulin Disease Mutations combination with a2d-1. Patches containing from 50 to several 100 channels þ Kaiqian Wang1, Jocelyn Lu1, Kamilla T. Larsen2, Michael T. Overgaard2, were recorded in 75 mM Ba2 and in the presence of 1 mM (-) Bay K 8644 Filip Van Petegem1. and held at several voltages ranging from 70 to 20 mV for 10 to 90 s. Po 1University of British Columbia, Vancouver, BC, Canada, 2Aalborg times the number of channels (NPo) were obtained by finding the most likely University, Aalborg, Denmark. combination of normal distributions shifted by the single channel currents Calmodulin (CaM) is a ubiquitous calcium-sensing protein involved in the amplitude that described the data. The relative weight of the different distribu- propagation of intracellular calcium signals and the regulation of events tions was then contrasted with what should be expected from a Poisson distri- ranging from muscle contraction to cell excitability. The human genome bution of opening levels. The number of channels (N) was estimated from the contains three CaM genes (CALM 1-3) which encode for protein with iden- noise analysis of hundreds of tail current traces at 40 mV following a depo- tical primary sequences. Despite the redundancy of CaM, single missense larizing pulse to þ80 mV. In the presence of a2d-1, the limiting slope was mutations in even one of the six alleles are associated with disease pheno- about 4 elementary charges, close to the sum of the voltage-dependencies of types such as catecholaminergic polymorphic ventricular tachycardia VSDII and VSDIII activation and clearly less than the sum of all VSDs and (CPVT) and early-onset severe long QT syndrome (esLQT). CPVT can thus consistent with the idea that only a subset of VSDs contribute to the effec- lead to stress- and exercise-induced arrhythmias and sudden cardiac death; tive charges for channel opening. When the a2d-1 subunit was absent the esLQT is characterized by a prolonged QT interval which can also result limiting slope was reduced by about one elementary charge. This modest in ventricular fibrillation. Despite the devastating genetic disorders associ- change can be accounted by a reduction in the electrical distance that gating ated with CaM mutations, the molecular mechanisms by which these muta- charges need to cross for channel opening. tions manifest into dominant disease phenotypes have yet to be elucidated. Funding: FONDECYT 3140590 (GC) and 1120864 (AN), R01GM110276 Here, we present the crystal structures of several CaM disease mutants, one (RO) and 16POST27250284 (NS). of which represents a novel CaM conformation not previously characterized. Significant structural changes are observed in both EF-hands III and IV of 541-Pos Board B306 the C-lobe. In particular, the mutation disrupts the calcium coordination Calmodulin and Stac3 Enhance Functional Expression of CaV1.1 network in EF-hand III and results in abolished calcium binding, leading Jacqueline Niu, Manu Ben Johny, David T. Yue, Takanari Inoue. to CaM that resembles an intermediate between the Ca2þ-CaM and Johns Hopkins University, Baltimore, MD, USA. apo-CaM states. In contrast, structures of other disease mutants revealed CaV1.1 is a prominent L-type voltage-gated calcium channel (VGCC) that CaM conformations that closely resemble the wild type structure, with little plays an integral role in mediating skeletal muscle excitation-contraction positional shift in the EF-hand helices of either the N- or C-lobe. These coupling. Unlike other homologous L-type channels (e.g. CaV1.3), in depth structures can help explain the diverse effects of CaM mutations and the biophysical analysis of CaV1.1 is challenging as functional expression of associated disease mechanisms, especially as CaM mutations have differen- these channels has been generally restricted to cell types with a muscular line- tial effects on the function of ryanodine receptor 2 and calcium-dependent age. Interestingly, in contrast to CaV1.3, the carboxy terminus of CaV1.1 has a inactivation of L-type calcium channels. low affinity for the calcium binding protein, calmodulin (CaM) suggesting that the loss of CaM pre-association may be responsible for the poor func- 539-Pos Board B304 tional expression of CaV1.1 in recombinant systems. Here, we explicitly Strontium and Barium in Aqueous Solution and an Ion Channel demonstrate that restoration of CaM to the channel complex enables func- Blocking Site tional expression of CaV1.1 in HEK293 cells. Further mechanistic analysis Mangesh Chaudhari, Susan Rempe. shows that CaM substantially enhances surface membrane trafficking of Sandia National Labs, Albuquerque, NM, USA. CaV1.1. In conjunction with recent studies that showed Stac3 (SH3 and Ion hydration structure and free energy establish criteria for understanding cysteine rich domain 3) adaptor proteins also enable CaV1.1 currents in re- selective ion binding in potassium (Kþ) ion chan- nels, and may be significant combinant systems (Polster et al (2015) PNAS 112:602), our results argue to understanding blocking mechanisms as well. Recently, we investigated the that multiple cell signaling molecules can evoke similar functional outcomes 2þ þ hydration properties of Ba , the most potent blocker of K channels among and points to redundancy in molecular pathways that orchestrate CaV1.1 sur- the simple metal ions. Here, we use a similar method of combining ab initio face expression. These results also suggest there may be an overlap between molecular dynamics simulations, statistical mechanical theory, and electronic channel regulation and trafficking of L-type VGCC with Stac3 and CaM. In structure calculations to probe the fun- damental hydration properties of all, our findings furnish a convenient platform to probe CaV1.1 function and Sr2þ, which does not block bacterial Kþ channels. The radial distribution of related pharmacology.

BPJ 7731_7735 Sunday, February 12, 2017 109a

542-Pos Board B307 Voltage-gated K Channels and Mechanisms of CaV1.3 (Cacna1d) Gain-of-Function De Novo Missense Mutations are Associated with CNS Disorders Voltage Sensing and Gating I Alexandra Pinggera1, Luisa Mackenroth2,Jo¨rg Striessnig1. 1Pharmacology and Toxicology, University of Innsbruck, Innsbruck, Austria, 544-Pos Board B309 2Institute for Clinical Genetics, Technical University Dresden, Dresden, Examining the Role of Phosphorylation on Interactions between the Germany. Cardiac Potassium Channel Alpha-Subunits hERG and KVLQT1 2þ 1 2 Cav1.3 belongs to the family of L-type voltage gated Ca channels. It is Medeea C. Popescu , Louise E.O. Darling . involved in many physiological functions, like hearing, sinoatrial node pace- 1Biochemistry Program, Wellesley College, Wellesley, MA, USA, making and hormone secretion. Moreover, it is expressed postsynaptically in 2Biological Sciences Department and Biochemistry Program, Wellesley neurons where it shapes neuronal firing, mediates gene transcription, and con- College, Wellesley, MA, USA. þ trols synaptic morphology and pruning. Recently we described two different KvLQT1 and hERG are the voltage-gated K channel a-subunits of the de novo Cav1.3 gain-of-function missense mutations in two patiens with autism channels which carry the cardiac repolarizing currents IKs and IKr,respec- spectrum disorders (ASD) suggesting that they present a strong risk for the tively. These currents function in vivo with some redundancy to maintain disorder. Here we strenghten this hypothesis by reporting the discovery and appropriate action potential durations (APDs) in cardiomyocytes. As such, characterization of a third Cav1.3 de novo missense mutation in a patient protein-protein interactions between hERG and KvLQT1 may be important with ASD and epilepsy, localized in a highly conserved region within the chan- in normal cardiac electrophysiology, as well as in arrhythmia and sudden nel’s activation gate. cardiac death. Previous phenomenological observations of functional, The mutation was identified by sequencing of the coding exons of 4813 genes mutual downregulation between these complementary repolarizing currents associated with known Mendelian disorders. We introduced the mutation into in transgenic rabbit models and cell culture have motivated our investiga- two functionally distinct C-terminal long (Cav1.3L) and short (Cav1.343s) splice tions into interactions between hERG and KvLQT1. These data suggest variants of the Cav1.3 a1-subunit. We co-expressed wild-type or mutant a1-sub- that a dynamic physical interaction between hERG and KvLQT1 modulates units with auxiliary b3 and a2d-1 subunits in tsA-201 cells and performed the respective currents. However, the mechanism by which HERG-KvLQT1 whole-cell patch-clamp recordings. interactions are regulated is still poorly understood. Phosphorylation is The mutation resulted in a pronounced gain-of-function in both splice thought to play a regulatory role in this process: modifying the phosphory- variants, which, next to other gating changes, was evident by enhanced cur- lation state of each the proteins has been shown to alter channel kinetics, rent densities and a negative shift of steady-state activation and inactivation and both hERG and KvLQT1 are targets of the Ser/Thr protein kinase leading to an increased window current at more negative voltages. Moreover, PKA, activated by elevated intracellular cAMP concentration. Through it significantly reduced Ca2þ-dependent inactivation in both splice variants classic biochemical assays and quantitative FRET approaches, we aim to whereas voltage-dependent inactivation was only affected when introduced characterize the effects of phosphorylation in regulating interactions be- into Cav1.343s. The observed gating changes are expected to affect neuronal tween KvLQT1 and hERG in cellular model systems. We have developed signaling and excitability. Together with previous findings we identify ion channel fusions to fluorescent proteins, which include hERG and recurrent Cav1.3 gain-of-function mutations as a strong risk factor for CNS KvLQT1 phosphonull and phosphomimetic mutants. We hypothesize that disorders presenting as ASD, with and without epilepsy. Existing L-type phosphorylation abrogates protein-protein interactions, as suggested by find- 2þ Ca channel blockers used as antihypertensives may provide a therapeutic ings that increased cAMP levels leads to decreased hERG-KvLQT1 interac- option for such patients. tion. This work potentially furthers our understanding of hERG-KvLQT1 Supported by Austrian Science Fund (FWF F44020, W11010). interactions and may elucidate mechanisms that underlie many types of arrhythmia as well as characterize novel interactions between two distinct 543-Pos Board B308 potassium channel families. Dual Effect of Palmitate on Voltage-Gated Calcium Channels and Insulin Secretion in Pancreatic Beta Cells of Rats 545-Pos Board B310 Neivys Garcı´a-Delgado1, Myrian Velasco-Torres1,2, Carmen Sanchez-Soto1, Monitoring Structural Reorganization of Calmodulin in Complex with the Marcia Hiriart2,3. C-Terminus of KCNQ Channels 1Inst Fisiologia Celular, Neuroscience Division, Universidad Nacional Carolina Gomis-Perez1, Eider Nunez-Viadero1, Ganeko Bernardo- Autonoma de Mexico, Mexico DF, Mexico, 2Centro de Ciencias de la Seisdedos1, Covadonga Malo1, Pilar Areso2, Alvaro Villarroel1. Complejidad, Universidad Nacional Autonoma de Mexico, Mexico, Mexico, 1Instituto Biofisika (CSIC, UPV/EHU), Leioa, Spain, 2Dpt. Farmacologı´a 3Inst Fisiologia Celular, Neuroscience Division, Universidad Nacional (UPV/EHU), Leioa, Spain. Autonoma de Mexico, Mexico, Mexico. Calmodulin (CaM) is an essential component of the non-inactivating A variety of signaling molecules modify voltage-gated calcium channels ac- voltage-dependent potassium channels conformed by Kv7 subunits, and me- tivity in pancreatic beta cells, among them are, free fatty acids (FFA). Acute diates current suppression upon intracellular calcium elevation. Despite exposure to FFA increases glucose-stimulated insulin secretion, while recent atomic-level information on CaM complexed to Kv7 domains, the chronic exposure increases basal insulin secretion, but decreases glucose- structural consequences upon calcium binding remains elusive. To obtain in- stimulated (Olofsson et al., 2004; Zhou and Grill, 1994). Preliminary studies sights on the structural changes caused by calcium, we have monitored in our laboratory showed that pre-incubation with 1 mM palmitate during FRET between the AB module tagged with the blue protein mTFP1 and 48-72 hours reduced barium currents in beta cells. In this work, we analyze CaM tagged with the yellow protein Venus using purified recombinant pro- the effect of different palmitate concentrations on calcium currents and in- teins. In addition, FRET has been monitored in CaM/AB complexes in sulin secretion in beta cells of adult male Wistar rats. Electrophysiological which the AB module was tagged with both fluorescent proteins. Significant recordings were performed using whole-cell voltage clamp technique. changes in energy transfer were observed in the presence of calcium, being Reverse hemolytic plaque assay measured insulin secretion by isolated more prominent for Kv7.1 than for Kv7.2 subunits. Thus, these data suggest beta cells. Pre-incubation with 0.25 and 0.5 mM palmitate during 48-72 h that calcium causes structural changes to different extent on each Kv7 increased the high voltage-activated (HVA) calcium current without isoform. affecting the LVA current. An acute 5 min pre-incubation with 0.5 mM Financed by grant BFU2015-66910-R from MINECO. palmitate also increased HVA current. In contrast, 24 h pre-incubation with 1 mM decreased both, LVA and HVA currents. A similar effect was 546-Pos Board B311 observed during an acute incubation. Chronic pre-incubation with 0.25, Novel Insights from Structural Analysis of Interactions of KCNQ KD 0.5 and 1 mM palmitate decreased the percentage of insulin secretory cells, Channels with Calmodulin immunoplaque area and insulin secretion index in a glucose-stimulated con- Crystal R. Archer1, Akash Bhattacharya1, Benjamin T. Enslow2, dition (15.6 mM). Chronic pre-incubation with 0.5 and 1 mM palmitate, also Alex B. Taylor1, Dmitri N. Ivanov1, Mark S. Shapiro3. decreased the secretion index at basal glucose (5.6 mM). However, acute 1Biochemistry, University of Texas Health Science Center, San Antonio, San pre-incubation with 1 mM palmitate increased the immunoplaque area in Antonio, TX, USA, 2School of Medicine, University of Texas Health Science 5.6 mM glucose. Together these results show that palmitate has a dual ef- Center, San Antonio, San Antonio, TX, USA, 3Cellular and Integrative fect, depending on time and concentration on the calcium currents and insu- Physiology, University of Texas Health Science Center, San Antonio, San lin secretion in beta cells. Partially supported by DGAPA PAPIIT-UNAM Antonio, TX, USA. Grants: IN213114, M Hiriart; IV100116 to A. Frank and M Hiriart, and Voltage-gated M-type Kþ channels, made by KCNQ subunits, regulate excit- IN211416 to M Velasco-Torres. ability in nerve and muscle. The gating of these channels are modulated by

BPJ 7731_7735 110a Sunday, February 12, 2017

receptors coupled to Gq/11 G proteins via several signals, such as calmodulin CP1 increases IKs currents by two-fold and alters voltage dependence and ki- (CaM) and the membrane phospholipid phosphatidylinositol 4,5-bisphosphate netics of activation and deactivation of IKs channels. The potentiation effect (PIP2), and protein kinases. Here, focusing on KCNQ4, we studied the struc- of CP1 on IKs currents has an EC50 of 7.4 mM. Consistent with targeting the tural and biochemical mechanisms of channel regulation by CaM, using PIP2 binding site,CP1 rescues the IKs current with the coexpression of CiVSP, isothermal titration calorimetry, microscale thermophoresis and nuclear mag- which depletes PIP2 and inhibits the currents. The KCNQ1/IKs specific blocker, netic resonance. As for a number of other ion channels, CaM directly interacts Chromanol 293B inhibits the enhanced current due to CP1 application, suggest- with the C-terminus of KCNQ channels, at their A & B helices. Using similar ing that CP1 substitutes for PIP2in activating IKs channels. In preliminary ex- biochemical and structural methods, we also studied the interaction of KCNQ perimetns with Guinea-pig ventricular myocytes, CP1 caused similar changes channels with PIP2 analogues and its cross-talk with CaM actions. Such cross- in chromanol sensitive IKs while also shortening action potential duration. talk is made more likely by two loci of PIP2 interactions with the C-terminus CP1 as an opener of the IKs channels may provide a novel therapy to treat con- near the A & B helices. Since it is still unclear how CaM and PIP2 binding cause gential and acquired LQT syndromes. physical rearrangements of the channels that affect gating, we probed the affin- ities of CaM for the A & B helices, asked whether CaM is constitutively bound 549-Pos Board B314 to the channels, and utilized our crystal structure of CaM bound to the KCNQ4 Pi(4,5)P2 Modulates Hysteresis and Pharmacology of KV7 Channels A & B helices to gain insight into CaM-induced structural rearrangements of Carlos A. Villalba-Galea. the C-terminus. To probe for potential cross-talk, we asked if Ca2þ binding Physiology and Pharmacology, University of the Pacific, Stockton, CA, USA. KV7 channels are critical components of the plasma membrane in many excit- to the CaM/C-terminal complex affects PIP2 interactions and thus, PIP2 regu- lation of channel gating. Supported by NIH grants R01 NS094461-01 and able cells. The main function of KV7 channels is to contribute to the mainte- R01NS043394-11 to M.S.S. and NRSA training grant F31 NS090887-02/ nance of the resting potential, thus regulating cellular electrical excitability. NIH to C.R.A. In Central Nervous System, the activity of the heteromeric KV7.2/KV7.3 chan- nel gives rise to M-currents. These Kþ-currents are subject to muscarinic 547-Pos Board B312 modulation as the phosphoinositide PI(4,5)P2, a signaling lipid dephosphory- KCNE1 and KCNE3 Modulate KCNQ1 Channels by Affecting Different lated during muscarinic activity, is required by KV7.2/KV7.3 channels to be Gating Transitions functional. On the other hand, Corbin-Leftwich and colleagues (JGP, 2016) 1 1 1 1 Rene Barro-Soria , Rosamary Ramentol , Sara I. Liin , Marta E. Perez , have recently reported that the deactivation rate of KV7.2/KV7.3 channels Robert S. Kass2, H Peter Larsson1. depends on the duration of activation. This observation revealed that this het- 1Physiology & Biophysics, University of Miami, Miami, FL, USA, eromeric channel displays a remarkable hysteretic behavior. In addition, it was 2Pharmacology, College of Physicians & Surgeons, Columbia University, shown that these channels have, at least, two open modes and that these modes New York, NY, USA. differ in stability, as deactivation from one mode is slower than the other, and in KCNE b subunits assemble with and modulate the properties of voltage- pharmacology, as the anticonvulsant Retigabine preferentially acts on the þ gated K channels. In the heart, KCNE1 associates with KCNQ1 to generate slower-deactivating mode. Since, (1) the hysteretic behavior of KV7.2/KV7.3 the slowly activating, voltage-dependent IKs current that controls the repo- depends of activity and, in turn, (2) activity depends on PI(4,5)P2, an clear larization phase of cardiac action potentials. By contrast, in epithelial cells next step was to determine whether PI(4,5)P2 can modulate the effect of the from the colon, stomach and kidney, KCNE3 coassembles with the a-sub- anticonvulsant Retigabine. In addressing this question, here, it is presented þ unit KCNQ1 to form apparent voltage-independent K channels important that each of the open modes of KV7.2/KV7.3 channels displays distinct apparent for controlling water and salt secretion. How KCNE1 and KCNE3 subunits affinities for PI(4,5)P2. Also, it was found that decreasing the PI(4,5)P2 concen- modify KCNQ1 channel gating so differently is largely unknown. Different tration either pharmacologically or enzymatically, reduces the effect of Retiga- molecular mechanisms have been proposed to explain the effects of KCNE1 bine on channel activity when applied in low doses. Furthermore, here it is and KCNE3 on KCNQ1 channels. Here, we use voltage clamp fluorometry shown that the hysteretic behavior of the KV7.2/KV7.3 channels does not to determine how KCNE1 and KCNE3 affect the voltage sensor and the gate emerge from being heteromeric, as the individual components display hyster- of KCNQ1. By separating S4 movement and gate opening by a mutation, we etic behavior as well. The work presented here provides evidence the leads show that KCNE1 affects both the S4 movement and the gate, whereas to propose that muscarinic modulation of M-current is likely dependent on KCNE3 directly affects the S4 movement and only indirectly affects the the hysteretic behavior of KV7.2/KV7.3 channels. gate in KCNQ1. Further, we show that a triple mutation in KCNE3 converts KCNQ1/KCNE3 channels into KCNQ1/KCNE1-like channels by intro- 550-Pos Board B315 ducing KCNE1-like effects on the KCNQ1 gate. Our results suggest that Molecular Mechanism of the Kv7.1-Channel Activator N-Arachidonoyl the difference between the effects of KCNE1 and KCNE3 on KCNQ1 is Taurine 1 2 2 that KCNE1 affects both the voltage-sensing domain and the gate, whereas Sara I. Liin , Rene Barro-Soria , H Peter Larsson . 1Clinical and Experimental Medicine, Linko¨ping University, Linko¨ping, KCNE3 primarily affects the voltage-sensing domain and only indirectly 2 affects the gate. Sweden, University of Miami, Miami, FL, USA. The cardiac IKs channel - formed by KV7.1 and KCNE1 - is important for the 548-Pos Board B313 timing of the cardiomyocyte repolarization. About 300 loss-of-function muta- CP1 Opens IKs Channels by Substituting PIP2 tions in the KV7.1/KCNE1 channel have been identified in patients with Long Moawiah M. Naffaa1, Xianjin Xu2, Hongwu Liang1, Guohui Zhang1, QT syndrome and cardiac arrhythmia. These arrhythmias are today primarily Hong Zhan Wang3, Junyuan Goa3, Ira S. Cohen3, Xiaoqin Zou4, treated with drugs that prevent the triggering of arrhythmic activity, such as Jianmin Cui1. beta-blockers, or with arrhythmia-terminating implantable defibrillator. 1Biomedical Engineering, washington university in st louis, st louis, MO, A different treatment strategy for Long QT syndrome caused by loss-of- 2 USA, Department of Physics and Astronomy, University of Missouri, function mutations in the KV7.1/KCNE1 channel would be to pharma- Columbia, MO, USA, 3Department of physiology and Biophysics, Stony cologically augment the channel function of these mutants, thereby shortening Brook University, Stony Brook, NY, USA, 4Dalton Cardiovascular Research the prolonged QT interval and lower the risk of arrhythmia development. Center, University of Missouri, Columbia, MO, USA. We have in previous studies observed that the fatty acid analogue N-arachido- The voltage-gated potassium channel KCNQ1 co-assembles with the auxiliary noyl taurine activates the wild-type KV7.1/KCNE1 channel and KV7.1/KCNE1 subunit KCNE1 to form the IKs channel in the heart. The IKs current, a slow de- channels with diverse Long QT syndrome mutations by shifting the conduc- layed rectifier Kþ current, plays a key role in repolarization of cardiac action tance versus voltage (G(V)) curve towards negative voltages. This potentials. Mutations in the KCNQ1 gene are associated with long QT suggests that N-arachidonoyl taurine may function as a general activator of (LQT) syndrome, atrial fibrillation and short QT syndrome, which lead to car- KV7.1/KCNE1 channels with diverse mutational defects. The molecular diac arrhythmias. It was shown that KCNQ1 and IKs channels require PIP2 mechanism of how N-arachidonoyl taurine activates the KV7.1/KCNE1 (Phosphatidylinositol 4,5-bisphosphate) for activation. This lipid acts as a channel is however not fully understood. In this project, we therefore use cofactor of the channel for the movements of the voltage sensor domain to voltage-clamp fluorometry and kinetic modeling to study the effect of N-arach- trigger the pore gate domain to open. Using the previously identified PIP2 idonoyl taurine on voltage sensor movement and channel opening in KV7.1/ binding site in a homology structural model of KCNQ1 as the target, an KCNE1 channels. We also test the importance of specific residues in the in-silico screening of chemical compounds in the Available Chemical Database channel’s voltage sensing domain for the ability of N-arachidonoyl taurine to (ACD, Molecular Design, Ltd.) and a subsequent experminetal testing on the activate the KV7.1/KCNE1 channel. An understanding of the molecular mech- IKs channels expressed in Xenopus oocytes identified CP1 as a novel IKs opener. anism of N-arachidonoyl taurine is critical to further improve this and related

BPJ 7731_7735 Sunday, February 12, 2017 111a

fatty acid analogues, which may inspire development of future KV7.1/KCNE1 but none of them can open VGIC in spite of membrane potential. Here, channel activators to treat congenital Long QT syndrome caused by mutations we report a cysteine is a ‘voltage- to ligand- gated’ switch for KCNQ2 chan- in KV7.1/KCNE1. nel. Substitutions with alanine (A) or other smaller volume amino acids in this position endow a ligand-gating to KCNQ2 channel in a measurable 551-Pos Board B316 range of membrane potential. Under constant 120 mV or even lower ML277 Opens KCNQ1 Channels by Selectively Enhancing the AO State membrane potentials, the mutant channels can be opened by small chemical Panpan Hou, Ling Zhong, Powei Kang, Zachary Beller, Kelli McFarland, ligands, such as ztz240 and ZnPy . Whereas, the wild type channel kept in Jingyi Shi, Jianmin Cui. close under the identical conditions. Further mechanism investigations Biomedical engineering, Washington University in St Louis, St. Louis, revealed that the volumn rather than other properties in this position is MO, USA. essential for the ligand induced opening, inferring that this position may þ a The KCNQ1 K channel subunit and the auxiliary subunit KCNE1 form the function as a baffle plate that restricts the upward movement of the S4 IKs channel in the heart that is important in controlling heart rhythm. In segment. Our study reports a ligand-gated opening of KCNQ2 channel for response to membrane depolarization, the KCNQ1 channel undergoes interme- the first time. The identification of the ‘voltage- to ligand-gated’ switch in diate open (IO) and activated open (AO) states that correspond to the stepwise KCNQ2 channels breaks through the traditional barrier between VGIC movement of the voltage sensor to the intermediate and full activation. IO and and LGIC. AO states showed different properties in voltage dependence of gating, perme- ation, and pharmacology. The association of KCNE1 suppresses IO but en- 554-Pos Board B319 hances AO, thereby radically alters properties of the channel to suit the Voltage and Ca2D Sensor Coupling Modulation by b Subunits in the BK physiological role of IKs in terminating the action potential. In this study, we Channel found that a recently identified KCNQ1 opener, ML277, increased the Yenisleidy Lorenzo1,2, Karen Castillo1, Gustavo Contreras1, KCNQ1 current by selectively enhancing the AO state without affecting the Willy Carrasquel-Ursulaez1, Carlos Gonzalez1, Ramon Latorre1. IO state. ML277 effects on KCNQ1 channels showed a time and voltage depen- 1Centro Interdisciplinario de Neurociencia de Valparaiso, Facultad de dence that coincide with that of the AO state. Voltage clamp fluorometry Ciencias, Universidad de Valparaiso, Valparaiso, Chile, 2Doctorado en studies revealed that ML277 did not change the voltage sensor movement Ciencias Mencio´n Neurociencia, Facultad de Ciencias, Universidad de but enhanced channel opening in the AO state, indicating an enhanced VSD- Valparaiso, Valparaiso, Chile. pore coupling selectively in the AO state. Consistently, ML277 was found to BK channels are modulated by b-subunits (b1-b4) in a tissue-specific 2þ increase the sensitivity of channel activation to PIP2, which is required for manner. The b1- and b2-subunits increase in the apparent Ca sensitivity mediating the VSD-pore coupling as shown in our previous studies. These re- of BK can be explained by a stabilization of the voltage sensor domain sults suggest that ML277 enhances the VSD-pore coupling specifically when (VSD) in its active configuration. However, whether b1 modifies the number the VSD is at the fully activated state to increase the AO state occupancy, of charges associated with the voltage sensor activation is still a matter of thereby increasing the KCNQ1 current. Given that the IKs channel primarily controversy. The purpose of our study is to determine the effects of the opens at the AO state, ML277 provides an example of drugs that can modulate presence of b-subunits on the gating charge in the presence of Ca2þ,and 2þ IKs channels with a high specificity. ML277 may also be used as an excellent to evaluate the coupling between Ca -binding and VSD activation. The tool to study the molecular mechanisms of how the IO and AO states are number of gating charges per channel was measured in BK channels formed determined in KCNQ1 channels. by a-subunit alone and with the different b-subunits in Ca2þ-free internal solutions. The maximum gating charge displaced was obtained from the 552-Pos Board B317 charge-voltage (Q-V) curve and the total number of channels in the patch Comprehensive Assessment of Disease Mutant Forms of the Human was determined using noise analysis. Furthermore, we evaluated the effect KCNQ1 Potassium Channel of b-subunits on the interaction between Ca2þ sensors and VSD in different Hui Huang, Keenan Taylor, Charles Sanders. Ca2þ concentrations. We found that the total number of charges per Biochemistry, Vanderbilt University, Nashville, TN, USA. channel was 4.4, 3.0 and 4.2 e0 for BKa,BKa/b1andBKa/b3b channels The voltage-gated potassium channel KCNQ1 is critical for the cardiac action respectively. Increasing intracellular [Ca2þ]inBKa (100 mM) promotes a potential. Mutations in KCNQ1 and its accessory protein KCNE1 are the most significant leftward shift (~-140 mV) of the Q-V curve. The calcium effect common cause of congenital long-QT syndrome (LQTS). There are a variety of on voltage sensor in BKa becomes apparent at [Ca2þ] R 1 mM(~V mechanisms by which a given mutation may cause KCNQ1 channel dysfunc- 30 mV). However, the leftward shift of the Q-V curve in BKa/b1 channels tion and prolonged activation potentials. The ideal treatment of patients becomes evident in the nanomolar [Ca2þ] range (100-500 nM). BKa/b3b harboring a KCNQ1 mutation is dependent on which specific mechanisms channels behave as BKa channels. We conclude that: a) b1-subunit not cause loss of function. In the present study, we have employed a multidisci- only modifies the resting-active equilibrium of the voltage sensor but also plinary approach to systematically investigate the specific effects of 51 decreases the total number of apparent gating charges; b) there is a strong KCNQ1 mutations on the channel structure, stability, trafficking, and (i.e. coupling between voltage and Ca2þ sensors, this coupling is increased in through collaboration) electrophysiological properties. The 51 mutations, the presence of b1-subunit. located in the voltage sensor domain (VSD), are disease causing, benign, or of unknown significance. High quality NMR spectra of the isolated wild type 555-Pos Board B320 VSD and of its mutant forms that are locked in fully activated state or in resting 2D state serve as reference spectra. The 1H-15N TROSY spectrum of each mutant Alpha-B Helix of RCK1 is a Major Transduction Pathway for Ca was collected and compared with the reference spectra to determine whether Activation of BK Channels 1 2 3 2 the mutation destabilizes the protein, or shifts the basal activated vs. resting Yanyan Geng , Zengqin Deng , Gonzalo Budelli , Alice Butler Butler , 2 2 2 1 state equilibrium. We have also expressed each mutant full length KCNQ1 Jianmin Cui , Peng Yuan , Lawrence Salkoff , Karl Magleby . 1University of Miami, Miami, FL, USA, 2Washington University, St. Louis, in HEK293 cells and quantitatively assessed its total protein expression and 3 cell surface expression using flow cytometry. These results will help elucidate MO, USA, Brandeis University, Waltham, MA, USA. BK (Slo1) type Kþ selective channels are activated by both depolarization the exact defects of each mutant associated with LQTS, potentially providing 2þ information that can be used to inform personalized treatment of LQTS subjects and intracellular Ca . BK channels are comprised of an integral- harboring KCNQ1 mutations. membrane core consisting of four lateral voltage sensor domains surround- This work was supported by NIH Grant RO1 HL122010. We also thank the lab ing a central pore-gate domain (S5-S6), and a large cytosolic domain (CTD) assembled from 8 RCK domains. The CTD (gating ring) is attached to the of Prof. Alfred George at Northwestern University for providing the cDNA for 2þ the KCNQ1-VSD mutants. core through four short S6-RCK1 peptide linkers. Ca binding to the CTD activates the channel. Crystal structures (Yuan et al. 2010, 2012) sug- þ 553-Pos Board B318 gest that Ca2 binding elevates the alpha-B helix in each RCK1 domain A Voltage- to Ligand- Gated Switch in Voltage-Gated Potassium Channels where it could push upwards against the core while simultaneously moving Xiaoping Pi, Qiang Ding, Zhaobing Gao. a rigid protein arm in RCK1 laterally and downward to pull on S6 through þ SIMM, Shanghai, China. the S6-RCK1 linkers. Ca2 activation thus may involve a synergistic push- To open voltage-gated ion channels (VGIC), the changes of membrane pull mechanism. We examined the contribution of the alpha-B helix to this potential, usually depolarization, is prerequisite. In contrast, ligand-gated hypothetical mechanism by introducing several discrete mutations into the ion channels (LGIC) open when ligands bind. VGIC often stay in the alpha-B helix. The mutation L390P in the alpha-B helix of mSlo1 greatly þ close state under hyperpolarize membrane potentials. Small chemical li- decreased Ca2 activation. The crystal structure of the hSlo1 CTD with gands have been found to be able to allosterically modulate VGIC, the L390P mutation indicated a discrete alteration of the alpha-B helix

BPJ 7731_7735 112a Sunday, February 12, 2017

2þ 2þ 2þ alone: Ca still bound to the Ca bowl and there were no other notable ethanol increased cbv1 Po by z3-fold. At 300 mMCa i, ethanol failed to differences in the CTD structure. L390P decreased Ca2þ activation through modify cbv1 Po in CLR-free bilayers but increased cbv1 Po in CLR- both high affinity Ca2þ binding sites, and also increased the voltage required containing (33 mol%) bilayers. The discrepancy in ethanol effect between for activation in 0 Ca2þ. Lengthening the RCK1-S6 linker of L390P by one native membranes and planar bilayers may likely arise from their difference residue in 0 Ca2þ decreased voltage activation, indicating that L390P did in proteo-lipid, including CLR, composition. Cbv1CRAC4T450F did not not remove all of the tension in the linker. Other alpha-B helix mutations respond to ethanol in CLR-free bilayers, but was activated by ethanol in were also consistent with push-pull activation. These observations suggest presence of 33 mol% CLR. Collectively, the similarity of ethanol modula- that an intact and rigid alpha-B helix plays a major role in transducing tion of BK channel activity studied with cbv1 vs. cbv1CRAC4T450F Ca2þ binding to pore opening in BK channels. Supported by R01 suggests that CRAC4 does not contribute to CLR modulation of ethanol ac- 2þ GM114694. tiononslo1athigh[Ca ]i. Support: R01AA023764 (AB), R37AA11560 (AD). 556-Pos Board B321 Carbon Monoxide May Regulate BK slo1 Channel Activity by Partially 558-Pos Board B323 Disrupting Heme Coordination Differential Expression of BK Channel Beta1 Subunits in Rat Mesenteric, Taleh Yusifov1, Nicoletta Savalli1, Antonios Pantazis1, Coronary and Cerebral Arteries Stefan H. Heinemann2, Toshinori Hoshi3, Riccardo Olcese1,4. Guruprasad Kuntamallappanavar, Shivantika Bisen, Anna Bukiya, 1Anesthesiology, Division of Molecular Medicine, UCLA, Los Angeles, CA, Alex Dopico. USA, 2Biophysics, Friedrich Schiller University of Jena, Jena, Germany, Pharmacology, University of Tennessee Health Center, Memphis, TN, 3Physiology, University of Pennsylvania, Philadelphia, PA, USA, Memphis, TN, USA. 4 2þ þ Physiology, UCLA, Los Angeles, CA, USA. Large conductance, Ca i/voltage-gated K (BK) channels regulate several The putative gasotransmitter CO is reported to activate large-conductance physiological processes (Yang et et al., 2015). In smooth muscle, BK com- Kþ channels (BK); however, the mechanism remains controversial. We plexes include channel-forming a and auxiliary b1 subunits (Brenner et al., recently found evidence that M691, within the RCK1-RCK2 linker of the 2000). BK b1 increases the channel’s apparent Ca2þ sensitivity (Brenner et BK C-terminus, coordinates heme together with the distal, conserved al.,2000) and is required for channel activation by lithocholate (LCA)(Bukiya heme regulatory motif (HRM), acting as a second axial ligand to the et al., 2009). Here, we studied the expression of BK a and b1 subunits in heme Fe atom. A similar binding configuration is observed in cytochrome mesenteric, coronary and cerebral (anterior, middle, posterior and basilar) c (CytC). Based on this and other lines of evidence, we have proposed arteries, and the contribution of such differential expression to LCA-induced that BK channels possess a CytC-like domain (BKCytCD). In CytC, CO as- artery dilation. Thus, we conducted quantitative real-time PCR, fluorescence sociates with heme by cleaving the Met80 S-Fe2þ bond; in this work, we are confocal imaging and patch-clamp electrophysiology on freshly isolated testing the hypothesis that BK channels bind CO by a similar mechanism. cerebral artery myocytes, and diameter determination of arteries pressurized We expressed and purified the intracellular C-terminal portion of the human in vitro. Data show that: 1) BK a mRNA is higher in cerebral anterior and BK channel (gating ring, GR). We first strengthened the evidence for the basilar arteries. In turn, b1 mRNA is higher in basilar and coronary arteries; participation of M691 in heme coordination by detecting an absorption 2) BK a protein levels at the myocyte membrane surface are similar across peak at 690nm, characteristic of the Met-Fe2þ bond, which was eliminated the different arteries whereas b1 levels are higher in basilar and coronary ar- by either mutation M691A or HRM disruption (C615S/H616R). We then teries; 3) BK channel basal activity is also higher in basilar and coronary artery probed for CO-induced GR conformational rearrangements using the 8-ani- myocytes; 4) Likewise, BK channels from basilar and coronary artery myo- lino-1-naphthalenesulfonic acid (ANS) fluorescent label in the presence of cytes are more responsive to LCA; 5) Finally, LCA-induced dilation of basilar heme: the wild-type GR/heme complex exhibited strong ANS fluorescence and coronary arteries is higher than dilation of other arteries. Our results reduction in response to CO, reporting structural transitions likely demonstrate a differential protein expression and functional impact on both underlying CO regulation of BK conductance. The GR-M691A/heme com- basal channel activity and its response to LCA, of BK b1 subunits across plex was unresponsive up to 1.2 mM of CO. However, in the presence of arteries from cerebral and systemic vasculatures. In particular, basilar and CO-releasing CORM-2 ([RuCO3CI2]2), the GR-M691A/heme complex ex- coronary artery myocytes show an increased biochemical and functional hibited b/a absorption bands (540, 570nm) characteristic of CO binding. presence of BK b1 subunits, suggesting that these vascular territories We propose that CO regulates BK conductance by inducing conformational are more susceptible to modulation via BK b1. Support: HL104631, rearrangements in its GR ligand-sensing domain. Specifically, CO substi- R37AA11560 (AMD); AHA Pre-doctoral Fellowship (GK). tutes M691 as the second heme axial ligand; cleavage of the M691-heme bond partially unfolds the BKCytCD, generating the optically-tracked 559-Pos Board B324 conformational changes. This action may relieve heme-induced inhibition Functional Coupling of BK Channels to NMDA Receptors in Hippocampal of BK opening. Dentate Gyrus Xin Guan, Jiyuan Zhang, Qin Li, Hui-Lin Pan, Jiusheng Yan. 557-Pos Board B322 Department of Anesthesiology and Perioperative Medicine, The University Role of the slo1 CRAC4 Motif in BK Channel’s Ethanol Sensitivity of Texas MD Anderson Cancer Center, Houston, TX, USA. Maria N. Simakova, Dasha Zaytseva, Shivantika Bisen, Alex M. Dopico, The large conductance calcium- and voltage-activated potassium channel (BK Anna N. Bukiya. channels) is widely expressed in the central nervous system. The physiological Pharmacology, UTHSC, Memphis, TN, USA. roles of BK channels and the calcium sources of their activation in mammalian Cholesterol (CLR) has been recognized for its ability to interact with ion brain remain not well-understood. Our proteomic and biochemical analyses of channels. Among ion channels that sense CLR presence are large conduc- BK channels and the calcium-permeable NMDA receptors and have found that tance, voltage/Ca2þ-gated Kþ (BK) channels. Functional BK channels result they form protein complexes in whole brain and various brain regions, consist- from tetrameric association of pore-forming slo1 subunits. CLR inhibition ing of the obligatory BK channel alpha-subunit and GluN1 subunits and at least of slo1 current may result from CLR-sensing by the slo1 cytosolic tail the regulatory GluN2A and GluN2B subunits. We examined the functional domain (CTD). CTD contains seven CLR-recognition amino acid consensus coupling of BK channels to NMDA receptors in mice hippocampal dentate (CRAC) motifs that contribute to the overall CLR sensitivity of the channel, gyrus. We observed that glutamate evoked outward currents of BK channels with the contribution by CRAC4 being studied (Singh et al., 2012). Alcohol in dentate gyrus granule cell soma at potentials more positive than 20 mV (ethanol) also modifies BK channel function, this effect resulting from an which were sensitive to BK channel-specific blocker paxilline and abolished 2þ orchestration of several factors that include [Ca ]i. In native membranes, by NMDA receptor antagonist AP-5. We also found that blockade of BK chan- 2þ ethanol increases slo1 channel open probability (Po) at [Ca ]i<20-30 mM nels by paxilline increased the amplitude of excitatory postsynaptic potential 2þ whereas reduces Po at [Ca ]i<30 mM. Both CLR-enrichment and CLR- (EPSP) of granule cells evoked by stimulation in the perforant path-granule depletion of native membranes modify ethanol action on BK channels. cell synapses, which was prevented by the presence of AP-5. We thus inferred Thus, we set to determine the role of CLR-sensing by the CRAC4 motif that BK channels were activated by NMDA receptor-mediated Ca2þ influx in in the slo1 channel’s ethanol sensitivity. Slo1 cloned from rat cerebral artery hippocampal dentate gyrus granule cells and the calcium-mediated functional myocytes (cbv1) and the cbv1CRAC4T450F were incorporated into POPE: coupling between these two types of channels plays a role in regulation of þ þ 2þ POPS (3:1 w/w) bilayers in 300/30 mM K i/K o gradient. At 0.3 mMCa i, synaptic transmission.

BPJ 7731_7735 Sunday, February 12, 2017 113a

TRP Channels I closed/open-state specific hydrogen bonds. By comparing the closed-state sim- ulations at 30C and 60C, we observed heat-activated conformational changes 560-Pos Board B325 in the MPD linker, the outer pore, and the TRP helix that resemble the closed- Binding of Extracellular Ca2D to the Specific Amino Acids is Required for to-open conformational changes, along with partial formation of the open-state Heat-Evoked Activation of TRPA1 specific hydrogen bonds. Some of the residues involved in the above key Erkin Kurganov1, Shigeru Saito1, Claire T. Saito1, Makoto Tominaga2. hydrogen bonds were validated in comparison with previous mutational 1Division of Cell Signaling, National Institute for Physiological Science, studies. Taken together, our MD simulations have offered rich structural and Okazaki-shi, Japan, 2Division of Cell Signaling, National Institute for dynamic details beyond the static structures of TRPV1, and promising targets Physiological Science, OIIB, Okazaki-shi, Japan. for future mutagenesis and functional studies of the TRPV1 channel. Transient receptor potential, ankyrin 1 (TRPA1) is a homotetrameric nonselec- 563-Pos Board B328 tive cation- permeable channel with six transmembrane domains handed by The Emerging Role of Human TRPV1 S1 S4 Sensing Domain in Channel cytoplasmic N and C termini. Although many TRP channel family members Gating contain the range of 3-6 ankyrin repeats (AR) within the N-terminal region, Minjoo Kim, Nicholas Sisco, Jacob Hilton, Wade Van Horn. TRPA1 is distinguished by having an unusually large number of such repeats Arizona State University, Tempe, AZ, USA. (16-17 ARs). A number of studies have shown that both intracellular and extra- þ Transient potential receptor ion channel vanilloid member 1, TRPV1, was cellular Ca2 is a key regulator of many TRP channels, including TRPA1. In þ initially identified as the capsaicin receptor, but is also modulated by various the previous study, we found that extracellular Ca2 , but not intracellular þ chemical and physical stimuli including elevated temperature, acidic pH, Ca2 plays an important role in heat-evoked activation of green anole 2þ endogenous ligands and regulatory proteins. TRPV1 functions as a primary TRPA1 (gaTRPA1). In this study, we focus on extracellular Ca -dependent heat sensor in humans. Recently determined structures of rat TRPV1 have pro- heat sensitivity of gaTRPA1 by comparing gaTRPA1 with other heat-activated vided a better understanding of channel architecture and the ligand gating. TRPA1s from rat-snake (rsTRPA1) and chicken (chTRPA1). We found that However, the mechanism of how thermal stimulus is integrated and converted rsTRPA1 and chTRPA1s are activated by heat with small inward currents in 2þ to channel gating remains poorly understood. The isolated S1-S4 sensing the absence of extracellular Ca . We identified several negatively charged domain of human TRPV1 (hV1-SD) was probed for its role in ligand gating amino acid residues (glutamate and aspartate) near outer pore vestibule in and thermosensing using biophysical techniques such as solution nuclear mag- gaTRPA1, chTRPA1 and rsTRPA1 for activation by heat in the presence of þ netic resonance (NMR) spectroscopy and far-UV circular dichroism. Here, we extracellular Ca2 . These results suggest that the neutralization of the acidic 2þ investigate the direct binding of capsaicin to the hV1-SD. In addition, we iden- amino acids by extracellular Ca is important for the heat-evoked activation tify that the hV1-SD undergoes reversible conformational change as a function of gaTRPA1, chTRPA1 and rsTRPA1. of temperature. Thermodynamic studies suggest that the hV1-SD provides the dominant TRPV1 driving force that underlies thermosensitivity. Further NMR 561-Pos Board B326 studies probe temperature dependent changes in this domain and suggest sim- Oligomeric and Thermal Stability of TRPA1 Coiled-Coil Domain by Poly- ilarities between TRPV1 ligand and temperature based activation. phosphates Gilbert Q. Martinez, Luke D. Cody, Sharona E. Gordon. 564-Pos Board B329 Physiology and Biophysics, University of Washington, Seattle, WA, USA. Inactivation in TRPV1 Ion Channels Human Transient Receptor Potential Ankyrin 1 (TRPA1) is a cation channel Leon D. Islas1, Ana Sanchez-Moreno1, Gisela Rangel-Yescas1, involved in the sensation of pain that is regulated by multiple stimuli including Ernesto Ladron de Guevara1, Tamara Rosenbaum2. temperature, a wide range of irritants, and oxidative stimuli. The importance of 1Physiology, School of Medicine, National Autonomous University of TRPA1 is underscored by the numerous studies of the channel as a target for Mexico, Mexico City, Mexico, 2Instituto de Fisiologia Celular, National novel pain therapies. The recent cyro-EM structure (Paulsen et al, 2015) pro- Autonomous University of Mexico, Mexico City, Mexico. vides a strong framework in which to understand the mechanisms underlying TRPV1 are cation-permeable ion channels involved in several physiological functional activation. The structure of TRPA1 revealed the presence of a C-ter- processes. TRPV1 can be activated by several, very different, stimuli. Chief minal coiled-coil domain that was hypothesized to require polyphosphates for among these is a characteristic activation by temperatures in the noxious range oligomeric structural stability. This is consistent with the requirement of poly- and activation by several ligands, including capsaicin, the pungent compound phosphates for functional channel expression assessed via electrophysiology that gives chilies their characteristic spiciness. Depolarizing voltages can (Kim and Cavanaugh, 2007). Furthermore, it has been speculated that the un- also activate these channels, although with a very small voltage-dependence folding of the TRPA1 coiled-coil helices in response to temperature could be (the associated valence of activation is at most 1 elementary charge, eo). In involved in thermal gating, as has been observed in bacterial sodium channels the absence of any other ligands, voltage behaves as a ‘‘partial agonist’’, pro- (Arrigoni et al, 2016). Here we examine the biochemical and biophysical prop- ducing a maximal open probability of less than 0.5 at extremely high voltages. erties of the coiled-coil of TRPA1 to determine the role of polyphosphates in Activation by voltage is more effective when combined with other activation oligomerization and their contributions to thermal stability. modes, including activating by capsaicin and temperature. It has been previ- ously shown that allosteric coupling models can describe this behavior, with in- 562-Pos Board B327 dependent voltage, temperature and ligand gating modules allostericaly Toward Elucidating the Heat Activation Mechanism of the TRPV1 Chan- coupled to a pore module. Here we show that in the presence of these activators, nel Gating by Molecular Dynamics Simulation TRPV1 channels show a marked inactivation behavior induced by high volt- Han Wen, Feng Qin, Wenjun Zheng. ages, which can be seen as time- and voltage-dependent reductions in the SUNY at Buffalo, Buffalo, NY, USA. magnitude of the outward currents and can be explained by models with inac- As a key cellular sensor, the TRPV1 cation channel undergoes a gating transi- tivation happening after the open state. High activating temperatures (47-60 oC) tion from a closed state to an open state in response to various physical and can also induce inactivation by itself, even at moderate voltages and this tem- chemical stimuli including noxious heat. Despite years of study, the heat acti- perature-dependent inactivation seems to be an irreversible process. Inactiva- vation mechanism of TRPV1 gating remains enigmatic at the molecular level. tion of TRPV1 has not been previously described; although careful Toward elucidating the structural and energetic basis of TRPV1 gating, we inspection of previously published TRPV1 current records show evidence of have performed extensive molecular dynamics (MD) simulations (with cumu- this phenomenon. This work supported by grants PAPIIT-DGAPA-UNAM lative simulation time of 3 microseconds), starting from the high-resolution No. IN209515 to LDI and No. IN200314 to TR and CONACYT Fronteras closed and open structures of TRPV1 solved by cryo-electron microscopy. In de la Ciencia No.77 to LDI and TR. the closed-state simulations at 30C, we observed a stably closed channel con- stricted at the lower gate (near residue I679), while the upper gate (near resi- 565-Pos Board B330 dues G643 and M644) is dynamic and undergoes flickery opening/closing. In Kinetic Analysis of Vanilloid-Induced Activation in TRPV1 Channel the open-state simulations at 60C, we found higher conformational variation Simon Vu, Bo Hyun Lee, Jie Zheng. consistent with a large entropy increase required for the heat activation, and University of California, Davis, Davis, CA, USA. both the lower and upper gates are dynamic with transient opening/closing. The capsaicin receptor, TRPV1 ion channel, is a polymodal nociceptor and Through ensemble-based structural analyses of the closed state vs. the open attractive drug target. Recent advances in cryo-electron microscopy revealed state, we revealed pronounced closed-to-open conformational changes the vanilloid-binding pocket in high resolution, which allowed atomic interac- involving the membrane proximal domain (MPD) linker, the outer pore, and tions mediating capsaicin binding to be proposed. In the current model, capsa- the TRP helix, which are accompanied by breaking/forming of a network of icin adopts a ‘‘tail-up, head-down’’ pose; key interactions include two

BPJ 7736_7739 114a Sunday, February 12, 2017 hydrogen bonds formed between the amide of capsaicin and Thr551 and be- capsaicin, and noxious temperatures above 42C. While recent cryo-EM struc- tween the hydroxyl on the vanillyl group and Glu571 (of mouse TRPV1). tures have revealed the binding pockets for capsaicin, double-knot toxin Guided by this model, the vanilloid-insensitive TRPV2 channel has been suc- (DkTx), and resiniferatoxin (RTx), no consensus has been reached on the struc- cessfully transformed to be vanilloid-sensitive with minimal alterations. In this tural basis of temperature activation. It was recently discovered that removal of study, we provide further evidence from kinetic and equilibrium analyses in sodium from the extracellular side of the membrane shifts a temperature-depen- support of the capsaicin binding model. Eliminating the key hydrogen bonds dent opening transition such that TRPV1 is open at room temperature, indi- by point mutations right-shifted the concentration-response relationship, which cating that sodium binding modulates activation by temperature. was achieved by accelerating the current OFF rate and decelerating the ON TRPV1’s closest relative, TRPV2, is similar in sequence and structure, and can rate. These observations are consistent with the expectation that mutations be rendered sensitive to the TRPV1-selective agonist RTx by only two point would weaken capsaicin binding. Surprisingly, though resiniferatoxin (RTX) mutations, suggesting that the two channels have similar underlying gating has been proposed to bind in a similar fashion as capsaicin, kinetic and equilib- mechanisms. In spite of these similarities, TRPV2 fails to respond to most rium analyses revealed that removing these crucial hydrogen bonds did not alter TRPV1 agonists, including temperatures near 42C, and TRPV2 is not acti- RTX activation kinetics as they did capsaicin, suggesting that RTX may behave vated by removal of extracellular sodium. To probe the link between tempera- differently. ture dependence and sodium sensitivity, and to localize the sodium ion binding site, we generated a series of chimeras in which transmembrane regions of 566-Pos Board B331 TRPV1 were transferred into TRPV2. Transfer of the entire transmembrane Rational Design and Validation of a Vanilloid-Sensitive TRPV2 Ion domain and TRP box of TRPV1 into TRPV2 resulted in chimeras that became Channel activated upon sodium removal, supporting the feasibility of our strategy. Fan Yang, Simon Vu, Vladimir Yarov-Yarovoy, Jie Zheng. Although none of chimeras in which small regions of TRPV1 were transferred Physiology and Membrane Biology, University of California, Davis, Davis, into TRPV2 were activated upon sodium removal, several exhibit potentiation CA, USA. when sodium is removed in the presence of subthreshold concentrations of Vanilloids activation of TRPV1 represents an excellent model system for agonist, indicating the successful transfer of the sodium binding site. Ongoing ligand gating of ion channels. Recent studies using cryo electron microcopy experiments are focused on determining the minimal TRPV1-like sodium bind- (cryo-EM), computational analysis and functional quantification revealed the ing site and characterizing the temperature-activation thresholds of these location of capsaicin-binding site and critical residues mediating ligand-bind- chimeric proteins to determine whether TRPV1-like temperature sensitivity ing and channel activation. To validate our current working model for capsaicin is conferred by the sodium-binding domain. activation, here we have successfully converted the vanilloid-insensitive TRPV2 channel to be resiniferatoxin-activated, using a rationally designed 569-Pos Board B334 minimal set of four point mutations (F467S-S498F-L505T-Q525E, termed Investigating the Putative Activation Gate in the Selectivity Filter of the TRPV2_Quad). We found that resiniferatoxin binds to TRPV2_Quad with TRPV1 Channel sub-micromolar affinity even though the ligand-induced open state is relatively Andres Jara-Oseguera, Kenton J. Swartz. unstable. Using Rosetta-based molecular docking, we observed a common Molecular Physiol/Biophys, NINDS, NIH, Bethesda, MD, USA. structural mechanism underlying vanilloids activation of TRPV1 and The TRPV1 cation channel functions as heat sensor and an integrator of painful TRPV2_Quad, where the ligand serves as a molecular ‘‘glue’’ that bridges stimuli in sensory neurons. The mechanism through which any of the multiple the S4-S5 linker to the S1-S4 domain to open these channels. Furthermore, known TRPV1 modulators induce the opening and closing of the pore is un- our analysis revealed previously unrecognized structural requirements needed known. In order to understand this, it is necessary to identify the regions of for capsaicin binding to TRPV1. These results should help guide the design the channel that directly open and close the pore, known as ‘‘activation gates’’. of drug candidate compounds for this important pain sensor. CryoEM structures of the TRPV1 channel in different closed and activated states revealed that the conduction pathway in the closed state presents two 567-Pos Board B332 constrictions, or activation gates, that have to expand in the activated state to Localization of the Temperature Sensors to the Pore Domain of the TRPV1 enable cation conduction. The first constriction is formed by Ile679 at the intra- Channel cellular half of the pore-lining S6 helices. The second constriction is located at Feng Zhang1, Sonya Hanson1,2, Andres Andres Jara-Oseguera1, the selectivity filter, and its function as a gate has not been tested experimen- Kenton Swartz1. tally. In order to determine whether the conducting and non-conducting confor- 1NINDS, NIH, Rockville, MD, USA, 2Computational Biology Program, mations of the selectivity filter are associated with the activation or deactivation Memorial Sloan Kettering Cancer Center, New York, NY, USA. of the channel, we have investigated the accessibility to external cadmium ions Temperature detection is key to animal survival, and thermo-TRP channels of cysteines introduced along the conduction pathway. We have introduced sin- play important roles in sensing environmental temperature in mammals. The gle cysteine substitutions starting at positions above the selectivity filter and all TRPV1 channel is a classic thermo-TRP channel that is also a detector of the way to the internal gate at Ile679. We have determined which introduced noxious stimuli, including acidosis, vanilloid compounds, venom toxins, mem- cysteines can coordinate externally applied cadmium ions leading to rapid cur- brane lipids and heat. Despite extensive investigation, the mechanisms of tem- rent block. We found that surprisingly few positions can lead to high-affinity perature sensing and the location of the temperature sensor in TRPV1 channels metal coordination. Moreover, those cysteines that coordinate metals can remains enigmatic. One candidate for housing the temperature sensor is the also form spontaneous disulfide bridges that are consistent with the structural outer pore of TRPV1, as mutants in this region, as well as occupancy by toxins data. Finally, preliminary experiments suggest that in the closed state, cadmium and sodium ions, have been shown to alter temperature-dependent activation. ions can still access cysteines below the selectivity filter, suggesting that the fil- Here we engineered chimaeras between TRPV1 and the Shaker Kv channel us- ter does not function as a gate. We are now investigating the effects of the ing available cryo-EM and X-ray structures to see if functional chimeras could multi-ion binding properties of the TRPV1 pore on the kinetics of cadmium be generated. Our biochemical analysis shows that a chimera containing the coordination. pore domain of TRPV1 in the background of the Shaker Kv channel can be robustly expressed in oocytes and traffic to the membrane surface. Electrophys- 570-Pos Board B335 iological recordings show that these chimeric channels can be activated by a The Mechanism of Regulation of TRPV6 Channels by PI(4,5)P2 TRPV1-selective pore targeting double-knot toxin and are permeable to cal- Marina Kasimova1, Aysenur Yazici2, Chike Cao2, Phanindra Velisetty2, cium ions similar to TRPV1. Remarkably, these chimeric channels are highly Vincenzo Carnevale1, Tibor Rohacs2. 1 2 temperature sensitive, being activated by temperatures above 40C, similar to Temple University, Philadelphia, PA, USA, Rutgers - New Jersey Medical the WT TRPV1 channel. Taken together, our results suggest that the pore School, Newark, NJ, USA. 2þ domain of TRPV1 houses the sensory apparatus underlying steep tempera- Transient Receptor Potential Vanilloid 6 (TRPV6) is a Ca selective ion chan- 2þ ture-dependent activation. nel playing important roles in intestinal Ca absorption, male fertility and can- cer development. TRPV6 is a member of the highly diverse TRP ion channel 568-Pos Board B333 family. The only known common functional feature among TRP channels is Transfer of TRPV1 Sodium Binding Site into TRPV2 their dependence on, and modulation by phosphoinositides, mostly phosphati- Katherine E. Huffer, Andre´s Jara-Oseguera, Kenton J. Swartz. dylinositol 4,5-bisphosphate [PI(4,5)P2]. Most TRP channels require factors National Institute of Neurological Disorders and Stroke, National Institutes of other than PI(4,5)P2 to open, which are often in complex interaction with Health, Bethesda, MD, USA. PI(4,5)P2 regulation, which hinders understanding of the molecular mechanism The transient receptor potential vanilloid 1 (TRPV1) channel is a nonselective of how PI(4,5)P2 opens these channels. TRPV6 is constitutively active, thus cation channel activated by a wide variety of agonists, including acid, toxins, devoid of these complexities, and therefore is an ideal model to gain molecular

BPJ 7736_7739 Sunday, February 12, 2017 115a

insight into regulation by PI(4,5)P2. We computationally docked PI(4,5)P2 to phase to isometric tension. The predicted rates, r, of quick recovery phase both TRPV6, and the heat and capsaicin sensitive TRPV1. We find that following the length step, were similar to observations. However, to obtain PI(4,5)P2 binds to TRPV1 mainly in the proximal C-terminus and in the S4- good fits to the observed T1-y nonlinear relationship required inclusion S5 linker, consistent with earlier predictions, and experimental data. In nonlinear crossbridge compliance (Kaya et al., Science 329:686-688, 2010). TRPV6, however, the closest interacting residues were in the proximal N-ter- In summary, the precise coupling between nonlinear strain dependent transition minus, close to the first transmembrane region and, similar to TRPV1, in the rates of the actomyosin cycle, nonlinear crossbridge compliance and elasticity S4-S5 linker. Introduction of a positive charge in the S4-S5 linker that is of myofilaments provides excellent quantitative description of the molecular in- missing from TRPV6, but present in TRPV1, markedly increased the affinity teractions and muscle fiber transient responses. of TRPV6 to PI(4,5)P2. Neutralizing a proximal N-terminal positively charged Supported by: R01 AR048776, HD048895 and W.Trust 085309 residue predicted to interact with PI(4,5)P2 markedly reduced current TRPV6 amplitudes, but neutralizing neighboring positively charged residues had no ef- 573-Pos Board B338 In Vivo fect. Our data will provide mechanistic insight into PI(4,5)P2 regulation of Increased Non-Uniformity in Sarcomere Length during a Tetanic TRPV6. Contraction Eng Kuan Moo, Timothy R. Leonard, Walter Herzog. 571-Pos Board B336 Human Performance Laboratory, University of Calgary, Calgary, AB, The Role of the Sensing Domain (S1-S4) in TRPM8 Temperature and Canada. Menthol Dependent Gating Introduction Parthasarathi Rath. The maximal, steady-state, isometric force produced by a muscle depends on its School of Molecular Sciences, Arizona State University, TEMPE, AZ, USA. sarcomere length (SL). In previous studies in single myofibrils, it was found The human transient receptor potential melastatin 8 (hTRPM8) ion channel is a that sarcomere length non-uniformities increased during activation and force primary sensor of environmental cold temperature. hTRPM8 is gated in a poly- production. However, single myofibrils lack much of the structural proteins modal manner by voltage, lipids, modulatory proteins, and chemical ligands, that provide stability to entire muscles, thus the observed SL non-uniformities including menthol and icilin. TRPM8 is also involved in human health and dis- in myofibrils might not occur in whole muscles. This study was aimed at inves- ease, playing roles in pathophysiologies including cancer, pain, obesity, and tigating the change in SL distribution during tetanic contractions in an intact diabetes. A better understanding of the molecular mechanism of channel acti- muscles of live mice. vation may prove useful in unlocking the TRPM8 therapeutic potential. Here, Methods we analyze the sensing domain (transmembrane helices S1-S4) as a function of Mice were anaesthetized using isoflurane. The proximal femur and foot of the temperature and menthol with circular dichroism (CD), and compare it with the left lower limb were clamped. The skin over the left tibialis anterior (TA) mus- S1-S6 transmembrane domain and full-length hTRPM8. Additionally, planar cle was opened and stretched to form a bath for a saline solution that allowed bilayer electrophysiology measurements of the S1-S6 transmembrane domain for imaging using a water-immersion objective. The TA was supra-maximally in presence of PIP2 and menthol indicate similar thermosensitivity when stimulated using a nerve cuff electrode on the sciatic nerve using 0.1ms square compared to the full-length protein. The temperature and menthol dependent wave pulses at 60Hz for 1s. SL were measured using second harmonic gener- conformational change of the sensing domain was further investigated by solu- ation microscopy for the fully stretched TA. Measurements were made for the tion nuclear magnetic resonance (NMR) spectroscopy. Narrowing down from passive and activated TA over an area of 160x3 mm2 in the mid-belly of the the full-length protein to the sensing domain, our data suggest that the S1-S4 muscle. sensing domain is important for both TRPM8 temperature- and menthol-depen- Results dent gating of the ion channel. SLs for the passive muscle were 2.5350.06mm (mean5sd). During contrac- tion, sarcomeres shortened by ~12% to 2.2450.12mm. The coefficient of vari- ation of the SLs doubled from 2.4% at rest to 5.2% during tetanic contractions. Skeletal Muscle Mechanics, Structure, and The range of SLs increased from the passive (2.36-2.71mm) to the active state Regulation (1.85-2.62mm). Conclusion 572-Pos Board B337 SL non-uniformity doubled during muscle activation and differed by more than Modulation of Fast Transients by Compliance of Crossbridges and 0.7 mm. The functional implications of these massive SL non-uniformities need Myofilaments to be explored, and the common practice of representing muscles with a single Srboljub M. Mijailovich1, Djordje Nedic2, Boban Stojanovic2, SL value needs to be reconsidered. Thomas C. Irving3, Michael A. Geeves4. 1Mechanical Engineering, Wentworth Institute of Technology, Boston, MA, 574-Pos Board B339 USA, 2Mathematics and Informatics, University of Kragujevac, Kragujevac, Myofibrillar Regulatory Mechanisms of Stretch Activation in Mammalian Serbia, 3Department of Biological Sciences, Illinois Institute of Technology, Striated Muscle Chicago, IL, USA, 4School of Biosciences, University of Kent, Canterbury, Joel C. Robinett, Laurin M. Hanft, Kerry S. McDonald. United Kingdom. Medical Pharmacology and Physiology, University of Missouri, Columbia, Understanding the coupling between actomyosin cycle and compliance of MO, USA. crossbridges and the myofilaments in the 3D sarcomere lattice is essential for Stretch activation is described as a delayed increase in force after an imposed interpretation of fast transient data and for extracting key parameters from stretch. Stretch activation is an essential process in the flight muscles of many the observations. This mechano-chemical coupling is complex due to the insects and is observed, to some degree, in mammalian striated muscles. The three-dimensional nature of myosin binding to actin, as well as its effect on mechanistic basis for stretch activation remains uncertain, although appears the strain-dependence of transition rates between actin-myosin states. In order to involve cooperative activation of the thin filaments (Campbell, K.S., Bio- to assess the effect of crossbridge compliance and myofilament elasticity on physical Journal, 2006). The purpose of this study was to address myofibrillar sarcomere contraction we implemented both linear and nonlinear crossbridge regulatory mechanisms of stretch activation in mammalian striated muscle. For compliance in the computational platform MUSICO (MUscle SImulation these studies, permeabilized rat slow-twitch and fast-twitch skeletal muscle fi- COde). In the model simulation we included nine state myosin cycle (Smith bers were mounted between a force transducer and motor, and a slack-re-stretch þ et al., Ann. Biomed. Eng.36(10):1624-40, 2008), up to 20 sarcomeres in series maneuver was performed over a range of Ca2 activation levels. Following and at least 500 myosin filaments per sarcomere. Stochastic binding in the 3D slack-re-stretch there was a stretch activation process that often resulted in a sarcomere lattice generates large variation of strains in time at any location transient force overshoot (PTO), which was quantified relative to steady-state 2þ along the filaments and up to 20% between the filaments. Overall the model isometric force. PTO was highly dependent upon Ca activation level and predictions are modulated by nonuniform distributions of crossbridge strains the magnitude was greater in slow-twitch fibers than fast-twitch fibers. In along the filament overlap and by variation of forces between the filaments both slow-twitch and fast-twitch fibers, force development involved a fast, 2þ 2þ due to the stochastic myosin binding process. The predicted T1-T2 transient re- Ca activation dependent process (k1) and a slower, less Ca activation sponses of the multi-sarcomere system strongly depend on the number of dependent process (k2). Interestingly, the two processes converged at low levels 2þ attached crossbridges. This dependence is strongly evident in traces of T1 vs. of Ca activation in both fiber types. PTO also contained a relaxation phase, 2þ the imposed length change, y, and to a much lesser degree of the T2-y responses. which progressively slowed as Ca activation levels increased and was þ After matching the observed value of isometric tension before release, the sim- more Ca2 activation dependent in slow-twitch fibers. We next investigated ulations closely recapitulate the rapid tension recovery, after the rapid drop in the effects of PKA-induced phosphorylation of MyBP-C on stretch activation tension to T1, to the early plateau, T2,followed by a slow tension redevelopment in slow-twitch fibers. Following PKA treatment, the magnitude of PTO more

BPJ 7736_7739 116a Sunday, February 12, 2017 than doubled, however, this only occurred at low levels of Ca2þ activation netics is unclear. Here, we used a spatially explicit, computational model of (i.e., ~25% maximal Ca2þ activated force). Overall, these results are consistent muscle force generation to examine how cross-bridge characteristics, such as with a model whereby stretch transiently increases the number of cross-bridges force per cross-bridge and ton, are affected by various rates of shortening and made available for force generation and PKA phosphorylation of MyBP-C en- lengthening (0.1-1 muscle lengths (ML) per second). We found that the force hances these stretch activation processes. per cross-bridge increased by ~15% when the sarcomere was lengthened at 0.1 ML/s, and by ~40% when lengthened at 1.0 ML/s. Conversely, force per 575-Pos Board B340 cross-bridge decreased during shortening, by ~25% at 0.1 ML/s and by Effect of Calcium on Myosin Binding to a Regulated Thin Filament from ~70% at 1.0 ML/s. Concurrent with these measurements, we found that myosin Single Molecule to Ensemble ton decreased by ~50-70% as the sarcomere was lengthened at 0.1-1 ML/s, Thomas Longyear1, Sam Walcott2, Edward P. Debold1. potentially due to increased cross-bridge strain or forced detachment. However, 1 Kinesiology, University of Massachusetts, Amherst, Amherst, MA, USA, myosin t increased by ~50% when the sarcomere was shortened at 0.1 ML/s, 2 on University of California, Davis, Davis, CA, USA. but decreased by ~50% when shortened at 1 ML/s. These data suggest that Muscle contraction is regulated via two processes that both contribute to acti- myosin attachment time is sensitive to length-dependent changes in cross- vation of the thin filament (actinþtroponinþtropomyosin). First, calcium bridge strain, and that the kinetics may be more sensitive to the rate of short- (Caþþ) displaces tropomyosin, which increases the availability of myosin ening than the rate of lengthening. While these results are model dependent binding sites on actin. Second, myosin binding accelerates the attachment of and rely upon parameter estimation to fit model predictions with empirical neighboring myosin molecules to the thin filament. The relative importance data from skinned fibers, these computational models help illustrate complex and underlying mechanisms of these two processes remain unclear, therefore system behavior underlying dynamic muscle contraction. These simulation re- we devised assays to separately quantify their contribution . Myosin binding sults indicate that sarcomere lengthening may increase the strain borne by in- only activates nearby myosins, therefore by varying myosin density we varied dividual cross-bridges and decrease myosin ton, and conversely, shortening the contribution of this process. Myosin-induced activation was eliminated by may decrease cross-bridge strain and prolong myosin ton. using a single myosin molecule in the laser trap assay to directly measure Caþþ’s effect on myosin’s interaction with a thin filament. Slightly increasing 578-Pos Board B343 the myosin density generated mini ensembles of myosin that experienced weak Cleavage of Skeletal Muscle Myosin Loops 1 and 2 Leads to a Decreased coupling . Increasing the myosin density further using a motility assay created Function large myosin ensembles with strong myosin-induced activation, and thin fila- Yu-Shu Cheng, Oleg Matusovskiy, Dilson Rassier. ment speed was measured at increasing ATP and Caþþ levels. The data Department of Kinesiology and Physical Education, McGill University, were analyzed with a mathematical model that de-convolves the effects of Montreal, QC, Canada. Caþþ-induced and myosin-induced activation. This analysis revealed (1) Introduction: There is controversy over the roles of loops 1 and 2 in skeletal how Caþþ affects myosin’s attachment to a thin filament; and (2) that myosin muscle myosin. The goals of this study were to investigate the effects of cleav- molecules are coupled over a Caþþ-independent distance L=400nm. Scaling age of loops 1 and 2 on the amino-acid structure of the myosin and to correlate up the model generated accurate predictions of isometric force production these changes with myosin function. Methods: Heavy meromyosin (HMM) and Caþþ binding to the thin filament previously observed in muscle fibers, was isolated from myosin purified from rabbit psoas, and treated with trypsin providing a molecular basis for how Caþþ and myosin contribute to filament for periods of time ranging between 0 and 30 minutes, which caused a cleavage activation. of loops 1 and 2. Specific cleavage of loop 1 was performed by adding actin that binds to myosin and protects loop 2, and cleavage of both loops was achieved 576-Pos Board B341 by an ATP-free treatment. A motility essay with a frictional load was used by Coordinated Variations in Myosin Light Chain and Troponin T Isoforms adding different concentrations of a-actinin (0.5 to 1.5mg/mL), and actin veloc- and in Maximal Shortening Velocity in Human Slow Skeletal Muscle ity was recorded at 30C in 1% nitrocellulose-treated coverslips. The myosin Fibers bands were cut from sodium dodecyl-sulfate polyacrylamide gel after trypsin Peter J. Reiser. proteolysis and subjected to tryptic cleavage. Myosin peptides of each band Biosciences, Ohio State University, Columbus, OH, USA. were identified by liquid chromatography-tandem mass spectrometry (LC- Recent measurements revealed a 1.7-fold range in maximal shortening velocity MS/MS). A model was developed to interpret the results and showed that the (Vmax) among a set of slow fibers from adult human skeletal muscle. The fibers loop 2 was damaged before loop 1 on myosin during actin motility. Results were examined on multiple gel formats to determine whether there were differ- and Discussion: Sliding velocity of actin filaments decreased consistently ences in myofibrillar protein isoform composition that might explain the vari- with increasing time during trypsin treatment. The decrease was dependent ation in Vmax. All of the fibers in this set were determined to express on the treatment: cleavage of loops 1 and 2 decreased the velocity from exclusively slow-type myosin heavy chain (MHC). The fibers were then exam- 4.05mm/sec to 2.24mm/sec after 4 minutes of enzymatic treatment, and cleav- ined on a gel format to reveal potential differences in myosin light chain (MLC) age of loop 1 decreased the velocity from 3.82mm/sec to 1.22mm/sec after 2 isoform composition. The faster fibers in this set expressed substantial amounts minutes of enzymatic treatment. These results suggest that loops 1 and 2 regu- of fast-type MLC1 (MLC1F), along with slow type MLC1 (MLC1b) and slow late the velocity of the myosin motor and the kinetics of the actomyosin type MLC2, and their mean Vmax was significantly higher (24%) than the interactions. slower fibers in this set, which had only trace or no detectable amounts of MLC1F. The slower fibers had greater amounts of a protein that is tentatively 579-Pos Board B344 identified as MLC1Sa, which others have reported to be expressed in human Force and Power of a Synthetic Myosin II-Based Machine skeletal muscle. The same gel format also indicated differences in troponin T Pasquale Bianco1, Irene Pertici1, Luca Melli2, Giulia Falorsi1, (TnT) isoform composition among the same fibers. Regression analysis re- Danut-Adrian Cojoc3, Tama´s Bozo´4, Miklos Kellermayer4, vealed that there are strong associations between the expression of specific Vincenzo Lombardi1. 1 TnT isoforms and the expression of specific MLC 1 isoforms among human Department of Biology, University of Florence, Sesto Fiorentino, Italy, 2 3 slow skeletal muscle fibers. Conclusion: coordinated variations in MLC iso- National Heart, Lung and Blood Institute, Bethesda, MD, USA, IOM-CNR, 4 form and TnT isoform appear to provide a mechanism to expand the range Trieste, Italy, Semmelweis University, Budapest, Hungary. of contractile properties of human slow muscle fibers that express exclusively The function as a collective motor of skeletal muscle myosin II is studied slow-type MHC. in vitro with a synthetic bio-machine, consisting of an ensemble of myosin mo- tors interacting with a single actin filament attached with the correct polarity to 577-Pos Board B342 a bead trapped in the focus of a Dual Laser Optical Tweezers (DLOT, Bianco et Computational Simulations of Load-Dependent Myosin Kinetics during al. Biophys. J.101:866-874, 2011). The motor ensemble provides the condition Muscle Shortening and Lengthening for cyclic interactions with the actin filament, allowing the development of Axel J. Fenwick, Bertrand C.W. Tanner. steady force and filament sliding. The mechanical outputs of the machine are Integrative Physiology and Neuroscience, Washington State University, measured by means of the DLOT, which acts as a force transducer (range Pullman, WA, USA. 0.5-200 pN and resolution ~0.3 pN), and a piezoelectric nano-positioner car- Muscle contraction results from myosin heads binding to the actin filament to rying the support for the myosin motors, which acts as a length transducer form cross-bridges, with contractile force dependent on the total number of (range 1-75.000 nm and resolution ~1 nm). Here is reported the performance cross-bridges, the force that each cross-bridge produces, and the myosin attach- of a first version of the machine, consisting of an ensemble of myosin motors ment time (ton). Muscle shortening and lengthening also influences contractile purified from frog skeletal muscle randomly adsorbed on the surface of a chem- force, though the direct effect of dynamic length changes on cross-bridge ki- ically etched single-mode optical fibre with diameter 4 mm. Isometric and

BPJ 7736_7739 Sunday, February 12, 2017 117a isotonic contractions are reproduced by the motor ensemble in solution with Medical School, Worcester, MA, USA, 3Oregon Health and Science physiological [ATP] (2 mM) and temperature 21 C. Following a drop in force University, Portland, OR, USA, 4Molecular Physiology & Biophysics from the maximum isometric value (F0) to a lower value (F), the actin filament Division of Cardiovascular Health and Disease, University of Cincinnati, slides at a constant velocity (V) which is larger the smaller the force, as ex- Cincinnati, OH, USA. pected from the in vivo force-velocity relation. Up to five F-V points for Myosin-binding protein C (MyBP-C) is a sarcomeric protein localized to each interaction can be determined, allowing the definition of the maximum po- discrete regions (C-zones) of myosin thick filaments in all striated muscles. Un- wer at F ~0.3 F0 (V ~2 mm/s) and demonstrating the unequalled ability of the like cardiac muscle with its specific MyBP-C isoform, mass spectrometry synthetic machine to define the power of native and engineered myosin II mo- shows that rat slow-twitch soleus (SOL) muscle expresses two different tors from striated muscle. Supported by IIT-SEED, Genova and ECRF, 2015 slow MyBP-C isoforms (sMyBP-C), having one (sMyBP-C1) or two N-termi- (Italy). nal inserts (sMyBP-C2), while fast-twitch extensor digitorum longus (EDL) ex- presses these same sMyBP-C isoforms and a fast MyBP-C isoform (fMyBP-C). 580-Pos Board B345 Do these MyBP-C isoforms differentially regulate skeletal actomyosin contrac- Dissecting Actomyosin Mechanochemistry using Blebbistatin as a Pharma- tility in a muscle-specific manner? To address this, we characterized Ca2þ- cological Tool regulated motion of SOL and EDL native actin-thin filaments (NTFs) over 1 2 1 Mohammad A. Rahman , Dilson Rassier , Alf Ma˚nsson . their corresponding native myosin-thick filaments, having the physiological 1Department of Chemistry and Biomedical Sciences, Linnaeus University, 2 complement of MyBP-C in the C-zone. At low calcium (pCa7.5) where Kalmar, Sweden, Department of Kinesiology and Physical Education, NTFs should be inhibited, motion occurred within the C-zone of both SOL McGill University, Montreal, QC, Canada. and EDL native thick filaments. Whereas, at high Ca2þ (pCa5), SOL and Muscle contraction results from cyclic interactions between actin and myosin EDL NTFs had initial fast velocities followed by ~30% slower velocities under turnover of ATP. Here, we use the myosin II-specific inhibitor blebbis- within the C-zone. These data suggest that one or more of these MyBP-C tatin as a pharmacological tool to elucidate this interaction. Particularly we can sensitize NTFs to Ca2þ and modulate contractility at high Ca2þ. To deter- consider the hypotheses that: 1. the effects of blebbistatin depend on the level 1 mine how each isoform contributes to these modulatory capacities, we bacte- of myosin regulatory light chain phosphorylation and 2. blebbistatin stabilizes rially-expressed N-terminal fragments (sMyBP-C1, sMyBP-C2, fMyBP-C) the start of the actomyosin power-stroke state, characterized by an absence of 2 and assessed their individual impact on SOL or EDL NTF motility over their inorganic phosphate (Pi) in the active site . In vitro motility assays (IVMAs) respective monomeric myosins. For the EDL where all three MyBP-Cs are ex- and skinned muscle fiber studies were performed using preparations from pressed, at pCa7.5 sMyBP-C1 was most effective at activating NTFs, while at m fast rabbit muscle. Blebbistatin (1 M) inhibited sliding velocity in the IVMA pCa5 all three reduced velocity by ~50%. For SOL, where only sMyBP-C iso- (~50%) independently of the RLC phosphorylation status, suggesting that the forms are expressed, at pCa7.5 sMyBP-C1 activated NTFs while at pCa5 phosphorylation dependence in muscle cells is fully attributed to myosin sMyBP-C2 reduced velocity ~40%. Our data suggest that the distinctive head-backbone interactions. The inhibitory effect was approximately half in mixture of MyBP-C isoforms expressed in the SOL and EDL is matched to conditions of reduced ionic strength from 130 to 60 mM, whereas the effect the muscle’s physiological demands. Each MyBP-C isoform possesses specific was similar at different MgATP concentrations (0.01 1 mM). Muscle fiber modulatory capacities that may be called upon under different contractile 5 5 data showed a reduced isometric force development by 36 11% and 89 states. Specifically, to fine-tune actomyosin contractility within the C-zone m m þ 5% at 2 M and 10 M blebbistatin, respectively. The force enhancement by Ca2 -sensitizing the thin filament and/or modulating velocity at maximal during an imposed stretch to the fiber during activation (10% of fiber length; activation. 1 length/s) was unaltered. An interpretation of the blebbistatin effects linked to a mecahano-kinetic model of muscle contraction is consistent with an 583-Pos Board B348 increased population of a start-of-power stroke actomyosin state without Pi Expression of Myosin Storage Myopathy Mutations in Drosophila Disrupts in the active site, and it is necessary for the assumption that blebbistatin stabi- Muscle Function, Myofibrillar Structure and Causes Defects in Thick lizes actomyosin states with bound Pi. Filament Assembly 1 2 2 581-Pos Board B346 Meera C. Viswanathan , Rick Tham , William A. Kronert , 3 2 2 Development and Phenotype Studies of a Slow Skeletal Troponin T E180 William Schmidt , Floyd Sarsoza , Adrianna S. Trujillo , 2 3 Nonsense Mutation Knock-In Mouse Line Sanford I. Bernstein , Anthony Cammarato . 1Johns Hopkins University, Baltimore, MD, USA, 2Molecular Biology Han-Zhong Feng, J.-P. Jin. 3 Physiology Department, Wayne State University, Detroit, MI, USA. Institute, San Diego State University, San Diego, CA, USA, School of A nonsense mutation in codon Glu180 of TNNT1 gene encoding the slow skel- Medicine, Johns Hopkins University, Baltimore, MD, USA. etal muscle isoform of troponin T (ssTnT) causes a recessively inherited severe Myosin storage myopathy (MSM) is a congenital skeletal muscle disorder b nemaline myopathy (Amish nemaline myopathy, ANM). Muscle biopsies of caused by missense mutations in the slow muscle/ -cardiac myosin heavy ANM patients showed a total loss of ssTnT. The present study developed chain (MHC) rod and is characterized by subsarcolemmal accumulations of b and characterized mice with the E180 nonsense mutation knocked-in the -MHC that have a hyaline appearance. Cases of MSM with cardiomyopathy Tnnt1 gene (ssTnT-KI). Homozygotes of ssTnT-KI mice survived to adult- have been reported. The mutations disrupt hydropathy or charge of residues hood. Histology studies revealed significant increases of centralized nuclei in in the heptad repeat, altering interactions that stabilize the rod’s coiled-coil di- the soleus muscle of ssTnT-KI when compared with age matched wild type mers and thick filaments. This potentially affects ordered myofibrillogenesis controls. Immunohistochemistry staining showed atrophic slow type 1 fibers and/or myofibrillar integrity causing myosin aggregation. We generated in ssTnT-KI mouse soleus muscle with severe inflammatory cells infiltration. Drosophila MSM models to perform biochemical, biophysical, physiological Western blots confirmed the absence of full length ssTnT with no ssTnT frag- and structural analysis of the effects of L1793P, R1845W, and E1883K muta- ments detectable, mimicking that seen in the muscle of ANM patients. Contrac- tions. Flight and jump ability were highly compromised in mutant homozy- tile properties of ssTnT-KI mouse soleus muscle measured in situ demonstrated gotes and heterozygotes. Confocal and electron microscopy revealed severe a decreased fatigue resistance. Significant expression of cardiac TnT is de- disruption of indirect flight muscle (IFM) morphology and myofibrillar tected in soleus muscle of ssTnT-KI mice, consistent with the presence of disarray and degeneration with hyaline-like inclusions in pupal muscles, active muscle regeneration. Decrease of slow type isoforms and increase of respectively. Mutant heterozygotes exhibited restrictive cardiac physiology fast type isoforms of other myofilament proteins with a significant hypertrophy and diastolic dysfunction, which worsened with age. Lifespans of MSM mu- of fast fibers in ssTnT-KI mouse soleus muscle indicate a potentially compen- tants were reduced relative to control. Thus, our Drosophila models pheno- satory adaptation to the loss of slow fiber functions. The ssTnT-KI mouse line copy afflicted patients’ muscle structure and function. To help identify the provides a model of ANM for pathogenesis, pathophysiology and therapeutic molecular basis of the disease, in vitro studies were performed using MHC pu- studies. rified from homozygous IFMs to evaluate the mutations’ effects without the confounding influence of wild-type myosin. Assembly assays demonstrated 582-Pos Board B347 a decreased ability of mutant MHC to polymerize, which resulted in shorter Skeletal Myosin-Binding Protein C Modulates Actomyosin Contractility in L1793P filament lengths. In addition, proteolysis experiments indicated an Isoform-Dependent Manner reduced stability of L1793P and E1883K filaments. We are currently deter- Amy Li1, Shane Nelson1, Kyounghwan Lee2, Samantha Previs1, mining if similar thick filament aberrations are present in heterozygotes, Karen Brack1, Michael Previs1, Suresh Govindan3, Sakthivel Sadayappan4, in vivo. Our results indicate that diminished ability of the mutant myosin Roger Craig2, David Warshaw1. to form stable thick filaments likely contributes to the dystrophic muscle 1Molecular Physiology & Biophysics, University of Vermont, Burlington, phenotype observed in humans. Supported by R01HL124091 (AC) and VT, USA, 2Cell and Developmental Biology, University of Massachusetts R21OD01561 (SIB).

BPJ 7736_7739 118a Sunday, February 12, 2017

584-Pos Board B349 tension, and contributes to active tension development. To determine if this A Simplified Flexible Chain Model of Calcium Regulated Myosin-Actin increased heterogeneity of titin isoforms within the TA had an effect on Interaction sarcomere length, we performed immunohistochemistry. An antibody to a-ac- Leonard P. Heinz, Rainer H.A. Fink. tinin was used to demarcate Z-lines in longitudinal sections of the tibialis Medical Biophysics Unit, Institute for Physiology und Pathophysiology, anterior muscle and sarcomere length was measured as the distance Heidelberg University, Heidelberg, Germany. between Z-lines. While average sarcomere length was not different between While the process of muscle contraction is relatively well understood at the iMSBmal1þ/þ and iMSBmal1-/- muscle, variation in sarcomere length was molecular level, the collective behavior of many molecular motors has increased following Bmal1 knockout in skeletal muscle. Further studies become an interesting topic for computer simulations in recent years. Duke’s with our model are currently ongoing and will lead to increased knowledge model has proven to provide a good description of the myosin-actin interac- of the importance of titin isoform maintenance in a variety of pathological tion and the Flexible Chain Model can be used to describe the regulatory conditions. behavior of the troponin-tropomyosin complex. Based on these models, we developed and implemented a simple computational motility assay that 587-Pos Board B352 covers calcium-regulated muscle activation and allows to study the coopera- Cacium-Induced SR Calcium Leak in Dysferlin-Null Murine Muscle tive effects of many motormolecules working in parallel as well as the rela- Fibers tionships between contraction speed and isometric force versus calcium Valeriy Lukyanenko, Joaquin Muriel, Robert J. Bloch. Physiology, University of Maryland, Baltimore, MD, USA. concentration. Like in Duke’s model, myosin heads are modeled using a sim- 2þ ple 3-state system corresponding to detached, attached and strongly attached Osmotic shock injury (OSI) decreases the amplitude of voltage-induced Ca - transients (VICTs) in dysferlin-null (A/J) but not control (A/WySnJ) myofibers to actin. The behavior of the troponin-tropomyosin complex, located at every þ th and also markedly increases sarcoplasmic [Ca2 ] (Kerr et al., Proc. Natl. Acad. 7 actin binding site, is treated using a simple 2-state model, allowing 2þ troponin to be attached or detached to actin. Both troponin and myosin control Sci. USA, 2013). We compared the effects of drugs that target L-type Ca - a continuous energy landscape that models the steric blocking of actin binding channels (LTCC: diltiazem, nifedipine, verapamil) and ryanodine receptors sites by tropomyosin. This troponin-potential is computed using radial basis (RyR1: dantrolene, tetracaine, S107) on A/WySnJ and A/J FDB myofibers in interpolation. It is locally reduced, if a myosin head binds to actin and culture to assess their effect on VICTs following OSI. We also examined A/J fibers transfected to express N-terminal Venus chimaeras of dysferlin increased, if a binding site is occupied by troponin, causing the system to 2þ act cooperatively. (V-Dysf). All Ca antagonists inhibited VICTs in A/J and A/WySnJ fibers at high concentrations, but 1-10 mM diltiazem specifically increased VICT am- 585-Pos Board B350 plitudes by ~15% in A/J fibers, restoring them to values close to controls. All Reconstruction of Functional Insect Flight Muscle Fibers with Rabbit the inhibitors at low concentrations improved recovery of VICTs in A/J fibers Skeletal Muscle Actin after OSI. The fact that inhibitors of the LTCC and of the RyR1 protect A/J Hiroyuki Iwamoto. fibers from OSI-induced loss of VICTs strongly suggests that the damage Res. & Util. Div., SPring-8, JASRI, Sayo-gun, Hyogo, Japan. caused to the VICTs by OSI is mediated by Ca2þ-induced SR Ca2þ leak Stretch activation (SA, delayed rise of force after stretch) is a mechanism through the RyR1. Consistent with this, injured A/J fibers produced Ca2þ essential for the fast wing-beat of insects with asynchronous flight muscles. waves, indicative of Ca2þ-induced Ca2þ release (CICR). Treatment of A/J fi- To determine which constituent protein(s) is (are) indispensable for SA, we bers with 10 mM S107 (stabilizer of RyR1-FKBP coupling that reduces Ca2þ have been conducting experiments to replace the endogenous actin of leak) or expression of V-Dysf both protected A/J fibers against the loss in bumblebee flight muscle with rabbit skeletal muscle actin, and we have amplitude of VICTs following OSI and prevented OSI-induced Ca2þ waves. demonstrated that actin filaments can be regenerated as judged from X-ray Our data suggest that, in the absence of dysferlin, OSI causes increased leak diffraction patterns (2016 annual meeting). In this experiment, the endoge- of SR Ca2þ through the RyR1, leading to CICR. We conclude that dysferlin nous actin is removed by gelsolin, and after this, rabbit G-actin is polymer- stabilizes the coupling of the LTCC and RyR1 to reduce Ca2þ leak when fibers ized in situ. To prevent spontaneous contraction, a myosin inhibitor must are mechanically stressed. Supported by the Jain Foundation, MDA and NIH be added to the solutions. For vertebrate skeletal and cardiac muscles, the (RO1 AR064268). preferred inhibitor is BDM (butanedione monoxime, Fujita et al., 1996). However, BDM is not an effective inhibitor for insect flight muscle. For 588-Pos Board B353 this reason, we used blebbistatin instead, and the actin filaments were suc- MG29 Interacts with Bin1 for Maintaining T-Tubule Structure in Skeletal cessfully restored. The problem is that the inhibition by blebbistatin is irre- Muscle Physiology and Regeneration versible, and it is not reversed by extensive washout or irradiation with Xinyu Zhou1, Kristyn Gumpper1, Xinxin Wang1, Junwei Wu1, Tao Tan1, blue light. Here we used a high concentration of BDM (100 mM) and Miyuki Nishi2, Hiroshi Takeshima2, Jianjie Ma1, Hua Zhu1. 1Department of Surgery, Davis Heart and Lung Research Institute, The Ohio repeated the experiments. The actin filaments were restored, and unlike in 2 the case of blebbistatin, actin-based layer line reflections were intensified af- State University, Columbus, OH, USA, Department of Biological ter washout of BDM and ATP. This indicates that the ability of the endoge- Chemistry, Kyoto University, Kyoto, Japan. nous myosin to form rigor complexes is not compromised by the use of Mitsugumin 29 (MG29), a member of the synaptophysin-like family proteins, BDM. We are currently trying to activate the flight muscle fibers prepared is a transmembrane protein mainly expressed in the t-tubule membranes of in this way. skeletal muscle. Electron microscopy analysis of skeletal muscle derived from mg29-/- mice revealed morphological defects of t-tubules, indicating 586-Pos Board B351 importance of MG29 in maintenance of muscle structure and function. To The Skeletal Muscle Molecular Clock Regulates Titin Isoform Expression determine the function of MG29 in muscle physiology and regeneration, we Lance A. Riley1,2, Xiping Zhang1,2, Karyn A. Esser1,2. performed co-immunoprecipitation and found that MG29 can bind to Bin1, 1Physiology and Functional Genomics, University of Florida, Gainesville, another t-tubular protein. Two-color STORM super-resolution imaging anal- FL, USA, 2Myology Institute, University of Florida, Gainesville, FL, USA. ysis confirmed co-localization of MG29 and Bin1 on t-tubules of skeletal The circadian clock transcription factors, BMAL1 and CLOCK, are funda- muscle. Interestingly, organized distribution Bin1 is severely disrupted in mental transcriptional regulators of cell time keeping and critical cell mg29-/- muscle. For testing the role of MG29 in muscle regeneration, we specific genes important for homeostasis. To determine the specific role of injured gastrocnemius muscle with cardiotoxin (CTX) and tracked muscle the molecular clock in adult skeletal muscle, our lab developed the inducible, repair and regeneration. Western blot showed that following CTX injury, skeletal muscle specific Bmal1 knockout (iMSBmal1-/-) mouse. Notably, MG29 protein levels were transiently reduced from day 1 to day 3, followed skeletal muscles from these mice exhibit decreased specific tension and by recovery associated with muscle regeneration. Protein level of Bin1 in wild reduced unstimulated tension developed during a fatigability test. To begin type muscle remained unchanged during the CTX-injury and recover process. to discern the molecular mechanisms that link the changes in the molecular Compared with wild type muscle, the mg29-/- muscle displayed delayed clock with changes in muscle function, we tested whether iMSBmal1-/- muscle regeneration with significantly reduced levels of Bin1 following CTX-induced would also exhibit changes in sarcomeric protein expression. We determined injury. Together our data suggest that functional interaction between MG29 that the tibialis anterior muscle of the iMSBmal1-/- mice exhibits a significant and Bin1 contribute to maintenance of t-tubule network and its remodeling þ þ increase (38% in iMSBmal1-/- mice vs. 19% in iMSBmal1 / mice) in ex- process associated with muscle injury and regeneration. Targeting the pression of a longer isoform of titin. It is established that titin, the giant fila- MG29/Bin1 complex might provide a potential effective approach for treat- mentous protein that maintains sarcomeric structure, underlies passive ment of muscle diseases.

BPJ 7736_7739 Sunday, February 12, 2017 119a

589-Pos Board B354 A small molecule modulator of cardiac myosin enzymatic function has been Phosphorylation of Myosin Increases the ATPase Activity of Relaxed identified (refered to as compound-A) and our characterization demonstrates Skeletal Muscle Fibers that this agent exerts its effect through the regulatory light chain (RLC) of Nariman Naber, Roger Cooke. myosin. Compound A inhibited the actin activated ATPase activity of cardiac Biochem/Biophys, Univ california, San Francisco, CA, USA. heavy meromyosin (HMM, derived from bovine heart), which contains both the Myosin in skeletal muscle is phosphorylated by a calcium-calmodulin depen- essential and regulatory light chains, whereas no inhibition was seen with dent kinase during periods of sustained activity. Phosphorylation of myosin bovine cardiac myosin subfragment-1 which does not contain the regulatory is known to destabilize the array of myosin heads bound to the core of the thick light chain in our preparations. The regulatory light chain from full length car- filament in relaxed muscle, known as the interacting heads motif. Myosin heads diac myosin was removed and the extent of inhibition was dramatically in this array are associated with a state with a highly inhibited ATPase activity, reduced. Binding of this small molecule to the RLC was assessed using differ- known as the Super Relaxed State (SRX). To determine whether phosphoryla- ential scanning fluorimetry (DSF) and found that binding occurs to the RLC. tion of myosin increases the ATPase activity, we measured the ATPase activity Compound A was further characterized in permeabilized rat cardiac muscle of relaxed, skinned skeletal fibers in the presence and absence of myosin phos- fibers and a dose-dependent decrease in maximal tension was observed. It phorylation. Fibers were phosphorylated, >50%, by including phosphatase in- has been demonstrated in animal studies that a small molecule modulator of hibitors in the glycerinating solution. Control fibers had an ATPase activity of myosin has applicability in treating hypertrophic cardiomyopathy, the identi- 0.040 5 0.004 s 1. Phosphorylated fibers had an ATPase activity of 0.060 5 fied regulatory light chain modulator described here suggests that the RLC 0.002 s 1, a 50 % increase. We have recently found a small molecule, piperine may be a potential new target for therapeutic applications in hypertrophic that destabilizes the SRX, increasing relaxed fiber ATPase activity. In the cardiomyopathies. presence of 100mM piperine the control fibers had an ATPase activity of 0.064 5 0.004 s1, the phosphorylated fibers had an ATPase activity of 592-Pos Board B357 0.057 5 0.001 s1. Thus both phosphorylation and piperine increase the Cardiac Myosin Strutural Kinetics Modulated by Small Molecules ATPase activity of resting fibers by destabilizing a fraction of myosin heads John A. Rohde, Hyun Cho, Lien Phung, David D. Thomas, in the SRX, 30-50%, but their effects are not additive, suggesting that they Joseph M. Muretta. are each affecting the same population of myosin heads. One candidate for Department of Biochemistry, Molecular Biology and Biophysics, University this population may be the ‘‘free’’ head of the interacting heads motif, which of Minnesota, Minneapolis, MN, USA. is known to be less stable than the other head. As myosin remains in a phos- Myosin’s lever arm undergoes structural changes that are coupled to its phorylated state for several minutes following a period of activity, the effect biochemical state during ATPase cycling in muscle. Structural states of myo- sin’s lever can be detected directly using the high-precision, high-throughput described above will contribute to the energetic cost of physical activity, partic- 2 ularly during light activity. technique of transient time-resolved FRET, (TR) FRET, revealing kinetics, thermodynamics, and allosteric coupling. We have previously used this tech- nique to investigate mechanochemical coupling in skeletal myosin and cardiac Cardiac Muscle Mechanics and Structure I myosin perturbed by the heart failure drug omecamtiv mecarbil. Here we inves- tigate other small molecule modulators of cardiac myosins mechanochemistry: 590-Pos Board B355 arachidonic acid, EMD 57003, and compounds developed by MyoKardia, un- Impact of Para-Nitroblebbistatin on Human Beta-Cardiac Myosin at the der investigation for treating hypertrophic cardiomyopathy. We find that one Molecular and Tissue Levels Myk compound dramatically decreases the amplitude of the fast phase of the Wanjian Tang1, Cheavar Blair2, Kenneth S. Campbell2, power stroke, but does not significantly affect the rate of this transition. These Christopher M. Yengo1. data suggest that the compound shifts the equilibrium constant for hydrolysis 1Cellular and Molecular Physiology, Pennsylvania State University College toward the pre-hydrolysis state. This is in sharp contrast to the effect of ome- of Medicine, Hershey, PA, USA, 2Physiology, University of Kentucky, camtiv mecarbil (Cytokinetics) under investigation for systolic heart failure, Lexington, KY, USA. which shifts the equilibrium constant toward the post-hydrolysis states. This Inherited cardiomyopathies are a common form of heart disease caused by mu- work was supported by NIH AR032961 & AR057220 (DDT), American Heart tations in sarcomeric proteins with beta cardiac myosin being one of the most Association Scientist Development Grant (JMM), and Graduate Excellence frequently affected genes. Since the discovery of the first cardiomyopathy asso- Fellowship-University of MN (JAR). ciated mutation in beta-cardiac myosin, a major goal has been to correlate the in vitro myosin motor properties with the contractile performance of cardiac mus- 593-Pos Board B358 cle. Mutations that cause hypertrophic cardiomyopathy are the most common Force-Dependent Recruitment of Cross-Bridges from the Myosin Off- form of the disease and are often associated with increased isometric force State can Contribute to Length-Dependent Activation Kenneth S. Campbell1, Paul Janssen2, Stuart G. Campbell3. and hyper-contractility. Therefore, the development of drugs designed to 1 decrease isometric force by reducing the duty ratio (the proportion of ATPase Physiology and Cardiovascular Medicine, University of Kentucky, Lexington, KY, USA, 2Physiology and Cell Biology, The Ohio State cycle time myosin spends bound to actin) has been proposed for the treatment 3 of hypertrophic cardiomyopathy. There is still no consensus about whether hy- University, Columbus, OH, USA, Department of Biomedical Engineering, pertrophic mutations in beta-cardiac myosin increase myofilament calcium Yale University, New Haven, CT, USA. Cardiac muscle cells develop more force if they are stretched before they are sensitivity as is commonly found in troponin mutations. We examine the 2þ impact of a small molecule drug proposed to decrease duty ratio (para-Nitro- activated. The Ca concentration that is required to produce half-maximal blebbistatin) using purified human beta cardiac myosin motor assays and force also decreases. These effects are known as length-dependent activation studies of permeabilized human cardiac muscle mechanics. We find that this and are thought to contribute to the Frank-Starling Mechanism. The precise drug reduces actin-activated ATPase and in vitro motility while not changing mechanisms underlying length-dependent activation remain unclear. Kam- the ADP release rate constant. Thus, the reduction in the in vitro sliding veloc- pourakis et al. (PMID 27162358) recently showed that increasing the length ity is likely due to a slowing of attachment rate. In muscle fiber studies we of cardiac muscle causes myosin head domains to adopt orientations that are more perpendicular to the thick filament axis. (There is an experimental found that para-Nitroblebbistatin reduces steady-state force and calcium sensi- 2þ 2þ tivity but does not change maximum contractile velocity and the rate of force trend at low Ca and a statistically significant effect at high Ca .) development. These results support a model in which the drug reduces the duty Increasing the extent of phosphorylation of cardiac regulatory light chain in- ratio without altering the kinetics of attached crossbridges. Thus, small mole- duces a similar but synergistic effect. These structural changes may represent cule drugs that target the crossbridge attachment rate are an attractive strategy myosin heads shifting from a super-relaxed myosin OFF state towards a con- since they are capable of reducing the force generating capacity and calcium ventional detached cross-bridge state. We developed a computer model of this sensitivity without altering contractile velocity. system using freely-available MyoSim software (http://www.myosim.org). Our initial simulations established the model parameters that govern cooper- 591-Pos Board B356 ative Ca2þ activation of the thin filament. Parameters were fitted to data by Modulation of Cardiac Myosin by a Small Molecule Agent that Targets the assuming a proportional relationship between the number of available binding Regulatory Light Chain sites (as predicted by the model) and the movement of troponin C in the Anu R. Anto, Raja Kawas, Robert Anderson, Marcus Henze, absence of bound cross-bridges (as assessed experimentally by Kampourakis Hector Rodriguez, Danielle Aubele, Jacques Mauger. et al.). Further calculations then fitted predictions for tension-pCa curves MyoKardia, South San Francisco, CA, USA. measured at short and long lengths and before and after treatment with myosin

BPJ 7736_7739 120a Sunday, February 12, 2017 light chain kinase to complementary experimental data. We discovered that [Ca2þ] required to generate half-maximal force) decreased for TpmD230N prep- the simulations could reproduce Kampourakis et al.’s experimental data if arations at both SL compared with WT preparations (with a larger effect at long the rate constant governing myosin’s transition from the OFF super-relaxed SL), and the increase in pCa50 with stretch from SL ~2.0mm to ~2.3mm was state to the conventional detached state increased with myosin light chain greater for WT compared to TpmD230N preparations (p<0.05). Together, these phosphorylation and also increased linearly with force. Simulations without results demonstrate an attenuated LDA in cardiac muscle of DCM TpmD230N the force-dependent transition did not exhibit length-dependent change in hearts and suggest a role for tropomyosin in modulating LDA. Funding: Ca2þ sensitivity. These simulations demonstrate that a mechanism of force- HL111197. sensitive myosin activation is capable of producing realistic length- dependent activation. 596-Pos Board B361 Cardiomyopathy Mutations in the Converter Domain of Human Beta- 594-Pos Board B359 Cardiac Myosin Impairs Mechanochemistry in the Presence and Absence Differences in Actomyosin Function in the Left and Right Ventricles of of Load Human Hearts Wanjian Tang, Shane D. Walton, William C. Unrath, Christopher M. Yengo. Sebasian Requena. Department of Cellular and Molecular Physiology, Penn State College of University of North Texas, Fort Worth, TX, USA. Medicine, Hershey, PA, USA. In both ventricles of the heart, actin is expressed from the same genes [1]. Missense mutations in human b-cardiac myosin are associated with inherited There are no differences in twitch duration, work performance and power cardiomyopathies, a leading cause of heart failure worldwide. The mecha- among the right (RV) and left (LV) ventricles in animals [2] So there is no nisms for how mutations alter myosin’s motor performance are still unclear. expectation that the properties of actin or myosin isolated from either ventricle We expressed and purified human beta-cardiac myosin subfragment 1 would be different. Nevertheless, the situation is more complex in human (M2b-S1) containing point mutations that are associated with hypertrophic hearts. The LV must pump more powerfully, because it has to overcome a (R723G) or dilated (F764L) cardiomyopathy. We demonstrate that both the larger resistance presented by the systemic system than the RV, which has to R723G and F764L mutations slow down the maximum actin-activated overcome a lower resistance offered by the pulmonary system. The question ATPase activity (20-30%). Direct measurements of the ADP release rate con- arises whether stronger pumping action of the LV is partially caused by the stant in the presence of actin using mant labeled ADP demonstrate R723G has LV actomyosin developing more force than the RV actomyosin. Such a ques- a 35% faster ADP release while F764L has a 20% slower ADP release tion is impossible to answer by making macroscopic measurements, (such as compared to wild-type. Since the actin-activated ATPase is reduced in both tension or ATPase activity) because the number of molecules involved in mutants, it suggests a slower actin-activated phosphate release rate constant such processes are enormous (of the order of 1011). To obtain molecular infor- or slower transition between actomyosin.ADP states. The in vitro motility mation, measurements must be taken from a few molecules. We measured var- assay demonstrates relatively small differences compared to wild-type, with iations in the polarization of fluorescence of a few actomyosin molecules a 5-10% higher actin sliding velocity for R723G and 5-10% slower velocity during the contraction cycle and obtained molecular information by calculating for F764L. We also examined the sliding velocity as a function of ADP con- its autocorrelation function using R (version 3.3.1). The autocorrelation curve centration, which provides information about the strain sensitivity of the mu- was fitted with a bi-exponential decay model in order to extract the rate con- tants. We found that both mutants were less sensitive to ADP inhibition of in stants using XPFIT (version 1.2.1) Alango Ltd. XPFIT employs the Inverse vitro actin sliding velocity suggesting reduced strain sensitivity. Therefore, we Laplace algorithm in order to numerically invert the decay time domain propose that both the hypertrophic R723G mutation and dilated F764L muta- data. The goodness of fit was assessed by chi-squared. The results suggest tion have slower ATPase kinetics in the absence of load, while the mechano- that actomyosin function is identical in both ventricles. Refs: 1. Wessels, chemistry in the presence of load indicates a reduced ability to adapt to higher M.W. and P.J. Willems, Mutations in sarcomeric protein genes not only lead loads. to cardiomyopathy but also to congenital cardiovascular malformations. Clin Genet, 2008. 74(1): p. 16-9. 2. Wikman-Coffelt, J., C. Fenner, A. Smith, 597-Pos Board B362 and D.T. Mason, Comparative analyses of the kinetics and subunits of myosins Maximal Force Increases at Physiological Temperature in Myocardial from canine skeletal muscle and cardiac tissue. J Biol Chem, 1975. 250(4): Strips from Non-Failing and Failing Human Hearts 1 2 2 p. 1257-62. Peter O. Awinda , Cheavar A. Blair , Maya E. Guglin , Kenneth S. Campbell2, Bertrand C.W. Tanner1. 595-Pos Board B360 1Integrative Physiology and Neuroscience, Washington State University, The Frank-Starling Mechansim is Attenuated by a Dilated Pullman, WA, USA, 2Division of Cardiovascular Medicine, Department of Cardiomyopathy-Associated Tropomyosin Mutation Physiology, University of Kentucky, Lexington, KY, USA. Joseph D. Powers1, Farid Moussavi-Harami1,2, Maria Razumova1, Heart failure contributes to 1 in 9 deaths in the United States. Contractile def- Jil Tardiff3, Michael Regnier1. icits at the myofilament level may contribute to the heart inadequately pump- 1Department of Bioengineering, University of Washington, Seattle, WA, ing blood throughout the body. Yet, few studies have investigated force USA, 2School of Medicine, Division of Cardiology, University of production and cross-bridge kinetics at physiological temperature in human Washington, Seattle, WA, USA, 3Department of Medicine, University of myocardium from non-failing and failing hearts. Using skinned myocardial Arizona, Tucson, AZ, USA. strips from the left ventricle free wall, we found that maximal Ca2þ-activated, Sarcomere length-dependent activation (LDA) of force development in cardiac isometric force was ~35% greater in non-failing vs. failing tissue at both 17 muscle is the cellular basis of the Frank-Starling mechanism, and is often and 37C(p<0.001 for condition effect at pCa 4.8 and 2.3 mm sarcomere blunted in heart failure. In patients with dilated cardiomyopathy (DCM), length). Moreover, increasing temperature from 17 to 37C increased LDA may be affected differently depending on the sarcomeric protein mutation maximal force by roughly 30% and 40% in non-failing and failing myocardial underlying the disorder, thus complicating strategies for therapeutic interven- strips, respectively (p<0.001 for temperature effect). Ca2þ-sensitivity of the tion. We investigated the effects of the DCM-associated tropomyosin mutation force-pCa relationship was reduced for non-failing vs. failing myocardial D230N D230N (Tpm ) on LDA by measuring force generation and twitch kinetics strips (p=0.03 for condition effect), with differences in pCa50 being greatest at short (~2.0mm) and long (~2.3mm) sarcomere lengths (SL) of intact and de- at 17C between non-failing and failing samples. These decreases in maximal membranated trabeculae/papillary muscles from hearts of a transgenic murine force and increases in Ca2þ-sensitivity of the force-pCa relationship that model (>6 months of age) containing the TpmD230N mutation. Both types of occurred with heart failure were not driven via differences in cross-bridge preparations were mounted between a force transducer and linear motor. Intact attachment or detachment kinetics, as kinetics measures were largely consis- trabeculae were perfused with oxygenated physiological solution (30C) and tent among non-failing and failing samples at each temperature. However, stimulated at 2Hz. Demembranated muscle preparations were bathed in phys- cross-bridge kinetics were 12-13 times faster at physiological temperature iological solutions (15C) containing varying Ca2þ concentrations and allowed vs. 17C in both non-failing and failing samples (p<0.001 for temperature ef- to generate steady-state force. As expected, for intact WT trabeculae, the twitch fect), thereby representing a Q10 of ~3.5 for cross-bridge attachment and force (Tp) and maximum rate of force development increased and the time to detachment rates in human myocardial strips. Thus, faster cross-bridge peak tension decreased when muscles were stretched from short to long SL cycling rates accompanied greater force production at physiological tempera- (p<0.02). In contrast, TpmD230N trabeculae did not show significant changes ture in both non-failing and failing samples. In addition, these data suggest in these parameters between short and long SL. There were no significant that heart failure compromises force production without significantly altering length-dependent changes in twitch relaxation kinetics for either WT or cross-bridge kinetics, both at sub-physiological and physiological tempera- D230N Tpm trabeculae. In demembranated muscle preparations, pCa50 (the tures in human myocardium.

BPJ 7736_7739 Sunday, February 12, 2017 121a

598-Pos Board B363 3Department of Pharmacology and Toxicology, Helwan University, Cairo, Structure and Nanomanipulation of the Titin M-Line Complex Egypt, 4Department of Surgery, The Ohio State University Wexner Medical Zsolt Martonfalvi, Dominik Sziklai, Marton Kovacs, Zsombor Papp, Center, Columbus, OH, USA. Miklos S. Kellermayer. Diastolic dysfunction occurs not only in diastolic heart failure, but also Semmelweis University, Budapest, Hungary. ubiquitously in systolic heart failure. A main determinant of diastolic passive Titin is a giant protein spanning between the Z- and M-lines of the sarcomere. tension is the elastic sarcomeric protein titin. The role of titin mechanics In the M-line the C-terminal region of titin overlaps with that of oppositely ori- in diastolic dysfunction is not clearly understood. Here we developed a ented titin from the other half of the sarcomere. Furthermore, titin-binding pro- novel approach to detect mechanical properties of the elastic domain of single teins such as myomesin and M-protein localize in the M-line so as to form a titin molecules from failing and non-failing human hearts. We further inves- complex. The M-line complex appears as a globular head-like structure in titin tigate the role of titin mechanics in heart failure by correlating clinical and preparations. The exact structure and the molecular arrangement within this biometric data from the corresponding human hearts, in conjunction with M-line complex is unknown. We analyzed the structure and stability of the diastolic function of isolated human cardiomyocytes. Using atomic force M-line complex by using atomic force microscopy (AFM). We mechanically microscopy, antibody specific tethering of the human native titin PEVK- dissected the M-line complex of single surface-adsorbed titin oligomers by distal Ig domain and the distal Ig domain alone was identified. Titin domains using AFM-based nanolithographic procedures. Titin was first deposited on were stretched and released at different frequencies based on ventricular mica so that the oligomers conformationally equilibrated on the surface. volume changes in the cardiac cycle. The nonlinear force tracings recorded Then the M-line complex was dissected by pressing the cantilever tip into from a series of linear stretch and release steps reflected titin viscoelasticity. the center of the globular head and moving the tip sideways in predetermined Mean titin domain passive tension was measured in the range of 21.2 to directions and with constant velocity (1 um/s) and pressing force (1 nN). 213.8 pN. The mechanical properties of the testing domains were significantly Finally, we scanned the surface so as to reveal the evoked changes. Loops of correlated with both the diastolic parameters measured in field-stimulated sin- filaments with lengths up to 400 nm were pulled out of the M-line complex. gle cardiomyoyctes, and the biometric data of the corresponding human The thickness of the filaments was one half that of native titin, suggesting hearts. Our approach identified the mechanical properties of human native that partial unfolding may have taken place as a result of nanodissection. cardiac titin domains, and their relationship to the diastolic function of Our results suggest that the titin M-line complex may have a higher order three human hearts, up to single cell level. The current study provided insights dimensional structure involving the packaging of participating filamentous into our understanding of cardiac relaxation kinetics in health and disease, molecules. Nanodissection may be used as a tool to investigate the internal contributing to the discovery of novel diagnostic and therapeutic strategies structure of stable biomolecular complexes. for heart failure. 599-Pos Board B364 601-Pos Board B366 Pre-Activation of Cardiomyocytes Determines Speed of Contraction: Role Modeling and Experiment to Determine the Role of Passive Stiffness on of Titin Mechanical (Strain Rate) Control of Relaxation Michiel Helmes1,2, Aref Najafi1, Martijn van der Locht1, Maike Schuldt1, Charles S. Chung. Ilse AE Bollen1, Max Goebel1, Coen Ottenheijm1, Jolanda van der Velden1,3, Department of Physiology, Wayne State University, Detroit, MI, USA. Diederik WD Kuster1. Diastolic dysfunction is increasingly linked to exercise intolerance and heart 1Physiology, VU Medical Centre, Amsterdam, Netherlands, 2Ionoptix Llc, failure-like symptoms. The serendipitous discovery of a rat with a sponta- Westwood, MA, USA, 3ICIN Netherlands Heart Institute, Utrech, neous mutation in the RNA Binding Motif-20 (RBM20) has led to new studies Netherlands. showing the effects of reduced titin-based passive tension. Decreased passive The giant myofilament protein titin has an extendable region that functions as a stiffness in the heart is associated with increased exercise tolerance, but also molecular spring. Cardiomyocytes have exquisite control over the length of ti- slowed crossbridge attachment and detachment rates. The influence on relax- tin, through splicing, enabling it to regulate passive stiffness. We hypothesized ation, and specifically mechanical control of relaxation by fast end systolic that titin as it sets the preload on the cardiomyocyte when stretched, together stretch, is not yet known. We utilized MyoSim, a computational modeling with diastolic Ca2þ pre-activates the cardiomyocyte during diastole and that environment, and experiments using intact trabeculae to investigate the rela- this pre-activation is a major determinant for force production in the subsequent tionship between titin-based passive stiffness and mechanical control of systolic phase. Via this route titin is thought to play an important role in active relaxation. We have previously obtained physiological myofilament parame- force development. Mutations in the splicing factor RNA binding motif protein ters by fitting a 2-state crossbridge model to experimental data using MyoSim. (RBM20) results in the expression of large, highly compliant titin isoforms. In These model parameters were held fixed except for passive stiffness, which the present study we aimed to investigate the effect of long, highly compliant was reduced by 50 and 75% to match passive stiffness in heterozygous and titin on the contractile properties of single cardiomyocytes. We measured single homozygous RBM20 mutant tissues. Isometric relaxation was slowed by 4 cardiomyocyte work-loops that mimic the cardiac cycle, in wildtype (WT) and and 5%, respectively, but relaxation rate became 19 and 23% more sensitive heterozygous (HET) RBM20 deficient rats. In addition we studied detergent- to mechanical control of relaxation (slope of relationship between relaxation permeabilized human patient samples that had known variations in titin based rate and end systolic strain rate). These data predict that titin based passive stiffness. At low pacing frequencies, myocytes isolated from HET left stiffness may modify relaxation rate. Trabeculae experiments using wild- ventricles were unable to produce normal levels of work (55% of WT), but this type, heterozygous, and homozygous RBM20 mutant rats, show no significant difference disappeared when diastolic calcium increased at high pacing fre- change in the mechanical control of relaxation. However, reduced titin stiff- quencies (>6 Hz). HET myocytes operated at higher SL to achieve the same ness was associated with a later time to peak contraction (shortening). level of work (2.1mm vs. 1.94mm at 6 Hz). In detergent-permeabilized cardio- Reducing crossbridge attachment and detachment rates in the computational myocytes isolated from human and rat heart we simulated cardiac twitches by model parameters replicates the experimental results. These data suggest transiently (0.5 s) exposing the cell to a physiological calcium concentration of that passive stiffness may play a role on mechanical control of relaxation pCa 5.7. Increasing pre-activation by bathing the cells in pCa 6.7 or pre- and confirm that crossbridge properties significantly contribute to early dia- stretching the myocyte increased the kinetics of force development and thus stolic relaxation. the total force development within a transient activation. This is consistent with our hypothesis that pre-activation can increase force development in a 602-Pos Board B367 time limited contraction such as a cardiac twitch. Pre-activation was pre-load Direct Observation of Strain Transmission through the Microtubule dependent as the sarcomere length to which the myocytes had to be stretched Network of Cardiomyocytes for equivalent levels of pre-activation varied with the compliance of titin. Matthew A. Caporizzo1, Brandon Kao2, Patrick Robison1, Alexey I. Bogush1, Benjamin L. Prosser1. 600-Pos Board B365 1Department of Physiology, The University of Pennsylvania, Philadelphia, A Novel Approach to Identify the Role of Single Molecule Titin Mechanics PA, USA, 2Department of Material Science, The University of Pennsylvania, in Human Heart Failure Philadelphia, PA, USA. Mei-pian Chen1,2, Nancy S. Saad1,3, Benjamin D. Canan1,2, Ahmet Kilic4, The stable microtubule (MT) network plays a significant role in regulating car- Peter J. Mohler1,2, Paul M.L. Janssen1,2. diomyocyte contractility and acts as a critical component for stretch-dependent 1Department of Physiology and Cell Biology, The Ohio State University, activation of intracellular Ca2þ release. Our previous studies suggest that force Columbus, OH, USA, 2Dorothy M. Davis Heart and Lung Research Institute, transmission though the MT network enhances Ca2þ sparks in response to me- The Ohio State University Wexner Medical Center, Columbus, OH, USA, chanical stress, yet stress transmission through the MT network has not been

BPJ 7736_7739 122a Sunday, February 12, 2017 observed or characterized. To this end we have combined high-speed sub- the two species, there were relatively small differences in solute penetration diffraction limit microscopy and atomic force microscopy (AFM) to directly below ~70kDa, and in the time-course of fluorescence recovery during observe deformation of the MT network in response to localized stress. The FRAP. These data suggest that gross differences in t-tubule structure do not radial spread of MT deflection and the timescale of network relaxation was have marked effects on the rate of diffusion within t-tubules. This work was quantified while directly probing myocyte mechanical properties. We find supported by the BHF and MRC that the normalized radial propagation of strain in the MT network is inversely proportional to cardiomyocyte stiffness. Further, MT strain propagation is Cell Mechanics, Mechanosensing, and Motility I decreased by a post-translational modification of the MT network, detyrosina- tion, which is increased in heart disease. To determine if MT deflections may be 605-Pos Board B370 transmitting forces capable of enhancing Ca2þ release, the length and time From Elasticity to Inelasticity in Cancer Cell Mechanics: A Loss of Scale dependence of calcium sparks were measured before, during and after localized Invariance mechanical stress. During the application of stress, a localized upregulation in Francoise Argoul. calcium sparks occured over the same length-scale that deformations in the MT Laboratoire Ondes et Matie`re d’Aquitaine, Talence, France. network propagated. These findings suggest that the stable MT-network may Soft materials such as polymer gels, synthetic biomaterials and living biolog- transmit mechanical signals through the cardiomyocyte that correlate with ical tissues are generally classified as visco-elastic or visco-plastic materials, Ca2þ release and depend on MT detyrosination. because they behave neither as pure elastic solids, nor as pure viscous fluids. When stressed beyond linear viscoelastic regime, cross-linked biopolymer 603-Pos Board B368 gels can behave nonlinearly (inelastically) up to failure. In living cells these Novel Roles for Obscurin Proteins in Cardiac Muscle networks exhibit high sensitivity to stress and propensity to local failure (plas- Patrick F. Desmond, Stephanie Myers, Anush Velmurugan, Matthew Klos, ticity). However, unlike synthetic passive gels, living cells can dynamically Yusu Gu, Nancy Dalton, Eric Devaney, Kirk Peterson, Ju Chen, compensate these failures thanks to ATP driven reparation mechanisms and Stephan Lange. recover a sustainable networked cytoskeleton architecture, as long as they Medicine, University of California San Diego, San Diego, CA, USA. are kept in a suitable culture environment. When the failure events are too Obscurin is a giant myofibrillar protein important for sarcomere structure, frequent or too strong or when the reparation mechanisms are not efficient, irre- signaling, and maintenance of sarcoplasmic reticulum (SR) organization. Sur- versible losses of mechanical homeostasis and chronic diseases such as cancer prisingly, obscurin knockout mice develop normally and only show signs of a may ensue. Here, we concentrate on chronic myelogenous leukemia (CML) as mild skeletal muscle myopathy. However, little is known about cardiac func- a model for stem cell cancer transformation. We compare the mechanical abil- tions of obscurin. We characterized cardiac roles of obscurin and found no ity of both primary (healthy donors and patients) and immature cells (TF1) changes to cardiac physiology. One explanation for the lack of a cardiac pheno- transduced by the CML oncogene BCR-ABL to respond to mechanical stresses type in obscurin knockout mice is that other obscurin protein family members (nano-indentation experiments). Our experimental study shows that primary possess a functionally redundant role. We hypothesized that obscurin-like 1 CML (CD34þ) hematopoietic cells encounter more frequent brittle failure (Obsl-1) is responsible for this redundancy, in part due to its shared ability to events than normal ones. This failure enhancement is also accompanied by a bind the sarcomeric proteins titin and myomesin. We generated double modification of the scale-invariant rheologic property of these cells. Interest- knockout (dKO) mice by crossing the obscurin-KO mice with a cardiac specific ingly, stiffening induced failure events are more visible on primary than on Obsl1-KO line to investigate the roles for these proteins in cardiac development TF1 model cells, pointing out that their cross-talk with their native microenvi- and function. Although a majority of the dKO-mice died after 12 months of age, ronment may also affect their transformation. Finally, combining these exper- microscopic and ultrastructural analysis failed to reveal overt changes to imental studies with RNA-seq analysis on both control and BCR-ABL sarcomere organization. Dramatic changes were observed, however, in SR transduced cells, we highlight the actin cytoskeleton signaling pathway as structure and function. Specifically we observed: 1.) loss of SR structural integ- significantly altered during CML transduction. rity and apparent loss of SR content, and 2.) alterations to Ca2þ transients and in References: the levels of Ca2þ handling proteins in KO animals. The majority of these B. Laperrousaz, et al. Physical Biology 13 (2016) 03LT01. changes were more severe in dKO-mice. Furthermore, we employed serial C. Martinez-Torres et al. Applied Physics Letters 108 (2016) 034102. blockface TEM technology to precisely measure the volume of the SR and 606-Pos Board B371 mitochondria and noticed significant differences between KO and control ani- Migration Behavior of Normal and Metastatic Human Mammary Cells mals. Physiologically, these mice develop a late onset cardiomyopathy charac- Josiah Low, Keith Bonin, Hyunsu Lee, Amanda Smelser, Martin Guthold. terized by increased systolic left ventricular internal dimension and alterations Physics, Wake Forest University, Winston-Salem, NC, USA. in hemodynamic properties at ~12 mo. of age. Our data suggests that obscurin Metastasis is the process by which cancer spreads from a primary tumor site and Obsl1 are not critical for sarcomerogenisis, however are important for þ to other sites in the body. Once a cancer has metastasized it is much more jointly organizing SR organization, cellular Ca2 handling, and long-term sur- difficult to treat, and a better understanding of metastasis is, thus, a critical vival in mice. goal in cancer research. Metastasis involves cell movement through the body, and the aim of our study was to explore the differences in motility be- 604-Pos Board B369 tween metastatic cells and noncancerous cells. Toward this end, we investi- Diffusion in the Transverse-Axial Tubule System of Cardiac Myocytes gated cell motility using migration assays, in which cells are grown in two 1 2 1 2 Cherrie H.T. Kong , Eva A. Rog-Zielinska , Clive H. Orchard , Peter Kohl , adjacent, but separate zones to confluency. After confluency is reached, the 1 Mark B. Cannell . barrier separating the two cell zones on a glass bottom dish is removed, 1Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, 2 and the motion of the two edges of the cells separated across the septum United Kingdom, Cardiac Biophysics and Systems Biology Group, Imperial gap is recorded. We hypothesized that cancerous human breast cells (MDA- College, London, United Kingdom. MB-231 cells) would exhibit a higher rate of migration and a lower degree Excitation-contraction (E-C) coupling in cardiac ventricular myocytes is criti- of coordinated movement than normal breast cells (HMEC cells). Migration cally dependent on the structure of transverse-axial (t-) tubules, which are in- assays were performed on both types of cells, and the migration into the vaginations of the surface sarcolemma. Many currents have been shown to gap between cell populations was recorded using time-lapse DIC microscopy. be preferentially located at the t-tubular membrane, including the L-Type cal- The resulting videos were processed using areal analysis and particle image cium current, Na-Ca exchange, tetrodotoxin-sensitive Na and steady-state K velocimetry (PIV), which allowed extraction of areal migration rates and ve- currents (Orchard, Pa´sek, and Brette. 2009. Exp. Physiol. 94: 509-519). It locity vector fields. An average migration rate of 242mm2/min was measured has been suggested that membrane folding within the t-system may introduce for HMEC cells, and a lower degree of coordination was observed in MDA a slow diffusion zone, which may have implications for ion balance for these cells compared to HMEC cells. currents during the E-C coupling cycle (Hong et al. 2014. Nat. Med. 20: 624-632.). To investigate diffusion in the t-tubule system, we examined t-tu- 607-Pos Board B372 bule structure in rabbit and mouse ventricular myocytes as two common exper- Adhesion-Controlled Proliferation Revealing Anti-Cancer Drug Resis- imental models, using electron tomography. Cells were also superfused with tance of Breast Cancer Cells solutions containing solutes of varying molecular weight to examine their pene- Soyeun Park. tration into the t-system. Fluorescence Recovery After Photobleaching (FRAP) College of Pharmacy, Keimyung University, Daegu, Korea, Republic of. was used to examine transport within the t-tubules of quiescent myocytes. We Mechanical interactions of cancer cells with surrounding environments play found that despite marked differences in t-tubule diameter and complexity in an important role in cancer progression. More evidence suggests that the

BPJ 7736_7739 Sunday, February 12, 2017 123a mechanical interplays between cancer cells and the extracellular matrix trigger The DNA damage response (DDR) is a collection of processes by which a cell adhesion-mediated signaling pathways that affect not only the cells’ mechan- identifies and repairs a site of DNA damage. Many factors can cause DNA dam- ical properties such as motility and rigidity but also the cell’s viability such age and initiate the repair machinery, such as UV, reactive oxygen species nu- as proliferation and apoptosis. In this study, we interrogated whether the cleases, or even drug treatments like etoposide. Recent studies of several cancer anti-cancer drug resistance of breast cancer cells can be discerned by moni- lines as well as immortalized epithelial cells (RPE-1) and primary dendritic toring the proliferation of breast cancer cells seeded on nanoscaffolds. The cells have convincingly characterized nuclear envelope rupture and cell death nanoscaffolds help systematically control the maturation of focal adhesions in migration through narrow channel. ATM kinase phosphorylates H2AX (to on well-defined nano-sized areas and distances. They were fabricated as gH2AX) as well as 53BP1 among other targets. A high dose of an ATM inhib- two-dimensional arrays of gold nanoislands on glass substrates using our itor (ATMi: KU-55933) increases migration-induced death of RPE-1 and bottom-up procedures combining nanosphere lithography and orthogonal HT1080 fibrosarcoma cells, particularly when combined with perturbations chemistry. By varying the size of nanospheres (300 - 1,000 nm) used for nano- to the nuclear lamina or envelope repair. However, ATMi at high doses affects lithography, the size and spacing of nanoislands were controlled. The MCF-7 other pathways and ATM’s roles beyond DNA repair now include a migration and MCF-7/ADR cells were investigated as the drug-sensitive and the drug- phenotype independent of DNA damage in MDA-MB-231 cells treated with resistant breast cancer cell lines, respectively. The difference in the siATM. Such results motivate dose-response assays here of ATMi in assessing doxorubicin-sensitivity of the two cell lines was confirmed by the MTT assay. relations between DNA damage and survival in pore migration of U2OS cells. The cell proliferation was determined from the phase contrast images taken Foci counts of phospho-ATM and of gH2AX (a target of ATM kinase) in 2D every 24 hours after the initial cell seeding on the nanoscaffolds. We found cultures were reduced by 50% at just 0.01 mM, consistent with pharmacological that the proliferation of the drug-sensitive breast cancer cells was highly studies of ATM. Surprisingly, comet assays showed no DNA breaks even at affected by the geometrical characteristics of the underlying nanoscaffolds. 10-32 mM. Only at these very high ATMi concentrations did cells die: in 2D Unlike the MCF-7 cells, the proliferation of the drug-resistant breast cancer cultures, 50% of cells die at 66 mM ATMi, and this decreases to 37 mM and cells was not noticeably affected by the nanoscaffolds for the first two days 14 mM in migration through 8 mm and 3 mm pores, respectively. However, of observation. We postulate that MCF-7/ADR cells showed the abnormal ATM knockdown does not increase cell death in constricted migration, sug- maturation of focal adhesions beyond the restricted area and thus continuously gesting that high dose effects of ATMi involves pathways independent of proliferated. We also observed a faster wound closure for MCR-7/ADR cells DNA damage. than MCF-7 cells. The increase in 2D motility of MCF-7/ADR could be re- sulted from higher levels of the traction force generated by the enhanced focal 610-Pos Board B375 adhesions. In conclusion, we found that the abnormal maturation of focal ad- Genomic Variation in an Osteosarcoma Cell Line Caused by Pore hesions via vinculin overexpression caused the uncontrolled proliferation and Migration resulted in the acquisition of the drug resistance in breast cancer cells. We sug- Jerome Irianto1, Yuntao Xia1, Charlotte R. Pfeifer1, Avathamsa Athirasala1, gest that the restoration of the normal maturation of focal adhesions might be a Jiazheng Ji1, Cory A. Alvey1, Manu Tewari1, Rachel R. Bennett2, promising way to sensitize the anti-cancer drug response from breast cancer Shane M. Harding3, Andrea J. Liu2, Roger A. Greenberg3, cells. Dennis E. Discher1. 1SEAS, University of Pennsylvania, Philadelphia, PA, USA, 2Graduate 608-Pos Board B373 Group/Department of Physics & Astronomy, University of Pennsylvania, Division Induced Dynamics in Non-Invasive and Invasive Breast Cancer Philadelphia, PA, USA, 3Cancer Biology, Abramson Family Cancer Ann-Katrine Vranso West1, Lena Wullkopf2, Amalie Christensen1, Research Institute, Perelman School of Medicine, University of Natascha Leijnse1, Jens Magelund Tarp1, Joachim Mathiesen1, Pennsylvania, Philadelphia, PA, USA. Janine Terra Erler2, Lene Broeng Oddershede1. Normal and diseased cells in vivo sometimes squeeze their nucleus through 1The Niels Bohr Institute, University of Copenhagen (UCPH), Copenhagen, tissues and basement membrane matrices. Recent studies has shown that con- Denmark, 2Biotech Research & Innovation Centre (BRIC), University of stricted migration give rise to an increase in GFP-53BP1 marked DNA dam- Copenhagen (UCPH), Copenhagen, Denmark. age and to die more frequently, but any effect on genome integrity is Cancerous cells pose a great threat when they gain the ability to metasta- unknown. Similar studies here with one of the cancer cell lines, U2OS oste- size, but little is known on why some cells gain the ability to invade osarcoma cells, show that such migration increases gH2AX foci, increases adjacent tissue while other cells are restricted to the primary tumor. We electrophoretic displacements of DNA from isolated nuclei, and changes have approached this topic by experimentally characterizing the division chromosome copy numbers. DNA breaks decrease over time and the cells induced dynamics of invasive and non-invasive breast cancer monolayers proliferate normally on rigid plastic, but lasting changes in chromosome using human and murine model systems. Particle image velocimetry copy number are revealed by standard DNA array methods. DNA repair fac- measurements of intrinsic velocities surrounding a dividing cell reveal a tors tend to be more cytoplasmic after migration, which is consistent with re- strong relation between tissue dynamics, such as vorticity and divergence, ports of migration-induced nuclear envelope rupture as is cell death due to and the invasive potential of the cell type. When a cell divides we inhibition of repair. Accumulation of mutations in cancer progression is often observe two distinct vortex pairs in the vorticity field surrounding the associated with the replication stress during cell division. The repair of dividing cell. Analyzing images over longer time scales reveals no long migration-induced DSBs through the mutation prone non-homologous end range interactions between the cancerous cells, and the interactions joining, could potentially provide an alternative path toward mutation observed surrounding a dividing cell are constricted to under one cell diam- accumulation, increasing intra- and inter-tumor heterogeneity. Genomic insta- eter away from the point of cytokinesis. This is to be expected as cancer bility is closely related to the development and progress of cancer, which is a cells are known to have decreased cell-to-cell interactions compared to major disease in the US and around the world. New cures to cancer will healthy cells. An increased intensity in the dynamics (velocity, vorticity benefit from new insights into basic mechanisms of nuclear processes in and divergence) of invasive monolayers compared to their non-invasive migration. counterparts, is apparent for both human and murine cell lines. These dy- namics can be simulated using a continuum model, and from this we extract 611-Pos Board B376 the characteristic force exerted by a dividing cell on the neighboring cells. Diffusive Behavior of Mismatch Repair Protein MSH2 in Cells at Different These values reveal a correlation between the force and the invasiveness of Stages of Cancer the breast cancer cells. Together, the model and experimental data suggest a Keith D. Bonin1, Justin Sigley1, John Jarzen2, Karin Scarpinato3, correlation between the dynamical properties of cells and their invasive Martin Guthold1, Tracey Pu1, Daniel Nelli1, Josiah Low1. potential. Further study of the difference in dynamics between invasive 1Physics, Wake Forest University, Winston-Salem, NC, USA, 2Medical and non-invasive cancers could help us understand the mechanisms govern- University of South Carolina, Charleston, SC, USA, 3OVPR, University of ing metastasis. Miami, Miami, FL, USA. Cellular interiors present media that are very complex as they encompass 609-Pos Board B374 a mixture of proteins, organelles, viscoelastic filaments, and chromatin Inhibition of a DNA Repair Kinase ATM Leads to Cell Death in 3D Migra- structures in the nuclear interior. The behavior of proteins in such environments tion Independent of DNA Damage reflects the cellular complexity. Here we present the results of studies on the Yuntao Xia, Jerome Irianto, Charlotte Pfeifer, Roger Greenberg, mobility, or diffusion, of free eGFP proteins, and of the important nuclear Dennis Discher. mismatch repair protein, MSH2. MSH2 was labeled with eGFP and the diffu- University of Pennsylvania, Philadelphia, PA, USA. sion of both the free eGFP and MSH2-eGFP was measured using Fluorescence

BPJ 7736_7739 124a Sunday, February 12, 2017

Recovery After Photobleaching (FRAP) on four distinct cell types that act as a (ECM) leading to reduced diffusion and convection of molecules through the model for the neoplastic cancer transformation of human mammary epithelial tumor micro-environment (TME). cells from non-cancerous to metastatic. We obtained several interesting results, such as a significant change in behavior for the first stage of cancer (immortal) 614-Pos Board B379 compared to other stages. We also observed a significantly lower value for Mechanical Properties of Normal Breast Cells and Metastatic Cancer Cells MSH2 diffusion (in both cytoplasm and nucleus) than what would be expected in Co-Culture by scaling from the eGFP values (using appropriate size ratios of the two pro- Hyunsu Lee, Amanda Smelser, Josiah Low, Martin Guthold, Keith Bonin. teins). Diffusion coefficients ranged from 14 to 24 mm2/s for EGFP and from 3 Physics, Wake Forest Univserity, Winston Salem, NC, USA. to 7 mm2/s for EGFP-MSH2. This material is based upon work supported by the The mechanical properties of cells play an important role in cellular processes National Science Foundation under Grant Number 1106105 (KB, KS, and such as cell migration, division, and mechanotransduction. Stiffness is an MG). We also thank Glen Marrs, director of the WFU Microscopic Imaging important mechanical property and can be measured using an atomic force Core Facility, and Anita McCauley, for their help with the confocal microscopy microscope (AFM) (to determine the Young’s modulus). Our previous studies setup. showed that normal breast cells (human mammary epithelial cells, HMEC) are stiffer than highly metastatic breast cancer cells (MDA-MB-231). Also, normal 612-Pos Board B377 breast cells in the center of a monolayer micro-colony are stiffer than cells Chemotherapy Impedes In Vitro Microcirculation and Promotes Migra- on the periphery and isolated cells which means that the microenvironment tion of Leukemic Cells with Impact on Metastasis significantly affects the mechanical properties of cells. However, the effect Sruti V. Prathivadhi-Bhayankaram1, Jianhao Ning2, Michael Mimlitz1, on stiffness when normal cells and cancer cells grow together has not Carolyn Taylor1, Erin Gross2, Michael Nichols1, Jochen Guck3, been well established. In this study, we investigate how the mechanical Andrew E. Ekpenyong1. properties of HMEC and MDA-MB-231 cells change in co-culture. Our 1Physics, Creighton University, Omaha, NE, USA, 2Chemistry, Creighton preliminary results show that both HMEC and MDA-MB-231 cells get > University, Omaha, NE, USA, 3Biotechnology Center, Technische significantly stiffer in co-culture (HMEC 1.3 kPa - 15 kPa; MDA-MB-231 > Universit€at Dresden, Germany. 0.59 kPa - 1.48 kPa). This study will offer an understanding of normal-to- Although most cancer drugs target the proliferation of cancer cells, it is metas- cancer cell interactions affect stiffness, and consequently how it might be corre- tasis, the complex process by which cancer cells spread from the primary lated to metastasis. tumor to other tissues and organs of the body where they form new tumors, 615-Pos Board B380 that leads to over 90% of all cancer deaths. Thus, there is an urgent need Kappa-Actin Alters Hepatocellular Carcinoma Physiology in Cirrhotic for anti-metastasis therapy. Surprisingly, emerging evidence suggests that Microenvironment certain anti-cancer drugs such as paclitaxel and doxorubicin can actually pro- Chi-Shuo Chen, Cheng-Yi Lin, Chi-Hung Ho, Wei-Chi Wu. mote metastasis, but the mechanism(s) behind their pro-metastatic effects Biomedical Engineering and Environmental Sciences, National Tsing Hua are still unclear. We used a microfluidic microcirculation platform which University, Hsinchu, Taiwan. mimics the capillary constrictions of the pulmonary and peripheral microcir- Hepatocellular carcinoma (HCC) is the fifth most common cause of cancer- culation, to determine if in-vivo-like mechanical stimuli can evoke different related mortality over the world, and liver cirrhosis was reported as the responses from cells subjected to various cancer drugs. We found that most important risk factor for HCC development. However, the roles of me- leukemic cancer cells treated with doxorubicin and daunorubicin, commonly chanotransduction in HCC have not been fully explored yet. Kappa-actin used anti-cancer drugs, have over 100% longer transit times through the de- (k-actin), a novel class of actin correlated with poor postoperative survival, vice, compared to untreated leukemic cells. Such delays in the microcircula- was selected as our study model. We investigated how k-actins regulate tion are known to promote extravasation of cells, a key step in the metastatic HCC cells using stiffness adjustable polymeric matrixes, which mimic the < cascade. Furthermore, there was a significant (p 0.01) increase in the cirrhotic microenvironment. Different cellular physiology, such as prolifera- chemotactic migration of the doxorubicin treated leukemic cells. Both tion, contact topography and 3-dimensional invasion, were observed in enhanced retention in the microcirculation and enhanced migration following microenvironment with different stiffness. Interestingly, with high k-actin chemotherapy, are pro-metastatic effects which can serve as new targets for expression, we noticed the significantly decrease of focal adhesions (FAs) for- anti-metastatic drugs. mation while substrate’s stiffness > 16 kPa. Photoactive microscopy showed lower stability of k-actin structures, and the unstable actin organization can 613-Pos Board B378 contribute to the decrease of FAs/Adherence junction (AJs) formations. The hERG1/Beta1 Interaction Compromises the Mechano-Reciprocity of Furthermore, the instability of AJs (E-cadherin) may correlate to the observed Pancreatic Cancer higher invasion of k-HCC in vitro. Traction force microscopy (TFM) was 1 2 1 Stefano Coppola , Annarosa Arcangeli , Thomas Schmidt . developed to further quantify the mechanical interactions at the ECM-cell/ 1Physics of Life Processes, Huygens-Kamerlingh Onnes Laboratory, Leiden 2 cell-cell interfaces. By measuring the deformation of polymeric gel substrates, University, Leiden, Netherlands, Department of Experimental and Clinical the force balance at the contact surfaces can be reconstructed. We observed the Medicine, University of Florence, Florence, Italy. þ ECM-cell traction force increased with the increasing substrate stiffness, and K channels encoded by the human ether-a`-go-go related gene (Kv11.1, or the traction force decreased with the k-actin expression, which consisted with hERG1) are frequently overexpressed in human cancers, including the highly the spatial pattern of FAs. In summary, we showed the expression of k-actin metastasizing pancreatic ductal adenocarcinoma (PDAC). HERG1 controls alters the formation of FAs/AJs in HCC, which can contribute to the observed the neoplastic cell biology by modulating intracellular signaling cascades, high cell proliferation and invasion of HCC. Furthermore, using photoactive coupled to cell adhesion, due to a functional cross-talk with integrin receptors. microscopy, we demonstrated the actin dynamics play the essential roles in The integrin-hERG1 interplay is bidirectional. Integrins both activate hERG1 FAs and AJs formation of HCC. These findings may contribute to our under- channels and alter hERG1 electrophysiology and, conversely, hERG1 channels standing about the influence of actins on HCC through mechanotransduction modulate integrin-mediated signaling. We hypothesized that hERG1 channels perspective. compromise the PDAC mechano-reciprocity, the ability to dynamically respond to externally applied forces by exerting forces, which enhances inva- 616-Pos Board B381 sion and compromises treatment. Is Shape of Cancer Cell Correlated with its Invasiveness? A combination of single-molecule microscopy and force measurements gives Elaheh Alizadeh1, Samanthe M. Lyons2, Jordan M. Castle3, us access on mechanical information at focal adhesion level, simultaneously Jacqueline Irene Foss2,4, Ashok Prasad1,2. to hERG1/b1 complex dynamics and localization. The correlated motion of 1Chemical and Biological Engineering, Colorado State University, Fort hERG and integrins suggest that both are localized in the same structure, prob- Collins, CO, USA, 2School of Biomedical Engineering, Colorado ably the focal adhesion as suggested by staining with vinculin. Force measure- State University, Fort Collins, CO, USA, 3Department of Biology, ments further suggest that the mechanical properties of PDAC cells are altered Colorado State University, Fort Collins, CO, USA, 4Mechanical Engineering, by the integrin-hERG1 interaction leading to an increase in cellular forces by Colorado State University, Fort Collins, CO, USA. 20% on coexpression of both proteins. Our results on how cancer-related over- This work is based on the hypothesis that shape of the cell on substrate is deter- expression of hERG1 potassium channels perturbs the force sensing machinery mined by the active mechanical properties of the cytoskeleton. Since invasive provides a new view on earlier findings which explain the chemo- and radio- cancer cells are believed to have altered mechanical properties compared resistance of PDAC mostly with the increased density of extracellular matrix with non-invasive cells, it follows that changes in shape of cancer cells may

BPJ 7736_7739 Sunday, February 12, 2017 125a correlate with the acquisition of invasive capacity. We try to understand on the cell’s ability to form cell-substrate adhesions. We will also provide data whether there is a correlation between cancer cell shape and its phenotypic showing a clear relationship between the aforementioned migratory character- state. To proceed, we first need to capture cell shape and quantify it. Cells istics and computed displacement and stress fields around migrating neutro- were cultured on glass substrates and then were fixed and stained for actin phils in collagen matrices. The results from our study show that neutrophils cytoskeleton. Images were processed to eliminate background noise. In order migrating in 3-D environments employ distinct mechanical mechanisms that to quantitatively describe cell’s shape, we chose a set of Zernike moments, depend on their ability to form adhesions. which are widely used in image recognition and are calculated from a set of orthogonal basis function. Since we are dealing with high dimensional 619-Pos Board B384 data we used principal component analysis (PCA) to examine changes in Novel Mechanism for Driving Amoeboid-Like Motility of Human Neutro- cell shape. We also used a machine learning neural network classifier to phils under an Electric Field, Based on Intracellular Proton Currents and distinguish between different cell populations. We use paired osteosarcoma Cytoplasm Streaming cancer lines which consist of a parental line that rarely undergoes metastasis Hagit Peretz Soroka1, Reuven Tirosh2, Murray Alexander3, Jolly Hipolito1, and a line derived from parental line that almost always does. Comparing Francis Lin1. 1 shape changes of invasive cancer line versus their paired low invasive cancer Physics and Astronomy, University of Manitoba, Winnipeg, MB, Canada, 2 3 line gives insight to correlation between cell shape and invasiveness. To under- Chemistry, Tel - Aviv University, Tel - Aviv, Israel, Physics, University of stand cytoskeletal mechanism behind this relation we perturbed cytoskeleton Winnipeg, Winnipeg, MB, Canada. with different pharmaceutical drugs which each targets different element Experimental studies have shown that actin filaments motion, cytoplasm of the cytoskeleton and looked at the patterns of drug effect on cell shape. streaming and muscle contraction can be reconstituted under actin-activated We also characterize shape changes of normal cells during their genetic ATP hydrolysis by soluble non-filamentous myosin fragments. Thus, biological step-wise transformation to cancer cells. [1]S. M. Lyons et. al. Biology motility was demonstrated without requiring conventional contractile protein Open 2016, 5, 289-299. [2]E.Alizadeh et. al. Integrative Biology, 2016, function. A possible alternative mechanism based on the integration of http://dx.doi.org/10.1039/C6IB00100A. directional proton current and cytoplasm streaming is proposed to drive amoeboid-like cell motility and muscle contraction. To test the cell motility 617-Pos Board B382 aspect of this mechanism, in the current study we employ a microfluidic device Stability on the Edge: Probing the Biophysical Mechanisms of Polarity to quantitatively characterize human blood neutrophil electrotaxis. Several Maintenance at the Leading Edge of Motile Neutrophil-Like Hl-60 Cells interesting predictions from this new model were confirmed in these experi- Rikki M. Garner1, Elena Koslover2, Julie Theriot3. ments. We demonstrated firstly, the direct correlation of cytoplasm streaming 1Biophysics Program, Stanford University, Stanford, CA, USA, 2Physics to neutrophil motility profiles; secondly, the electric field and cell activation Dept., The University of California, San Diego, San Diego, CA, USA, stage dependent directional neutrophil orientation and migration; and thirdly, 3Biochemistry Dept., Microbiology and Immunology Dept., and the Howard the migration memory of neutrophil electrotaxis. These results support the Hughes Medical Institute, Stanford University, Stanford, CA, USA. importance of active intracellular force for driving amoeboid-like cell motility, Cell motility is an essential cellular function. With motility being such a ubiq- with implications for physiological processes such as cell-cell migratory inter- uitous process, cells are outfitted with a vast yet well-conserved set of proteins actions and metabolic activation. This basic integrative working hypothesis is that self-organize in order to form protrusions with a surprising range of shapes expected also to give raise to new applications in bioenergetics and immuno- and sizes. These protrusions are formed by the coordinated force of thousands logical diagnosis and therapy. of actin polymers growing against the membrane at the leading edge. How are thousands of nanometer-sized actin filaments able to form a stable leading edge 620-Pos Board B385 several orders of magnitude larger, in time and space, than the assembly of its Macrophages are Sensitive to Substrate Elasticity during Phagocytosis individual components? I seek to answer this question by analyzing protrusion Wolfgang Gross, Franziska Zecherle, Kathrin Weidner-Hertrampf, fluctuations at the leading edge of lamellipodia, thereby quantifying the limits Holger Kress. of self-correction of the actin network. Using a cycle of theoretical modeling University of Bayreuth, Bayreuth, Germany. and experimental perturbation, I will tease apart the possible mechanisms for Phagocytosis, the internalization of objects like living bacteria and dead cells self-correction and the subsequent effects on the stability of the leading edge by macrophages is a main function of the innate immune system. After the of motile cells. detection of foreign objects by membrane receptors, this process is driven by the reorganization of the actin cytoskeleton, which leads to a protrusion of 618-Pos Board B383 the membrane around the target. Although many molecular players have Understanding the Mechanics of Neutrophil Migration in Three-Dimen- been identified in the past, there is still little known about the role of mechanics sional Extracellular Matrices during this process in general and about the role of the mechanical cellular Joshua Francois1, Ruedi Meili2, Juan Carlos del Alamo3, Richard Firtel2, environment in particular. Juan C. Lasheras3. In this work we investigate the influence of the underlying substrate rigidity on 1 Bioengineering, University of California, San Diego, La Jolla, CA, USA, the phagocytic uptake efficiency and the uptake velocity. We cultured murine 2 Section of Cell and Developmental Biology, University of California, San J774 macrophages on thin polymer gels with different stiffnesses in the phys- 3 Diego, La Jolla, CA, USA, Mechanical and Aerospace Engineering, iological range. The uptake efficiency of antibody-coated microparticles was University of California, San Diego, La Jolla, CA, USA. quantified with secondary antibody staining and the uptake speed was measured While much research has been dedicated to the identification of the cascade of in live cell experiments by using optical tweezers. We found that the uptake ef- specific biochemical processes involved in the recruitment of neutrophils, ficiency as well as the uptake velocity depend on the rigidity of the substrate. much less is known about the mechanical events driving their migration; in Furthermore, we observed that cells were able to adapt to the various substrate particular, how they generate the necessary traction forces to migrate across stiffnesses over time. In addition to these cellular measurements, we also pre- three-dimensional (3-D) extravascular spaces and the importance of forming sent a novel calibration technique that enables the simultaneous characteriza- cell-substrate adhesions during this process is unclear. In this study, we inves- tion of the elastic modulus and the Poisson’s ratio of thin gel layers. The tigate the importance of cell-substrate adhesions on the mechanics of 3-D technique requires only a standard epifluorescence microscope and spherical neutrophil motility in collagen gels using Elastographic 3D Force Microscopy indenters. (E3DFM). We used wild type neutrophil-like differentiated human promyelo- Our results on the phagocytic uptake velocity and efficiency support the hy- cytic leukemia (dHL-60) cells and talin 1 knockout dHL60 cells, which were pothesis that phagocytosis is a mechanosensitive process. Our findings might unable to engage their integrins, as our model systems. Both cell lines were contribute to an understanding of the complex interplay between the immune embedded in collagen matrices containing fluorescent micro-beads. Neutrophil system and disease states that come along with changes in tissue rigidity like motility was induced via the introduction of the neutrophil chemokine formyl- cancer and atherosclerosis. Methionyl-Leucyl-Phenylalanine (fMLP) in a custom build device. Both Confocal and Fluorescent microscopy techniques were used to image the move- 621-Pos Board B386 ment of the embedded micro-beads as well as fluorescently labeled cells. Par- Investigating Actin Mechanics during Phagocytic Uptake and Transport ticle Image Velocimetry (PIV) and Finite Deformation Theory were used to Konrad Berghoff, Steve Keller, Holger Kress. compute displacement fields in the collagen matrices. Stress fields in the University of Bayreuth, Bayreuth, Germany. matrices were computed using our E3DFM method. We will present data Phagocytosis, the internalization and consecutive digestion of biological mate- showing that morphological changes and migratory patterns differed depending rial by macrophages is a major part of the innate mammalian immune response.

BPJ 7736_7739 126a Sunday, February 12, 2017

Fc gamma receptor-mediated uptake is driven by actin recruitment to the inter- sion by intensifying its magnitude to values >19 pN and spatially reorganizes nalization site and consecutive formation of membrane protrusions around the the location of receptor forces to the kinapse, the zone located at the trailing target object. Molecular motors and cytoskeletal elements contribute to the sub- edge of migrating T cells, thus demonstrating crosstalk between TCR and sequent transport of phagosomes inside the cells. Although a large number of LFA-1 receptor signaling. Finally, T cells display a dampened and poorly spe- molecules that are involved in the phagocytic uptake and the phagosomal trans- cific response to antigen agonists when forces are chemically abolished or port are identified by now, the mechanics of these processes are largely un- physically ‘‘filtered’’ to a level of 12 pN using mechanically labile DNA known. We therefore investigated the mechanics of phagocytic uptake and tethers. Therefore, we conclude that T cells tune receptor mechanics with pN phagosomal transport by using live cell microscopy in combination with optical resolution to create a checkpoint of agonist quality necessary for specific im- and magnetic tweezers with a focus on the role of actin filaments. We hypoth- mune response. esize that the actin recruitment during phagocytic uptake leads to a transient and localized increase of the stiffness of the uptake region. 624-Pos Board B389 To test this hypothesis we induced Fc gamma receptor-mediated phagocytosis Mechanics of Blood Cells with Marginal Band: Competition between in J774A.1 mouse macrophages by offering the cells target microbeads coated Cortical Tension and Rigidity with immunoglobulin-G. During the uptake we measured local cell stiffness Serge A. Dmitrieff, Adolfo Alsina, Mathur Aastha, Ne´de´lec Franc¸ois. changes with a ‘blinking optical traps’ technique executed via periodic inten- CBB Nedelec Lab, EMBL, Heidelberg, Germany. sity modulation of the optical force. Preliminary data indicates that that the The fast blood stream of animals is associated with large shear stresses. Conse- stiffness of the uptake region increases temporarily during the uptake. In addi- quently, blood cells have evolved a special morphology and a specific internal tion, we investigated the influence of actin filaments on the phagosomal trans- architecture allowing them to maintain their integrity over several weeks. Non- port by magnetic tweezers-based bead displacement assays and cytochalasin mammalian red blood cells, mammalian erythroblasts and platelets in particular treatment of the cells. have a peripheral ring of microtubules, called the marginal band, that flattens Our measurements are expected to identify characteristic length- and time- the overall cell morphology by pushing on the cell cortex. scales for the variation of cell mechanical properties during phagocytosis and We modeled how the shape of these cells stems from the competition between to contribute to a more comprehensive understanding of this medically relevant marginal band elasticity and cortical tension. We predict that the diameter of process. the cell scales with the total microtubule mass, and verify the predicted law across a wide range of species. Our analysis also shows that the combination 622-Pos Board B387 of the marginal band rigidity and cortical tension increases the ability of the B Cell Antigen Extraction is Regulated by Physical Properties of Antigen cell to withstand forces without buckling. Eventually, we show that the buck- Presenting Cells ling of the marginal band observed during platelet activation is caused by a Katelyn M. Spillane, Pavel Tolar. rapid increase of the cortical tension. The Francis Crick Institute, London, United Kingdom. B cells play a critical role in immune responses by producing antibodies against 625-Pos Board B390 the foreign pathogens that they encounter. Their responses are initiated when Multiscale Modeling of Red Blood Cells Squeezing through Submicron the B cell receptor (BCR) binds antigen on the surface of an antigen- Slits presenting cell (APC) in a cell-cell contact known as the immune synapse. Huijie Lu, Zhangli N. Peng. This event triggers the B cell to internalize, process, and present the antigen Aerospace and Mechanical Engineering, University of Notre Dame, Notre to helper T cells, which provide signals that are required for full B cell activa- Dame, IN, USA. tion and clonal selection. High affinity B cells receive more T cell help than low A multiscale model is applied to study the dynamics of healthy red blood cells affinity B cells do, suggesting that affinity discrimination during antigen extrac- (RBCs), RBCs in hereditary spherocytosis, and sickle cell disease squeezing tion and internalization is essential for high-affinity antibody responses. The through submicron slits. This study is motivated by the mechanical filtration mechanisms of antigen recognition, discrimination, and uptake remain a topic of RBCs by inter-endothelial slits in the spleen. First, the model is validated of debate, with physical extraction through mechanical forces and enzymatic by comparing the simulation results with experiments. Secondly, the deforma- liberation through lysosome secretion proposed as efficient ways for B cells tion of the cytoskeleton in healthy RBCs is investigated. Thirdly, the mecha- to acquire antigen. To investigate B cell extraction mechanisms, we developed nisms of damage in hereditary spherocytosis are investigated. Finally, the DNA-based nanosensors to interrogate antigen extraction from both artificial effects of cytoplasm and membrane viscosities, especially in sickle cell disease, substrates and live APCs. We show that B cells acquire antigen primarily are examined. The simulations results provided guidance for future experi- through mechanical force, and resort to enzymatic liberation only if force- ments to explore the dynamics of RBCs under extreme deformation. dependent extraction fails. The use of mechanical force renders B cells sensi- tive to the physical properties of the APCs. We find that stiff APCs promote 626-Pos Board B391 strong B cell pulling forces and stringent affinity discrimination, while flexible Rolling Adhesion of Malaria-Infected Red Blood Cells APCs allow B cells to extract low-affinity antigen using weak forces. Thus, B Anil Kumar Dasanna. cell antigen extraction is regulated by the physical properties of immune syn- Heidelberg University, Heidelberg, Germany. apses, suggesting that distinct physical properties of APCs may support The clinical symptoms of the malaria disease appear when healthy red blood different stages of B cell responses. cells are invaded by the malaria parasites during the blood stage of the life cycle. The whole infection of the blood cell by the malaria parasite Plasmo- 623-Pos Board B388 dium falciparum takes about 48 hrs and proceeds through the three stages of Revealing the Mechanical Basis of T Cell Signaling ring, trophozoite and schizont. During this process, the infected red blood Khalid Salaita. cells (iRBCs) increasingly develop adhesive protrusions, so-called knobs, on Chemistry, Emory University, Atlanta, GA, USA. their surface. These knobs cause iRBCs to adhere to endothelial cells in the Because T cells are highly migratory and antigen recognition occurs at an inter- microvasculature, preventing their clearance by spleen and liver, but also membrane junction where the T cell physically contacts another cell, there are leading to capillary obstruction. We first present how exactly the shape of long-standing questions of whether T cells transmit defined forces to their re- iRBCs change during the time course along with their geometrical features ceptors and whether chemo-mechanical coupling influences immune function. such as volume and surface area using confocal microscopy and image pro- I will discuss the development of DNA-based gold nanoparticle tension sensors cessing. We then discuss how these changes in shape and knob details through to provide, to our knowledge, the first pN tension maps of individual T cell re- out the blood stage affect the rolling adhesion of iRBCs on endothelial cells ceptor forces during T-cell activation. We show that naı¨ve T cells harness cyto- using both flow chamber experiments and adhesive dynamics simulations. In skeletal coupling to transmit 12-19 pN of force to their receptors within particular, we will discuss how the spatial organization of the knobs on the seconds of ligand binding and preceding initial calcium signaling. CD8 core- surface of the iRBCs lead to different dynamics states, such as flipping mo- ceptor binding and lymphocyte-specific kinase signaling are required for tion, transient or stable rolling adhesion and firm adhesion, and how these antigen-mediated cell spreading and force generation. Lymphocyte function- states are related to parameters such as kinetic rates of ligand-receptor bonds associated antigen 1 (LFA-1) mediated adhesion modulates TCR-pMHC ten- and cell elasticity.

BPJ 7736_7739 Sunday, February 12, 2017 127a

Transporters and Exchangers I hill’ movement of other coupled ions, protons, metabolites, -or electrochemical gradients. The focus is on how two variations, driven by 627-Pos Board B392 electric potential, and proton gradients, can drive anion transport, in highly Markov State-Based Quantitative Kinetic Model of Sodium Release from homologous family members? the Dopamine Transporter 1 1 1,2 630-Pos Board B395 Asghar Razavi , George Khelashvil , Harel Weinstein . Substrate Binding to Serotonin Transporters Reduces Membrane 1Physiology and Biophysics, Weill Cornell Medical College of Cornell 2 Capacitance University, New York, NY, USA, Physiology and Biophysics, Institute for Verena Burtscher, Matej Hotka, Walter Sandtner. Computational Biomedicine, New York, NY, USA. Center for Physiology and Pharmacology, Medical University of Vienna, The dopamine transporter (DAT) belongs to the neurotransmitter/sodium sym- Vienna, Austria. porter (NSS) family of membrane proteins that are responsible for reuptake of Monoaminergic signal transmission is terminated by reuptake of monoamines neurotransmitters from the synaptic cleft to terminate a neuronal signal and via the closely related transporters for dopamine (DAT), norepinephrine (NET) enable subsequent neurotransmitter release from the presynaptic neuron. and serotonin (SERT). SERT is a secondary active transporter that harnesses The release of one sodium ion from the crystallographically determined so- the chemical energy stored in the gradients of Naþ,Cl- and Kþ to transport dium binding site Na2 had been identified as an initial step in the transport 5-HT uphill against an opposing concentration gradient. cycle which prepares the transporter for substrate translocation by stabilizing The transport cycle of SERT is complex and encompasses several partial- an inward-open conformation. We have constructed Markov State Models reactions, some of which can directly be monitored utilizing measurements (MSMs) from extensive molecular dynamics simulations of human DAT of currents mediated by SERT. However, these measurements are limited to (hDAT) to explore the mechanism of this sodium release. Our results quantify ionic conditions that support currents but fail to provide insights at conditions the release process triggered by hydration of the Na2 site that occurs concom- at which SERT is electrically silent. itantly with a conformational transition from an outward-facing to an inward- We therefore performed capacitance measurements during rapid serotonin facing state of the transporter. The kinetics of the release process are application and observed that substrate binding to SERT decreases the mem- computed from the MSM, and transition path theory is used to identify the brane capacitance. This effect was absent in control cells. most probable sodium release pathways. An intermediate state is discovered Our analysis suggests that the reduction in membrane capacitance occurs as a on the sodium release pathway, and the results reveal the importance of consequence of the adsorption of 5-HT to a charged residue in the binding site various modes of interaction of the N-terminus of hDAT in controlling the of SERT. This process eliminates negative extracellular surface charges. pathways of release. Surface charge elimination is accompanied by a change in the surface potential, 628-Pos Board B393 concomitantly affecting the transmembrane potential. The change of the latter Oligomerization of Human Dopamine Transporter (hDAT) then gives rise to a change in the measured membrane capacitance. The Kumaresan Jayaraman, Harald H. Sitte, Thomas Stockner. substrate-induced decrease in membrane capacitance was still present, when Institute of Pharmacology, Medical University of Vienna, Vienna, Austria. omitting co-substrates. This is consistent with our hypothesis that the reduced Monoamine transporters of the SCL6 family are found on presynaptic neu- capacitance resulted from serotonin binding (adsorption). At sodium-free rons and terminate the neurotransmission by the reuptake of neurotransmit- conditions, the affinity for serotonin was decreased which was reflected by a smaller capacitance reduction. Our findings suggest random order binding re- ters. The family of monoamine transporters includes the transporters for þ dopamine (DAT), serotonin (SERT) and norepinephrine (NET) which actions of substrate and co-substrates and that serotonin and Na bind in a couple substrate transport with ion gradients of sodium and chloride. cooperative manner. Dysfunction of these transporters can lead to clinically important disease Financial support was given to WS by the Austrian Funds FWF (P28090-B27). states, for instance to depression. It has been shown that monoamine trans- porters and other members of the family exist in oligomeric form in cells 631-Pos Board B396 and studies reported that different oligomeric sizes are present at the level Exploring Ligand-Binding Kinetics in the S2 Site of MhsT by Atomistic oftheplasmamembrane.Thereisawealth of reports regarding the residues Simulations and Markov Models critical for oligomerization, however, the binding orientation and possible Ara M. Abramyan1, Cathy Xue2, Lei Shi1. 1Molecular Targets and Medications Discovery Branch, NIDA-IRP/NIH, hierarchies of the many putative oligomerization interfaces are poorly 2 understood. In this study, we extensively scrutinized the oligomeric forms Baltimore, MD, USA, Department of Computer Science & Department of of the human DAT using molecular dynamics simulations and applying Mathematics, Yale University, New Haven, CT, USA. the MARTINI coarse grained force field. Two monomers of DAT were in- Neurotransmitter:Sodium Symporters (NSS) terminate neurotransmission serted into a POPC membrane in random orientations. Subsequently, the as- through sodium-driven reuptake of cognate neurotransmitters, and sembly of the monomers were analyzed over 2 ms time for 500 independent traverse between outward-open and inward-open states. NSS include seroto- systems resulting in a total of 1ms simulation time. The convergence of the nin and dopamine transporters, which are targets for antidepressants and whole ensemble is quantified by the interaction energy plot, the number of abused psychostimulants. Crystallographically, whereas both substrates conformers and their orientations is shown by the density plot. This study and inhibitors have been found to bind the central binding site (denoted indicates that DAT forms dimers through four distinct orientations. These S1) of the NSS proteins, it has been shown that inhibitors for LeuT and results allowed us to propose a testable hypothesis of residues located within SERT can also bind to a binding cavity in the extracellular vestibule the oligomeric interface. (denoted S2). Based on computational and experimental studies in LeuT, ithasbeenfoundthatsubstratescanbindtoS2aswell,andsuchbinding 629-Pos Board B394 triggers the intracellular release the substrate and Naþ from S1. However, Two Mechanisms in One Family: Packaging of Glutamate into Synaptic whether such a role of S2 substrate is common for other NSS remains Vesicles against a Proton Gradient, a Transporter Driven by Membrane unclear. Potential, versus a Homolog Driven Downhill with Proton Gradients The newly available crystal structure of MhsT, a bacterial NSS homolog, Robert M. Stroud. which was solved in an inward-occluded state, provides a more suitable Biochemistry & Biophysics, University of California, San Francisco, San starting point to study the substrate induced conformational changes, Francisco, CA, USA. including the S2 substrate induced transition from inward-occluded to Vesicular transporters for glutamate depend on membrane potential as inward-open state transition. Here, we use extensive molecular dynamics driving force. The crystal structure of a close homolog (23% identity) in simulations combined with hidden Markov model (HMM) analysis to the same superfamily has been determined, and transport assays used to investigate the feasibility and kinetics of the substrate L-tryptophan binding confirm that it conducts its anionic substrate driven by proton gradients as to S2 of MhsT. Based on HMM analysis, we identified transition binding po- a proton symporter. How then can the vesicular cousin drive its anionic sub- sitions in the extracellular vestibule that form potential substrate binding strate against the normal proton gradient? Transmembrane channels and fa- pathways, and the conformational changes near the S2 site associated cilitators can be highly selective in mediating the transport of nutrients and with the two most populated bound states. Our findings shed light on the ions across membranes ‘downhill’, down their concentration gradient. S2 substrate binding kinetics and the induced conformational changes, Transporters can drive and concentrate nutrients or ions ‘uphill’ (energeti- which may be critical in triggering the subsequent events in the transport cally) across membranes achieving gradients of >1000:1 driven by ‘down- mechanism.

BPJ 7740_7745 128a Sunday, February 12, 2017

632-Pos Board B397 1Cardiovascular Research Institute and Department of Pharmaceutical Computational Investigation of the Serotonin Transporter Conformation Chemistry, UCSF, San Francisco, CA, USA, 2Department of Pharmaceutical and Reset Mechanism Chemistry, UCSF, San Francisco, CA, USA, 3Department of Physiology, Emily M. Benner, Jeffry D. Madura. UCLA, Los Angeles, CA, USA, 4Department of Chemistry and Molecular Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA, USA. Biology, University of Gothenburg, Go¨teborg, Sweden, 5Department of The aid of monoamine transporter (MAT) proteins in terminating the effect Structural Biology, Stanford, Stanford, CA, USA. of neurotransmitters in the synaptic cleft is crucial for the maintenance of Sodium-dependent glucose transporters (SGLTs) are members of the proper neurotransmitter concentrations. Termination of effect is achieved large solute carrier (SLC) family of proteins that exploit the sodium ion via the process of reuptake, where the proteins bind neurotransmitters to a concentration gradient to transport a myriad of small molecules across central substrate binding site and transport the substrate to the other side the plasma membrane. In humans, there are six SGLT subtypes labeled of the membrane through a permeation pathway. The MAT family of pro- 1-6 that are expressed widely in the small intestine, kidney, lung, muscle, teins includes the serotonin transporter (SERT), which is responsible for and brain. Due to their role in sugar reabsorption, SGLTs are currently the reuptake of serotonin from the synaptic cleft. SERT, as well as the other exploited as drug targets for the treatment of type 2 diabetes, especially MAT proteins are implicated in several psychological disorders, including hSGLT2, which is responsible for 98% of glucose reabsorption in the depression, anxiety, and addiction. Treatments for these disorders are kidneys. Current inhibitors are chemical derivatives of the naturally focused on the inhibition of the MAT system, especially SERT. These occurring small molecule phlorizin, which is expressed in the bark of drugs are known as selective serotonin reuptake inhibitors, which act fruit trees, such as apple and pear. The structural basis of binding is by binding to the substrate site of SERT, preventing the reuptake of seroto- not known, in part, because high-resolution structures of mammalian nin. Recent work in our lab has focused on identifying the complete SGLTs do not exist. However, the inward-facing structure of the bacterial transport mechanism of SERT computationally, employing both a single homologue from vibrio parahaemolyticus (vSGLT) has been solved bilayer and dual bilayer system to investigate this phenomenon. The work both in apo and in complex with galactose, and our collaborators recently presented here represents the data collected from our most recent dual solved the outward-facing structure of a closely related homologue (unpub- bilayer system. Herein we utilized the newly crystallized human SERT pro- lished). Here we combine homology modeling, virtual screening techniques tein (PDB 5I6X), embedded in POPE lipid membranes. The dual bilayer and molecular dynamics simulations to achieve two goals: 1) model setup allows the system to maintain ion concentrations on either side of the outward-facing state of SGLTs, and 2) predict the binding mode of the membrane when periodic boundary conditions are implemented. phlorizin and its derivatives hSGLT1 and 2. As a result, the spectroscopic Ions were added to either side of the membrane at physiological concentra- data (double electron-electron resonance) probing outward facing state tions, keeping the membrane potential at around 70mV, representative of of SGLTs validate our homology model and mutagenesis studies testing resting potential. Understanding more fully the complete transport process, binding to hSGLT1 and 2 are in agreement with our predicted binding with specific attention paid to the reset mechanism, can aid in drug discov- modes. ery and design for treatments that better alleviate symptoms of depression and related disorders. Presented here are the molecular dynamics and 635-Pos Board B400 conformational data from our ongoing simulations of the dual bilayer Uptake Dynamics in the LacY Membrane Protein Transporter SERT system. Dari Kimanius1, Stephen White2, Erik Lindahl3, Ronald Kaback4, Magnus Andersson5. 633-Pos Board B398 1Stockholm University, Stockholm, Sweden, 2University of California Irvine, Characterizing Outward- to Inward-Facing Transition Pathway of Irvine, CA, USA, 3KTH Royal Institute of Technology/Stockholm Dopamine Transporter University, Stockholm, Sweden, 4University of California Los Angeles, Zhiyu Zhao1, Emad Tajkhorshid2. Los Angeles, CA, USA, 5KTH Royal Institute of Technology, Stockholm, 1Center for Biophysics and Quantitative Biology, UIUC, Champaign, IL, Sweden. USA, 2UIUC, Champaign, IL, USA. Membrane protein transporters govern important cellular processes and The dopamine transporter (DAT) belongs to the family of neurotransmitter are therefore central to human health. To accomplish transport, these sodium symporters (NSSs), which harness transmembrane electrochemical proteins rearrange their structures to alternatively expose an internal ionic gradients to transport neurotransmitters against their chemical gradi- binding site to either side of the membrane. Recent advances in protein ents. Dysregulation of DAT is associated with serious neurological disor- structural determination methods have resulted in a steadily increasing ders, such as Parkinson’s disease, depression, anxiety and epilepsy. As number of high-resolution structures of membrane transporters trapped with all transporters, substrate translocation through DAT follows the in different intermediate states. However, to understand the under- alternating-access mechanism in which the protein swiches between lying transport mechanism, the molecular details of uptake and release outward-facing (OF) and inward-facing (IF) states. The details of these need to be determined. We have used specialized simulation hardware to structural changes and their coupling to chemical events such as substrate simulate uptake of galactoside sugar into the Lactose permease (LacY) of and ion binding events remain elusive. In the present study, we charactrized Escherichia coli. The extended brute-force simulation revealed large-scale large-scale transition from the OF state to IF state using all-atom molecular structural rearrangements, lipid and amino acid interactions, and hydration dynamics simulations of membrane-bound models of DAT. As all crystal associated with sugar uptake. The free energy landscape of sugar entry structures of DAT are in the OF state, the initial phase of the study included was determined by parallel bias-exchange metadynamics simulations and modeling of a stable IF structure of DAT in the context of a membrane using identified a global free energy minimum that coincided with the crystallo- the bacterial sodium-coupled leucine symporter (LeuT) as a template. graphic binding site and also a local free energy minimum in the larger Furthermore, equilibrium simulations performed in this phase revealed a periplasmic cavity of LacY. Together, our observations show a putative novel sodium binding site located between TM3 and TM8 helices, which molecular mechanism for sugar uptake in this prototype membrane are elements involved in coupling of protein structural changes to substrate transporter. binding and translocation. Using the orientations of helics TM1e/TM8e and TM1i/TM8i as collective variables, and employing two dimensional bias- exchange umbrella sampling and string method with swarms of trajectories, 636-Pos Board B401 we characerize a structural transition pathway between the OF and IF states Elevator-Like Mechanism of Transport in the EIIC Glucose Superfamily of DAT. The results of this study provide a deeper understanding of the of Transporters 1 1 2 3 2 functional mechanism of DAT, wiht implications to all members of the Zhenning Ren , Yin Nian , Jumin Lee , Jason McCoy , Wonpil Im , 1 NSS family. Ming Zhou . 1Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, 634-Pos Board B399 TX, USA, 2University of Kansas, Lawrence, KS, USA, 3Broad Institute, Structural Insights into Sodium-Dependent Sugar Transporters and their Cambridge, MA, USA. Inhibition Mechanism The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is Paola Bisignano1, Chakrapani Kalyanaraman2, Chiara Ghezzi3, crucial for sugar uptake in bacteria. It has a membrane embedded compo- Ernest M. Wright3, Jeff Abramson3, Aviv Paz3, Matthew P. Jacobson2, nent, EIIC, that translocates a sugar from the extracellular to the intracel- Rosmarie Friemann4,5, Michael Grabe1. lular side of the cell. Before the sugar is released into the cytosol, a

BPJ 7740_7745 Sunday, February 12, 2017 129a cytosolic EIIB protein binds to EIIC and transfers a phosphate group to the grained and all-atom molecular dynamics (MD) simulations, we character- incoming sugar that prevents the sugar from escaping the cell and at the ized the membrane deformation induced by GltPh trimers in all possible same time primes the sugar for entering metabolic cycles. Little is known permutations of inward-facing and outward-facing protomer states. The sim- concerning how EIICs recognize and transport carbohydrates or how an ulations show that when a protomer is in the outward-facing state, the sur- EIIC coordinates with EIIB to achieve phosphate transfer. Crystal structures rounding membrane is largely unperturbed. However, in the inward-facing of a maltose transporter bcMalT[1] and a N-diacetylchitobiose transporter state, the transport domain induces a strong deformation on the lipid bilayer, bcChbC[2] were solved recently, and the two structures appear to be in bending its average plane by ~10 A˚ along the direction perpendicular to the different states of a transport cycle: bcMalT in an outward facing state membrane plane. This perturbation extends radially for ~50 A˚ , but remark- and bcChbC in an inward facing state. The bcChbC structure provides a ably, it is largely localized around each of the transport domains in the template to build a model of bcMalT in an inward facing state, and vise vicinity of the protein, i.e. the membrane shape is restored near the versa. To examine the models, we designed pairs of cysteine residues that protomer-protomer interfaces. The specific protein-lipid contacts that are distant in the crystal structures but are predicted to move close to explain this local deformation are identified from all-atom MD simulations. each other in the alternate conformation. Several pairs of cysteines in in In summary, MD simulations demonstrate that the lipid membrane both bcMalT and bcChbC can be crosslinked by micromolar concentrations favors the outward-facing state of GltPh over the inward-facing state, owing of mercury, indicating that these residues can move close to each other. We to the long-range curvature deformation induced by the latter. However, then solved the structure of the T280C/E54C bcMalT double cysteine these simulations also show that this large deformation does not imply mutant in the crosslinked state to 3.6 A˚ resolution, and the structure is a membrane-mediated protomer cross-talk, explaining the mystifying indeed in an inward-facing conformation. The new structure illustrates the absence of measurable cooperativity among protomers in this trimeric large-scale movement of a structurally conserved domain in bcMalT, and transporter. shows that EIIC employs an elevator-like mechanism for substrate translo- cation. Further analyses suggest how the inward facing conformation could 639-Pos Board B404 interact with an EIIB protein to achieve phosphate transfer. The structures Protein-Protein Interaction between Sodium-Coupled Monocarboxylate also provide a solid starting point for investigating the dynamics of the Transporter 1 (SMCT1) and PDZ Domain-Containing Ring Finger EIIC protein using spectroscopic approaches. Domain 3 (PDZRN3) Reference Yusuke Otsuka. [1]. Mccoy JG, Ren Z, Stanevich V, et al. The Structure of a Sugar Transporter Pharmacology, Chiba University Graduate School of Medicine, Chiba-shi, of the Glucose EIIC Superfamily Provides Insight into the Elevator Mechanism Japan. of Membrane Transport. Structure. 2016;24(6):956-64. Sodium-coupled monocarboxylate transporters (SMCTs) are membrane [2]. Cao Y, Jin X, Levin EJ, et al. Crystal structure of a phosphorylation- proteins which transport lactate on the apical side of kidney. In addition coupled saccharide transporter. Nature. 2011;473(7345):50-4. to renal lactate reabsorption, it is known that both SMCTs and urate/anion 637-Pos Board B402 transporter1 (URAT1) bind to PDZK1 and that SMCTs and URAT1 func- Substrate-Induced Conformational Change in LeuT tionally cooperate to transport urate. This indicates that these transporters Yuan-Wei Zhang1, Lucy R. Forrest2, Gary Rudnick1. reabsorb urate by exchange for sodium. Our recent study revealed that 1Pharmacology, Yale University, New Haven, CT, USA, 2NINDS, NIH, SMCT1 (SLC5A8), a higher-affinity transporter than SMCT2 (SLC5A12), Bethesda, MD, USA. bound to PDZ domain-containing RING finger domain 3 (PDZRN3) in a LeuT is a prokaryotic amino acid transporter that has been used extensively yeast two hybrid screening. But there is less information available on the as a model for neurotransmitter transport. We recently demonstrated that interaction between SMCT1 and PDZRN3. In this study, we elucidated the conformational change induced by Naþ ions requires the Na2 site this protein-protein interaction between them to resolve the regulation observed in LeuT crystal structures. We observed this conformational mechanism of serum urate level. We performed coimmunoprecipitation change using LeuT in E. coli membranes, in the absence of detergent, study to confirm the binding between SMCT1 and PDZRN3, and performed 3 by a decrease in reactivity of a single cysteine (Y265C) in the cytoplasmic [ H] nicotinate uptake study to reveal whether there was a functional change permeation pathway. We now show that this effect of Naþ is observed by this binding, using HEK293 cells transiently transfected with SMCT1, its whether Kþ or NMDGþ is used as a control ion. In the presence of Naþ, mutant lacking the PDZ motif and PDZRN3. Coimmunoprecipitation study addition of alanine, a substrate, induces the reverse conformational revealed that the wild type SMCT1, but not its mutant lacking the SMCT1 change, opening the cytoplasmic pathway and increasing Cys-265 reactivity. C-terminal PDZ motif, directly bound to PDZRN3. In uptake study, there Three mutations in the substrate binding site each altered the affinity of was no significant difference in amount of nicotinate uptake via SMCT1 LeuT for leucine and alanine, but did not interfere with the conformational and its mutant lacking the PDZ motif, in the presence of PDZRN3 or not. change induced by Naþ. One of the mutations also blocked the substrate- These results showed the protein-protein bind between SMCT1 and dependent conformational change. The results suggest a mechanism by PDZRN3, but no functional interaction. This suggests that PDZRN3 regu- which substrate interactions with the central binding site of LeuT reverse lates urate reabsorption via URAT1 by preventing SMCT1 from binding the ability of Naþ to stabilize outward-facing conformations of this model to PDZK1. transporter. 640-Pos Board B405 638-Pos Board B403 It Runs in the Family: Determining the Transport Mechanism of Sodium/ Dicarboxylate Symporter hNaDC3 Membrane Remodeling by GltPh in the Inward- and Outward-Facing Conformations Explains Lack of Protomer Cooperativity Alissa J. Becerril1, Cristina Fenollar-Ferrer2, Lucy R. Forrest2, Wenchang Zhou1, Claudio Anselmi1, Horacio Poblete1, Ali Karimi2, Joseph A. Mindell1. 1 Lucy Forrest2, Jose Faraldo-Gomez1. Membrane Transport Biophysics Unit, NINDS, NIH, Bethesda, MD, USA, 2 1National Heart, Lung and Blood Institute, NIH, Bethesda, MD, USA, Computational Structural Biology Unit, NINDS, NIH, Bethesda, MD, USA. 2National Institute of Neurological Disorders and Stroke, Bethesda, Members of the divalent anion:Na(þ) symporter (DASS) family play im- MD, USA. portant roles in mammalian physiology, transporting divalent anions, Membrane-embedded proteins can induce the remodeling of the adjacent including Krebs cycle intermediates and sulfate, across the plasma mem- lipid bilayers by promoting curvature, altering the membrane thickness brane. These transporters may be key contributors to determining urinary and/or exposing hydrophobic groups. When these proteins undergo a citrate levels, which, in turn, may affect kidney stone formation; they also function-related conformational transition, the energy cost associated have been implicated in metabolic regulation in both drosophila and with these membrane perturbations adds up to the free energy of each of mammals. It is therefore important to understand the relationships in these the protein states and can therefore modulate their functional mechanisms. proteins between their structure and functional mechanisms. Though no Here, we study the case of the Naþ-coupled aspartate transporter from structures are yet available for mammalian DASS family members, a crystal structure of a bacterial homolog, vcINDY, has been determined. Pyrococcus horikoshii (GltPh). GltPh has been crystallized as a trimer both þ in an inward- and outward-facing conformations. In this outward-to- We recently demonstrated that vcINDY, a Na -coupled succinate trans- inward conformational exchange, the so-called transport domain porter, utilizes a dramatic ‘‘elevator’’ mechanism to transport substrate, moves ~20 A˚ across the membrane, relative to the seemingly rigid trimeri- involving a large-scale vertical movement of a protein domain perpendicular zation/scaffold domains, so as to expose the substrate and Naþ-binding sites to the plane of the lipid bilayer membrane. Here, we sought to determine to either the cytoplasm or the extracellular space. Using large-scale coarse- whether a mammalian family member utilizes a similar transport

BPJ 7740_7745 130a Sunday, February 12, 2017 mechanism, specifically human NaDC3. With ~30% identity between the vealed altered Ca2þ homeostasis which might underlie the functional vcINDY and hNaDC3, we hypothesized that a similar mechanism could impairments. be responsible for the functioning of hNADC3. Homology modeling with Our results reveal the specific contributions of MICU1 in neuronal Ca2þ ho- vcINDY suggested that several pairs of residues are brought into proximity meostasis and provide some clues to the neuronal pathogenesis in MICU1 upon substrate translocation. We prepared a series of double mutants loss of function patients. introducing cysteines at positions predicted to be brought together in the outward-facing state of the protein and expressed them in Xenopus 643-Pos Board B408 laevis oocytes. Using two-electrode voltage clamp and disulfide cross- D IP Receptor Isoform Dependence of the ER-Mitochondrial CA2 - linking, we investigated whether inhibition of transport activity observed 3 Transfer in Mammalian Cells upon formation of disulfides is consistent with the proposed mechanism of Mate Katona1, Kamil Alzayady2, David I. Yule2, Gyorgy Hajnoczky1. transport. 1Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA, 2Department of Pharmacology and Mitochondria in Cell Life and Death I Physiology, University of Rochester, Rochester, NY, USA. 2þ IP3 receptors (IP3Rs) mediate Ca release from the ER to the cytosol and can 2þ 641-Pos Board B406 relay Ca signals locally to mitochondria. The IP3R family is composed by Cyclophilin D Acetylation Regulates Cardiac Myocyte Differentiation three isoforms (type 1,2 and 3) showing heterogeneous structure, function and tissue distribution. In some paradigms specific IP3Risoformswereshownto George A. Porter, Jr., Morgan L. Albert, Kathia R. Fantauzzi-Nieves, þ Gisela Beutner. be enriched close to and support Ca2 transfer locally to the mitochondria. Pediatrics, University of Rochester Medical Center, Rochester, NY, USA. Until recently, a mammalian system allowing systematic evaluation of each INTRODUCTION: Protein acetylation is an important regulator of isoform has remained unavailable. The establishment of IP3Rtripleknock cellular function. Acetylation of cyclophilin D (CyPD) at lysine 166 increases out (TKO) HEK cells and rescue with different IP3R isoforms tagged with its ability to open the mitochondria permeability transition pore (PTP), which a common biochemical tag provided new opportunities to test the hypothesis that IP3R isoforms are equal in terms of coupling to the mitochondria. To is an important regulator of mitochondrial metabolism and myocyte differen- þ tiation. HYPOTHESIS: Acetylation of CyPD controls PTP activity, mito- study Ca2 coupling between ER and mitochondria we used WT HEK cells chondrial structure and function, and myocyte differentiation in the and TKO cells acutely and stably rescued by the different isoforms of 2þ developing heart. METHODS: We use immunoblotting and immunoprecipita- IP3Rs. Ca -signaling was induced by an IP3-generating agonist, carbachol. 2þ 2þ To measure changes in mitochondrial Ca ([Ca ]m) we transfected cells tion to determine the global protein acetylation state and that of CyPD during þ embryonic cardiac development in wild type (WT) and CyPD-null mice. We with the genetically-encoded Ca2 sensor CEPIA targeted to the mitochon- 2þ 2þ also re-expressed WT and mutant CyPD in CyPD-null embryonic and drial matrix (mtCEPIA), changes in cytosolic Ca ([Ca ]c) were measured neonatal cardiac myocyte cultures to determine effects on mitochondrial by FURA-2 AM simultaneously. WT HEK cells showed a marked increase in 2þ 2þ [Ca ]c followed by a fast rise in [Ca ]m with the coupling time of structure and function and myocyte differentiation. RESULTS: Immunoblot- þ ting revealed that global protein acetylation was high in the embryo but fell 0.94 50.22 s (n=56) while TKO cells showed no changes in [Ca2 ]c and 2þ during later development. In contrast, CyP-D was highly acetylated in the [Ca ]m to agonist stimulation. Acute and stable type 1, 2 or 3 IP3Rrescue 2þ 2þ TKO cells regained [Ca ]c responses and also showed a [Ca ]m increase. early embryonic heart, but this decreased dramatically during subsequent þ development. When re-expressed in cultures of CyPD-null myocytes, WT Quantitative analysis of the Ca2 transfer efficiency to the mitochondria CyPD recapitulated the WT cell phenotype of fragmented mitochondrial and the localization of each IP3R isoform relative to the mitochondria are structure, low mitochondrial membrane potential (Dcm), and decreased differ- currently pursued. In summary, we have validated a new model for the study 2þ entiation. Re-expression of a CyPD acetylation mimic mutant (K166Q) had of IP3R-mediated local Ca signaling between the ER and mitochondria, al- similar or greater effects on these parameters than WT CyPD, while an inac- lowing us to determine the specific function of each isoform in these tivating (R96G) and de-acetylation mimic (K166R) CyPD mutant did not. processes. CONCLUSION: These data suggest that myocyte differentiation during car- diac development is associated with a decrease in overall protein acetylation, 644-Pos Board B409 perhaps because of the changes that occur in metabolism as the heart develops. Mitochondrial Calcium Uptake and Matrix Calcium Buffering in Skeletal However, specific acetylation of CyPD appears to be particularly important Muscle for maturation of mitochondrial function and regulation of cardiac myocyte Valentina Debattisti, Gyorgy Csordas, Erin Seifert, Gyorgy Hajnoczky. differentiation. MitoCare, Thomas Jefferson University, Philadelphia, PA, USA. Many clues suggest that mitochondrial calcium uptake plays an essential 642-Pos Board B407 role in muscle function. Mitochondria can contribute to shaping of þ þ The Role of MICU1 in Neuronal Mitochondrial Calcium Homeostasis the sarcoplasmic [Ca2 ] transients by their Ca2 handling and are providing and Function energy for muscle contraction and relaxation by their ATP production. þ Adam Bartok1, Melanie Paillard1, Valentina Debattisti1, David Weaver1, Mitochondrial calcium handling involves uptake, release and Ca2 chelation þ Ashley Tyburski2, Lianteng Zhi3, Hui Zhang3, Melanie Elliott2, in the matrix, which allows storage of vast amounts of Ca2 . Recent iden- þ Gyo¨rgy Hajno´czky1. tification of the molecular machinery of the Ca2 uniporter (mtCU) created þ 1Pathology, Anatomy and Cell Biology, Thomas Jefferson Unversity, an opportunity to target specific mechanisms of Ca2 handling and address þ Philadelphia, PA, USA, 2Neurological Surgery, Thomas Jefferson Unversity, their function in Ca2 homeostasis and contractile function. To elucidate þ Philadelphia, PA, USA, 3Neuroscience, Thomas Jefferson Unversity, the role of mitochondrial Ca2 uptake in skeletal muscle (SM) function þ Philadelphia, PA, USA. we ablated MICU1, the Ca2 sensing regulator of mtCU in mouse. Mitochondrial Ca2þ homeostasis controls intracellular calcium signals, MICU1 ablation in SM resulted in impaired gatekeeping and attenuated þ cellular energy production and mechanisms of cell death. We and others Ca2 uptake and resulted in less endurance exercise performance when have shown that MICU1 is an intermembrane space protein that provides challenged with fatigue protocols. However, in this mouse and in cell lines, Ca2þ sensitivity to Ca2þ uptake by the mitochondria through the mitochon- upon MICU1 ablation protein levels of other components of the mtCU and þ drial Ca2þ uniporter. Specifically, MICU1 is required both for protecting Ca2 chelation in the matrix were also altered. To overcome the compensa- mitochondria from Ca2þ uptake when the cytoplasmic [Ca2þ] is low and tory effects we decided to apply two strategies. First, we induce acute for proper transmission of the short-lasting Ca2þ signal from the ER/SR to MICU1 knock out using a tamoxifen-inducible CRE-system. Secondly, we the mitochondria. Human loss of function mutation of MICU1 has been linked also targeted the mitochondrial phosphate (Pi) carrier (PiC) in a similar to learning difficulties, skeletal muscle weakness, fatigue and motoric manner. Pi transport into mitochondria is crucial for both mitochondrial þ impairment. ATP synthesis and Ca2 chelation in the matrix. To quantitatively and þ In this study, we describe and validate a neuron-specific MICU1 KO simultaneously measure Ca2 uptake and matrix buffering, mitochondria model in mice. Proteomic analysis of the mitochondria confirms loss are isolated from SM for fluorometry are loaded with furaFF/AM and of MICU1 and indicates a possible compensatory mechanism for the loss are incubated in the presence of rhodFF. SM mitochondria acutely þ of MICU1 by other constituents of the uniporter. Behavioral tests depleted for PiC show a decreased Ca2 chelation capacity in the matrix þ performed on the animals showed decreased motoric function progressing and unexpectedly, take up more Ca2 than the control. When challenged with age. Measurements of cytoplasmic and mitochondrial Ca2þ signals in an incremental exercise test, PiC-ablated mice could only run for in cultured cortical neurons and Ca2þ uptake by isolated mitochondria re- 8.551.7 min, whereas control mice ran for the full 20 min, showing

BPJ 7740_7745 Sunday, February 12, 2017 131a impaired performance. The effects of acute MICU1 depletion in SM on 647-Pos Board B412 D mtCU composition, mitochondrial Ca2þ handling and SM function are A Theoretical Study on the Roles of Ca2 in the Energy Metabolite currently evaluated. Thus, our results provide genetic evidence for the Stability during Cardiac Workload Transition contribution of mitochondrial Ca2þ homeostasis to SM function and are ex- Ayako Takeuchi1, Ryuta Saito2, Yukiko Himeno3, Satoshi Matsuoka1. pected to determine the function and mechanism dependent on specifically 1University of Fukui, Fukui, Japan, 2Mitsubishi Tanabe Pharma on MICU1 and PiC. Corporation, Saitama, Japan, 3Ritsumeikan University, Ninamikusatsu, Japan. 645-Pos Board B410 The heart is a continuously working pump that is energetically driven by Mitochondrial Calcium Flux Contributes to Arrhythmia in Mouse Heart hydrolysis of ATP. Since the amount of ATP in the heart is small, ATP during Acute Myocardial Infarction synthesis must increase so as to adapt to a rise in cardiac ATP demand. An Xie, Hong Liu, Anyu Zhou, Guangbin Shi, Samuel C. Dudley, Jr. Therefore, the cardiac energy metabolites such as ATP, phosphocreatine, Rhode Island Hospital, providence, RI, USA. ADP and NADH are kept relatively constant during physiological cardiac Introduction: Acute myocardial infarction (AMI) is associated with workload transition. Ca2þ has been implicated to be one of the regulators lethal ventricular arrhythmias. One important mechanism for these arr- of the constancy, though its detailed roles are not yet clarified. Here, we hythmias is delayed afterdepolarizations resulting from Ca2þ overload in 2þ constructed and analyzed a detailed mathematical model of cardiac mito- border zone. Mitochondria participated in Ca homeostasis, and the chondria which includes oxidative phosphorylation, substrate metabolism mitochondrial Ca2þ uniporter (MCU) mediates mitochondrial Ca2þ entry. 2þ and ion/substrate transporters, based on experimental literatures, for the pur- We tested whether mitochondrial Ca flux played a role in ventricular ar- pose of elucidating the roles of Ca2þ-dependent regulation mechanisms in rhythmias during AMI. Methods: Hearts and ventricular cells were isolated þ/- the metabolite constancy. from CD1 wild type (Wt) and MCU knock down (MCU ) mice. Whole cell The model well reproduced the experimental data on Ca2þ- and inorganic current-clampwasemployedtorecordaction potentials (APs). Cytoplasmic 2þ 2þ phosphate (Pi)-dependencies of oxygen consumption, NADH level, and mito- Ca and mitochondrial Ca were sampled by loading Indo-1 AM and chondrial membrane potential, under the in vitro condition of isolated mito- Rhod-2 AM (recorded synchronously with APs) respectively. Electric chondria with malate/glutamate used as substrates. When the model was pacing was used to induce arrhythmia. MCU mRNA and protein expression incorporated into a simple cardiac cell model and malate/glutamate were were evaluated by Western blotting & Real-Time PCR. Optical mapping þ/ used as substrates, the metabolite constancy could not be maintained at higher was used to detect arrhythmias in the intact heart. Results: In MCU workload with cytosolic Ca2þ below physiological range ( ~ 0.1 mM). Pi and 5 5 mice, the mRNA (from 1.00 0.02 to 0.42 0.03 ) and protein expression NADH levels became stable during workload transition with higher cytosolic (from 0.73 5 0.07 to 0.33 5 0.04)ofMCUwassubstantiallyreduced,with 2þ 2þ 2þ Ca . Interestingly, the Ca dependency of NADH was influenced by the peak mitochondrial Ca transients (F/Fo) prominently decreased from composition of cytosolic mitochondrial substrates. That is, the Ca2þ-depen- 5 5 0.21 0.02 to 0.14 0.03. Infarct size was similar in the two models: dency of NADH almost disappeared and energy metabolites became more Wt (48% 5 4%) and MCUþ/- (44% 5 2%) mice. Two of eight of Wt þ/- stable during workload transition under the in vivo condition that malate, micediedsuddenlywithin1hafterAMI,butnoneofMCU mice died glutamate, pyruvate, citrate and 2-oxoglutarate were used as substrates. It suddenly in this period. Compare to the Wt mice, pacing induced signifi- þ/- was revealed that composition of mitochondrial substrates is important for cantly less ventricular arrhythmias in MCU mice during the first metabolite constancy during cardiac workload transition, and Ca2þ has stage of coronary artery ligation as VT/VF duration decreased from only a minor role under physiological conditions. These results help us under- 5 5 þ/- 0.38 0.09 s in Wt mice to 0.12 0.05 s in MCU mice. Consistent standing the regulation mechanisms of cardiac energy metabolism under the with a role of mitochondria in ischemic ventricular rhythms, inhibition of physiological conditions. mitochondrial NCX by CGP-37157 (i.p. 0.4mg/100g) in both Wt and MCUþ/ mice could abolish ventricular arrhythmias evoked by electric pac- ing during AMI. Furthermore, VT/VF was detected only in Wt mice at 648-Pos Board B413 2D infarction border zone by optical mapping experiment. Finally, after AMI, Reciprocal Polarization of the Mitochondrial Ca Uniporter and D 2D action potential durations were longer in Wt mouse hearts/cardiomyocytes NA -CA Exchanger in Cardiac Muscle than those in MCUþ/- mouse hearts/cardiomyocytes. Conclusion: Our Sergio De la Fuente1, Shey-Shing Sheu2, Gyorgy Csordas1. 1 study suggest a role for mitochondrial Ca2þ handling in arrhythmic MitoCare Center, Pathology Anatomy and Cell Biology, Thomas Jefferson 2 risk during AMI. Reducing mitochondrial Ca2þ cycling improved University, Philadelphia, PA, USA, Center for Translational Medicine, arrhythmic risk. Thomas Jefferson University, Philadelphia, PA, USA. Control of myocardial energetics by Ca2þ signal propagation to the mito- 646-Pos Board B411 chondrial matrix includes local Ca2þ delivery from sarcoplasmic reticulum Regulation of Carbon Substrate Utilization by Cardiac Mitochondria (SR) ryanodine receptors (RyR2) to the inner mitochondrial membrane Santosh K. Dasika, Sunil M. Kandel, Daniel A. Beard. (IMM) through the Ca2þ uniporter (mtCU). Ca2þ is extruded by the Naþ- MIP, University of Michigan, Ann Arbor, MI, USA. Ca2þ exchanger (NCLX), predominantly active in excitable tissues such Mitochondria play key roles in central metabolism, not only by synthesizing as heart, and/or the Hþ-Ca2þ exchanger preferentially active in non- ATP via oxidative phosphorylation, but also by synthesizing key intermedi- excitable cells. We have recently reported that the mtCU is strategically ates that serve as anaplerotic entry points in numerous pathways. Because positioned in cardiac muscle at the mitochondria-junctional SR association reactions producing and/or consuming several of the intermediates in the in order to support more efficiently the local Ca2þ signals. In those studies tricarboxylic acid cycle that are transported across the mitochondrial we established a comparative abundance profile for a range of mitochondrial inner membrane (pyruvate, a-ketoglutarate, citrate) are regulated in part proteins between the mitochondrial and SR fractions of mouse and rat heart by calcium concentration, we hypothesize the calcium is a key regulator homogenates. While mtCU constituents were equally distributed (MCU, of entry and exit of substrates into and out of the TCA cycle. We set out MICU1) or enriched in the SR fraction (EMRE), the NCLX was strongly to first refine this hypothesis through following time courses of consumption (multiple fold) enriched in the mitochondrial fraction. We thus hypothe- of various carbohydrate based substrates (including complex I substrates sized, that NCLX was excluded from the mtCU hot spots at the dyad junc- such as pyruvate and citrate, and the complex II substrate succinate) and tions and that it would be localized at areas where mitochondria associate matching data on rates of oxidation and NADH production to simulations with the network SR. Indeed, percoll purification of the crude mitochondrial of mitochondrial metabolic kinetics. Specifically, model simulations were fraction that decreased the abundance of MCU and EMRE lead to an fit to data on total NAD(P)H and oxygen consumption flux at different enrichment of NCLX. Mitochondrial 45Ca2þ uptake assays using isolated respiratory states and with varying inorganic phosphate concentrations. mitochondria or SR fraction with vs. without NCLX inhibition (pharmaco- The resulting identified model is used to predict how calcium regulates logical or Naþ removal) revealed a robust Naþ-dependent efflux working carbon substrate utilization by mitochondria. These hypotheses are tested against the uptake in the mitochondrial but not the SR fraction. Direct using in vitro experiments with purified mitochondria. By comparing the measurement of the fractional Ca2þ release from the two membrane experimental data with citrate alone to combination of pyruvate, citrate fractions also pointed toward much more efficient NCLX-mediated Ca2þ and malate as the substrates, we conclude that transport of citrate into the extrusion in the mitochondrial fraction than in the SR. Thus, mitochondrial matrix is limited in the absence of malate. The rate of reduction of Ca2þ uptake and Naþ dependent extrusion seem to be reciprocally polarized NAD is slower with pyruvate alone as substrate compared to when malate to optimize both the efficacy of receiving local Ca2þ signals at the is present. The role of glutamate dehydrogenase in the context of cardiac en- dyadic interface and of feeding back the uptake Ca2þ to the SERCA pumps ergetics is investigated. of the network SR.

BPJ 7740_7745 132a Sunday, February 12, 2017

649-Pos Board B414 (bithionol) and inhibition (quinidine) of Slo2 channels with patch clamp Effect of Arsenic on Intracellular Calcium & Redox Homeostasis of isolated mitochondrial inner membranes (mitoplasts) from wildtype and Rafaela Bagur Quetglas1,Pe´ter Va´rnai2, Gyorgy Csorda´s1, Slo2.1-/- mice, to demonstrate the presence of Slo2.1 activity in cardiac Gyorgy Hajno´czky1. mitochondria at the single channel level. Additional analysis of these 1Pathology, Thomas Jefferson University, MitoCare Center, Philadelphia, Slo2.1-/- mice at the whole animal, heart, cardiomyocyte, and mitochondrial PA, USA, 2Physiology, Faculty of Medicine, Semmelweis University, level has revealed a metabolic phenotype comprising differential substrate Budapest, Hungary. preference under stress conditions, indicating a functional relationship be- A range of environmental agents causes tissue injury that has been attributed tween mitochondrial Kþ channels and the regulation of mitochondrial meta- to reactive oxygen species (ROS). Altered ROS production is commonly bolism. These data demonstrate a role for Slo2.1 in the regulation of cardiac originated from the mitochondria and is associated with dysregulation mitochondrial function. of calcium (Ca2þ) homeostasis and mitochondrial apoptosis. However, the causative pathways remain largely unknown because the fundamental 652-Pos Board B417 b roles of mitochondria in cell survival, cell signaling and dynamics are Mitochondrial Respiration and ROS Emission From -Oxidation in the commonly mediated through local communication between mitochondria Heart: An Experimental Computational Study and other organelles, which have been difficult to directly monitor. Our Miguel A. Aon, Steven J. Sollott, Sonia Cortassa. aim is to develop fluorescent protein-based tools allowing sensitive Laboratory of Cardiovascular Science, National Institute on Aging/NIH, and specific measurements of ROS and [Ca2þ] at the mitochondria- Baltimore, MD, USA. endoplasmic reticulum (ER) interface. Applying these novel tools, we Lipids are main fuels for cellular energy and mitochondria their major have studied the effects of environmental stress caused by arsenicon oxidation site. Yet unknown is to what extent the fuel role of lipids is influ- local mitochondria-ER communication and its impact for cell signaling, enced by their intrinsic uncoupling effects, and how this affects mitochon- and cell physiology in primary mouse hepatocytes. Either acute addition drial energetics, redox balance and the emission of reactive oxygen b (30 mM) or prolonged, overnight exposure (3 mM) to sodium arsenite has species (ROS). -oxidation of palmitoyl CoA (PCoA) in isolated heart mito- induced an increase in ROS. Upon prolonged arsenic exposure, these alter- chondria from Sham and streptozotocin-induced type 1 diabetic (T1DM) ations were accompanied by a significant decrease in the cytoplasmic guinea pigs (GPs) were comparatively analyzed. Parallel high throughput calcium signal to an IP3-linked agonist: a decrease was observed in the frac- measurements of the rates of oxygen consumption and H2O2 emission as tion of responsive cells, in the amplitude of the response as well as in the a function of PCoA concentration were performed. We found that PCoA < sustained phase. Regarding the acute effect of arsenic, we observed a pro- concentration 200 nmol/mg mito protein resulted in low H2O2 emission, gressive increase of the basal cytoplasmic [Ca2þ] with a decrease in the increasing thereafter in Sham and T1DM GPs under both states 4 and 3 amplitude of the IP3-linked calcium signal. Neither acute nor prolonged respiration with diabetic mitochondria releasing higher amounts of ROS. arsenic exposure impaired mitochondrial calcium uptake. Nevertheless, In both control and diabetic mitochondria, respiratory uncoupling and > we documented increased mitochondrial calcium content in the prolonged ROS overflow in both states 4 and 3 respiration occurred at PCoA 600 arsenic exposure group. In conclusion, the results suggest that an nmol/mg mito prot., leading to extensive matrix swelling, NAD(P)H pool > initial component of the arsenic-induced hepatocyte injury is increased oxidation, impairment of the state 4- 3 transition concomitantly with ROS, which is associated with impaired local mitochondria-ER calcium decreased activity of matrix glutathione/thioredoxin ROS scavenging communication. systems, and enhanced H2O2 emission. The experimental data could be simulated with a computational model of mitochondrial b-oxidation of 650-Pos Board B415 long-chain fatty acids, including main energy-redox and ionic processes. Effect of Rutin on Mitochondrial Respiration and Cellular Energy Status Results indicate that in a demand-led organ like the heart, mitochondria b Jeong-Soo Park, Icksoo Lee. can increase ROS release as a function of the rate of -oxidation dependent Dankook University, Cheonan, Korea, Republic of. respiration, but impairment of mitochondrial function would only happen Rutin, a phenolic compound found in many plants is known to have anti- after a certain threshold of lipid concentration is attained. Keeping low oxidative and anti-inflammatory properties. It has been reported that rutin res- levels of intracellular lipid appears to be crucial for mitochondria and cells cues the cells from the challenges such as ethanol, methylmercury, radiation, to maintain ROS within physiological levels compatible with signaling and hydrogen peroxide, and other cytotoxic chemicals. It is also suggested to reliable energy supply. enhance mitochondrial biogenesis in obese rats. 653-Pos Board B418 Cytochrome c oxidase is a rate limiting enzyme in mitochondrial electron trans- Detection of Reactive Oxygen Species in Live Cell Mitochondria port chain and consequently influences on mitochondrial membrane potential Zhen Luo, Jixiang Liu, Qin Zhao, Yunting Xi, Ruogu Peng, Jinfang Liao, and therefore the ATP production. Also its electron donor cytochrome c is Zhenjun Diwu. well known to participate in type 2 apoptosis in cells. AAT Bioquest Inc., Sunnyvale, CA, USA. Here it is shown that rutin elevates the activity of cytochrome c oxidase in hu- Oxidative stress in mitochondria has received intensive attentions recently man breast cancer cells. The mitochondrial respiration and cellular energy because it is the most important source of intracellular reactive oxygen species levels also increase by rutin treatment. We propose that the beneficial cytopro- (ROS) and is considered as a major contributor to some public health-related tective effects of rutin might due to, at least in part, the upregulated mitochon- diseases, such as asthma, atherosclerosis, diabetic vasculopathy, osteoporosis, drial enzyme, cytochrome c oxidase which would enhance mitochondrial a number of neurodegenerative diseases and Down’s syndrome. However, respiration and therefore cellular energy levels. the existing probes have difficulties to target live cell mitochondria and distinct *This research was supported by Basic Science Research Program through the the individual ROS. National Research Foundation of Korea(NRF) funded by the Ministry of Edu- We have developed a new family of mitochondrial ROS fluorescence cation(NRF-2014R1A1A2058722). probes, which not only can specifically localize in mitochondria, but also can selectively detect the different ROS species, including superoxide, 651-Pos Board B416 hydroxyl radical and hydrogen peroxide in multiple Ex/Em wavelengths Detection of a Bithionol Sensitive Potassium Channel in Cardiac channels. Mitochondria Absent in Slo2.1 Knockouts Cancer cells stained with the novel fluorescent probes showed negligible fluo- Charles O. Smith. rescence in absence of ROS stimulants. In contrast, OxiVision Blue-loaded Biochemistry, University of Rochester, Rochester, NY, USA. cells with hydrogen peroxide treatment displayed strong blue fluorescence in þ Mitochondrial K channels are important mediators of cell protection mitochondria. MitoROS OH580-loaded cells with Fenton reaction (to induce against stress, however less is known about their physiologic roles. Using hydroxyl radical) showed strong red fluorescence in mitochondria. MitoROS mouse knockout models our lab has recently reported the absolute require- 520-loaded cells exhibited strong green fluorescence in mitochondria after þ þ ment of the Na -activated-K channel Slo2.1 for volatile anesthetic precon- treated with superoxide stimulants. All these probes have also been success- ditioning against cardiac ischemia reperfusion injury [PMID: 26845140]. fully used with a flow cytometer for the quantitative detection of ROS in live Functional examination of this channel in isolated mitochondria using a cells. þ Tl flux assay demonstrated the presence of a bithionol-sensitive mito- In conclusion, these probes are mitochondrion-targeted ROS probes, which can þ chondrial Tl uptake in wildtype cardiac mitochondria that was absent in be used for monitoring the exogenous and endogenous changes of ROS levels in those from Slo2.1 knockout. Herein we combine pharmacologic activation living cells in real time by fluorescence imaging and or with a flow cytometer.

BPJ 7740_7745 Sunday, February 12, 2017 133a

654-Pos Board B419 passaged in bulk to determine whether unique combinations of mutations A Standardized Method to Quantify ER-Mitochondrial Interfaces in became fixed in the population. Using high depth RNA sequencing, we Electron Mircographs demonstrate the distribution of all possible amino acid substitutions from David Weaver, Adam Bartok, Gyorgy Csordas, Gyorgy Hajnoczky. initial production in the laboratory, replication in the host, and passaging in Thomas Jefferson University, Philadelphia, PA, USA. the presence or absence of neutralizing antibody. These results demonstrate The interaction sites between endoplasmic reticulum (ER) and mitochondria a method for surveying fitness landscapes that may be broadly applicable to are signaling hubs in the cell implicated in calcium transfer, lipid meta- virus-host evolution. bolism, autophagy, and cell death among other functions. Transmission electron microscopy (TEM) is commonly used to visualize the geometry 657-Pos Board B422 of these interfaces, but metrics used to describe and compare between con- Chiral Vortex Dynamics on Membranes is an Intrinsic Property of FtsZ ditions, as well as the techniques of measurement are not standardized. Here Driven by GTP Hydrolysis we describe a pair of scripts for ImageJ that allow for rapid, reproducible Diego A Ramirez-Diaz1, Daniela Garcia-Soriano1, Ana Raso2, Mario Feingold3, Germa´n Rivas2, Petra Schwille1. and flexible quantification of interface geometries. One script is focused 1 2 on measuring the physical apposition of the membranes while the other Max Planck Institute of Biochemistry, Munich, Germany, Centro de Investigaciones Biolo´gicas, Consejo Superior de Investigaciones Cientı´ficas considers the potential exposure of the outer mitochondrial membrane 3 (OMM) to calcium released from the ER. In both cases, the user simply (CSIC), Madrid, Spain, Department of Physics, Ben Gurion University, Beer traces the OMM and nearby ER membranes; the script bins the interface dis- Sheva, Israel. tances and returns the length of the OMM that participates in an interface of The primary protein of the bacterial Z ring guiding cell division, FtsZ, has a given gap width, as well as the total OMM and ER lengths. A ‘score’ recently been shown to engage in intriguing self-organization together with based on the inverse-square of the distance is also generated as an one of its natural membrane anchors, FtsA. When co-reconstituted on flat sup- attempt to make a single value representing ‘interface-ness’. The additive ported membranes, these proteins assemble into dynamic chiral vortices nature of these measures allows cell-wise totals to be calculated for a whose diameters resemble the cell circumference. These dynamics are due given TEM section. While this approach standardizes the measurement to treadmilling polar FtsZ filaments, supposedly destabilized by the co- technique for a number of parameters of ER-mitochondrial interfaces, it re- polymerizing membrane adaptor FtsA, thus catalysing their turnover. Here mains to be seen which parameters best correspond with physiological we show that FtsA is in fact dispensable and that the phenomenon is an changes in different models. intrinsic property of FtsZ alone when supplemented with a membrane anchor. The emergence of these chiral dynamic patterns occurs at intermediate FtsZ 655-Pos Board B420 surface densities, in agreement with theoretical predictions, and beyond a Control of Mitochondrial Structure and Antioxidant Response by the threshold GTP concentration. The interplay of membrane tethering, GTP ATPase Inhibitory Factor 1 Define a Novel Potential Oncogenic Mechanism binding, and hydrolysis promotes both, the assembly and the destabilization Danilo Faccenda1, Junji Nakamura2, Gurtej K. Dhoot1, Mauro Piacentini3, of FtsZ polymers, leading to the observed treadmilling dynamics. Notably, Masasuke Yoshida2, Michelangelo Campanella1. the vortex chirality is defined by the position of the membrane targeting 1Department of Comparative Biomedical Sciences, Royal Veterinary sequence (mts) and can be inverted when attaching it to the opposite end of College, London, United Kingdom, 2Department of Molecular Biosciences, FtsZ. This reveals the vectorial character of the filament-supported membrane Kyoto Sangyo University, Kyoto, Japan, 3Department of Biology, University system consisting of three orthogonal directions, filament polarity, curvature, of Rome Tor Vergata, Rome, Italy. and membrane attachment. The ATPase inhibitory factor 1 (IF1) is an ubiquitously expressed mitochon- 658-Pos Board B423 drial protein that blocks the reversal of the F1Fo-ATPsynthase, preventing dissipation of cellular ATP and ischaemic damage. Many human cancers ex- Engineering of Chimeric Proteins to Enhance Immunogenicity for the Production of High-Affinity Specific Monoclonal Antibodies press high levels of IF1, which suppress cell death and enhance tumour cell Sienna Wong, M. Moazzem Hossain, Rong Liu, J.-P. Jin. invasion and chemoresistance. In this study, we assessed the effect of IF1 over-expression on mitochondrial redox balance and apoptotic cristae remod- Wayne State University, Detroit, MI, USA. A wide range of biomedical research and diagnostic applications utilize high- elling. We found that IF1 maintains ATP levels under apoptosis and reduces glutathione (GSH) loss and inactivation of peroxiredoxin 3 (Prx3). This cor- affinity antibodies, the production of which depends on effective immuno- relates with inhibition of the metallopeptidase OMA1-mediated processing of gens. However, some proteins are intrinsically weak immunogens incapable the pro-fusion dynamin related protein optic atrophy 1 (OPA1), impeding of eliciting a strong immune response for the production of useful antibodies. To remedy this problem, weak immunogens are conjugated to exogenous car- cristae remodelling and apoptosis completion. IF1 therefore has a pivotal anti- oxidant activity that hinders the OMA1/OPA1-dependent deconstruction of rier proteins to increase immunogenicity and provide an adjuvant effect. In the mitochondrial morphology and cellular demise. The data presented here high- present study, we tested an alternative strategy of using chimeric fusion pro- tein constructs to enhance immunogenicity. We first applied the N-terminal light a dual regulatory activity of IF1 on both mitochondrial bioenergetics and structure, resulting in increased proliferative capacity of tumour cells and domain of glucose-regulated protein 96 (GRP-NTD), a protein with newly significantly contributing to the molecular signalling of mitochondria in identified activities in autoimmune diseases, as a carrier to present several cancer. experimentally identified weak immunogens: cardiac troponin C (cTnC), the N-terminal peptide of cardiac troponin T (N69), and the C-terminal peptide of calponin 2 (C85). Antigen peptides were engineered to the N-terminus of Emerging Techniques and Synthetic Biology the carrier, expressed in E. coli, large scale purified, and injected intramuscu- larly into adult mice without adjuvants. The effects of immunization were as- 656-Pos Board B421 sessed by ELISA titration of serum samples after three immunizations and/or Site Saturation Mutant Viruses Evolve Neutralizing Antibody Resistance the outcome of high-affinity specific monoclonal antibodies (mAb) in hybrid- in a Microfluidic Cell Culture System oma development. The results demonstrate significantly enhanced immunoge- 1 2 1 1 Jared D. Evans , Audrey E. Fischer , Susan Wu , Peter M. Thielen , nicity for cTnC and N69, but not C85. Based on an observation that cloned Thomas S. Mehoke1, Ashok Sivakumar1, Joshua T. Wolfe1. 1 2 mouse ApoE protein served as an effective carrier for the production of REDD, JHUAPL, Laurel, MD, USA, AOS, JHUAPL, Laurel, MD, USA. mAb against a small peptide, we constructed a C85-ApoE fusion protein Viruses constantly evolve to overcome host and environmental pressures dur- for hybridoma development. Surprisingly, C85-ApoE immunization was not ing their replication process. These evolutionary adaptations are generally effective for C85 but yielded high-affinity mAbs against mouse ApoE, an considered to be limited to a particular subset of beneficial mutations. We endogenous protein abundant in host plasma. The results suggest a potentially constructed a series of replication-competent mouse norovirus (MNV-1) new strategy of using chimeric immunogens to improve the production of site-saturation libraries to test how these viruses evolve in response to a specific antibodies. neutralizing antibody. Residues in the A’-B’ (E296) and E’-F’ (V378, A382, D385) loops of the MNV-1 capsid protruding (P) domain were mutated 659-Pos Board B424 using site directed mutagenesis. Viruses were evolved in the presence and A Multiscalar Framework describes Fluorescence and FRET of Fluctu- absence of monoclonal neutralizing antibody A6.2 in a droplet microfluidic ating Molecular Species and Resolves Kinetic Networks device such that starting virus populations at low multiplicity of infection Thomas-Otavio Peulen1, Oleg Opanasyuk1, Suren Felekyan1, could replicate over several passages in the absence of large population effects Stanislav Kalinin1, Hugo Sanabria2, Claus A.M. Seidel1. such as purifying selection. Droplet evolved viruses were subsequently 1Physical Chemistry II, Heinrich Heine University, Dusseldorf,€ Germany,

BPJ 7740_7745 134a Sunday, February 12, 2017

2Physics and Astronomy, CU School of Health Research, Clemson the potential of a novel artificial lipid membrane system, the droplet in- University, Dusseldorf,€ Germany. terface bilayer (DIB), as a platform for a spatially segregated compart- A combination of multi parameter fluorescence detection (MFD) with mentalised nanoreactor. DIB technology serves as a simple method for structure based fluorescence models is presented, to capture and describe producing small aqueous compartments bounded by lipid membranes. The fluorescence and FRET between a donor (D) and acceptor (A) dye of incorporation of membrane transport proteins between the aqueous com- fluctuating macromolecules over more than five orders of magnitude from partments will allow distinct chemical environments to be maintained picoseconds to seconds with A˚ ngstro¨m resolution. The presented top and for the controlled separation of substrates and products. The nanoreac- down approach combines molecular models with established fluorescence tor design will aim to recreate the segregated microenvironments and techniques such as time correlated single photon counting, burst integrated enhanced catalytic efficiencies found in biological processes in a synthetic fluorescence lifetime analysis, filtered fluorescence correlation spectroscopy setting. (fFCS), and photon distribution analysis in a joined framework and thus facilitates the analysis and interpretation of fluorescence experiments. 662-Pos Board B427 Fluorescence and FRET on the picoseconds to nanosecond regime is Cell Line Phenotypic Enrichement based on Migration and Morphology 1 1 1 2 described by combining atomistic models with a coarse grained representa- Joannie Roy , Loı¨c Binan , Javier Mazzaferri , Camille Lehuede , 2 3 2 tion of the dyes. Their conformational space is quantified by coarse grained Se´bastien Tabarie`s , Giuseppina Ursini-Siegel , Peter Siegel , Claudia Kleinman3, Santiago Costantino1. accessible volume simulations while Brownian dynamics simulations 1 capture transient effects of FRET and fluorescence quenching. Assuming Maisonneuve-Rosement Hospital, Montreal University, Montreal, QC, Canada, 2Goodman Cancer Research Centre, McGill University, Montreal, dynamic quenching and FRET are decoupled, the first two modes of the 3 DA-distance distribution are determined for single molecules to conve- QC, Canada, Lady Davis Institute for Medical Research, McGill University, niently reveal macromolecular kinetics on the milli- and sub-millisecond Montreal, QC, Canada. kinetics by MFD histograms. Structural models are projected to yield Cell Labeling via Photobleaching (CLaP) is an optical technique parametric equations of single-molecule observables. This serves as a recently developed to cross-link fluorescent molecules to the surface of visual guide to analyse MFD- histograms. Numeric integration of the chem- individual cells, by focusing a low-power laser on their membrane. ical master equation analyses MFD-histograms and quantifies in com- Using CLaP, it is possible select single cells or a sub-population based on bination with fFCS kinetic networks of dynamically exchanging visual criteria without the need of molecular markers. This technology macromolecular conformations in the sub-microsecond to millisecond can also be exploited for inducing transient cell adhesion to culture sub- regime. A joint analysis of multiple fluorescence decays by structure based strates, which are resistant to dissociation agents such as EDTA or trypsin. patterns resolved chemical equilibria in live cell. In future, such holistic ap- Since only the illuminated cells remain attached to the dish surface, CLaP proaches may exploit the information contained in the fluorescence signal can be exploited to tailor the spatial distribution of cells in a culture dish, and connect dynamic molecular structural models with kinetic and equilib- andalsotoenrichtheproportionofachosenphenotypewithinproliferating rium networks of biomolecules to picture molecular machines in living cells. cells. Breast cancer cell line MDA-MB-231 is known for its potential to me- tastasize to secondary sites. Among the various factors influencing 660-Pos Board B425 their metastatic ability, epithelial to mesenchymal transformation and its Sub-Microsecond-Scale Dynamics in the Type-1 Ryanodine Receptor reverse, mesenchymal to epithelial transformation, are required for Observed with CMOS-Integrated Electrophysiology cells to leave the tumor primary site, migrate and establish metastases at Andreas J.W. Hartel1, Peiji Ong1, Indra Schroeder2, Oliver Clarke3, secondary sites. Here we assess the heterogeneity in motility and Siddharth Shekar1, Hunter M. Giese3, Andrew R. Marks4, morphology of these cancer cells via time-lapse microscopy, automated Wayne A. Hendrickson4, Ken L. Shepard1. segmentation and tracking. We use CLaP as a way to select clones of 1Electrical Engineering, Columbia University, New York, NY, USA, proliferating cells based on morphological and behavioral characteristics, 2Membrane Biophysics Group, Dept. of Biology, Technische Universit€at to enrich cell cultures of specific phenotypes. We validate which of Darmstadt, Darmstadt, Germany, 3Biochemistry and Molecular Biophysics, these traits are preserved after cell division, and which ones are affected Columbia University, New York, NY, USA, 4Physiology and Cellular by exposure to different conditioned media promoting mesenchymal to Biophysics, Columbia University, New York, NY, USA. epithelial transformation. Conventional electronics typically limit the available temporal resolution for single ion-channel measurements, making fast channel gating events 663-Pos Board B428 (<10ms) undetectable. Our group recently developed a platform that allows Zlock, a Broadly Applicable Optogenetic Method, Controls Cofilin in high-bandwidth conductance measurements by directly interfacing a sus- Living Cells 1 1 1 2 2 pended membrane containing the ion-channel under study on a custom Orrin J. Stone , Neha Kaul , Hui Wang , Ved P. Sharma , Robert J. Eddy , John S. Condeelis2, Klaus M. Hahn1. complementary-oxide-semiconductor (CMOS) preamplifier chip. A passiv- 1 2 ation layer with a 20 mm aperture is deposited on the CMOS-chip surface UNC Chapel Hill, Carrboro, NC, USA, Anatomy and Structural Biology, and a small suspended lipid bilayer is formed within the aperture. This Albert Einstein College of Medicine, New York City, NY, USA. allows us to decrease the total capacitances at the input of the amplifier The spatial and temporal organization of protein activity controls the flow to less than 3pF, enabling us to measure high conducting ion-channels at of information through signaling networks. Optogenetics, the regulation of bandwidths up to 1MHz. Here we present single ion-channel conductance protein activity with light, has proven very useful to dissect the role of measurements of the skeletal muscle ryanodine receptor (RyR1) at up to spatio-temporal control by inducing specific protein activities with seconds 500kHz bandwidth. We observe microsecond current states which we asso- and microns resolution in living cells. However, current methods are not ciate with very fast channel gating events. Further analysis of the recorded applicable to many important proteins. Here we describe Zlock, a broadly data by extended beta-distribution-based analysis reveals closed-state applicable optogenetic method to control protein or small-peptide activity flickering with time scales as short as 50ns. New high-speed CMOS ampli- in living cells using modifications of Zdark (Zdk), a small protein that binds fiers provide a new tool for high-bandwidth studies of ion-channel dynamics only to the dark state of the LOV2 domain from Phototropin. The LOV2 in vitro. domain undergoes a reversible conformational change induced by 400- 500 nm light. Zdk, derived by screening a library of Z domain variants of 661-Pos Board B426 protein A, binds selectively to the dark state of LOV2 with dark Kd <100 Droplet Interface Bilayers as a Platform for a Spatially Segregated nanomolar, and lit Kd > 10 micromolar. In the ‘‘Zlock’’ system, Zdk and Nanoreactor LOV2 are fused to the N- and C- termini of a target protein or small- Maxwell P. Allen-Benton. peptide. In the dark, they bind to one another and cover or distort the active Chemistry, King’s College London, London, United Kingdom. site. Upon irradiation they release one another in less than a second, freeing Compartmentalisation is a defining feature of biological processes. Within the active site for target interaction. We have engineered Zdk for eukaryotic cells, organelles serve as segregated microenvironments for the reversible intramolecular binding needed in the Zlock design. We specific biochemical processes. These microenvironments allow distinct have successfully applied Zlock to generate photoactivatable analogs of chemical environments to be maintained within the cell and concentrate important cytoskeletal regulators including cofilin and Alpha Tubulin Ace- enzymes involved in catalytic cycles in the same space. This concentration tyltransferase 1 (ATAT). Photoactivation of cofilin and ATAT in live cells of enzymes and substrates within a small segregated environment contrib- controls cell protrusion and microtubule acetylation, and has demonstrated utes to the great efficiency of biochemical processes. I aim to investigate a clear role for cofilin in regulating directionality during cancer cell

BPJ 7740_7745 Sunday, February 12, 2017 135a migration. Current work is focused on ‘caging’ photoactivatable small- terize the temperature response of clock neurons in Drosophila to tem- peptide inhibitors of Cdc42, PP1, JNK and Gqa, to control endogenous pro- perature modulation. We show that a selective group of clock neurons teins with light. responds to temperature changes and that dorsal neurons (DNs) are excited by cooling and inhibited by warming. We further investigated Neuroscience: General, Computational, and the physiological input pathway of temperature sensing into the circadian Experimental Approaches and Tools I clock. We find that arista and chordotonal organs are both critical factors that contribute to the response of circadian neurons to tempe- 664-Pos Board B429 rature modulation. Our work reveals that clock neurons respond to Noise Induced Hearing Enhancement: Clinical and Machine Learning temperature changes through multiple temperature input pathways, suggest- Studies ing a complex network similar to the entrainment of circadian clocks by Kang-Hun Ahn, Woo Seok Lee. light input. Department of Physics, Chungnam National University, Daejeon, Korea, Republic of. 667-Pos Board B432 The addition of a certain amount of background noise has been shown to TRP Channel Function in iPSC-Derived Sensory Neurons improve the hearing of pure-tone sounds. The underlying mechanism of Laura Vangeel. this counter-intuitive phenomenon is believed to be stochastic resonance, KULeuven, Leuven, Belgium. where the threshold level of a pure-tone signal is lowered by adding an Using somatic cells to generate induced pluripotent stem cells (iPSC) is an appropriate amount of background noise. Here, to investigate whether back- established method in research and has multiple applications and advan- ground noise can aid the hearing of more complex sound such as the human tages. An increasing amount of cell types have been successfully differenti- voice, we perform hearing tests with Korean syllables, and find that syllable ated from iPSCs, including hematopoietic cells, cardiomyocytes, smooth recognition is enhanced with noise for rarely used syllables. Similar tests muscle cells, pancreas, liver and renal tissue. Directing differentiation into with a machine-learning speech recognition shows that the same enhance- neuronal cells has the great benefit of bypassing the problematic isolation ment arises only when the system is insufficiently trained, corresponding of human neuronal cells. Recently, a protocol using dual SMAD inhibition to hearing a rare syllable. The overall phenomenon looks similar to the was shown to drive differentiation into sensory neuron-like cells. While stochastic resonance but any successful model should explain that the expression of canonical markers of sensory neurons has already been vali- enhancement arises in perceptual processes as it depends on the level of dated, an in-depth characterization of sensory TRP channels in these training and the syllables. induced neurons is still lacking. In this study, we use qPCR, Fura-2-based microfluorimetry and patch-clamp experiments to evaluate the expression 665-Pos Board B430 and function of the sensory TRP channels at different time points during Stress-Induced Differential Regulation Leads to Decoupling of the Activity the differentiation toward a sensory neuron phenotype. We not only confirm between mPFC and Amygdala expression of the sensory channels TRPV1, TRPM8 and TRPA1, but also Mohammed Mostafizur Rahman1,2, Sumantra Chattarji1,2. demonstrate for the first time strong molecular and functional expression 1Centre for Brain Development and Repair, Institute for Stem Cell Biology of TRPM3 in iPSC-derived sensory neurons. Interestingly, we found a and Regenerative Medicine, Bangalore, India, 2National Centre for temporarily increase of TRPM3 responses at an early time point in differen- Biological Sciences, Bangalore, India. tiation, which might indicate a role for this channel in the development of Exposure to severe stress leads to the development of psychiatric disorders. sensory neurons. To conclude, we were able to produce sensory neurons us- Clinical studies have shown that three brain areas involved in learning and ing iPSCs, and validated functional expression of TRP channels important in memory—the hippocampus, amygdala and medial prefrontal cortex somatosensation. This approach has the potential to investigate the develop- (mPFC)—undergo distinct changes with stress disorders. While the hippo- ment of sensory neurons in vitro, and to explore the cellular physiology and campus and mPFC show impairment in structural and functional changes, pharmacology of TRPM3 and other sensory TRP channels in a human the amygdala shows an enhancement. Despite these three brain regions context. Moreover, it may open the door to generate patient-derived neurons having strong anatomical connections, most of these studies focus on indi- for disease modeling and target validation. vidual brain regions. However, recent studies have shown that these connec- tions between regions have strong functional implications. The connectivity 668-Pos Board B433 between the mPFC and the amygdala has recently been shown to be crucial Effect of Spatial Complexity on Dopaminergic Signaling Revealed from for fear expression (Likhtik et al., 2014). The effect of stress on the func- Multiscale Simulations tional connections between these regions is poorly understood. Therefore, Cihan Kaya1, Mary H. Cheng1, Ethan R. Block2, Alexander Sorkin2, we performed in-vivo local field potential recordings from the mPFC and James R. Faeder1, Ivet Bahar1. the amygdala in awake behaving rats during fear expression. We found 1Computational and Systems Biology, University of Pittsburgh, Pittsburgh, that stress differentially regulates the activity in the mPFC and the amygdala PA, USA, 2Cell Biology, University of Pittsburgh, Pittsburgh, PA, USA. during fear expression. Consistent with cellular findings, the activity in the Efficient clearance of neurotransmitters from the synapse by dopamine amygdala is upregulated by stress during fear expression. However, the ac- transporters (DATs) is critical to regulating dopamine (DA) signaling tivity of the mPFC is unaffected by stress during fear expression. We also in the central nervous system. Despite significant advances in the field, found that stress causes a decoupling between the activity in the amygdala we still lack a complete mechanistic understanding of DA transport events. and mPFC. Interestingly, an earlier study showed that stress strengthens the First it is still unclear how molecular structure and dynamics affect coupling between the hippocampus and the amygdala (Ghosh et al., 2013). cellular neurosignaling events. Second, the structural and stochastic proper- Therefore, although chronic stress impairs structure and function in both the ties of the cellular environment, including the morphology of the synaptic hippocampus and mPFC, the interactions of these two areas and the amyg- regions, and the heterogeneous distribution of transporters on the cell dala appear to be affected in a contrasting fashion. Functional connectivity membrane may affect the efficiency of neurotransmitter transport, and no gets stronger from amygdala to hippocampus but it gets disrupted between realistic simulations of the dopaminergic signaling has been carried out to mPFC and amygdala. Future studies need to focus on mechanisms involved date. We adopted a multiscale methodology to examine the effects of in these connectivity changes. spatial complexity and firing patterns on DA reuptake by DATs. We used a kinetic scheme derived from our earlier molecular, together with high 666-Pos Board B431 resolution images dopaminergic neurons from fluorescence spectroscopy Temperature Sensation and Integration in the Drosophila Circadian Clock and electron microscopy, to reconstruct in silico the simulation environment Chang Jiang, Swathi Yadlapalli, Andrew Bahle, Pramod Reddy, mediating DA signaling. Overall, our model provides a framework to Edgar Meyhofer, Orie Shafer. investigate the effect of variations in different neuronal properties to University of Michigan, Ann Arbor, MI, USA. gain a better understanding of the modulation of DA signaling in the central Circadian clocks are entrained by zeitgebers, environmental cues such as nervous system. Our results highlight the significance of considering light and temperature that adapt living organisms to the physical rhythms the realistic geometry as well as the spatial heterogeneities from ex- of the earth. Although temperature has been shown to be a major zeitgeber periments as opposed to adopting well-mixed assumptions. The and can entrain the circadian clock of Drosophila, the neural and molecular computing platform also permits us for the first time to gain a quantitative mechanisms by which circadian clocks respond to temperature remain understanding of the effect of psychostimulants and antidepressants on DA poorly understood. In our work, we use in vivo calcium imaging to charac- signaling.

BPJ 7740_7745 136a Sunday, February 12, 2017

669-Pos Board B434 mediating synaptic transmission. According to EOD-linked whole-fish O2 Sensitivity and Robustness in an Axon Guidance Signaling System consumption (for fish with EODs between ~300-500 Hz) ATP demand per Brendan A. Bicknell1, Peter Dayan2, Geoffrey J. Goodhill1. AP grows exponentially with frequency. The unimodal task, continual firing, 1Queensland Brain Institute, University of Queensland, Brisbane, Australia, and simple homogeneous structure of the EO render it especially suitable for 2Gatsby Computational Neuroscience Unit, University College London, probing excitable system energetics in relation to molecular, cellular and London, United Kingdom. tissue features. We develop a model, Epm, to depict currents at the electro- Correct functioning of the brain requires the orderly wiring of billions of cells cyte’s energy-dissipating Excitable posterior membrane. In situ, 3Na/2K- during development. A major mechanism that mediates this is the guidance of ATPases (pumps) counteract the dissipation of electrocyte ENa (and EK). axons to their targets by extracellular chemical gradients. Although well stud- Using Epm we calculate AP frequency-dependent ‘‘Naþ-entry budgets’’ for ied, the computational rules by which even simple guidance decisions are synaptic activation (pulsatile and/or steady-state, with/without noise). Com- made are not yet understood at a biophysical level. The task of a growing parison of Epm-calculated ATP consumption (inferred from total Naþ-entry) axon to estimate a direction signal is immense, as unavoidable biological against published EOD-linked whole-fish O2 consumption suggests that noise corrupts the measurement at all levels of processing. Here, we show EOD-linked energy demands external to electrocytes (neuronal, circulatory how the modulation of growth by a balance of positive and negative feedback etc) exceed, several-fold, those of electrocyte excitability per se. can explain the remarkable chemotactic sensitivity observed in vitro.We Well-understood conductance processes (as modeled by Epm) proved fully perform a detailed analysis of the experimental data of ref. [1] which charac- adequate for generating sustained APs (including during jamming avoidance terizes the neurite growth of ~2500 rat dorsal root ganglia explants in very responses) from 200-600 Hz. By contrast, although we computationally shallow gradients of nerve growth factor. Constrained by these data, we impose the equivalent of fast stimulatory variations in [ACh], even at the construct a model chemical signaling system for growth and guidance in bottom of this frequency range the means by which synaptic transmission the developing brain. In the model, amplification of the gradient signal occurs machinery so reliably achieves the requisite fast variations is a mystery. via paracrine signalling between cell bodies within the ganglion, while robust- The simple Eigenmannia EO therefore continues to emerge as a fascinating ness is conferred by the dynamics of receptor trafficking. The model gives a model system for studying not only the energetics but the subcellular and unified and quantitative account of experimentally observed behavior, and broader-level dynamics of high frequency excitability. yields testable predictions with implications for understanding brain develop- ment and repair after injury. 672-Pos Board B437 [1] Mortimer, D. et al (2009) A Bayesian model predicts the response of axons Analyzing and Modeling the Dysfunction of Inhibitory Neurons in to molecular gradients. Proc. Natl. Acad. Sci. U.S.A, 106(25), 10296-10301. Alzheimer’s Disease Carlos M. Perez1, Ghanim Ullah1, Jokubas Ziburkus2. 670-Pos Board B435 1Physics, University of South Florida, Tampa, FL, USA, 2Biology Mathematical Modeling and Analyses of Interspike-Intervals of Sponta- and Biochemistry, University of Houston, Houston, TX, USA. neous Activity in Afferent Neurons of the Zebrafish Lateral-Line Alzheimer’s disease (AD) is characterized by the abnormal proteolytic pro- Sangmin Song1, Ji Ah Lee2, Ilya Kiselev1, Varun Iyengar1, Josef G. Trapani1, cessing of amyloid precursor protein, resulting in increased production of a Nessy Tania2. self-aggregating form of beta amyloid (Ab). Several lines of work on AD pa- 1Biology, Amherst College, Amherst, MA, USA, 2Mathematics and tients and transgenic mice with high Ab levels exhibit altered rhythmicity, Statistics, Smith College, Northampton, MA, USA. aberrant neuronal network activity, reflected in clusters of hyperactive neu- Temporal patterns of spontaneous activity may vary between sensory systems rons, network hyperexcitability, and spontaneous epileptic activity. Recent such as the auditory, vestibular, and lateral line systems due to differences in studies highlight that abnormal accumulation of Ab changes intrinsic proper- physiology at the level of hair cells. In the absence of stimuli, hair cells ties of inhibitory neurons, including their inability to reliably produce action display spontaneous synaptic vesicle fusion and neurotransmitter release, potentials, one of the main reasons underlying the impaired network activity. which lead to action potential (spike) generation in innervating afferent neu- Specific cellular mechanisms leading to the interneuron dysfunction are not rons. As a result, features of synaptic transmission and the innervation of hair completely understood. Using extended Hodgkin-Huxley (HH) formalism cells will affect the timing of spontaneous spike trains. We analyzed sponta- model in conjunction with patch-clamp experiments, we investigated the neous spiking recorded from the lateral line of zebrafish, and found that the mechanisms leading to the impaired activity of interneurons. Our detailed distribution of interspike-intervals (ISI) had an ‘‘L-shape’’ that decayed faster analysis indicates that increased conductance of Naþ leak channels recreates and had a wider tail than a typical exponential distribution commonly several observed inhibitory neuron behaviors in models of AD, including observed in other models of spike timing. Additionally, successive ISI’s in failure to reliably produce action potentials, smaller action potential ampli- the lateral line recordings tended to have positive serial correlation, i.e., suc- tude, increased resting membrane potential, and higher membrane depolariza- cessive ISI pairs were either short/short or long/long. This pattern contrasts tion in response to a range of stimuli in a model of APP-swe/PSEN1DeltaE9 previous findings from the auditory system where ISI’s tended to have nega- (APdE9) AD mice. Increasing the conductance of hyper-polarizationactivated tive serial correlation presumably due to the effects of synaptic depletion. cyclic nucleotide-gated (HCN) ion channels (Ih) could account for most of the Computational models of spike trains that included the calcium-dependency reported dysfunctions. However, the extent of changes required to reproduce of neurotransmitter release at the ribbon synapse of hair cells were able to these observations render such changes unphysiological. We investigate and generate ISI distributions consistent with those we acquired experimentally. discuss other pathways that would lead to some but not all observations within These simulations suggested that fluctuations in total calcium channel activity, HH formalism. including both the number and cooperativity of channels in the population, are a primary contributor to serial correlations in hair-cell evoked spike trains. 673-Pos Board B438 Given the difference in innervation pattern between auditory and vestibular/ Denied Access of Ions and Molecules to Axons in the Development of lateral line hair cells, we further modeled the effects of single versus multiple Alzheimer’s Disease synapses on the temporal patterns of spontaneous spike trains. Altogether, our Wade Dauberman, Samuel Breit, Shaohua Xu. findings provide evidence for how physiological similarities and differences Biology, Florida Institute of Technology, Melbourne, FL, USA. between the auditory, vestibular, and lateral line systems can account for Self-assembly of amyloid-beta peptide and the formation of fibers and differences in spontaneous activity. Furthermore, our computational methods plaques in extracellular space of the brain is a characteristic of Alzheimer’s allow for future characterization of mechanisms that underlie spontaneous ac- disease. Whether and how fibers and plaques cause neurodegeneration tivity in these different sensory systems. remains unclear. Recently, we reported that amyloid fibers can further aggregate and form gels. Gels are known to eliminate bulk flow which is 671-Pos Board B436 required for the circulation of ions and molecules essential for neuronal A Model for Assessing ATP Demands of Sustained High Frequency Firing function. To understand how restriction of ions or molecules affects Bela Joos1, Michael R. Markham2, John E. Lewis3, Catherine E. Morris4. neurons, we examined the gels’ role in the propagation of nerve fibers’ 1Physics, University of Ottawa, Ottawa, ON, Canada, 2Biology, University of action potential. Unlike amyloid fibers, fibrin, which forms gels fast, within Oklahoma, Norman, OK, USA, 3Biology, University of Ottawa, Ottawa, ON, a couple of minutes, was used to simulate amyloid fiber gels. We found a Canada, 4OHRI, Ottawa, ON, Canada. 40% reduction of the compound action potential when fibrin gel was The continuous electric organ discharge (EOD) of the weakly electric fish, formed outside frog sciatic nerve fiber epineuriums, a porous membrane Eigenmannia, reflects action potentials (APs) fired by the EO’s muscle- wrapping around axon bundles. A 70% reduction of the action potential derived electrocytes. EODs enable electrosensing and communication. AP fre- was recorded when fibrin was formed inside the epineurium. Fibrin encap- quency is neurally controlled, with acetylcholine-gated channels (AChRs) sulating individual axons was verified under a confocal microscope when

BPJ 7740_7745 Sunday, February 12, 2017 137a

fluorescein-labeled fibrinogen was injected. Gelation also changes viscosity, aromatic stacking of the fullerene hexagonal rings with the Phe rings, as well which affects the diffusion of ions and molecules. An approximate 1.42-fold as the outstanding biocompatibility. Furthermore, we found that the bare increase of microviscosity during fibrin gelation was revealed using a carbon nanotube could dissociate the preformed fibrils to some extent. molecular rotor. Glycerol caused a concentration dependent reduction of Our results provide novel clues for studying interaction in amyloid/carbon- the compound action potential. These results imply that the formation of material system and seeking amyloidosis inhibitors with carbon nanomateri- amyloid plaques surrounding neuronal processes initially disrupt the propa- als, which may have important potentials in the development of drug gation of action potential and then trigger the cascade of events leading to candidates against Alzheimer’s disease. neuronal death seen in Alzheimer’s disease. Denied access of the neurites to nutrients, signaling molecules, and/or waste drainage pathways might 676-Pos Board B441 be responsible for the plaque’s effect on the action potential and underlie Ultrasound-Enhanced Molecular Therapy for Axon Neurogenesis the pathogenesis of Alzheimer’s disease. The methods introduced in the pa- Asis Lopez, Damir Khismatullin. per for the study of gelation’s effect on action potential may have broad Tulane University, New Orleans, LA, USA. applications. Between 28M - 55M individuals experience spinal cord injuries (SCIs). In the U.S. alone, this amounts to an estimated 180k-230k individuals. In the central 674-Pos Board B439 nervous system (CNS) after an injury to a nerve, the environment prevents the Biodistribution of Insulin-Nanogels in Mouse: A Preliminary Study for the healing/regrowth of neurons and myelin, because myelin inhibitors are Treatment of Alzheimer’s Disease present, growth factor is not re-expressed, and glial tissue scars very quickly. 1 2 3 Daniela Giacomazza , Pasquale Picone , Laura Ditta , Knowing that nerves need to be regenerated quickly, research has been done 3 4 1 M. Antonietta Sabatino , Valeria Militello , P. Luigi San Biagio , to discover an optimal treatment for nerve regeneration. Unfortunately, no 2 2 5 5 Laura Cristaldi , Domenico Nuzzo , Antonella Amato , Flavia Mule , effective method currently exists to stimulate nerve fiber regeneration (i.e., Clelia Dispenza3, Marta Di Carlo2. 1 2 3 neurogenesis). To address this gap, we propose a non-invasive method to IBF, CNR, Palermo, Italy, IBIM, CNR, Palermo, Italy, DICGM, stimulate neurogenesis through low-intensity focused or unfocused ultra- University of Palermo, Palermo, Italy, 4DCF, University of Palermo, 5 sound irradiation. This method is new and has high potential as a transforma- Palermo, Italy, STEBICEF, University of Palermo, Palermo, Italy. tive rehabilitation approach for people with disabilities due to SCIs. A A growing body of evidence shows that Insulin, Insulin Receptor (IR) and IR combination of therapeutic methods that mitigate the inhibitory microenvi- signaling are involved in brain cognitive functions and their dysfunction is ronment at the site of the injury and restore the regeneration potential of implicated in Alzheimer’s disease (AD) neurodegeneration. Thus, adminis- corticospinal axons are required for achieving effective functional recovery tration of insulin could be a strategy for AD treatment. For this aim we after SCI. We developed an in vitro system integrating focused ultrasound have designed, synthesized and characterized a nanogel system (NG) that (FUS) with neuron axon dynamics for neurogenesis and an approach using has been conjugated to insulin molecules (NG-In) to deliver the protein a 3D microenvironment. Dorsal root ganglion (DRG) neurons are used for into the brain, as a tool for the development of a new therapy against AD. our studies and microsurgically removed at day 15 and E18 Sprague Dawley In our preclinical study in mice, intraperitoneal injection of fluorescent- cortical neurons from BrainBits, LLC. To determine if stimulation of axon labeled NG has allowed to determine the biodistribution of NG vs time in growth is occuring, we measure density and growth (distance) using phase the whole body and its clearance through the kidneys and bladder. Further- contrast imaging and a custom MATLAB code for image analysis. We tested more, we have observed that mice injected with nanogel did not experience a variety of operating parameters to identify optimal conditions that induce stress, discomfort, nor mortalities have been recorded during the observation axon neurogenesis by ultrasound. We demonstrated that ultrasound can stim- time. Thus, we may conclude that, under our experimental conditions, nano- ulate DRGs. gels did not cause any toxic effects and they are eliminated in urine. The administration of NG-In through the intranasal route to study its brain distri- bution has been done by fluorescence analysis and Western blot. Data have Molecular Dynamics I shown that insulin signaling is improved in the different brain areas when the protein is conjugated with nanogels with respect to the free insulin. In 677-Pos Board B442 addition, the histopathological analysis of the nasal cavity shows no signifi- Asymmetric Membrane Models for the PM And TGN of Yeast, An cant change in epithelial cell lining indicating that the nano-solution is well All-Atom Molecular Dynamics Study tolerated in mouse. These results indicate that the synthesized NG-In was a Viviana Monje-Galvan, Jeffery B. Klauda. suitable carrier for insulin delivery in the brain having a higher efficiency Chemical and Biomolecular Engineering, University of Maryland, College than free-insulin. Park, MD, USA. We previously examined symmetric membrane models for the plasma (PM) 675-Pos Board B440 and trans-Golgi-network (TGN) membranes of Saccharomyces cerevisiae Studies of the Interaction between Ab Peptides and Carbon Nano- (yeast) (Biochem 54:6852-6861). Although diverse in lipid nature and re- Materials flecting sterol compositions characteristic to those organelles, our models Dongdong Lin, Ruxi Qi, Luogang Xie, Shujie Li, Guanghong Wei, were not an accurate enough representation as they lacked sphingolipids Xinju Yang. (SM), relevant for membrane structure and dynamics. Once SM were State Key Laboratory of Surface Physics and Physics Department, Fudan parametrized for the CHARMM 36 lipid force field (BJ, 107:134-145), University, Shanghai, China. we included them in the new models and added lipid composition asymme- The aggregation of amyloid-b peptides (Ab) is considered as one of the try to better reflect the complexity of these bilayers. The new models mainly possible causes of the Alzheimer’s diseases (AD). How to suppress include inositol phosphoceramide (IPC) and mannose-(inositol-P)2-ceramide the formation of toxic Ab aggregates has been intensively concerned over M(IP)2C lipids, both present in the non-cytosolic leaflets of the PM and the past several decades. Increasing evidence shows that whether carbon TGN. We present a comparison study between the previous PM model nano-materials could suppress or promote the aggregation depend on their and the more complex ones using common membrane properties such as physicochemical properties. However, as amyloid fibrillization is a complex surface area per lipid, bilayer thickness, deuterium order parameters, lipid process, their interaction dynamics remains elusive. In this presentation, we diffusion constants, and lipid cluster formation. The last three properties have investigated the interaction between Ab(16-22) peptides/full-length will serve to investigate lipid phase coexistence and leaflet coupling in Ab peptides and three kinds of carbon nanomaterials (carbon nanotube, membrane order and diffusion. Our simulation trajectories were at least fullerene, and graphene) by utilizing atomic force microscopy (AFM), elec- 2ms long for each of the three models (old-PM, PM, and TGN), for a total trostatic force microscopy (EFM), Thioflavin T (ThT) fluorescence, com- of 15 ms of simulation data run in the Anton machine at the Pittsburgh bined with molecular dynamic (MD) simulations. Our experimental results Supercomputing Center. demonstrate that all of the three carbon nano-materials could suppress the process of Ab fibrillation. EFM and ThT fluorescence results further 678-Pos Board B443 confirmed that the Ab peptides are strongly absorbed on the surface of carbon PI(4,5)P2 Binds to Phospholipase C Delta 1 in a Cholesterol Concentration nano-materials by forming non-fibrillar aggregates. At the molecular scale, it Dependent Manner: Perspective on Implications to PI(4,5)P2-Binding is found that carbon nano-materials dramatically inhibit the formation of Proteins ordered b-sheet-rich structures as the strong hydrophobic and aromatic stack- Sami Rissanen1, Lauri Salmela1, Ilpo Vattulainen1,2, Tomasz Ro´g1,2. ing interactions between carbon nano-materials and Ab peptides. In compar- 1Tampere University of Technology, Tampere, Finland, 2University of ison, the fullerenes exhibit the most excellent inhibitory ability by the strong Helsinki, Helsinki, Finland.

BPJ 7740_7745 138a Sunday, February 12, 2017

Phosphoinositide, a phosphorylated form of phosphatidylinositol (PI), plays an 681-Pos Board B446 important role in, e.g., cell signaling and membrane trafficking. Phosphatidyli- Beyond Lateral Pressure Profiles: Local Stress and the Traction Vector in nositol 4,5-bisphosphate (PI(4,5)P2) found in, for instance, the plasma mem- MD Simulations brane and the Golgi apparatus is known to bind to a number of proteins. Juan M. Vanegas1, Alejandro Torres-Sanchez2, Marino Arroyo2. 1 2 How the PI(4,5)P2-binding proteins recognize their substrate and how the bind- Dept. of Physics, University of Vermont, Burlington, VT, USA, Dept. of ing is regulated and fine-tuned remains unclear. In this work, we hypothesize Applied Mathematics III, Universitat Polite`cnica de Catalunya- that the increasing cholesterol concentration along a path from the nucleus to- BarcelonaTech, Barcelona, Spain. wards the plasma membrane may be one of the key regulators in this process. Lateral pressure or stress profiles are routinely used to understand the me- To consider this hypothesis, we employed both atomistic and coarse-grained chanical behavior of lipid membranes from molecular dynamics (MD) sim- (CG) molecular dynamics simulations to study the binding of PI(4,5)P2 with ulations. However, the 1-dimensional stress profiles are not adequate to phospholipase C delta 1 (PLC) that is an important signaling enzyme known understand the multidimensional mechanical state of complex systems to bind PI(4,5)P2. Unbiased CG MARTINI simulations in POPC/PI(4,5)P2 where there are membrane-protein and protein-protein interactions. Further- and POPC/PI(4,5)P2/cholesterol mixtures were complemented by umbrella more, the fact that the microscopic stress from MD simulations is not sampling simulations to measure the free energy of PI(4,5)P2 binding to uniquely defined is a theoretical consideration that is most often ignored, PLC. The final structures of the PLC-PI(4,5)P2 complexes were fine-grained which has acute practical consequences when atomistic models are consid- to atomistic detail and simulated over microseconds to explore the details of ered. I will present our recent work (Phys. Rev. Lett. 114, 258102, 2015 and the binding process. J. Chem. Theory Comput. 10, 691, 2014) on the development of objective In essence, we observed deeper free energy minima for the binding of 3D local stress calculations and its applications to lipid bilayers, mechano- PI(4,5)P2 to PLC in the cholesterol-rich membrane system, compared to sensitive channels, and fibrous proteins. I will show how popular definitions cholesterol-free membranes, thus supporting the underlying hypothesis of the microscopic stress violate mechanical equilibrium and present an that PI(4,5)P2 bindingtoPLCandpossiblyalsootherPI(4,5)P2-binding unambiguous and physically sound definition based on the central decompo- proteins is strengthened by increasing cholesterol concentration. The sition of forces from multi-body potentials. I will also demonstrate the use atom-scale features such as hydrogen bonding patterns and lipid-specific in- of the traction vector as a powerful tool to visualize the local stress tensor on teractions that dictate the binding preference are discussed in detail in the interfacial surfaces (such as the surface of a protein embedded in a presented work. membrane). 679-Pos Board B444 682-Pos Board B447 STRD MARTINI: Simulating Quasi-2D Hydrodynamics with Chemically Molecular Dynamics Study on Lipid Wrapped Carbon Nanotube as an Detailed Lipid Models Artificial Membrane Channel Andrew Zgorski, Edward Lyman. Moon-Ki Choi, Hyunki Kim, Youngjin Kim, Kyunghoon Kim, Moonki Kim. Physics and Astronomy, University of Delaware, Newark, DE, USA. Mechanical engineering, SungKyunKwan University, Suwon, Korea, Hydrodynamic interactions are an important component of lipid membrane Republic of. dynamics. A proper molecular dynamics simulation of lateral diffusion in a Carbon nanotube (CNT) is an ideal membrane channel because of its hydropho- membrane requires coupling to hydrodynamic flows in the surrounding sol- bic inner pore and prominent transport properties like typical biological vent. In the low Reynolds number limit, the interactions mediated by these channels. These CNT porins can transport water, small ions, and DNA. flows are long-ranged. Camley, et al recently generalized the Saffman- CNT-membrane interaction simulation shows that CNTs having a length Delbruck result to periodic boundary conditions, and showed that hydrody- similar to the thickness of lipid bilayer can be inserted into membrane and namic interactions introduce significant error into lateral diffusion of lipids sustain angle almost perpendicular to membrane. Much longer ones can also and proteins in typical molecular dynamics simulations. The system sizes be inserted into membrane but they are buried inside hydrophobic region of required to overcome the finite size effect are computationally prohibitive membrane (1). However, recent experimental research elucidated that longer when using explicit solvent models in both all-atom and coarse-grained CNTs wrapped by lipids can be inserted into lipid-bilayer and maintain their simulation. Implicit solvent models offer a potential solution, but entirely angle within 15 degrees (2). neglect hydrodynamic momentum transport. To remedy this, we have sup- In this study, we performed molecular dynamics simulation for investigating plemented the Dry MARTINI implicit solvent model with an efficient meso- interaction between lipids wrapped CNT and lipid-bilayer. Martini coarse- scopic particle-based hydrodynamic model called Stochastic Rotation grained force field was used to reduce computational cost significantly. Dynamics (SRD). Our implementation allows for fine control over fluid Also, wrapping process was analyzed by using the coarse-grained carbon properties of the solvent, such as viscosity and Reynolds number, and in- nanotube (CG-CNT) and the 1,2-dioleoyl-sn-glycero-3-phosphocholine cludes a thermostat for the solvent that produces canonical energy fluctua- (DOPC) in various CNT length and surface adsorption density, respectively. tions without interfering with the mesoscopic hydrodynamic flows. The Furthermore, we performed insertion simulation to see how these properties approach, dubbed ‘‘STRD Martini’’, has been implemented in an in-house affect insertion process and finally compared CG-CNT’s insertion angles version of Gromacs v 5.1. with experimental data. As a result, CNT insertion takes longer time in gen- eral as the density of wrapped lipid increases. Also, we realized that CNT can 680-Pos Board B445 maintain its insertion angle almost perpendicular to lipid-bilayer at a certain Molecular Views of a Eukaryotic Plasma Membrane Model critical density. Karelia H. Delgado-Magnero, Gurpreet Singh, Valentina Corradi, Finally, its insertion mechanism is fully discussed with respect to interaction D. Peter Tieleman. energy between the wrapped CG-CNT and lipid membrane. Biological Science, University of Calgary, Calgary, AB, Canada. (1) Lelimousin, M., and M. S. P. Sansom. 2013. Small 9:3639-3646. Plasma membranes are composed of hundreds of lipid species and different (2) Geng, J., K. Kim, J. F. et al. 2014. Nature 514:612-þ. types of membrane proteins. Lipid-lipid and lipid-protein interactions play a crucial role in regulating cell membranes functions and are involved in 683-Pos Board B448 many diseases when altered. Despite advances in experimental methods, Molecular Dynamics Studies Support Elevator Type Transport Mecha- the interplay between lipids and proteins is not completely understood. In nisms in the Glucose EIIC Superfamily Transporters this context, computer simulations have become a powerful tool to investi- Jumin Lee1, Zhenning Ren2, Ming Zhou2, Wonpil Im1. gate the dynamic behavior of plasma membrane components and their inter- 1Lehigh University, Bethlehem, PA, USA, 2Baylor College of Medicine, actions at a near atomic resolution. The goal of our research is to work Houston, TX, USA. towards large-scale modeling of plasma membranes using computer simula- The phosphoenolpyruvate-dependent phosphotransferase system (PTS) pro- tions. In this study, we placed ten different types of eukaryotic membrane vides a distinct sugar transportation pathway in bacteria. Because PTS does proteins in a membrane model containing various lipid species found in not exist in eukaryotes, it is considered a novel target for antibiotics develop- the plasma membrane of eukaryotic cells. In our simulation setup, mem- ment. Among the components of PTS, the enzyme IIC (EIIC), a membrane brane proteins are present in different ratios, for a total of ca. 150 protein transporter protein, is particularly important since it transports a sugar through molecules, embedded in more than 60,600 lipids. The protein-membrane the bacterial cell membrane. In 2011, Cao et al. first determined the crystal system was prepared using the Martini force field, with in house software. structure of chitobiose-specific EIIC (bcChbC) in an inward-facing occluded Using molecular dynamic simulations, we investigate the local lipid envi- state. In addition, we have recently determined the crystal structure of ronment around proteins, and provide a molecular view of a realistic mem- maltose-specific EIIC (bcMalT) in an outward-facing occluded state. The brane model. crystal structures suggest an ‘‘Elevator’’ type transportation mechanism, but

BPJ 7740_7745 Sunday, February 12, 2017 139a the accurate mechanism of how EIIC transports a sugar has not been clearly mutant, pathway disruption was observed, thereby explaining its resistance demonstrated yet. To investigate more detailed and accurate transportation to certain antibiotics. The conductance calculated from the simulations mechanisms, we performed collective variable-based steered molecular agreed very well with measured conductances. Other successful examples dynamics (CVSMD) simulations. Our simulation shows the spontaneous of CompEL include the discovery of an unexpected ionic pathway in the transportation of the sugar toward the opposite side of the membrane with anti-microbial peptide dermcidin as well as the identification of a novel con- the preserved H-bonding interactions, supporting the ‘‘Elevator’’ type trans- duction mechanism in the potassium channel KcsA. We finally investigated port mechanism. A set of cross-linking experiments was carried out on the the conductance and the gating mechanism of pentameric ligand-gated ion basis of the CVSMD model structures, and we were able to acquire a few channels such as GLIC. We constructed a reaction coordinate from two of stable cross-linked structures. crystal structures, which have been proposed to represent the open and closed states of the channel, respectively. Constraining the pore to specific 684-Pos Board B449 positions on that reaction coordinate allowed us to sample the conductivity Molecular Basis of GLUT4 in Glucose Transport: Atomistic Molecular at these positions. CompEL confirms that the assumedly open structure is Dynamics Study indeed conducting and cation-selective (with conductances in the pS range, 1 2 3 1,3 Chetan S. Poojari , Job Roodhuizen , Fabio Lolicato , Tomasz Ro´g , comparable to published experimental values), whereas the closed structure 1,3 Ilpo Vattulainen . is indeed non-conducting. Moreover, conductance values steadily increase 1Department of Physics, Tampere University of Technology, Tampere, 2 along the closed-to-open transition and are correlated with the amount of Finland, Biomedical Engineering department, Eindhoven University of water in the pore. Technology, Eindhoven, Netherlands, 3Department of Physics, University of Helsinki, Helsinki, Finland. 686-Pos Board B451 The glucose transporter 4 (GLUT4) is one of the most important glucose Modeling Membrane Associated Proteins through Neutron Reflectivity transporter proteins for the absorption of glucose from the plasma circula- Augmented Molecular Dynamics tion after a meal or during exercise. It is present in skeletal muscle, adipose Bradley W. Treece1, Mathias Loesche1, Frank Heinrich1, tissue cells, cardiac muscle, and it has also been found in brain cells. It plays Arvind Ramanathan2. a significant role in the development of various diseases such as type 2 1Physics, Carnegie Mellon University, Pittsburgh, PA, USA, 2Computational diabetes, cancer, and cardiac diseases. Inside a cell, GLUT4 is transported Science, Oak Ridge National Laboratory, Oak Ridge, TN, USA. towards the cell membrane upon an insulin stimulus, leading to a 10- to The structural characterization of membrane-associated proteins on fluid 40-fold increase in the glucose uptake. In spite of its importance, the molec- lipid bilayers remains a challenge to most modern biophysical techniques. ular mechanism of glucose transport by GLUT4 is still not clear. There is no Neutron reflectivity (NR) has emerged as a method that provides sub- crystal structure available for the GLUT4 protein either. However, using nanometer resolution of proteins in a functional lipid environment. Interpre- existing structural information of the other solved glucose transporter tation of NR data gives an envelope structure related to the distribution of structures, we have modeled and validated the GLUT4 structure in protein density along the membrane normal direction and further refinement three conformations: the outward-open, outward-occluded, and inward- using structural information may yield a full atomistic description of the open conformation. The GLUT4 models with glucose bound were subse- protein-membrane complex. However, for flexible or intrinsically disor- quently embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine dered protein domains, such information is often not available or multiple (POPC) membrane and simulated over microsecond time scales using conformational states may contribute to the average density profile as unbiased atomistic molecular dynamics simulations. In addition, we also resolved by NR. Thus, characterization of such systems requires more elab- carried out random acceleration molecular dynamics simulations to explore orate approaches. We previously demonstrated that molecular dynamics all possible pathways for glucose transport across the membrane. Our (MD) simulations can provide a full atomistic interpretation of NR results simulation studies revealed specific GLUT4 residues that captured the in cases where only partial internal protein structure is available, but such conformational changes in GLUT4 structure on glucose binding. Overall, simulations are often plagued by long equilibration times. Here we present the study provides atomistic level structural information on glucose a procedure to steer MD simulations toward configurations that reproduce transport and also provides one with ideas for the development of therapeu- experimental NR results. Biasing potentials are calculated through a com- tic agents blocking the function of GLUT4. This development work is parison of the one-dimensional densities from NR data with the evolving important given that cancer cells express elevated levels of glucose density profile derived from the MD trajectory at each time step. This results transporter proteins and depend on increased glucose uptake for in steering forces that direct molecular conformations of the protein on the proliferation. bilayer toward the experimental results. Steering becomes weaker as the density profiles match more closely, disappearing entirely for matched den- 685-Pos Board B450 sities. The structure is guided toward the desired configuration, rather than Ions in Action - Studying Ion Channels by Computational Electrophysi- rigidly confined to the experimental density. Here we show the application ology in GROMACS of our method to model peptide and small protein systems, also discussing 1 1 2 Carsten Kutzner , R. Thomas Ullmann , Bert L. de Groot , the efficiency of the procedure and potential merits and pitfalls in its 3 1 Ulrich Zachariae , Helmut Grubmueller . application. 1Theoretical and Computational Biophysics, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany, 2Computational Biomolecular 687-Pos Board B452 Dynamics, Max Planck Institute for Biophysical Chemistry, Goettingen, Characterization of Apolipoprotein Mimetic Peptides on Nascent High Germany, 3Physics, University of Dundee, School of Science and Density Lipoproteins Engineering, Dundee, United Kingdom. Mohsen Pourmousa, Rafique Islam, Denis Sviridov, Scott Gordon, Ion channels play a fundamental role in maintaining vital electrochemical B. Scott Perrin, Jr., John Stonik, Alan T. Remaley, Richard W. Pastor. gradients across the cell membrane and in enabling electrical signaling in National Institutes of Health, Bethesda, MD, USA. cells. Understanding their functional mechanism is crucial for facilitating A nascent high-density lipoprotein (HDL) is a discoidal bilayer composed of drug design on this important class of membrane proteins. Key characteris- phospholipids, cholesterols, and two apolipoproteins that form a scaffold tics of ion channel function that are commonly quantified experimentally that stabilizes the assembly. Apolipoprotein mimetic peptides are short are ionic permeation rates and selectivities. The Computational Electro- synthetic peptides that share features of apolipoproteins and have potential physiology (CompEL) protocol allows the investigation of ion channels in therapeutic value based on their ability to form and stabilize nascent HDL. GROMACS all-atom molecular dynamics simulations. By employing an A key question in designing mimetic peptides is why some are more ion/water exchange protocol in a double-membrane simulation setup, a efficient than others. We characterize the properties of four mimetic pep- steady state with a continuous flow of ions through the channels is tides rich in the amino acids E, L, and K (called ELK peptides), using a achieved. The recorded ion permeation events directly reveal conductance, combination of Molecular Dynamics simulations and experimental tech- selectivity, and rectification behavior, which all allow a straightforward niques. Experiments show that the hydrophobic and neutral ELKs have a comparison to experimental quantities. In addition, the ionic pathways significantly higher ability to form nascent HDLs than the positive or nega- through the channels are readily revealed. CompEL is available in tive peptides. An in silico model of a discoidal bilayer was developed by GROMACS and has a negligible impact on the simulation performance introducing a water slab perpendicular to the bilayer head group surface, even in parallel. leading to acyl chains of edges exposed to water. Simulations on this model Recent application to the bacterial channel PorB resulted in conductance and discoidal bilayer with peptides indicate that hydrophobic and neutral ELKs selectivity values and revealed the pathways of traversing ions. For a stabilize the assembly by forming scaffolds at the edges in a picket fence

BPJ 7740_7745 140a Sunday, February 12, 2017 arrangement; the adjacent peptides are held together by strong salt-bridges. two different things with an ordinary microscope. One of the drawback of such In contrast, the positive and negative ELKs diffuse to the head group a technique is the long preparation made of several stages, i.e. immunostaining, surface, do not form effective scaffolds, and fail to stabilize the discoidal gelation, digestion and expansion. They are really crucial steps for a good im- assembly. Hence, the simulations provide a structural rational for the exper- aging post-expansion. imental observations, and provide an avenue for computer based design of In our work we present a comparison between ExM and stimulated emission apolipoprotein mimetic peptides. depletion (STED) nanoscopy(3). Our aim is to study the e possible combination of STED and ExM as a method to further enhance the final resolution achiev- 688-Pos Board B453 able. We will in particularly take advantage of the use of separation of photons Computational and Experimental Studies of Gold Nanoparticle Templated by lifetime tuning (SPLIT) STED (4). HDL-Like Nanoparticles for Cholesterol Metabolism We show application of these methods from single fixed cells to slices of fixed 1 2 2 2 Cheng-Tsung Lai , Wangqiang Sun , Rohun U. Palekar , C. Shad Thaxton , mouse retina tissue. 1 George C. Schatz . We are also interested in applying the approach to high-density compartments 1Department of Chemistry, Northwestern University, Evanston, IL, USA, 2 like the cell nucleus to decipher the high-order structure organization of chro- Department of Urology, Northwestern University, Chicago, IL, USA. matin-DNA. High-density lipoprotein (HDL) is involved in the transport and metabolism 1. Chen, F., P.W. Tillberg, and E.S. Boyden. 2015. Optical imaging. Expansion of phospholipids, triglycerides, and cholesterol. Mimics of HDL are being microscopy. Science. 347: 543-548. explored as potential therapeutic agents for removing excess cholesterol 2. Chozinski, T.J., A.R. Halpern, H. Okawa, H.-J. Kim, G.J. Tremel, R.O.L. from arterial plaques. Gold nanoparticles (AuNPs) functionalized Wong, and J.C. Vaughan. 2016. Expansion microscopy with conventional with apolipoprotein A-I (apoA-I), and with the lipids 1,2-dipalmitoyl- antibodies and fluorescent proteins. Nat Meth. 13: 485-488. sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3- 3. Bianchini, P., C. Peres, M. Oneto, S. Galiani, G. Vicidomini, and A. Diaspro. phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (PDP PE) have 2015. STED nanoscopy: a glimpse into the future. Cell Tissue Res. 360: been demonstrated to be acceptors of cellular cholesterol. However, detailed 143-150. structural properties of this functionalized HDL AuNP are not well under- 4. Lanzano, L., I. Coto Hernandez, M. Castello, E. Gratton, A. Diaspro, and stood. We combined coarse-grained and all-atom MD simulations to simu- G. Vicidomini. 2015. Encoding and decoding spatio-temporal information late the self-assembly of lipids and cholesteryl ester on the AuNP/apoA-I for super-resolution microscopy. Nature Communications. 6: 6701. construct to gain insights into its structural properties, and further make comparisons with experimental results. We find that lipids are oriented 691-Pos Board B456 differently in regions with and without apoA-I. We also find that in this Quantitative Microstructure Analysis of Casein Network Dynamics using functionalized HDL AuNP, the distribution of cholesteryl ester maintains STED Microscopy with Relation to Macroscopic Gel Properties a reverse concentration gradient that is similar to the gradient found in Zachary J. Glover, Adam Cohen Simonsen, Jonathan Brewer. native HDL. Incubating the HDL AuNP with cholesterol and lecithin: MEMPHYS, FKF, University of Southern Denmark, Odense, Denmark. cholesterol acyltransferase (LCAT) demonstrate the HDL AuNP is able to Casein proteins are the main structural element in many fermented dairy efficiently activate LCAT and form esterified cholesterol from free choles- products. Understanding how protein functionality is affected during mod- terol and phospholipid. ern food processing, such as filtration and spray drying, is key to product and process optimization. Advances in Super Resolution Microscopy have 689-Pos Board B454 provided novel tools to investigate protein microstructures. Herein, Stimu- High-Throughput Thermodynamics of Drug-Membrane Interactions from lated Emission Depletion (STED) Microscopy has been used to image Multiscale Simulations casein micelle aggregation, gel formation and network coarsening over Roberto Menichetti, Kiran Kanekal, Kurt Kremer, Tristan Bereau. time. Protein structures have been resolved to > 100 nm. Native skim Theory Group, Max Planck Institute for Polymer Research, Mainz, Germany. milk and reconstituted skim milk powder were used to produce gelled sys- The number of small organic molecules is overwhelmingly large–so large, tems. Gelation was induced either through rennet addition and acidification that most of it remains unexplored. Computer simulations offer an appealing with GDL. Images have quantitatively analyzed to monitor the development framework to probe many of these compounds without the need to synthe- of casein network over time, and the fractal dimensions of the different sys- size them in the laboratory. The main hurdles preventing a high-throughput tems have been determined. Label free Coherent Anti-Stokes Raman Spec- characterization of many small molecules relies on the time investment to troscopy (CARS) Microscopy was used to verify that the presence of parametrize the force field—a process that typically requires significant hu- fluorophore did not interfere with the gelation process during STED micro- man intervention—and extensive sampling requirements. We address these scopy. Image data has been directly correlated with dynamic texture issues by first sampling from the coarse-grained Martini model, for which analysis. we developed an automated parametrization protocol for small molecules. We demonstrate the ability to resolve casein micelle structures on the The resulting potential-of-mean-force (PMF) curves for the insertion of scale of a single micelle, and extract valuable information that directly small molecules in lipid membranes show excellent agreement for a number relates to the macroscopic properties of a gelled system. This work pro- of benchmark cases. Our framework allows us to run high-throughput mo- vides the basis for the use of STED microscopy to analyze other colloidal lecular dynamics simulations and estimate tens of thousands of relative structures. We present a framework to extract meaningful information free energies of different small molecules in a lipid bilayer. They provide that directly relates to the macroscopic properties and functionality of useful data to explore structure-property relationships from computer the system. simulations. Optical Microscopy and Super-resolution 692-Pos Board B457 A Novel STED Microscope with Nanometer Axial Sectioning Imaging: Novel Approaches and Analysis I Iva´n Coto Herna´ndez1, Siddharth Sivankutty2, Nicolas Bourg3, Sandrine Le´cart3, Guillaume Dupuis3, Sandrine Le´veˆque-Fort1. 690-Pos Board B455 1Univ. Paris-Sud, Universite´ Paris-Saclay, CNRS, Institut des Sciences Combining Expansion Microscopy and STED Nanoscopy for the Study of Mole´culaires d’Orsay (ISMO), Paris, France, 2Aix-Marseille Universite´, Cellular Organization CNRS, Institut Fresnel, Marseille, France, 3Universite´ Paris-Sud, Centre de Isotta Cainero1,2, Michele Oneto1,2, Luca Pesce1,2, Giulia Zanini1,2, Photonique BioMe´dicale (CPBM), Paris, France. Luca Lanzano`1,2, Alberto Diaspro1,2, Paolo Bianchini1,2. The axial resolution of conventional Stimulated emission depletion (STED) 1Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy, 2University of microscopy is limited by diffraction to about 500 nm. Overcoming this lim- Genoa, Genoa, Italy. itation usually comes at the cost of complex instrumentation, as 3D confine- Expansion microscopy (ExM) is a novel method that allows super-resolution ment usually implies to combine two STED beams. By trying to combine imaging with conventional microscopes(1, 2). It consists in soaking the cells with TIRF excitation this decrease the lateral performances. In this work with a polymer, inducing the polymerization to form a dense meshwork we reported a straightforward approach to restrict the axial extension of throughout the cell, cross-linking the fluorophores to the polymer and, after the STED microscope by only modifying the detection path, thus keeping digestion of cellular protein, rehydrating of the sample. The swelling of optimal lateral resolution. This new membrane imaging takes advantage the polymer gel led to a fourfold isotropic stretching of the sample. Therefore, of the supercritical angle (SAF) emission of fluorophore that allows one it increases the distance between two objects that otherwise couldnot be seen as to discriminate their position regarding the glass coverslip. Indeed only

BPJ 7740_7745 Sunday, February 12, 2017 141a when fluorophore are closed to the interface, their evanescent near field can structures at spatial resolutions up to an order of magnitude better than pre- become propagative and appears above the critical angle. This SAF emis- vious optical techniques allowed. These localization microscopy techniques sion represents up to 50% of the emission collected by the objective for a work by acquiring the positions of single fluorophore molecules with fluorophore at the interface. By filtering out of the undercritical emission diverse methods of varying complexity, but they all share the requirement in a conjugated plane of the back focal plane of the objective lens, only that in order to create quality images, the cellular structures being studied the SAF emission is detected. It allows nanometric axial sectioning of fluo- they must be labeled at quite high densities. Such dense labeling is not rescent emitters close to the water-glass interface; simply by adding a SAF always experimentally achievable nor desirable, most especially for live module in the detection path of all STED microscopes. This STED-SAF im- cell imaging studies. However, it is still possible to learn much about the plementation reduces the complexity and cost of its early implementation underlying structures in more sparsely labeled samples using correlative and as side effect, the photobleaching and light dose sent to the sample is analysis. In this study, we use an information theory framework to represent reduced since not extra STED beam is needed for isotropic resolution. In the process of building a localization microscopy image and develop a this work we show for the first time the coupling of STED and SAF micro- ‘‘completeness index’’ for the resulting images that aids in image scopy and we highlight the benefits of this implementation by imaging quality assessment. We develop a test with human observers to identify fluorescent beads and sub-cellular structures. The image quality of raw the baseline range of completeness index values required for identification data is improved by using deconvolution techniques. Finally the specific of features commonly found in fluorescence images of cellular structures. importance of the method towards the implementation of Tomography- We then use simulated and real localization microscopy datasets to deter- STED microscopy is discussed. mine how to maximize the information gained from correlation analysis of datasets with low completeness and determine guidelines to avoid introducing data processing artifacts into the data. We also confirm the 693-Pos Board B458 application of bootstrapping methods to estimate the uncertainty of the Well-Characterised Time-Gated Detector Photon Flux Resolves the measurements taken from correlations with these sparsely labeled datasets. Ultrastructure of DNA-Damage Nuclear Bodies with G-STED Nanoscopy Finally, we apply the analysis an example of TIRF-based localization Kok-Lung Chan1, Esther Garcia Gonzalez2, Sergi Padilla-Parra3, 2 imaging; the distribution of actin-modifying proteins around the site of Jorge Bernardino de la Serna . HIV-entry into a T-cell. 1Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom, 2Central Laser Facility, Science and Technology Facilities Council, Harwell-Oxford, Didcot, United Kingdom, 3The Wellcome Trust 695-Pos Board B460 Centre for Human Genetics, University of Oxford, Oxford, United Kingdom. Super-Resolution Imaging of Organelle Membrane Contact Sites via Widely spread commercial STED nanoscopes, and low-cost custom-built Far-Red Hyperspectral Localization Microscopy systems are generally equipped with continuous-wave STED lasers Adriano Vissa1, Maximiliano Giuliani2, Peter K. Kim3, Christopher M. Yip2. (cwSTED) combined with pulsed excitation lasers (peLaser), and time- 1BIochemistry, University of Toronto, Toronto, ON, Canada, 2IBBME, gated detection. This modality, termed gated-STED (gSTED), permits using University of Toronto, Toronto, ON, Canada, 3Biochemistry, University of lower cwSTED powers reducing background noise, at the expense of low Toronto, Toronto, ON, Canada. intensity images. gSTED critically depends on the photon-counting detec- The communication and exchange of material between subcellular organ- tors sensitivity, and photon-flux optimization to the temporal gate. The com- elles is critical to many cellular processes. Areas of close apposition mercial system employed in this work utilise an electronic trigger-box to between organelles, termed membrane contact sites (MCS), have recently synchronise the peLaser with time-gate hybrid detectors (gHyD). Unfortu- become a focus of study for intracellular communication. Through enrich- nately, commercial gSTED set-ups do not provide an accurate detection ment of specific proteins and protein complexes, these sites are implicated temporal gate characterization. Hence, users blindly select the time-gated in the exchange of lipids and metabolites such as calcium. As MCS window with none a priori knowledge of neither the shape, nor the delay characteristically display inter-membrane distances of 10-30 nm, electron relative to the peWLL. As a result, often the acquisition of sharp, highly- microscopy is often used to visualize these phenomena. However, the resolved images turns into seemingly random events. Here, we report how molecular specificity of the proteins involved in MCS is of great interest. to measure the gate shape in our gSTED microscope. Our gate starts at In order to address these questions more closely, we have developed a 2.5ns and last 4.5ns when exciting at 488nm, depleting at 592nm, and hyperspectral localization microscopy technique to spectrally distinguish with both notch filters. The effective gate shape is highly affected by the far-red fluorophores using a Versachrome thin film tunable filter (Sem- fluorescence emission and detection, as well as the optical elements in the rock) integrated into the emission path of an Olympus IX-83 dual deck in- pathway. For instance, neither a detection window from 500nm and above verted TIRF microscope. Using this hyperspectral 3D Direct Stochastic 560nm, nor short lifetime dyes yield sharp, highly-resolved images. Further- Optical Reconstruction Microscopy (dSTORM) imaging approach, we more, a thorough study on how different peLaser and cwSTED power ratios, were able to spectrally and spatially distinguish between Alexa 647 and and different time-gates impact the lateral resolution and image signal-to- Alexa 680 labels in our studies of the nanoscale spatial distribution of noise allowed generating resolution maps showing a priori knowledge to ER and peroxisome membranes under WT and MCS knockdown condi- best gSTED performance. Our method allowed resolving the nuclear bodies tions and the clustering of MCS proteins at key sites of membrane (NB) ultrastructure, a previously unresolved sub-nuclear DNA assembly in apposition. human cells. They spontaneously appear in ~10% of G1/early S phase cells, and it is suggested to be DNA damage sheltering centre for transgenera- tional DNA lesions originated in the previous S phase. Our method revealed 696-Pos Board B461 the NB organisation in high detail, showing that NB contains multiple Towards Quantitative High-Throughput 3D Localization Microscopy chromatin looped structures in the periphery. To gain further details of Joran Deschamps, Markus Mund, Jonas Ries. the NB inner core, we extended the chromatin by pre-extracting cells prior Cell biology and biophysics, EMBL, Heidelberg, Germany. to staining. Compared to the previous condition, we observe chromatin fibre Single-molecule localization microscopy (SMLM) has become a widely bundles of approximate 15-40nm diameter, and can now resolve a higher- used technique to observe cellular structures at the scale of tens of nanome- order chromatin organisation reminiscent of a molecular plait-like sub-struc- ters. However, major bottlenecks are still faced and limit the scope of appli- ture. In conclusion, a well-characterised time-gate, and cwSTED/peWLL cations. Here we present new technologies to overcome three of the major power ratio in commercial gSTED nanoscopes allows resolving the NB limitations of SMLM in throughput, quantitative counting of proteins and ultra-structure with a consistent resolution (within ~15nm) and possibly 3D resolution. Indeed, SMLM is an intrinsically slow method with acquisi- other biologically-relevant structures. tion and data analysis times in the minute to hour range. This severely limits the possibility to acquire a sufficient number of experiments in quantitative studies. To nevertheless achieve high throughput in SMLM, we automated 694-Pos Board B459 the entire workflow of data acquisition and analysis. This enabled us to Localization Microscopy Theory and Practice for Data Analysis on continuously acquire new experiments over the course of days and analyze Sparsely Labeled Samples them in real time. Another limitation concerns the inhomogeneous photo- Brian T. DeVree, Sarah L. Veatch. dynamics of molecules across the field of view, which is a consequence Biophysics, University of Michigan, Ann Arbor, MI, USA. of the Gaussian intensity profile in microscopes using lasers for excitation. In the last several years, the development of myriad localization-based Such inhomogeneity impedes accurate protein counting. To tackle this issue, fluorescence microscopy techniques has enabled the study of cellular we developed a homogeneous illumination scheme with the help of a

BPJ 7740_7745 142a Sunday, February 12, 2017 multimode fiber and a speckle reducer. This simple illumination achieves a in biology is to decipher the principles of organization of what can be con- very high uniformity and results in homogeneous measured localization pre- sidered, by analogy with human-created libraries, the cell’s primary unit of cisions across the field of view, removing statistical bias from quantitative information storage and retrieval. In this respect, the development of super- studies. Finally, we present the use of adaptive optics to improve the 3D res- resolved fluorescence microscopy has provided a new toolbox to peer into olution of supercritical angle localization microscopy (SALM), with the aim the nucleus. For instance, super-resolution microscopy has been recently to reach isotropic localization precision in the first few hundreds of nanome- applied to the investigation of nanoscale chromatin architecture, revealing ters above the coverslip. Altogether, these improvements open the way to nucleosome higher-order organization into heterogeneous ‘clutches’ and better sampled and more precise structural studies in superresolution epigenetically dependent folding motives [Ricci M et al, Cell, (2015); microscopy. Boettiger A et al, Nature (2016)]. However, dynamic properties of proteins in the nucleus are also critical for their function [Misteli T, Cell (2007)]. Fluorescence Correlation Spectroscopy 697-Pos Board B462 (FCS) has been used to map the dynamics of several proteins in the nucleus Versatile Super-Resolution Calibration Standard for Quantifying Protein with diffraction-limited spatial resolution. Here we combine a novel STED- Copy Number based super-resolution method [Lanzano’ L et al, Nat Commun (2015)] Francesca Cella Zanacchi1, Carlo Manzo2, Angel Sandoval Alvarez3, with FCS to measure protein dynamics in the nucleus with an improved Nathan D. Derr4, Maria Garcia Parajo2, Melike Lakadamyali1. spatial resolution of about 100 nm. We sample several positions within the 1AFIB group, ICFO-Institut de Ciencies Fotoniques, Barcelona, Spain, nucleus by performing line scanning. The measured spatial and temporal 2SMB group, ICFO-Institut de Ciencies Fotoniques, Barcelona, Spain, heterogeneity of the dynamics, quantified by a recently introduced algorithm 3ICFO-Institut de Ciencies Fotoniques, Barcelona, Spain, 4Smith College, [Scipioni L et al, Biophys J (2016)], is discussed in relation to nuclear Northampton, MD, USA. organization. Single molecule based super-resolution microscopy offers a unique opportu- nity for quantifying protein copy numbers with nanoscale resolution [1,2]. 699-Pos Board B464 While fluorescent proteins have been extensively characterized for quantita- Analysis of Fibrous Spatial Point Patterns from Single-Molecule Super- tive imaging using calibration standards, similar calibration tools for small Resolution Microscopy Data organic fluorophores used in conjunction with immunofluorescence are Ruby Peters, Dylan Owen, Juliette Griffie. lacking. King’s College London, London, United Kingdom. Within this framework, the development of methods able to access a precise Unlike conventional microscopy methods which generate pixelated molecular counting of protein copy numbers is essential, clearing the way to images, SMLM techniques produce images comprising of a list of molec- address several biological questions using super-resolution techniques such ular coordinates - a spatial point pattern (SPP). SMLM methods therefore as stochastic optical reconstruction microscopy (STORM). necessitate a statistical approach to data analysis. SPPs are routinely The development of a suitable calibration method represents the best analyzed using robust cluster analysis algorithms to quantify molecular way to address the challenges of molecular counting using super- clustering across diverse biological systems. The analysis of fibrous resolution [3,4]. Within this project, we demonstrate that DNA origami SPPs however, such as those derived from components of the cyto- in combination with GFP antibodies is a versatile platform for quantifying skeleton, remains relatively understudied. Here, we present statistical protein copy number in immunofluorescence based super-resolution micro- methodology based upon a variant of Ripley’s K function to quanti- scopy. We show that this calibration method, besides quantifying the tatively assess fibrous patterns generated by SMLM. We demonstrate average protein copy number in a cell, allows determining the abundance the technique using various simulated fibrous spatial arrangements of various oligomeric states. Furthermore, we apply this calibration method and extract spatial descriptors of the pointillist input. We further demon- to quantify nucleoporins (NUP107) [5] and molecular motors (dynein inter- strate the technique on experimental data acquired using the image recon- mediate chain) [6] in vivo. Overall, we provide a versatile strategy for struction by integrating exchangeable single-molecule localization (IRIS) quantifying a large number of proteins of interest using various labeling approach to SMLM. We quantitatively assess the fibrous distribution of approaches. filamentous actin at the T cell immunological synapse, whose structure 1. Durisic, N., et al., Single-molecule evaluation of fluorescent protein photo- has been shown to be important for cell morphology, polarization and activation efficiency using an in vivo nanotemplate. Nat Methods, 2014. 11(2): activation. p. 156-62. 2. Ulbrich, M.H. and E.Y. Isacoff, Subunit counting in membrane-bound pro- teins. Nat Methods, 2007. 4(4): p. 319-21. 700-Pos Board B465 3. Schmied, J.J., et al., DNA origami-based standards for quantitative fluores- Expanding the Spectral Resolution of Single-Molecule Localization cence microscopy. Nat Protoc, 2014. 9(6): p. 1367-91. Microscopy with Bodipy-Based Photoswitchable Fluorophores 4. Jungmann R. et al. Quantitative superresolution imaging with qPAINT Amy M. Bittel, Ashley Davis, Tao Huang, Xiaolin Nan, Summer L. Gibbs. Nature methods doi:10.1038/nmeth.3804 (2016) Biomedical Engineering, Oregon Health & Science University, Portland, 5. Szymborska, A., et al., Nuclear Pore Scaffold Structure Analyzed by Super- OR, USA. Resolution Microscopy and Particle Averaging. Science, 2013. 341(6146): Single-molecule localization microscopy (SMLM) has become an impor- p. 655-658. tant tool for studying molecular biology. Through the use of photoswitch- 6. Derr, N.D., et al., Tug-of-war in motor protein ensembles revealed able fluorophores, SMLM can accurately localize individual molecules with a programmable DNA origami scaffold. Science, 2012. 338(6107): with 10-20 nm resolution, an order of magnitude better than conventional p. 662-5. fluorescence microscopy. While SMLM succeeds in locating individual molecules, it is limited to 4-color emission based imaging due to the standard bandpass filter technology used to generate multicolor images, 698-Pos Board B463 restricting the number of molecular entities that can be simultaneously Heterogeneity of the Nuclear Environment Investigated by Superresolu- localized in a single sample. The development of multi-spectral super- tion Microscopy and Fluorescence Correlation Spectroscopy resolution microscopy (MSSRM) improves the spectral resolution of Luca Lanzano01, Melody Di Bona1,2, Lorenzo Scipioni1,3, SMLM enabling up to 20 color imaging in a single sample. However, Maria J. Sarmento1, Enrico Gratton4, Giuseppe Vicidomini5, MSSRM is currently restricted by the spectrally limited conventional Alberto Diaspro1,2. fluorophores with adequate photoswitching for SMLM. Herein, we de- 1Nanoscopy, Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy, signed, synthesized and validated a series of novel BODIPY-based fluoro- 2Department of Physics, University of Genoa, Genoa, Italy, 3Department of phores with appropriate photoswitching for SMLM that span the visible Computer Science, Bioengineering, Robotics and Systems Engineering, spectrum permitting high-resolution, multi-color images using our MSSRM. University of Genoa, Genoa, Italy, 4Biomedical Engineering, University of The BODIPY-based probes were selected from a 110 member library California, Irvine, CA, USA, 5Molecular Spectroscopy and Microscopy, of compounds synthesized through modification of a core BODIPY FL Nanophysics, Istituto Italiano di Tecnologia, Genoa, Italy. scaffold with diverse aromatic rings using a solid phase synthetic plat- Genomes are more than one-dimensional entities purely defined by their form. All BODIPY-based probes were characterized for absorption and linear DNA sequences [Misteli T, Cell (2007)]. A long standing challenge emission properties as well as key photoswitching properties to facilitate

BPJ 7740_7745 Sunday, February 12, 2017 143a selection of the ideal probes for SMLM and MSSRM. Through labeling via DGS-NTA(Ni), representing a system with adjustable surface densities known cellular structures with selected BODIPY-based probes, we and diffusion characteristics. Our data suggests the existence of several demonstrated that multiple fluorophores could be accurately resolved prolonged bright and dark states resulting in off times up to several seconds. when excited by the same laser line due to varied length Stokes shifts. Only about 66% of PS-CFP2 molecules were identified in a single image. The developed BODIPY-based probes for SMLM will be beneficial in Merging localisations from consecutive images increased this fraction to advancing understanding of cellular spatial organization by enabling 77%. Although the maximum number of localisations per PS-CFP2 was the localization of an increased number of molecular compounds greater than 10 in only ~3% of the cases, the influence of these molecules simultaneously. on the resulting image is rigorous. Including this blinking statistics in simula- tions and evaluation using popular clustering algorithms, such as Ripley’s K function, showed no differences between randomly distributed data containing 701-Pos Board B466 blinks versus clustered non-blinking data. We conclude, that SMLM methods DNA-Paint Imaging of DNA-Origami Rings Mimicking Biologically possess limitations for investigating protein clusters. Merging can be used for Relevant Structures the correction of suboptimal bleaching, however, for considering blinking of Arunima Chaudhuri1, Yang Yang1, Kenny Kwok Hin Chung1, 1 2,3 1 1 fluorophores, this correction is - by the nature of the imaging method itself - Zhao Zhang , Fredric Pincet , Shyam Krishnakumar , Chenxiang Lin , inherently limited. Appropriate ways to tackle the investigation of possible James E. Rothman1, David Baddeley1. 1 clustering include varying the labelling density and multi-color colocalization Department of Cell Biology, School of Medicine, Yale University; techniques. Nanobiology Institute, School of Medicine, Yale University, New haven, CT, USA, 2Department of Cell Biology, School of Medicine, Yale University, New Haven, CT 06520; Nanobiology Institute, School of Medicine, Yale 703-Pos Board B468 University, New Haven, CT 06520, New haven, CT, USA, 3Laboratoire de A Quantitative Platform for Super-Resolution Microscopy Imaging Physique Statistique, Ecole Normale Supe´rieure, Paris Sciences et Lettres Ottavia Golfetto1, Devin L. Wakefield1, Eliedonna E. Cacao1, Research University; Laboratoire de Physique Statistique, Universite´ Paris Kendra N. Avery1, Raphael Jorand1, Steven J. Tobin1, Ronald M. Clinton1, Diderot Sorbonne Paris Cite´; Laboratoire de Physique Statistique, Sorbonne Jennifer Gutierrez1, Yuelong Ma1, Daniel Ganjali2, Athanasios Sideris2, Universite´s, Paris, France. David A. Horne1, John C. Williams1, Tijana Jovanovic-Talisman1. Single-molecule localization microscopy methods such as DNA-PAINT 1Molecular Medicine, Beckman Research Institute-City of Hope, Duarte, CA, (point accumulation for imaging in nanoscale topography), utilize successive USA, 2Mechanical and Aerospace Engineering, University of California, localizations of single molecules over time to reveal of biological structures Irvine, Irvine, CA, USA. with photon-limited resolution1,2. However, visualization of sub-diffracted Pointillistic super-resolution microscopy techniques provide nanometer discrete bimolecular assemblies still remains a challenge. ‘‘DNA origami’’ scale spatial resolution and single molecule sensitivity. Thus, they are excel- has emerged as powerful nano-templating technique capable of generating sta- lent tools for probing the lateral organization of molecules, especially in ble self-assembled scaffold for positioning of molecular species of interest biological environments. Additional information on such organization can with nanometer precision3,4. Recent studies of DNA-PAINT have focused be obtained by subjecting super-resolution data to quantitative analyses. on imaging different molecular species, such as fluorophores and proteins, These powerful approaches are able to quantify the extent to which mole- positioned on synthetic grid-like DNA nanostructures1,4. These grids are cules localize into clusters and the size/occupancy of clusters. However, well suited for determining the resolution limit of sparsely labelled points quantitative super-resolution approaches typically encounter two major (e.g. one fluorophore per 5x5 nm2). However, biological structures often challenges. First, it is difficult to determine optimum imaging parameters have a much higher local density and complex structure, making imaging and characterize, under these conditions, the fluorescent probe’s biophysical more challenging. In this study, as a proof-of-concept, we explored imaging properties. Second, it is challenging to identify efficient labeling strategies of circular ‘‘ring-like’’ DNA-origami nanostructures of dimension mimicking for detecting endogenous proteins, particularly those that yield stoichio- small bio-molecular structures (25-50 nm inner diameter), like membrane re- metric and site specific labeling. To efficiently characterize fluorescent ceptors, ion channels etc. Using a TIRF-based imaging technique, we have probes, we have developed an easily implementable nano-biology assay. optimized the fluorophores density and its positioning on DNA-origami rings Using chemistry compatible with super-resolution imaging (i.e. producing to reconstruct ‘‘doughnut’’ shaped images and have been able to resolve inner minimal background signal), proteins were covalently immobilized to diameter of ~25 to 50 nm. We have carried out these analyses on glass sur- glass surfaces with specific orientations. Interrogating such surfaces re- faces and also on supported bilayers. Our study utilizing DNA rings provides vealed important features of both optical highlighter proteins (paGFP challenging targets for DNA-PAINT technology and underscores the use of and pamCherry1) and proteins labeled with organic fluorophores. To size-templated nanostructures, as approximates for bio-molecular complexes, efficiently label endogenous receptors, we leveraged a unique cyclic to optimize imaging and analysis protocols independent of biological sample peptide called meditope, which binds within the Fab framework of engi- preparation. neered monoclonal antibodies. A fluorescent meditope was complexed 1. Dai et al. (2016) Nat. Nanotechnol. 11:798-807. with the engineered trastuzumab Fab. This complex was used to image 2. Baddeley, D. (2015) Nat. Methods 12:1019-1020. both SK-BR-3 and BT-474 cells, where we delineated the distribution of 3. Yang et al. (2016) Nat. Chem. 8:476-483. epidermal growth factor receptor 2 (HER2). According to our results, the 4. Schlichthaerle et al. (2016) Curr. Opin. Biotechnol. 39: 41-47. majority of HER2 receptors were monomers as expected. However, a fraction of receptors were organized as oligomers, and more significantly in BT-474 cells that are oncogene-addicted. This demonstrates that our 702-Pos Board B467 method is capable of detecting subtle differences in endogenous receptor Characterization of PS-CFP2 for Reliable Super-Resolution Microscopy organization and thus offers a powerful platform for quantitative super- 1 2 2 € 1 Benedikt Rossboth , Rene Platzer , Johannes Huppa , Gerhard Schutz , resolution imaging. Mario Brameshuber1. 1Institute of Applied Physics, TU Wien, Vienna, Austria, 2Institute for Hygiene and Applied Immunology, Medical University of Vienna, Vienna, 704-Pos Board B469 Austria. Estimating the PSF from Single Molecule Data Single molecule localization microscopy (SMLM) methods constitute an Kenny K.H. Chung, David Baddeley. elegant way to infer information about cellular organization at high resolution, Cell Biology Department/Nanobiology Institute, Yale University, New e.g. distinguished structures such as the cytoskeleton or the nuclear pore com- Haven, CT, USA. plex. Hence, their application to identify (nano-)clustering on the plasma A common method of extracting 3D information in localization microscopy is membrane, which is widely acknowledged to be a common feature of many to use PSF engineering techniques where depth information is encoded in the proteins, is well reasoned. Identifying those, however, ultimately converges PSF shape. These techniques are very sensitive to aberrations and poor estima- to quantitation of SMLM images. Overcounting artefacts caused by multiple tion of the experimental PSF will not only reduce localization precision but also observations of the same fluorophore molecules due to blinking or incomplete distort the reconstructed structure (e.g. by introducing a z-dependent lateral bleaching can be easily introduced. In this study we aimed at characterizing shift). Sub-resolution beads are typically used as a PSF reference, but are an the blinking behaviour of the commonly used fluorescent protein, PS-CFP2. inexact match for a single fluorophore in terms of size and spectral properties, Recombinant, his-tagged PS-CFP2 was anchored to supported lipid bilayers and fail to capture sample-induced aberrations.

BPJ 7740_7745 144a Sunday, February 12, 2017

We have established a method which eliminates the need for an external 707-Pos Board B472 PSF reference and directly estimates the experimental PSF from single Methods to Eliminate Localization Bias and Reduce Localization Error in molecule data collected during a localization trial. Although individual sin- Localization Microscopy Datasets with Non-Uniform Background Fluo- gle molecule data have poor signal-to-noise, we can employ a PSF model rescence parameterized in terms of Zernike polynomials and ensemble fitting to esti- Thomas Shaw1, Sarah L. Veatch2. mate the PSF. For each choice of PSF parameters, we perform a 3D fit to all 1Applied Physics, University of Michigan, Ann Arbor, MI, USA, the single molecules in our training set and use the summed residuals as a 2Biophysics, University of Michigan, Ann Arbor, MI, USA. figure of merit. As Zernike modes are nearly independent, we were able Single molecule localization microscopy has become a standard tool for to perform fitting of each Zernike mode sequentially, drastically reducing nanoscale imaging of biological systems. However, improper treatment of the computation required. non-uniform background fluorescence can degrade image accuracy and We have validated this method on both simulated data and bead-derived PSF precision. In particular this can be problematic in cases where the structure data, with either an astigmatic or biplane 3D model. On experimental STORM being imaged is correlated in space with high background fluorescence. data, a small subset of single molecule images was sufficient for PSF extrac- Various methods have already been implemented for correction of tion. The use of a PSF extracted directly from the single molecule data led to background fluorescence. Here, we demonstrate that these background an overall improvement in z precision for the complete data set. Although estimation and correction procedures lead to both systematic and random er- here we have demonstrated a post-acquisition approach, further speed improve- ror in emitter localization, under experimentally relevant background condi- ments may allow for on-the-fly extraction to provide feedback for adaptive tions. Part of these errors result from background estimation procedures, and optics. part from fitting procedures that incorrectly model (or fail to model) the presence of background. We find that background estimation procedures from the literature may overestimate local background in the presence of 705-Pos Board B470 a high density of foreground fluorophores, and show implications of this Robust Nonparametric Descriptors for Clustering Quantification in bias for systematic localization errors when these estimates are used for Single-Molecule Localization Microscopy background correction before or during emitter fitting. We have developed 1 2 1 Shenghang Jiang , Sai Divya Challapalli , Yong Wang . a novel estimator of background fluorescence that uses selective temporal 1Department of Physics, University of Arkansas, Fayetteville, AR, USA, 2 averaging to reduce this bias. We further show that standard background Microelectronics and Photonics Graduate Program, University of Arkansas, correction procedures for emitter fitting produce experimentally relevant Fayetteville, AR, USA. systematic and random localization errors, even when the background is Single-molecule localization microscopy (SMLM) has been utilized broadly exactly known. We implement two new background-corrected fitting algo- in imaging biological molecules in various biological systems, allowing rithms that have no systematic error and less random error than standard quantitative analyses on the spatial organizations and patterns of these mol- procedures. We use simulations to compare the statistical performance of ecules. However, parameters are needed in many of the currently available these methods to that of earlier methods, and demonstrate improvements methods or algorithms, likely introducing subjective bias in the analyses. in images of biological systems. Here, we report a robust nonparametric descriptor, J’(r) , for quantifying the spatial organization of molecules in single-molecule localization micro- 708-Pos Board B473 scopy. J’(r) , based on nearest neighbor distribution functions, does not The Characterization of Cellulose Nanostructure using Super-Resolution require any parameter as an input for analyzing point patterns. We show Fluorescence Microscopy that it displays a valley shape in the presence of clusters of molecules, Mouhanad Babi. and the characteristics of the valley reliably report the clustering features McMaster University, Hamilton, ON, Canada. in the data. More importantly, the position of the J’(r) valley (rJ’m) depends Cellulose - a major and critical component of plant cell walls - constitutes exclusively on the density of clustering molecules. Therefore, it is ideal for the largest component of Earth’s biomass and is an attractive raw material direct measurements of clustering density of molecules in single-molecule to exploit in the production of biodegradable and renewable products, localization microscopy. such as biocomposites, biofuels and other biomaterials. Manufacturing these products often entails the chemical or biochemical depolymerization of cellulose, a process that is limited by its crystalline structure. To better un- 706-Pos Board B471 derstand these manufacturing processes and improve their efficiency, we Perturbation Upon Observation: User Defined, Nanoscale Labeling of require insight into the nanoscale structure of cellulose and the mechanism Super-Resolution Images of its depolymerization. In this work, direct stochastic optical reconstruction Ninning Liu, Mingjie Dai, Peng Yin. microscopy (dSTORM) was used to study the structure of fluorescently- Wyss Institute, Boston, MA, USA. labelled bacterial microcrystalline cellulose (BMCC) at the nanoscale. A basic process to investigate biomolecular systems is observing and manipu- Super-resolution imaging unveiled regular repeating patterns of high and lating single molecules within their native context. While many recent ad- low fluorophore density regions on BMCC microfibrils that are hypothesized vances in super-resolution microscopy methods (e.g. STORM, PALM, to represent disordered and crystalline regions of cellulose. Grafting cellu- PAINT) have allowed observation of molecular architecture down to the scale lose using different dyes or labelling reactions produced similar patterns, individual components (~5 nm), there has not been a parallel development of evidencing that the fluorescent patterns are labeling chemistry-independent manipulation methods at a comparable scale. Current light-based manipulation and are instead encoded within the native cellulose structure. The length techniques, including laser capture micro-dissection or focused laser crosslink- of the dark regions was measured and their distribution was found to corre- ing, are still diffraction-limited, and therefore incapable of selective targeting late with the length of cellulose nanocrystals produced by a short hydrolysis below a ~200 nm sized area. treatments, as observed using transmitted electron microscopy. The length Here we present a nanoscale, targeted manipulation method based on the of the microfibril dark regions were also determined to be dependent on PAINT (point accumulation for imaging in nanoscale topography) principle. the concentration of dye during the grafting procedure, strongly suggesting Briefly, PAINT utilizes a fluorescent probe that transiently and repetitively that the observed labelling patterns are due to intervening crystalline and binds to imaging targets. Under TIRF illumination, these transient binding disordered regions of cellulose microfibrils. A fluorescently-labeled cellu- events will appear as apparent blinks that can be localized and reconstructed lase - CBHI-Cy5 - bound to labelled BMCC was imaged in the same way into a super-resolution image. In contrast with localization-based methods and the results show that the enzyme does not exhibit any preferential such as STORM or PALM, the fluorescent probe in PAINT is freely diffusing, binding to either the crystalline or disordered cellulose regions. Understand- with a reasonable expectation of only one probe bound to an imaging target ing the nanostructure of cellulose will allow us to improve cellulose within a diffraction-limited area at any given time. This makes a light- manufacturing processes and give insight into how plant cells assemble, induced targeting technique possible, whereby a laser pulse will be introduced restructure and degrade their cell wall. at the precise moment when a probe binds to a pre-determined region of interest. 709-Pos Board B474 This active management of the PAINT process will utilize our previously Quantitative Analysis of Membrane Protein Clustering from Live-Cell, developed DNA-PAINT super-resolution imaging technique combined with a Single-Molecule Super-Resolution Microscopy Data fast, photo-activated crosslinking chemistry. Currently, we have demonstrated Juliette Griffie1, Dylan Owen1, Patrick Rubin-Delanchy2, Garth Burn1. multi-target, single-molecule labeling of points on a DNA nanostructure test- 1King’s college London, London, United Kingdom, 2University of Oxford, board with 30 nm spatial discrimination. Oxford, United Kingdom.

BPJ 7740_7745 Sunday, February 12, 2017 145a

To address the increasing need for a precise characterisation of the molecular beam light-sheet microscopy can be comprised by the non-uniformity of illumi- organisation in cells, a new family of fluorescence microscopy techniques, nation, especially in thick, highly scattering and absorbing tissues. To provide a super-resolution, has arisen. Single molecule localisation microscopy more uniform illumination, light-sheet excitation from both sides of the sample (SMLM) in particular, allows the localisation of fluorophores with a precision is adopted, which results in higher system complexity and optical alignment of 10-30 nm, hence revealing the cells’ nanoscale architecture at a molecular difficulties. Here we introduce the concept of a digital micromirror device level. Over recent years, 2D SMLM has been extended to 3D and live-cell (DMD)-based Airy beam light-sheet microscope (DMD-ALS) that can acquisitions, unravelling cellular machinery at the nano-scale. Using such compensate the intensity loss caused by scattering and absorption in biological tools, it has been confirmed that modification of molecular spatio-temporal tissues. In our modelling, DMD-ALS can provide thin and uniform illumination organisation at the plasma membrane, particularly molecular clustering, is over a large range, thus eliminating the necessity of dual excitation. The an important component of cell signalling. Despite the advances in imaging attenuation-compensating Airy beam is generated by phase-modulating a methodology, analysis tools which are able to accurately and objectively Gaussian laser beam with a DMD displaying the Lee hologram. The fluores- quantify these phenomena remain limited, especially for 3D and live-cell cence intensity profile along the optical axis is measured and used for the calcu- SMLM data. Here, we present novel model-based Bayesian cluster analysis lation of DMD spatial pattern in real time. This feedback adaptive procedures technique suitable for 2D SMLM as well as its extension to 3D and live- ensures optimal attenuation compensation for a variety of biological tissues cell analysis. We demonstrate the validity of these approaches on simulated with different optical properties, without human intervention. Taking advan- data sets. We further show that the transition from limited visual inspection tage of the fast frame-rate (~32kHz) of DMD, we expect that the adaptive of the data to the extraction of quantitative descriptors provides a unique control algorithm converges within 50 ms. insight into key biological processes. Using the presented analysis tools, we demonstrate the importance of clustering for T cell activation and the trig- 712-Pos Board B477 gering of an immune response. Analysis of live-cell PALM data demonstrates Light Sheet Microscopy by Dual Line Scanning of Two Bessel Beams the importance of molecular clustering and cluster dynamics at the membrane James Werner, Pengfei Zhang, Elizabeth Phipps, Peter Goodwin. during T cell activation. Our analysis, based on live-cell single-molecule Los Alamos National Laboratory, Los Alamos, NM, USA. super-resolution data is the first to provide real time descriptors of the clus- We have developed a light-sheet microscope that uses confocal scanning of tering, as well as a detailed tracking of cluster dynamics and interactions at dual-Bessel beams for illumination. A digital micro-mirror device (DMD) is the membrane. placed in the intermediate image plane of the objective used to collect fluores- cence and is programmed with two lines of pixels in ‘on’ state such that the 710-Pos Board B475 DMD functions as a spatial filter to reject the out-of-focus background gener- Selective Plane Illumination Microscopy in the Conventional Inverted ated by the side-lobes of the Bessel beams. The optical sectioning and out-of- Microscope Geometry focus background rejection capabilities of this microscope were demonstrated Per Niklas Hedde, Leonel Malacrida, Enrico Gratton. by imaging of human A431 cells whose actin was fluorescently stained. The Biomedical Engineering, University of California Irvine, Irvine, CA, USA. dual-Bessel beam system enables twice as many photons to be detected per During the last decade, selective plane illumination microscopy (SPIM) imaging scan, which is useful for low light applications (e.g. single molecule has proven to be one of the most suitable techniques for three- localization) or imaging at high speed with superior signal to noise. While dimensional time lapse imaging. By confining the excitation light to a sheet, demonstrated for two Bessel beams, this approach is scalable to a larger number SPIM combines axial sectioning capability with minimal light exposure and of beams. fast, camera-based image acquisition [1]. However, the typical arrangement of two objective lenses perpendicular to each other provides a number of 713-Pos Board B478 challenges in terms of instrument design and sample geometry, especially Single Molecule Fluroescence Approaches to Plasma Membrane if the use of high numerical aperture (NA) lenses is desired. A popular Biophysics approach is to dip into the sample container from the top, both lenses at a Philip R. Nicovich. 45 degree angle with respect to the sample plane [2,3]. Instead, our new University of New South Wales, EMBL Australia Node in Single Molecule þ design is based on a regular inverted microscope where the sample is illu- Science ARC Centre of Excellence in Advanced Molecular Imaging, minated from the side via an accessory. A custom designed chamber is Sydney, Australia. used to allow side illumination. This way, all microscope ports remain The plasma membrane is the fundamental component of a cell. Not only is the available for other purposes and there is unrestricted access from the top. plasma membrane the outer barrier of the cell, it also regulates many cellular Without the need of dipping into the sample container, smaller sample vol- functions including endocytosis, exocytosis, receptor signalling and cell migra- umes (< 1 ml) can be realized and the use of high NA lenses is facilitated. tion. As essential as the plasma membrane is, methods to capture the structure Also, isolation of optics and sample allows imaging of sealed sample con- of and the proteins within the membrane on biologically-relevant length scales tainers when demanded, e.g., for samples treated with potent toxins. Further, remain lacking. A particularly vexing issue is whether membrane rafts - regions in this design, the orientation of the imaging plane is parallel to the surface of locally-ordered lipids and associated proteins observed in model membrane of the sample container which is desirable for flat samples where it maxi- systems - are present in intact cells. Much of the previous evidence for these mizes the field of view. rafts relies on invasive biochemical techniques and the little imaging evidence < Work supported in part by NIH grants P50 GM076516 and P41 GM103540. available shows that these regions are small ( 100 nanometres) and short- < [1] Huisken, J. et al., Optical Sectioning Deep Inside Live Embryos by Selec- lived ( 100 milliseconds). tive Plane Illumination Microscopy. Science 305, 1007–1009 (2004). While still limited by the diffraction limit of visible light, single-molecule fluo- [2] Hedde, P.N. et al., Rapid Measurement of Molecular Transport and Inter- rescence imaging allows such small and transient features to be detected. We action inside Living Cells Using Single Plane Illumination. Sci. Rep. 4, 7048 will present three new approaches to investigating these structures in intact (2014). membranes. The first is a spectral fluorescence correlation spectroscopy [3] Hedde, P.N. et al., 3D fluorescence anisotropy imaging using selective approach demonstrating the presence of regions of local membrane order and plane illumination microscopy. Opt. Express 23, 22308–22317 (2015). disorder within live cell membranes. These regions have lifetimes of 5 to 25 ms and through simulations we can show that both probe diffusion and 711-Pos Board B476 domain diffusion give rise to the observed fluctuations in emission intensity. Light-Sheet Microscopy using Attenuation-Compensating Airy Beam The second technique is a self-calibrating image analysis method that is Cong Liu, Yen-Liang Liu, Tim Yeh. capable of capturing the coordination of local differences in membrane order Biomedical Engineering, University of Texas at Austin, Austin, TX, USA. with membrane protein distribution within intact cells. This approach demon- Diffraction-free light fields such as Airy beam have attracted significant strates the presence of protein sorting within intact cells as well as the effect of attention due to their ability to retain their transverse intensity profile during expression level on effective protein sorting. The final technique is a 3D super- propagation. This ability is highly desirable for light-sheet microscopy whose resolution method to map the nanometer-scale topography of the plasma field of view (FOV) is usually limited by the Rayleigh range of the Gaussian membrane. We will show initial results demonstrating our ability to map these beam used to create the light sheet. However, the extended FOV of Airy topographical features and their correlation with protein sorting.

BPJ 7740_7745 146a Sunday, February 12, 2017

Optical Microscopy and Super-resolution Eukaryotic Elongation Factor 1A2 (eEF1A2) is an isoform of the alpha subunit of eEF1A complex. Differently from the A1 isoform, the expression of eEF1A2 Imaging: Applications to Cellular Molecules is restricted to brain, heart and skeletal muscle. eEF1A2 is overexpressed in tu- mors, including multiple myeloma (MM), prostate, pancreas, and ovarian, and 714-Pos Board B479 has also an oncogenic behavior favoring tumor cell proliferation while inhibit- A Fast and Reliable Online-System for Platelet Viability Studies ing apoptosis. Thus, eEF1A2 is an interesting target for cancer treatment. Pli- Michael D. Brodesser1, Sandra Mayr1, Fabian Hauser1, Johannes Breuss2, tidepsin (AplidinÒ, APL) is an antitumor agent, originally isolated from the Michael Aspetsberger3, Andreas Hangler3, Lukas Bindreiter3, marine tunicate Aplidium albicans, which is being tested in MM patients in a Daniela Borgmann4, Stephan Winkler4, Christian Gabriel5, phase III pivotal trial in combination with dexamethasone and a phase I trial Eleni Priglinger5,6, Jaroslaw Jacak1, Birgit Plochberger1. in combination with bortezomib and dexamethasone. HeLa-WT and HeLa- 1 Medical Engineering, University of Applied Sciences Upper Austria, Linz, APLR (resistant to AplidinÒ) cell lines have been previously described. 2 3 Austria, Medical University of Vienna, Vienna, Austria, Catalysts GmbH, HeLa-APLR present lower eEF1A2 levels than HeLa-WT cells. Herein we 4 Linz, Austria, School of Informatics, University of Applied Sciences Upper reveal the interaction of Plitidepsin with eEF1A2 using a fluorescent AplidinÒ 5 Austria, Hagenberg, Austria, AUVA Research Center, Ludwig Boltzmann derivative, APL-DMAC as a FRET donor. We stably transfected HeLa-WT and Instiute for Experimental and Clinical Traumatology, Vienna, Austria, HeLA-APLR cells with plasmids encoding GFP-tagged eEF1A2 (FRET 6 Austrian Cluster for Tissue Regeneration, Vienna, Austria. acceptor), and selected clones expressing physiological levels of the fusion pro- Platelets are blood cells with the capability to adhere to injured tissues and blood teins. We applied the FLIM-phasor approach [Redford and Clegg 2005; Dig- vessels after trauma. Self-adherence is also known in thrombosis and inflamma- man et al. 2008], to localize and quantify APL-DMAC molecular species tion. Platelets release factors triggering the activation of other platelets, leuco- and to measure FRET efficiencies, DMAC - GFP, thus identifying different cytes and endothelial cells near the site of adherence. Here we show a fast and eEF1A2-APL complexes [Losada et. al. 2016]. reliable detection system for the activation status of platelets, even in early stages. In particular, the method allows the classification of the cell state, the viability and its reversibility under selected environmental parameters. An opti- 717-Pos Board B482 cal microscope in combination with a contrast enhancing technique like Differ- Image Correlation Spectroscopy based Assay to Investigate G-Protein ential Interference Contrast and single molecule fluorescence microscopy is Coupled Receptors utilized for grading unique features like e.g. cell morphology, cytoskeletal Nader Danaf. arrangement, protein diffusion and clustering. The online-system observes adhe- Chemie und Pharmazie, Ludwig Maximilian University, Munich, Germany. sion of platelets at controlled temperatures and buffer/surface conditions. G-Protein coupled receptors (GPCRs) coordinate and regulate several cellular Focusing on platelet apheresis concentrates labeled with a fluorescent antibody mechanisms and vital human activities such as behavior and sensing. Hence, to the activation receptor CD62p, we observed a distinct change in protein diffu- GPCRs constitute a superfamily of membrane proteins that encode approxi- 1 sion and clustering depending on activation state and associated morphological mately 4 - 5 % of the entire human genome. Interestingly, few GPCRs repre- changes. A systematic correlation between microtubule/actin cytoskeleton- and sent a huge pharmacological interest making them the target of ~ 50% of the morphological change was made. Activation steps were triggered either by tem- prescribed drugs on the market. GPCRs, being ubiquitos and diverse, made perature or buffer conditions on cleaned glass and epoxysilane-coated surfaces. these membrane proteins an interesting system to study and understand. In order to allow a classification of activation states of platelets in real-time, fea- Most current assays lack the ability to provide the essential physiological tures of diffusion, cluster properties and morphology were cross-correlated. The experimental conditions, which enables in vivo studies addressing the GPCR- feature analysis system, useful for the classification of platelets in a clinical ligand interactions. Here, we provide an image correlation spectroscopy based context, might prove applicable also to other specimen (e.g. pathology samples). assay that monitors and investigates GPCRs in live cells on a single molecule basis. The presently developed assay utilizes fluorescence and image correla- 715-Pos Board B480 tion methods2,3 to investigate several aspects of the GPCR membrane proteins. Visualizing Signaling Complexes in Filamentous Fungi Raster image correlation spectroscopy, allows to scan and determine diffusion Alexander W.A.F. Reismann1, Lea Atanasova2, Alexander Lichius2, coefficients of the receptors, was implemented to study the behavior of Sabine G. Gruber3, Susanne Zeilinger2, Gerhard J. Schuetz1. different GPCRs, like the b2-adrenergic and neurotensin receptors, before 1Institute of Applied Physics, TU Wien, Vienna, Austria, 2Institute of and after the binding of a certain ligand. Other image correlation spectroscopy Microbiology, University of Innsbruck, Innsbruck, Austria, 3Institute of Food techniques would be implemented to investigate the behaviour of these recep- Science, University of Natural Resources and Life Science Vienna, Vienna, tors at the membrane. For example, to study the associaton of GPCRs in the Austria. membrane, number and brightness would be a feasible analysis to perform in Due to its mycoparasitic lifestyle, the filamentous fungus Trichoderma atroviride order to question the degree of oligomerization the GPCRs would show in is a widely used biofungicide for the control of fungal plant pathogens in today’s the membrane. agriculture. However, the exact signaling mechanisms regulating the mycopara- 1 a) Rosenbaum, D. M; Søren, S. G. F.; Kobilka, B. K. Nature 2009, 459, 356- sitic attack of fungal preys are not yet fully understood. The sensing and location 363; b) Bjarndo´ttir, T. K; Gloriam, D. E.; of its prey fungi, such as Botrytis cinerea, is mediated by membrane receptors Hellstrand, S. H.; Kristiansson, H.; Fredriksson, R.; Schio¨th, H. B Genomics which react to small chemical substances secreted by the prey. Single molecule 2006, 88, 263-273. techniques have hardly been used in filamentous fungi and can open up efficient 2 Foo, Y. H.; Naredi-Rainer, N; Lamb, D. C.; Ahmed, S.; Wohland, T. Biophys. possibilities to observe these molecular processes in great detail. These methods J. 2012, 102, 1174-1183. can be used under different physiological conditions by adding specific stimuli or 3 a) Muller,€ B. K.; Zaychikov, E.; Br€auchle, C.; Lamb, D. C. Biophys. J. 2005, by co-cultivating Trichoderma with a prey fungus and detect behavioral differ- 89, 3508-3522; b) Hendrix, J.; Schrimpf, ences. In this study, we examine Gpr1, a G-protein coupled receptor, and its pu- W.; Ho¨ller, M.; Lamb, D. C. Biophys. J. 2013, 105, 848-861. tative interaction partner Sur7, which are known to contribute to the molecular signaling process, using single molecule techniques. By using strains expressing Sur7-mEOS3.2 fusion proteins, we performed PALM measurements and gener- 718-Pos Board B483 ated detailed localization maps in growing hyphae. Analyzing single molecule Single Molecule Imaging Reveals Dysferlin-Mediated Recruitment of trajectories of GFP tagged Sur7, we furthermore provide insights into the Phosphatidylserine in Cell Membrane Repair Lu Zhou1, Volker Middel1, Uwe Str€ahle1, G. Ulrich Nienhaus1,2. mobility of the protein in vivo. In the future, we will apply AF647-labeled nano- 1 2 bodies to perform STORM within fixed hyphae expressing Sur7-GFP fusions. Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany, University The obtained insights into the properties of the involved proteins will lead to a of Illinois at Urbana-Champaign, Urbana, IL, USA. deeper understanding of the molecular organization in mycoparasitic fungi. Dysferlin (Dysf) has been implicated as a key protein for membrane repair in muscle cells, although its precise biological function is still unknown. Dysf 716-Pos Board B481 is a transmembrane protein, and its rapid recruitment of Dysf to membrane le- Flim-FRET Imaging of Plitidepsin-eEF1A2 Complexes in Live Cancer sions has been observed; direct observation of Dysf transport has so far re- Cells using the Phasor Approach mained elusive [1]. Moreover, interaction of Dysf with various other proteins Carolina Garcia1, Alejandro Losada2, Miguel A. Sacristan1, has been investigated for many years to understand its role in cell membrane Jose M. Molina-Guijarro2, Juan F. Martinez-Leal2, Carlos M. Galmarini2, repair; however, studies addressing the role of phospholipids, the main compo- M. Pilar Lillo1. nents of the plasma membrane, are still scarce. 1Quimica Fisica Biologica, IQFR-CSIC, Madrid, Spain, 2Biologia Celular y We have observed fast transport of plasma membrane-derived Dysf in living Farmacogenomica, PharmaMar S.A., Colmenar Viejo. Madrid, Spain. zebrafish by using fluorescence loss in photobleaching (FLIP) and single

BPJ 7746_7748 Sunday, February 12, 2017 147a molecule localization imaging [2]. In addition, fluorescence imaging of human silica and localized with a mean precision of 25 nm to resolve structural fea- cells and zebrafish has revealed rapid phosphatidylserine (PS) accumulation in tures. Further co-localization studies on proteins of the Cingulin family when the repair patch in a Dysf-mediated fashion. Single molecule trajectory analysis extracted from the biosilica using a combined two-color PALMþSTORM provides evidence that Dysf facilitates PS translocation from adjacent mem- approach revealed characteristic protein filaments with distinct protein specific brane regions to the site of lesion. patterns. The enhanced microscopy techniques introduced here for diatoms will [1] A. Lek et al., ‘‘Calpains, cleaved mini-dysferlinC72, and L-type channels aid in elucidating the molecular mechanism of silica biomineralization as well underpin calcium-dependent muscle membrane repair’’, J. Neurosc., 2013, as other aspects of diatom cell biology. 33, 5085-94. [2] Middel, V., Zhou, L., Takamiya, M., Beil, T., Shahid, M., Roostalu, U., Grabher, C., Rastegar, S., Reischl, M., Nienhaus, G. U., & Str€ahle, U., ‘‘Dys- 721-Pos Board B486 ferlin-Mediated Phosphatidylserine Sorting Engages Macrophages in Sarco- Quantitative Super-Resolution Microscopy Detects HER2 Reorganization lemma Repair’’, Nat. Commun., 2016, 7, 12875. Following Meditope-Antibody Treatment Devin Wakefield, Raphael Jorand, Cindy Zer, John Williams, Tijana Jovanovic-Talisman. 719-Pos Board B484 Beckman Research Institute at the City of Hope, Duarte, CA, USA. Rotation of Single Cell Surface Molecules Examined via Polarized FCS We combine quantitative super-resolution microscopy and meditope technol- Measurements using Quantum Dot Probes ogy to investigate the effects of cross-linked antibodies on the organization of Domgmei Zhang1, Peter W. Winter1, Deborah A. Roess2, cancer receptor targets. A cyclic peptide, known as a meditope, has been B. George Barisas1. recently discovered to bind within the Fab framework of Cetuximab and 1Department of Chemistry, Colorado State University, Fort Collins, CO, various meditope enabled monoclonal antibodies (memAbs). In this work, USA, 2Department of Biomedical Sciences, Colorado State University, Fort several divalent, Fc-based meditope ligands were generated with different Collins, CO, USA. linkers (linear sequences with 10, 20, or 30 amino acids). BT-474 cells Rotational motions of cell surface molecules are of particular interest given were incubated with these meditopes, together with memAb Trastuzumab, the sensitivity of such motions to molecular size and aggregation state. The and subsequently fixed for dSTORM imaging of human epidermal growth time-autocorrelation function (TACF) of fluctuations in fluorescence polariza- factor receptor 2 (HER2). The spatial organization of HER2 was character- tion from asymmetric quantum dots (QD) such as Molecular Probes’ Qdot655 ized through pair-correlation (PC) analysis, using streamlined Matlab code labeling cell surface molecules indicates the rotational correlation times with minimal user input and methods to reduce bias in selecting regions for (RCT) of the QD-containing complexes. Type I Fcε receptors (FcεRI) and in- analysis. According to our results, HER2 organization strongly depends on sulin receptors (IR) on 2H3 cells, subjected to various treatments potentially meditope linker length. Importantly, an increase in linker length was found affecting receptor rotation, including polyvalent DNP-BSA, methyl-b-cyclo- to correlate with an increase in the proportion of HER2 oligomers. PC anal- dextrin, cytochalasin D or paraformaldehyde, were labeled by Qdot655s. ysis further revealed that these HER2 oligomers organize within clusters Over 700 individual QD were examined. Side-by-side vertically(v)- and ranging from approximately 14 to 25 nm in radius. Ultimately, exploring horizontally(h)-polarized fluorescence images of QD-labeled cells were ob- the molecular organization of receptors with our approach serves as an impor- tained simultaneously as image stacks at 10 ms per frame by means of a tant step toward optimizing a diversity of ligands with potential therapeutic Princeton Instrument Dual-View. To minimize apparent polarization TACF purposes. arising from QD lateral diffusion, v- and h-substacks were aligned to sub- pixel accuracy. From fluorescence in identical regions around each QD in 722-Pos Board B487 each stack, intensity and polarization fluctuation TACF were calculated. 3D Orbital Tracking of Single Gold Nanoparticles: A New Approach to Given the blinking of QD, experimental parameters such as g-factor, camera Study Vesicle Trafficking in Chromaffin Cells background, etc. were carefully optimized for each QD to minimize Manuela Gabriel1, Jose Moya-Diaz2, Fernando D. Marengo2, cross-correlation between polarization and intensity fluctuations. This could Laura C. Estrada1. otherwise appear as intensity fluctuation TACF contributing to the apparent 1Quantum Electronics Laboratory, Physics Department, University of polarization fluctuation TACF. Using these techniques, the initial polarization Buenos Aires, Buenos Aires, Argentina, 2Institute of Physiology, Molecular fluctuation TACF for FcεRI averages about 0.0015 and the geometrically- Biology and Neuroscience, University of Buenos Aires, Buenos Aires, averaged RCT is about 55-85 ms, both independent of cell treatment. More Argentina. limited results on IR appear similar. Absence of treatment effects on magni- Endocytosis and subsequent vesicle recycling serves to keep constant the tudes or decay rates of polarization TACF is puzzling. Previous pools of transmitter-containing vesicles ready for release in neurons and time-resolved phosphorescence anisotropy studies on FcεRI suggest limited endocrine cells. The study of these processes has been carried out by using presence of orientational relaxations slower than 1 ms. However, the absence different experimental approaches, like electrophysiological measurements of treatment effects here suggests such slow reorientation may be a property of and single photon fluorescence microscopy. However, the diverse experi- the membrane itself, perhaps reflecting large-scale fluctuations of mesoscale mental limitations of these techniques restricted a detailed and high resolved membrane regions. Supported by NSF grant MCB-1024668 and NIH grant study of the dynamics of vesicle trafficking after endocytosis in the whole CA175937 to BGB. cellular volume. Multiphoton microscopy provides optical sectioning for high-resolution imaging. In biological systems, most multiphoton micro- 720-Pos Board B485 scopy studies have relied on two-photon excited fluorescence (TPEF) to pro- Super-Resolution Imaging Reveals Protein-Templated Patterns for Bio- duce images. Because of their strong brightness and imaging durability, silica Formation metallic NPs have been recently introduced as labels in fluorescence micro- Philip Gro¨ger, Nicole Poulsen, Jennifer Klemm, Nils Kro¨ger, scopy. The use of TPEF and metallic nanoparticles in combination provides a Michael Schlierf. noninvasive, spatially localized, in vivo characterization for biological sam- B CUBE – Center for Molecular Bioengineering, TU Dresden, Dresden, ples. In this work, we tracked single gold nanoparticles after endocytotic Germany. internalization in mouse chromaffin cells stimulated with high potassium. The intricate, genetically controlled biosilica nano- and micropatterns pro- We use an orbital-scanning tracking method in a two-photon absorption mi- duced by diatoms are a testimony for biology’s ability to control mineral for- croscope. In the first place, we compare constitutive and active internalization mation (biomineralization) at the nanoscale and regarded as paradigm for of gold nanoparticles (AuNPs), assessing the number of internalized AuNPs nanotechnology. Several recently discovered protein families involved in and evaluating its dynamics in terms of velocity and displacement after stim- diatom biosilica formation remain tightly associated with the final biosilica ulation. In the second place, we evaluate these parameters pharmacologically structure. Determining the locations of biosilica-associated proteins with high blocking proteins classically involved in the development of endocytotic pro- precision is therefore expected to provide clues to their roles in biosilica cess. Our results show that endocytosis of AuNPs was much more efficient morphogenesis. To achieve this, we introduce single-molecule localization mi- when exocytosis was induced with high K in comparison with constitutive croscopy to diatoms based on photo-activated light microscopy (PALM) to cycling. In addition, the dynamics of AuNPs had a strong dependence on cla- overcome the diffraction limit. We identified six photo-convertible fluorescent thrin dependent endocytosis as well as on cortical actin polymerization. This proteins (FPs) that can be utilized for PALM in the cytoplasm of Thalassiosira study shows that the combination of 3D orbital tracking and AuNPs is an pseudonana. However, only three FPs that share a common molecular interesting tool for the study of vesicle trafficking after endocytosis in live conversion-mechanism were also functional when embedded in diatom bio- cells.

BPJ 7746_7748 148a Sunday, February 12, 2017

723-Pos Board B488 or cisplatin to induce cellular stress. To obtain maps of protein diffusion and Super-Resolution Imaging of DNA Replisome Dynamics in Live Bacillus directional local motion the fluorescence Diffusion Tensor Imaging (fDTI) Subtilis method, inspired by the Diffusion Tensor imaging in the MRI field, was Yilai Li1, Jeremy W. Schroeder2, Yi Liao3, Lyle A. Simmons1, used. This powerful technique provides maps of molecular diffusivity and Julie S. Biteen1. protein interactions in the context of their endogenous environments. The 1University of Michigan, Ann Arbor, MI, USA, 2University of Wisconsin, fDTI is based on spatial pair correlations between adjacent pixels in a given Madison, WI, USA, 3University of Chicago, Chicago, IL, USA. number of radial angles that are explored in a given point to determine the DNA replication happens in all living organisms, and assures that the genome is anisotropic diffusion. The time points for the pCF correlation function are accurately copied and maintained. The replisome is the molecular machine in calculated in a log scale using by dividing the tau axis in 32 segments equally cells that replicates DNA, and it is composed of several different proteins, spaced in the log scale. Thus diffusion and connectivity maps are obtained by including DNA polymerases, which directly synthesize DNA by adding nucle- calculating pair correlation functions of each voxel with all the neighbor vox- otides. Although the bacterial replisome has been studied extensively in vitro, els. For this work we show changes in molecular diffusion and alterations of little is known about the dynamics and architecture of replisome components protein connectivity post activation of the cell stress response mechanism and in vivo. Here we use Bacillus subtilis, a Gram-positive bacterium commonly are able to construct molecular connectivity maps of p53. This type of anal- found in soil, as a model organism in which to study the architecture and ysis can provide a well-informed picture of spatial temporal p53 regulation dynamics of several replisome components in vivo. Photoactivated localization throughout the living cell. This work is supported in part by NIH grants microscopy (PALM) and single-molecule tracking give a resolution of 20- P50 GM076516 and P41 GM103540. 40 nm, far below the diffraction limit of conventional microscopy, enabling us to localize and track every single protein molecule. In our study, we inves- 726-Pos Board B491 tigated the dynamics of a number of replisome components under different con- Association of Endophilin B1 with Cytoplasmic Vesicles ditions, including the DNA polymerases PolC and DnaE, and the b-clamp Jinhui Li1, Barbara Barylko2, Joachim D. Mueller1, Joseph Albanesi2, loader DnaX. We can watch the real time behavior of different replisome com- Yan Chen1. 1Physics, University of Minnesota, Minneapolis, MN, USA, ponents during the DNA synthesis process, and study them quantitatively. Sur- 2 prisingly, our investigations have revealed that all these replisome components UT Southwestern Medical Center, Dallas, TX, USA. are highly dynamic and exchange more rapidly than previously expected, and Studying protein association with cytoplasmic vesicles within the live cell re- we characterize the molecular scale distribution of each replisome component mains a general challenge for optical microscopy techniques. Here, we show within the dynamic replication machinery. that fluorescence fluctuation microscopy offers an effective approach to tackle this problem by using endophilins as a model system. Endophilins are SH3- 724-Pos Board B489 and BAR domain-containing proteins implicated in membrane remodeling Single-Molecule Tracking Reveals Altered Dynamics of a Transcription and vesicle formation. Endophilins A1 and A2 promote the budding of Regulator Expressed at Similar Levels from Different Gene Expression endocytic vesicles from the plasma membrane, whereas endophilin B1 has Systems been implicated in vesicle budding from intracellular organelles, including Chanrith Siv1, David J. Rowland1, Victor J. DiRita2, Julie S. Biteen3. the trans-Golgi network and late endosomes. We previously reported that 1Biophysics, University of Michigan, Ann Arbor, Ann Arbor, MI, USA, endophilins A1 and A2 exist almost exclusively as soluble dimers in the 2Microbiology & Molecular Genetics, University of Michigan, Ann Arbor, cytosol. Here we present results of fluorescence fluctuation spectroscopy an- East Lansing, MI, USA, 3Chemistry, University of Michigan, Ann Arbor, alyses indicating that, in contrast, the majority of endophilin B1 is present Ann Arbor, MI, USA. in multiple copies on small, highly mobile cytoplasmic vesicles. Formation The development of fluorescent proteins and their application in live-cell imag- of these vesicles was enhanced by overexpression of wild-type dynamin 2, ing have not only validated previously known molecular processes but have but suppressed by expression of a catalytically inactive dynamin 2 mutant. also unraveled new biological mechanisms. Though the functionality and sta- Using dual-color heterospecies partition analysis, we identified the epidermal bility of fusions to fluorescent proteins are routinely characterized, there is growth factor receptor (EGFR) on EndoB1 vesicles. Moreover, a proportion no strict agreement in the community on how to express these fusion proteins of EndoB1 vesicles also contained caveolin, whereas clathrin was almost un- in cells. Here, we measure differences in the single-molecule trajectories of a detectable on those vesicles. These results raise the possibility that endophilin transcription regulator protein, TcpP, depending on its expression method in B1 participates in dynamin 2-dependent formation of a population of transport Vibrio cholerae. TcpP was fluorescently labeled with the photoactivatable fluo- vesicles distinct from those generated by A-type endophilins. This work has rescent protein PAmCherry at its C-terminus and was either expressed off of the been supported by a grant from the National Institutes of Health (R01 native TcpP promoter in the chromosome or from an arabinose-inducible pro- GM64589). moter PBAD. Even though we detected similar levels of tcpP-pamcherry mRNA transcripts and TcpP-PAmCherry protein concentrations in vitro with both 727-Pos Board B492 expression methods, single-molecule tracking of the protein in live V. cholerae Characterization of Ire1 Interactions and Dynamics with Quantitative reveals differences which can obfuscate biological interpretation of the role of Super-Resolution Microscopy TcpP in toxin production. In the plasmid-induced strain, we observed (1) more Elizabeth M. Smith, Ragnar Stefansson, Maria Paz Ramirez Lopez, slowly diffusing TcpP-PAmCherry molecules, (2) an at least a five-fold in- Elias M. Puchner. crease in copies of TcpP-PAmCherry, contrary to the in vitro western results, Physics, University of Minnesota, Minneapolis, MN, USA. and (3) the absence of a very rapidly diffusing population that is observed Quantitative Super-Resolution Microscopy is a powerful technique that can be upon endogenous expression. In addition, we compared the dynamics of used to study biological processes below the diffraction limit. In this work, we TcpP-PAmCherry in V. cholerae to TcpP-PAmCherry in wildtype Escherichia employ our intracellular calibrated Photoactivated Localization Microscopy coli, in which background TcpP should not have any relevant biological activ- (PALM) technique to perform quantitative molecular counting of proteins ity. Our findings suggest that single-molecule tracking of proteins provides a involved in the unfolded protein response (UPR). The UPR is a signaling very sensitive assay to detect subtle differences in protein dynamics—and pathway which dynamically regulates endoplasmic reticulum (ER) protein thus activities—that are hidden in vitro. folding capacity in response to cellular stress. As is true with many signaling pathways, the spatiotemporal organization of the UPR-specific biomolecules 725-Pos Board B490 is an inherent feature of the pathway activation and downstream response. Spe- Fluorescence Diffusion Tensor Maps of p53 Activation Acquired with cifically, in response to stress, Ire1 (a bifunctional transmembrane kinase/ SPIM endoribonuclease) oligomerizes and forms discrete signaling clusters which Michelle A. Digman, Lukas Rottsch€afer, Enrico Gratton, Per Niklas Hedde. recruit and splice an mRNA encoding a transcription activator. Using PALM Biomedical Engineering, University of California, Irvine, Irvine, CA, USA. in conjunction with traditional fluorescence microscopy we characterize the in- The transcription factor, p53, can activate the DNA repair or apoptotic teractions and dynamics of Ire-1 at wild type expression levels in yeast cells. pathway in response to cellular stress. Specific binding sites must be acces- Specifically, we quantify the oligomeric state, of Ire1 under stressed and sible to active either pathway. Thus, specific chromatin location as well as unstressed conditions and track the motion of Ire1 during signaling activity accessibility are required for stable tetramers of p53 to activate either to study the kinetics of cluster formation. Finally we perform colocalization ex- response pathway. To gain information regarding p53 fast dynamic binding periments with downstream UPR biomolecules to further characterize the role interaction and diffusion to localized nuclear regions, we used single plane of Ire1 signaling centers in control of gene expression. This study provides illumination microscopy (SPIM) to acquire high speed frame rate images insight into the spatiotemporal organization of Ire1 and its downstream partners with a time resolution below 1 ms. Cells were treated with either anisomyocin in the signaling response of the UPR.

BPJ 7746_7748 Sunday, February 12, 2017 149a

728-Pos Board B493 sues, or entire organisms. In the last few decades, new experimental methodol- Diffusion of DNA-Binding Species in Thenucleus: A Transient Anomalous ogies were introduced that are able to unveil details on a length scale that is a Subdiffusion Model tiny fraction of the wavelength of light, thus moving spatial resolution far Michael J. Saxton. beyond the diffraction limit set by Ernst Abbe’s equation. Still, the enormous Biochemistry & Molec Med, University of California, Davis, CA, USA. wealth of information available today from optical microscopy measurements Recent single-particle tracking experiments have measured the distribution of on living samples is often underexploited. We argue that spatiotemporal fluo- dwell times of DNA-binding species diffusing in living cells: CRISPR-Cas9, rescence correlation spectroscopy (spFCS) can enhance the performances of TetR, and LacI [Knight, Science 2015; Normanno, Nat Commun 2015; Caccia- current nanoscopy methods and provide further insight into dynamic molecular nini, Faraday Disc 2015]. The observed distribution, a truncated power law, im- processes of high biological relevance (Di Rienzo, C. et al. Biophys J, 111, 679- plies transient anomalous subdiffusion, in which diffusion is anomalous at short 685, 2016). We present exemplary biological applications of spFCS to measure ^ times (mean-square displacement proportional to t a, a < 1) and normal at long dynamic molecular parameters well below the diffraction limit in a standard times (MSD proportional to t) [Saxton, Biophys J 66 (1994), 70 (1996), 92 optical setup, including the measurement of the nanoscale displacement of (2007)]. Monte Carlo simulations are used to characterize the time-dependent GFP in the cell cytoplasm (Di Rienzo, C. et al. Nat comm 5, n.5891, 2014), diffusion coefficient D(t) in terms of the exponent a, the crossover time, D(0), and of Transferrin Receptor-GFP (TfR-GFP) sub-diffraction confinement on and D(N), and these quantities in terms of the dwell time distribution. The the plasma membrane of live cells (Di Rienzo, C. et al. PNAS, 110 (30), simplest interpretation is that the dwell time is an actual binding time to 12307-12312, 2013). Also we discuss how standard super-resolution methods, DNA. One alternative interpretation is that the dwell time is the period of 1D which are intrinsically endowed with high static spatial resolution properties, diffusion on DNA in the standard model combining 1D and 3D search. The can take advantage of the resolution improvements that are accessible through model has several implications for cell biophysics. (1) The initial anomalous spFCS to describe dynamical molecular processes. Finally, we argue that by us- regime represents the search of the DNA-binding species for its target DNA ing spFCS we can definitely integrate the arsenal of methods at our disposal to sequence. (2) Non-target DNA sites have a significant effect on search kinetics. investigate living matter at the nanoscale. False positives in bioinformatic searches are potentially rate-determining in vivo. For simple binding, the search would be speeded if false-positive se- 731-Pos Board B496 quences were eliminated from the genome. (3) Both binding and obstruction Bacterial Type 3 Secretion Systems: High-Throughput 3D Single-Molecule affect diffusion. The proper controls for obstruction are GFP as a calibration Tracking of Sorting Platform Proteins in Live Cells Julian Rocha1, Andreas Diepold2, Judith P. Armitage2, Andreas Gahlmann3. standard among laboratories and cell types, and the DNA-binding species 1 with the binding site inactivated. (4) Overexpression of the DNA binding species Department of Chemistry, University of Virginia, Charlottesville, VA, USA, 2Department of Biochemistry, University of Oxford, Oxford, United reduces anomalous subdiffusion because the deepest binding sites are occupied 3 and unavailable. (5) The model provides a coarse-grained phenomenological Kingdom, Departments of Chemistry and Molecular Physiology & description of diffusion of a DNA-binding species, useful in larger-scale Biological Physics, University of Virginia, Charlottesville, VA, USA. modeling of kinetics and FRAP. (Supported in part by NIH grant GM038133). Bacterial secretion systems are large biomolecular assemblies that rely on static and transient interactions between individual molecular subunits. A central 729-Pos Board B494 example is the Type 3 Secretion System (T3SS) which consists of both the Spatial Dynamics of SIRT1 Relates to Metabolic Transitions in the Cell static membrane-embedded needle complex and the much more dynamic cyto- Nucleus plasmic sorting platform. Single-subunit turnover in the sorting platform and Suman Ranjit1, Lorena Aguilar-Arnal2,3, Chiara Stringari1,4, the resulting structural heterogeneity have made it challenging to decipher Paolo Sassone-Corsi2, Enrico Gratton1. the molecular-level mechanism of Type 3 secretion. Live-cell single-molecule 1Biomedical Engineering, University of California Irvine, Irvine, CA, USA, super-resolution microscopy is ideally suited to measure spatial locations and 2Center for Epigenetics and Metabolism, University of California Irvine, trajectories of individual molecular subunits with nanoscale precision. Extract- Irvine, CA, USA, 3Institute for Biomedical Research, UNAM, Mexico, ing meaningful biological results, however, requires characterizing the entire Mexico, 4Laboratory for Optics and Biosciences, Ecole Polytechnique, Paris, distribution of molecular behaviors, which in turn, necessitates a large number France. of individual measurements. Here, we apply high-throughput aberration-cor- þ SIRT1 is a NAD -dependent deacetylase functioning as metabolic sensor of rected 3D single-molecule localization microscopy to quantitatively measure cellular energy and it adapts different biochemical pathways to the changes the diffusion behaviors of over 100,000 individual T3SS sorting platform pro- in the environment. SIRT1 substrates include histones and proteins related to teins. The single-molecule trajectories reveal multiple diffusive populations in enhancement of mitochondrial and antioxidant protection. Fluctuations in intra- the bacterial cytoplasm suggesting the pre-formation of functionally important þ cellular NAD levels regulate SIRT1 activity, yet the exact pathway SIRT1 higher-order molecular complexes. By providing information on the spatiotem- þ enzymatic activity impacts NAD levels and its intracellular distribution re- poral regulation of protein function in living cells, our results complement mains unclear. Here, we demonstrate that SIRT1 determines the nuclear orga- recent structural and biochemical findings that the cytoplasmic T3SS sorting nization of protein bound NADH. Using multiphoton microscopy in live cells, platforms contain large pod-like structures and that cytoplasmic C-ring proteins we show that free and bound NADH are compartmentalized inside of the nu- may pre-assemble into oligomeric complexes prior to binding to the T3SS sort- cleus, and its subnuclear distribution depends on SIRT1. Importantly, SIRT6, ing platforms. a chromatin-bound deacetylase of the same class does not influence NADH nu- clear localization. In addition, using fluorescence fluctuation spectroscopy, 732-Pos Board B497 especially phasorFCS in single living cells, we reveal that NADþ metabolism Molecular Tattoo: Subcellular Confinement of Drug Effects In Vivo with in the nucleus is linked to subnuclear dynamics of active SIRT1. SIRT1 dif- Two-Photon Microscopy 1 1 ´ 2 1 fuses faster on the periphery of nucleus and the diffusion is slower in the center. Bogla´rka Va´rkuti , Miklo´s Ke´piro´ , Anna A. Rauscher ,La´szlo´Ve´gner , ´ 1 1 1 ´ 1 Comparison of results from phasorFCS and autofluorescence FLIM divulge a Aron Zsigmond , Vanda Imrich , Szilvia Ra´ti ,Ada´m I. Horva´th , relationship between NADþ metabolism, NADH distribution and SIRT1 activ- Ma´te´ Varga3, Miklo´s S. Kellermayer4, Malnasi-Csizmadia Andras1. 1Department of Biochemistry, MTA-ELTE Molecular Biophysics Research ity in the nucleus of live cells, and leads off to decipher links between nuclear 2 3 organization and metabolism. Group, Budapest, Hungary, Printnet Ltd., Budapest, Hungary, Department of Genetics, Eo¨tvo¨s University, Budapest, Hungary, 4Department of 730-Pos Board B495 Biophysics and Radiation Biology, MTA-SE Molecular Biophysics Research Spatiotemporal Fluctuation Analysis: A Powerful Tool for the Future Group, Budapest, Hungary. Nanoscopy of Dynamic Molecular Processes Technological resources for sustained local control of molecular effects within Francesco Cardarelli1, Enrico Gratton2, Fabio Beltram3, organs, cells, or subcellular regions are currently unavailable, even though such Carmine Di Rienzo3. technologies would be pivotal for unveiling the molecular actions underlying 1Nanomedicine, Center for Nanotechnology Innovation at NEST, Istituto collective mechanisms of neuronal networks, signaling systems, complex Italiano di Tecnologia, Pisa, Italy, 2Department of Biomedical Engineering, machineries, and organism development. We present a novel optopharmacolog- Laboratory for Fluorescence Dynamics, University of California at Irvine, ical technology named molecular tattooing, which combines photoaffinity label- California, US, Irvine, CA, USA, 3Center for Nanotechnology Innovation at ing with two-photon microscopy.Moleculartattooing covalently attaches a NEST, Istituto Italiano di Tecnologia; NEST, Scuola Normale Superiore and photoreactive bioactive compound to its target by two-photon irradiation Istituto Nanoscienze-CNR, Pisa, Italy. without any systemic effects outside the targeted area, thereby achieving A major challenge of present and future biophysics is to quantitatively study subfemtoliter,long-term confinement of target-specific effects in vivo. As we how biomolecules dynamically fulfill their physiological role in living cells, tis- demonstrated in melanoma cells and zebrafish embryos, molecular tattooing is

BPJ 7746_7748 150a Sunday, February 12, 2017 suitable for dissecting collective activities by the separation of autonomous and single-molecule observation while preserving the complexes in a near-native non-autonomous molecular processesin vivo ranging from subcellular to organ- live cell environment. Methods such as stepwise photobleaching can then ism level. Since a series of drugs are available for molecular tattoo, the technol- be used to study the subunit stoichiometry of membrane receptors, or for cyto- ogy can be implemented by a wide range of fields in the life sciences. Supported solic complexes, this strategy greatly facilities fluorescence correlation spec- by the Hungarian Research and Innovation Fund (VKSZ_14-1-2015-0052). troscopy based methodologies which can provide a quantitative assessment of cytoplasmic oligomerization state. We present examples that illustrate 733-Pos Board B498 the advantages of the method for both membrane and intracellular cytosolic in- Human Subcutaneous Adipose Tissue Adipocytes Demonstrate Two Phys- vestigations. The method, called Single Protein Recovery After Dilution iological States: Insulin Responsive or Insulin Refractory (SPReAD), is a simple and versatile means of extending the concentration Chad D. McCormick, Hang Waters, Ludmila Bezrukov, Brad Busse, range of single molecule measurements to what are often more normal cellular Andrew Demidowich, Paul S. Blank, Jack A. Yanovski, levels with minimal perturbation of protein complex stoichiometry. Joshua J. Zimmerberg. NICHD, NIH, Bethesda, MD, USA. 736-Pos Board B501 Insulin resistance is a precursor to Type II Diabetes. Peripheral insulin resis- Visualizing Heterogeneous Single-Molecule Dynamics of Molecular tance, as exemplified by the response of tissues like adipose to an insulin chal- Assemblies in Live Cells lenge, is a tissue average traditionally monitored by glucose uptake assays. We Michael Lacy1, David Baddeley2, Julien Berro1. hypothesize that these assays may not reflect cellular level heterogeneous 1Molecular Biophysics and Biochemistry, Yale University, West Haven, CT, behavior and instead report a weighted response of insulin responsive and re- USA, 2Cell Biology, Yale University, West Haven, CT, USA. fractory adipocytes. We developed new assays to monitor the adipocyte insulin Molecular assemblies can have highly heterogeneous dynamics within the cell. response in the context of ex vivo tissue samples. Within one hour of biopsy Classic fluorescence microscopy methods, such as FRAP (Fluorescence Recov- from subjects recruited to the NIH Clinical Center, we tested if AKT phosphor- ery After Photobleaching), have been invaluable to characterize these dynamics ylation, one of the major signaling nodes of the canonical insulin signaling at the micrometer scale. However, it has been particularly difficult to charac- pathway, is sufficient to monitor the number of insulin responsive or refractory terize molecular heterogeneities inside diffraction limited zones within multi- cells in fixed human tissue. Immunostaining revealed two adipocyte popula- molecular assemblies in live cells. We have developed a novel fluorescent tions: low pAKT cells, primarily seen in the absence of insulin stimulation, labeling and imaging protocol, called Single Molecule Recovery After Photo- and high pAKT cells, primarily seen in tissue from healthy subjects after insulin bleaching (SMRAP), which has allowed us to reveal the heterogeneous dy- stimulation. The fraction of tissue with a large pAKT response to insulin cor- namics of the eisosome, a multi-protein structure on the cytoplasmic face of relates well with the fraction of tissue with GLUT4, the insulin-stimulated the plasma membrane in fungi. By fluorescently labeling only a small fraction glucose transporter, localized in the adipocyte plasma membrane. The pAKT of cellular Pil1p, the core eisosome protein in fission yeast, we were able to fraction also supports a two-component model: insulin responsive versus insu- visualize whole eisosomes before photobleaching and, after photobleaching, lin refractory adipocytes, rather than a graded continuum of insulin responses, the recovery of individual Pil1p molecules that bound to the structure with when the pAKT fraction is matched with each subject’s insulin sensitivity in- ~30 nm precision. Further analysis of these sparsely labeled, dynamic struc- dex (SI), calculated using the insulin-modified frequently sampled intravenous tures allowed us to show that Pil1p turnover is spatially heterogeneous. We glucose tolerance test (FSIVGTT). These results agree with previous work from observed that Pil1p molecules from the cytoplasm bind and unbind at the our lab using isolated cells in which the healthier SI value a subject has, the ends of eisosomes, but not along the interior, supporting a new model of the greater fraction of their subcutaneous adipocytes respond to insulin. eisosome as a dynamic filament. We expect our new SMRAP method will be easily and broadly applicable to any molecular assembly in the cell, since it 734-Pos Board B499 only requires sparse labelling of proteins of interest and a standard TIRF setup Single Cell Examination of Membrane Fluidity and Cellular Respiration with single molecule detection capabilities. Krishna Ojha, John Ertle, Michael C. Konopka. Chemistry, The University of Akron, Akron, OH, USA. Single-Molecule Spectroscopy I Bulk measurements of oxygen consumption rates by bacteria have previously been shown to be related to the cells’ average membrane fluidity (as measured 737-Pos Board B502 by the diffusion of fluorescently-labeled molecules). Within a bacterial popula- Single-Molecule Counting Applied to Immunoassays tion, there is also significant cell-to-cell variation in both the diffusion coeffi- Patrick J. Macdonald, Qiaoqiao Ruan, Kerry M. Swift, Sergey Y. Tetin. cient of membrane probes and oxygen consumption rates of individual cells. Abbott Laboratories, Libertyville, IL, USA. One possibility is the diffusion of ubiquinone, an electron carrier in the electron We investigated the sensitivity of single-molecule TIRF counting in diagnostic transport chain (ETC), is being limited by the fluidity of the membrane which is immunoassay applications. This work focused on using single-molecule tech- causing the heterogeneity. niques purely for detection, with the assay itself taking place on a separate plat- We describe Fluorescence Recovery After Photobleaching (FRAP) measure- form with the detectable label being eluted for single-molecule measurement. ments to monitor cell-to-cell variation in membrane diffusion coefficient. Such an approach both limits the amount of background in the final measure- These results are compared to two approaches to look at what influence it might ment and sets up a universal detection procedure for a potentially wide variety have on respiration at the single cell level. The first uses a fluorescent indicator of immunoassays. We took advantage of the low volume required for single- of the activity of the ETC. The second directly measures the consumption of molecule measurements and demonstrated a sample reloading approach to oxygen by individual cells using a phosphorescent Pt-porphyrin dye. further concentrate the sample on the single-molecule surface. SM reload- ing—for a biotin-streptavidin surface capture reaction—is a remarkably robust 735-Pos Board B500 procedure, independent of starting concentration, and so can be used for assay- An Improved Single Molecule Imaging Vivo Method for In Vivo Stoichio- ing unknown samples. We tested model assays with single-molecule detection metric and Functional Analysis of Protein Complexes to substantiate this approach and demonstrate the potential of single-molecule 1 2 3 4 Warren R. Zipfel , Avtar Singh , Maria Sirenko , Alexander Song , counting for diagnostic applications. Paul Kammermeier5. 1Biomedical Engineering, Cornell University, Ithaca, NY, USA, 738-Pos Board B503 2Applied Physics, Cornell University, Ithaca, NY, USA, 3Cornell University, Comparing Antibody-Antigen Binding in Serum Versus Buffer with Fluo- Ithaca, NY, USA, 4Princeton University, Princeton, NJ, USA, 5University of rescence Correlation Spectroscopy Rochester Medical Center, Rochester, NY, USA. David Ortiz, Isabel Yannatos, Abhinav Nath. Elucidating the composition and stoichiometry of membrane bound and cyto- Medicinal Chemistry, University of Washington, Seattle, WA, USA. solic protein complexes is critical to understand their biological function and Therapeutic proteins or ‘biologics’ such as monoclonal antibodies (mAbs), the underlying molecular mechanisms involved. Existing techniques used to antibody-drug conjugates (ADCs) or Fc-fusions comprise a growing percent- determine subunit stoichiometry in single molecule experiments may signifi- age of drugs in the development pipeline. A majority of techniques used to cantly bias experimental results due to the need for either extremely low characterize these drugs require dilution into standard buffers, conditions that expression levels required to obtain concentrations suitable for single molecule do not reflect the complexity of crowded biological environments such as imaging, or in the case of protein complex isolation for in vitro characteriza- serum. Here, we use fluorescence correlation spectroscopy (FCS) to directly tion, perturbation of the normal stoichiometric relationships by the isolation assess the effect of biological solutions on the binding affinity of mAbs to method used. Here we present an alternative approach in which protein com- antigen. FCS is a single-molecule technique that measures the mean time a plexes are assembled at physiological concentrations and subsequently diluted labeled particle takes to diffuse across a small (~1fL) confocal volume. Diffu- by conditionally controlled cell fusion to obtain protein levels suitable for sion time is directly proportional to hydrodynamic radius, thus binding can be

BPJ 7746_7748 Sunday, February 12, 2017 151a tracked with changes in diffusion time of the larger mAb-antigen complex. Neuronal SNAREs (synaptobrevin, syntaxin, SNAP-25) are the minimal fusion With this approach, we measured the binding affinities of Alexa-488 labeled machinery, but require synaptotagmin, complexin, Munc13, and Munc18 for streptavidin and anti-streptavidin immunoglobulin G2 (IgG2) in buffer and fast Ca2þ-triggered neurotransmitter release. Munc13s have a central role in neat serum, and found that binding is ~3-fold tighter in serum versus buffer. synaptic vesicle priming through their MUN domain. Using single molecule Through control experiments in viscosity-matched sucrose solution, we found FRET we identified two distinct roles of the MUN domain in SNARE complex that differences in viscosity may account for some but not all of this effect. assembly for efficient Ca2þ-triggered fusion events. Prior to SNARE complex Serum contains high concentrations of co-solutes of various sizes including assembly syntaxin adopts a closed conformation tightly bound to Munc18. We albumin and g-globulins. We tested the effects of macromolecular crowding found that the MUN domain changes the conformation of the linker region of on binding using physiological concentrations of bovine serum albumin as syntaxin when bound to Munc18 providing a nucleation site for ternary well as polymeric crowders, and found that crowding also partially accounts SNARE complex formation. Interestingly we found an additional role of the for the tighter binding of the streptavidin-IgG2 pair in serum. Conversely, MUN domain where it cooperates with Munc18 to ensure properly assembled serum does not affect the binding affinity of anti-streptavidin IgG1 to strepta- SNARE complex, which resulted in a dramatic increase in Ca2þ-triggered vidin. Accurate characterization of therapeutic proteins in relevant conditions is fusion efficiency and Ca2þ-sensitivity. integral to assessing their safety and efficacy. The implications of these results with respect to drug development are discussed. 742-Pos Board B507 Monitoring Small Molecule and G-Quadruplex Interactions and Kinetics 739-Pos Board B504 using Single Molecule FRET In Vitro Binding of 6S RNA Mango to RNA Polymerase by Two Photon Parastoo Maleki. Fluorescence Cross Correlation Spectroscopy Kent State University, Kent, OH, USA. S. Shyam Sundar Panchapakesan1, Eric J. Hayden2, Peter Unrau1, Telomestatin and oxazole telomestatin derivatives (OTD) are small molecules Matthew L. Ferguson3. stabilizing G-quadruplex (GQ) structures and are prominent due to their anti- 1Department of Molecular Biology and Biochemistry, Simon Fraser cancer drug potential. Despite the observation of enhanced thermodynamic sta- University, Burnaby, BC, Canada, 2Department of Biological Science, Boise bility imparted by such small molecules on GQ, the underlying dynamics of State University, Boise, ID, USA, 3Department of Physics, Boise State small molecule-GQ interactions are not known. To have a better understanding University, Boise, ID, USA. of these interactions we employed single molecule Fo¨rster resonance energy The assembly of RNA and protein complexes is a fundamental process for life transfer (smFRET) to study the system, where we utilized a Cy5-labeled but has been difficult to study both in vitro and in vivo. Recently a novel OTD (L1Cy5-7OTD). These studies demonstrate that interactions of this small aptamer based method to fluorescently label RNA molecules has been molecule with GQ are dynamic in terms of binding kinetics and possibly in reported(Dolgosheina et al. 2014). Here we report the successful utilization terms of rotational freedom. The Cy5 fluorophore has enabled monitoring of the RNA Mango þ thiazole orange derivative to measure the in vitro binding and quantifying binding, dwell, and dissociation of a single L1Cy5-7OTD of 6S RNA to RNA Polymerase from E. coli. By two photon Fluorescence molecule as it interacted with GQ, which to our knowledge has not been Cross Correlation Spectroscopy, we are able to independently measure the mo- demonstrated for any GQ stabilizing small molecule before. We show that lecular brightness, diffusion coefficient and concentration of 6S RNA Mango, L1Cy5-7OTD remains bound to GQ for tens of seconds, with significantly RNA Polymerase GFP and complex as we increase RNA Polymerase concen- longer dwell times and higher binding frequencies for more stable GQ. In addi- tration giving us an estimate for the equilibrium binding constant of the com- tion, we propose L1Cy5-7OTD to have at least two preferred primary binding plex in vitro. This experiment demonstrates the utility of RNA Mango for both orientations and is able to transition between these orientations while it remains in vitro and in vivo single molecule experiments. stacked on a G-tetrad. Dolgosheina, Elena V., Sunny C. Y. Jeng, Shanker Shyam S. Panchapakesan, Razvan Cojocaru, Patrick S. K. Chen, Peter D. Wilson, Nancy Hawkins, Paul A. Wiggins, and Peter J. Unrau. 2014. ‘‘RNA Mango Aptamer-Fluorophore: 743-Pos Board B508 A Bright, High-Affinity Complex for RNA Labeling and Tracking.’’ ACS Engineering ClpXP for Single-Molecule Protein Sequencing Chemical Biology 9 (10): 2412-20. Mike Filius, Jetty van Ginkel, Chirlmin Joo. BioNanoScience, Delft University of Technology, Delft, Netherlands. Proteins are vital in all biological systemsastheyareinvolvedinalarge 740-Pos Board B505 number of structural and functional pathways. Mass sprectometric tech- Single-Molecule Fluorescence Study of RNA Recognition by Viral RNAi niques have been proposed to provide valuable tools for complete proteomic Suppressors studies of the human proteome. However, current mass spectrometers lack Mohamed Fareh1, Jasper van Lopik1, Iason Katechis1, Ronald van Rij2, the sensitivity for determining the least abundant proteins within a cell. A Chirlmin Joo1. single-molecule method that analyses the proteins molecule by molecule 1Kavli Institute of Nanoscience, Bionanoscience Department, Delft may have the sensitivity to shed light of the least abundant proteins within University of Technology, Delft, Netherlands, 2Department of Medical a single cell. The major challenge of protein sequencing lies in the fact Microbiology, Radboud University Nijmegen Medical Centre, Radboud that protein sequences consist of 20 different amino acids. Here, we aim Institute for Molecular Life Sciences, Nijmegen, Netherlands. to develop a novel protein sequencing technique that could identify proteins RNA interference (RNAi) is an indispensable antiviral defense mechanism in in- based on their fingerprint at the single-molecule level. In the novel method, sects including mosquitoes that transmit human diseases such as dengue or Zika proteins will be identified based on two types of amino acids only instead of fever. To escape the cellular defense, viruses employ various proteins called viral all 20 types. In the present study we demonstrate since-molecule FRET suppressors of RNAi (VSRs). VSRs are thought to suppress the RNAi pathway at detection of an acceptor labeled protein substrate by a donor labeled protein several different levels. This interaction precludes the recognition and elimina- analyzer ClpXP. This highly sensitive approach holds promise to detect the tion of viral RNA, leading to viral survival and proliferation in detriment of the least abundant proteins of a cell, that can have high value for biomedical host organism. Despite a decade of research on VSRs, how VSRs antagonize sciences. RNAi remains incompletely understood. Here we employed single-molecule fluorescence techniques to investigate how VSRs inhibit viral RNA recognition by Dicer-2, a key enzyme in RNAi antiviral defense. Our single-molecule data 744-Pos Board B509 showed that the majority of VSRs tested directly interact with double-stranded Investigating the Mechanism of Ultra-Fast Energy Transfer between RNA (dsRNA), a viral replication intermediate. The stable binding inhibits Venus Oligomers using Time-Resolved Anisotropy, Fluorescence Correla- recognition of viral dsRNA by Dicer-2 and consequently suppresses the RNAi tion Spectroscopy, and Photon Antibunching Youngchan Kim1, Grace H. Taumoefolau1, Tuan A. Nguyen1, antiviral pathway. The length of the double-stranded stem region is a crucial 1 2 1 feature in forming a stable interaction between viral dsRNA and VSRs. Henry L. Puhl , Paul S. Blank , Steven S. Vogel . 1NIH/NIAAA, Rockville, MD, USA, 2NIH/NICHD, Bethesda, MD, USA. FRET experiments using Fluorescent proteins (FPs) typically assume that indi- 741-Pos Board B506 vidual FPs act independently (i.e. weak coupling limit). However, several Dual Role of Munc13 in Regulating SNARE Assembly for Fast Neuro- recent studies have observed that assemblies of FPs can act as a single photon transmitter Release source, indicating that FPs in these complexes interact in the strong coupling Ucheor Brandon Choi. limit and may explain our observation of ultra-fast (<6 ps) energy transfer be- Molecular and Cellular Physiology, Stanford University, Stanford, CA, USA. tween FPs in Venus oligomers. To investigate the mechanism of this ultra-fast

BPJ 7746_7748 152a Sunday, February 12, 2017 energy transfer, time-resolved anisotropy was used to measure the dynamics of DNA at all developmental stages, but binding to DNA changes from transient energy transfer between fluorescent molecules, fluorescence correlation spec- to stable within the first twelve cell division cycles. This kinetic transition troscopy (FCS) to measure the number of fluorescent molecules diffusing in temporally coincides with the delayed onset of transcription in the Zebrafish the focal volume, and photon antibunching (AB) to determine the number of embryo. Our single molecule experiments reveal a potential mechanism of independent fluorescent emitters in the focal volume. Because the number of an important step in Zebrafish embryo development. fluorescent molecules measured by FCS is not necessarily equal to the number of independent emitters, a comparison of these values can provide insight into 747-Pos Board B512 the coupling strength required for ultra-fast energy transfer. We studied three A Hidden Markov Model Approach to Measure Two-State Diffusion of FP constructs; Venus monomers (V1), Venus dimers separated by a 5 flexible Thermobifida Fusca Cellulases 1 2 amino acid linker (V2, ~2 nm separation), and Venus dimers separated by a Markus Rose , Jose Moran-Mirabal . 1Department of Physics and Astronomy, McMaster University, Hamilton, semi-rigid linker containing 4 fibronectin type III domains (VFNV, ~14 nm 2 separation). Ultra-fast energy transfer and homo-FRET was only observed in ON, Canada, Department of Chemistry, McMaster University, Hamilton, V2. Thus, close proximity, even via a flexible linker, is correlated with ultra- ON, Canada. fast energy transfer. FCS and AB measurements of systematic dilutions of these The hydrolysis of crystalline cellulose by cellulases is a bottleneck in the gen- three constructs revealed that while VFNV has two independent emitters per eration of fermentable sugars, which is an important step in biofuel production. molecule, V2 has less than two and because V1 is a monomer, each V1 mole- However, the detailed interaction between the enzymes, i.e. catalytic domain cule detected must have one independent emitter. These results suggest that (CD) and carbohydrate binding module (CBM), and cellulose remains unclear. ultra-fast energy transfer requires strong coupling between FPs, and implicates In this work, the motion of Thermobifida fusca cellulases Cel5A, Cel6B and proximity as an essential factor in the mechanism. Cel9A on cellulose fibrils is observed through single-molecule fluorescence im- aging. A tracking algorithm provides trajectories, which display distinct sta- 745-Pos Board B510 tionary states as well as states of fast motion. A hidden Markov model, Plasma Membrane Organization and Dynamics is Probe and Cell Line implemented with a Markov chain Monte Carlo algorithm, separates the motion Dependent: An Imaging FCS Study into states of fast and slow diffusion and calculates the corresponding state- 1 2 3 2 switching probabilities. The results show a slow diffusion coefficient Thorsten Wohland , Shuangru Huang , Shi Ying Lim , Anjali Gupta , 4 2 1 1 2 1 Nirmalya Bag2. D1=10 um s and a fast diffusion coefficient D2=10 um s , with calcu- 1 lated state-switching probabilities of p12=4% and p21=45% respectively. D1 Biological Sciences and Chemistry, National University of Singapore, 1 Singapore, Singapore, 2Biological Sciences, National University gives an average diffusion speed of 20nm s which corresponds to the local- of Singapore, Singapore, Singapore, 3Chemistry, National University of ization uncertainty and therefore incorporates immobility as well as processive Singapore, Singapore, Singapore. hydrolysis. Although different binding combinations of CBM, CD and linker to The plasma membrane is a complex assembly comprising hundreds of different cellulose make for a complex system, we propose a simplified model of three lipids and proteins. Its properties are temperature dependent and change from observable states: A) a bound stationary state where the cellulase CD is cell line to cell line. This raises the question whether measurements at different engaged and directly interacts with the cellulose, resulting in limited motion temperatures and within different cell lines are comparable even when using and a slow diffusion coefficient, B) a bound state where the CD is disengaged, the same probe. Here, we investigate the dynamics and organization of five but the enzyme is otherwise loosely bound to the cellulose, e.g. via the CBM, different cell lines - Chinese hamster ovary (CHO-K1), Hela, neuroblastoma leading to a mobile state with a faster diffusion, C) a completely disengaged (SH-SY5Y), fibroblast (WI-38) and rat basophilic leukemia (RBL-2H3) - using enzyme that can freely diffuse - a state that is too fast for observation. With this approach we have developed a method to correlate the observed single- up to four different lipid probes. For the outer membrane leaflet we use DiI-C18, a common liquid disordered marker and GFP-GPI, a GFP tagged glycosylphos- molecule motion with the underlying interaction between cellulases and the phatidylinositol anchored protein as a marker for the liquid ordered phase. For cellulose substrate. the inner membrane leaflet we use PH-PLCd-RFP, a PIP2 binding domain, and PMT-GFP, a GFP tagged plasma membrane targeting domain that had been Micro- and Nanotechnology I shown to be sensitive to the cytoskeleton. We applied Imaging Fluorescence 748-Pos Board B513 Correlation Spectroscopy (imaging FCS) on a Total Internal Reflection Micro- Biomolecule Transport across Droplet Interface Bilayer Networks scope (TIRFM) which provided diffusion coefficients (D), the Arrhenius acti- Heather E. Findlay, Grant Pellowe, Paula J. Booth. vation energy for diffusion (E ), and the FCS diffusion law intercept (t ), Arr 0 Chemistry, Kings College London, London, United Kingdom. which report on membrane fluidity, molecular packing, and diffusion mode An important aspect of biochemical reactions as they occur in vivo is the of the probes (free, domain, or hop diffusion), respectively. The combination sequential and spatial control that arises from the compartmentalisation pro- of these parameters is unique for each of the probes. Our results showed, vided by the membranes of cells and organelles within them. Integral mem- that each of the labels is characterized by a unique set of parameters (D, brane proteins control the traffic of ions and biomolecules across these E , t ). Furthermore, the parameters do not stay constant for a particular Arr 0 membranes and the lipid composition of the bilayer in turn influences the struc- probe or change in a concerted manner, but can change individually from ture, function and stability of the proteins. Artificial systems such as Droplet cell to cell line, indicating that the organization and mode of diffusion of these Interface Bilayers are being developed that have the potential to replicate molecules depends strongly on the environment. This variability has important this feature, where sub-microlitre aqueous droplets are surrounded by a lipid implications for membrane measurements in general and the comparability of monolayer and assembled together to form bilayer regions at the connections. measurements is only given in a particular cell line and at a given temperature. Here, we seek to functionalise a simple droplet network by the incorporation of different channels and transporters and by varying the selection of the compos- 746-Pos Board B511 ite synthetic phospholipids, allowing for the selective transport of substrates be- Single Molecule Imaging of Transcription Factor-DNA Interactions in tween compartments. Zebrafish Development Matthias Reisser1, Shai R. Joseph2, Nadine L. Vastenhouw2, 749-Pos Board B514 J. Christof M. Gebhardt1. Hierarchically Layered Platform for the Formation of Free-Standing 1Department of Physics, Ulm University, Ulm, Germany, 2Max Planck Lipid Bilayer Membrane Institute of Molecular Cell Biology and Genetics, Dresden, Germany. Hyunil Ryu1,2, Sangbaek Choi1,2, Sun Min Kim2,3, Tae-Joon Jeon1,2. Cellular tasks such as transcription rely on stochastic interactions of biomole- 1Biological Engineering, Inha University, Incheon, Korea, Republic of, cules. Thus, single molecule methods are beneficial in revealing the kinetic and 2Biohybrid Systems Research Center (BSRC), Inha University, Incheon, structural underpinnings of these tasks, and consequently single molecule Korea, Republic of, 3Department of Mechanical Engineering, Inha tracking has been developed for live cells and organisms. Yet, following indi- University, Incheon, Korea, Republic of. vidual fluorescently labeled biomolecules throughout the development of an Free-standing lipid membranes are commonly used in biophysical applications organisms is still challenging. and they often require physically and chemically controlled conditions. They Here, we establish single molecule tracking of genetically encoded fluorescent are traditionally formed over small apertures in Teflon thin film by manually proteins in live developing Zebrafish embryos using reflected light-sheet micro- ‘‘painting’’ a droplet of organic solvent containing lipid molecules. However, scopy. In addition, we design a novel time-lapse acquisition scheme allowing as the conventional technique requires highly skilled expertise, its use has for fast quantification of biomolecular interaction kinetics. We use our methods been limited to only the research field. To resolve these issues, frozen mem- to monitor and quantify the interactions between the general transcription fac- brane precursor with expedited self-assembly (MPES) that uses hydrophobic, tor TBP and DNA during embryo development. We find that TBP interacts with porous material film to absorb solvent for expediting self-assembly was devised

BPJ 7746_7748 Sunday, February 12, 2017 153a with a few limitations (Choi et al., Journal of Visualized Experiments, e54258- transmembrane ion flux. Here, we show that the reaction between the azide e54258, 2016). In this study, we have improved and optimized the MPES using of the peptide and the DBCO of the lipids increases the potency of the pore- a hierarchically layered platform. forming peptide, resulting in pore-forming activity at peptide concentrations approximately ten times lower than the usual concentration required for activity 750-Pos Board B515 without conjugation. We also show that the peptides and the lipids are indeed Dynamics and Energetics of Phage T4 Injection Machinery linked covalently by observing channel activity for several hours after replac- 1 2 2 Ameneh Maghsoodi , Anupam Chatterjee , Ioan Andricioaei , ing the buffer solution in the aqueous compartments. This work suggests that Noel C. Perkins1. 1 click reactions could be used for targeting cellular membranes after their Department of Mechanical Engineering, University of Michigan, Ann site-specific activation with a bio-orthogonal conjugation reagent. Arbor, MI, USA, 2Department of Chemistry, University of California, Irvine, Irvine, CA, USA. 753-Pos Board B518 Bacteriophage T4 is one of the most common and complex tailed viruses from Identifying Peptide-Peptide Interactions with Lysenin Nanopores the family Myoviridae, and it infects E. coli using a highly efficient contractile Christopher A. Thomas1, Nisha Shrestha1, Juliette Tinker1, genome delivery machine. Phage T4 possesses a multi-protein capsid contain- Devon Richtsmeier1, Sheenah Bryant1, Xinzhu Pu2, Daniel Fologea1. ing the genomic DNA and a long contractile tail assembly that consists of a 1Department of Physics/Biomolecular Sciences Graduate Program, Boise rigid tail tube surrounded by a six helical-stranded sheath. The tail assembly State University, Boise, ID, USA, 2Biomolecular Research Center, Boise transmits the genomic DNA from the capsid to the host during injection. While State University, Boise, ID, USA. the atomistic structure of phage T4 has been studied extensively using Cryo- Numerous peptides have been identified as biomarkers, providing valuable in- EM and X-Ray crystallography, the dynamics of the injection process is not formation about diseases such as cancer, heart disease, rheumatoid arthritis, well understood. Questions remain regarding the energetics of injection as Alzheimer’s, and kidney disease. Similarly, the analysis of peptides has well as the pathway and time scale of the large conformational change of the emerged as a powerful tool for various diagnostic, therapeutic, and biomedical sheath. In this study, we employ a dynamic multi-scale model to address these applications. In this context, there is a tremendous need for investigating and fundamental questions. To this end, a two-stage solution process is employed; characterizing peptides beyond the established methods such as mass spectrom- first, a molecular dynamics (MD) simulation is carried out to estimate the etry, fluorescence spectroscopy, and immune staining. These methods can be elastic properties of the sheath strands. Second, a continuum model is devel- labor intensive, time-consuming, require sophisticated instruments, and can oped for the entire-assembled T4 injection machinery using the elastic con- suffer from low sensitivity. As an alternative approach, we propose to use stants estimated from MD. The resulting multi-scale model simulates the the biological nanopore lysenin for peptide identification and characterization dynamics of the sheath represented by six interacting helical strands that are at the single-molecule level. Lysenin is a nonameric pore-forming protein ex- coupled to the capsid represented by a massive cylinder. Dynamic simulations tracted from the earthworm E. foetida which self-inserts large conducting pores reveal the time scale, the pathway of sheath contraction, and the energetics into natural and artificial lipid bilayer membranes containing sphingomyelin. driving the injection process. While these results are specific to T4 as an Using the resistive-pulse technique, we investigated the translocation of the example, the resulting model and methodology may also inform the future Trp-Trp-His-Pro-Cys peptide, which can exist either as a monomer or dimer design of nanotechnology injection devices. due to the cysteine group that can form a disulfide bond. The electronic signa- 751-Pos Board B516 tures generated during the translocation of the peptide through single lysenin Using Magnetic Field to Purify Membrane Proteins in Supported Cell channels indicate the ability of lysenin to differentiate between the monomer Plasma Membranes and dimer form. Similarly, translocation of another peptide, His-Met-Trp- Kai-Hung Hsiao, Ling Chao. Trp-Met, which lacks cysteine and hence is only present as a single monomer, National Taiwan University, Taipei City, Taiwan. presented the electronic signature of a single monomer. Mass spectrometry Processing and separating membrane proteins in their native bilayer environ- analysis of the samples collected in the presence of large populations of lysenin ment still remains challenging. The conventional methods to purify membrane channels for an extended time period confirmed the translocation of peptides proteins for characterize require detergents or harsh chemicals, which may across the artificial bilayer. These results indicate that lysenin pores can be cause the membrane protein to denature. Here, we developed a supported used for peptide detection, identification, characterization, and hence can plasma membrane platform and a magnetic tweezer to purify the target mem- contribute to various biomedical and therapeutic applications. brane proteins. We deposited giant plasma membrane vesicles (GPMVs) directly derived from Hela cells on a polymer cushioned support and filled 754-Pos Board B519 the region which was not covered by the GPMV patch with artificially prepared Sensing ssDNA Molecules with Single Lysenin Channels lipid membrane in order to form a continuous membrane platform. We added Philip Belzeski, Nisha Shrestha, Sheenah Bryant, Juliette Tinker, magnetic beads coated with antibodies to fish the target membrane proteins and Christopher Alex Thomas, Devon Richtsmeier, Daniel Fologea. used the developed magnetic tweezer to move the beads with the membrane Department of Physics/Biomolecular Sciences Graduate Program, Boise proteins to the desired region for the collection and purification purpose. We State University, Boise, ID, USA. applied a polymer cushion at the support to prevent some membrane proteins In this work we exploit the use of wild-type lysenin as a stochastic sensor for from being stuck to the support. The continuous membrane allowed us to characterization of ssDNA molecules. Lysenin, a potent pore-forming toxin, move the membrane proteins in the 2-D bilayer platform so that the membrane self-oligomerizes into lipid membranes to form large conductance pores. The proteins can be always kept in their native bilayer environment, which reduces large size of the pore (~3 nm) is deemed suitable for translocation of charged the possibility for them to denature or aggregate. Our preliminary result showed macromolecules driven by electric fields. In our experiments, we exploited the that we can collect a native membrane protein in Hela Cells, aquaporin3, from biophysical properties of lysenin channels to build a sensing device based on our supported plasma membrane platform. the long-revered resistive pulse technique, largely used for single molecule analysis by employing either synthetic or biological nanopores. We inserted 752-Pos Board B517 a single lysenin channel into a planar bilayer lipid membrane composed of Targeting Specific Membranes with the Pore-Forming Peptide Cerato- neutral lipids (to avoid voltage-induced gating at positive voltages) and bathed toxin a using Click Chemistry by 1M KCl solutions. Addition of 69 nt DNA molecules into the reservoir sit- Simon F. Mayer1, Aziz Fennouri1, Jerry Yang2, Michael Mayer1. uated at the trans side of the protein channels did not induce any transients of 1Adolphe Merkle Institute, Fribourg, Switzerland, 2Department of Chemistry the open current irrespective of the orientation of the transmembrane electric and Biochemistry, University of California San Diego, San Diego, CA, USA. field, indicative of absence of translocation. However, addition of ssDNA to Targeting specific cellular membranes with selective pore-forming molecules the reservoir bathing the cis side of the protein yielded multiple transients in is a major component of the innate immune response of all mammals. Here the open current, resembling the translocation pattern reported for synthetic we explore the potential of a pore-forming peptide to specifically target lipid and biological nanopores. In addition, the transients have been observed only membranes that were activated by a click reagent. With this idea in mind we when the electric field orientation was such that the DNA molecules were elec- modified ceratotoxin A (CtxA), a 36-amino acid peptide found in the medfly trophoretically driven toward the nanopore. The analysis of the translocation Ceratitis capitata, at its N-terminal region to bear an azide group. When intro- events yielded a sharp distribution of the current blockages induced by the pass- duced into a solution in contact with a lipid bilayer containing dibenzocyclooc- ing molecules. In contrast, the dwell time distribution, although indicative of tyne (DBCO) moieties on the lipid headgroups, a spontaneous alkyne-azide translocation speeds much lower than other nanopores, was relatively broad, cycloaddition covalently links the peptide to the bilayer. CtxA and its modified suggesting strong interactions between the channel and ssDNA molecules. version (CtxA-azide) form pores according to the barrel-stave model similar to PCR performed on the translocated molecules and electrophoresis demon- alamethicin, displaying well-defined characteristic conductance states for strated that the DNA molecules may thread lysenin pores. This work may

BPJ 7746_7748 154a Sunday, February 12, 2017 contribute to further development of single molecule biosensors, fast DNA and concentration and storage of scarce biomolecular species, on-demand chemical protein sequencing, And single molecule characterization. reactions and nanopore sensing. 755-Pos Board B520 758-Pos Board B523 Solid State Nanopore Kit for Real-Time Analysis of DNA and Other Anomalous Ionic Conductance in Carbon Nanotube Nanochannels Analytes Steven F. Buchsbaum1, Shirui Guo1, Eric Meshot1, Preston Hinkle2, Federico Thei1, Michele Rossi1, James Yates2, Marco Bennati1. Anh Pham1, Zuzanna Siwy2, Francesco Fornasiero1. 1Elements SRL, Cesena, Italy, 2Nanopore Solutions LDA, Oeiras, Portugal. 1Physical and Life Sciences, Lawrence Livermore National Laboratory, Recent improvements in nanofabrication techniques have greatly increased the Livermore, CA, USA, 2Physics, University of California, Irvine, Irvine, reproducibility and stability of nanopores fabricated in ultrathin dielectrics such CA, USA. as silicon nitride or silicon oxide. These nanopores are now suitable for a wide Simulations and experimental studies have reported an unusually high ionic range of accurate and reliable molecular analyses. conductance in carbon nanotube (CNT) nanochannels. The origin of this phe- Here we present a compact, portable and easy to use nanopore kit, intended for nomenon is, however, poorly-understood: literature reports often disagree in laboratory experiments and development of prototype devices. Experiments the magnitude of the different transport mode contributions to the measured range from simple nanopore conductance and stability determination, to the ionic current and even in what ions are actually carrying the current; moreover, more complex analysis of translocations of DNA and other analytes (resistive results obtained with single pore measurements differ frequently from those pulse sensing/nanopore spectroscopy). The kit consists of: with membranes containing billions of open CNT channels, i.e. the average -A Teflon flow-cell with two 1.0 ml chambers, hosting a central cassette con- CNT behavior. Toward shedding light on these phenomena, we fabricated a taining the silicon chip with the nanopore. novel nanofluidic platform having a >30-nm wide, FIB-nanomachined silicon -A miniaturized low-noise and high bandwidth amplifier. nitride nanopore (SiNx) in series with vertically-aligned sub-5 nm carbon nano- The entire kit is contained within a 16x10x5 cm Faraday cage and connects to a tubes, the number of which can be controlled from one to billions. By employ- laptop via a USB cable. ing this platform with only one or a few open CNTs, we observed giant ionic The real-time analyses are performed using the amplifier control software, currents in CNT channels and a power-law increase of conductance with enabling a fast characterization of nanopore conductance, production of I/V KCl concentration (G ~ cn, n=0.1-0.4), a dependence that seems to be unique curves and production of histograms for blockade sizes and dwell times. of CNT pores. A few literature reports attributed this giant ionic current in The kit was tested under different conditions, using nanopores in the range from CNTs to a strong electro-osmotic flow. To quantify electro-osmosis in CNT 5 to 50nm. A 3,000bp fragment of DNA was successfully translocated on a pores, we investigated translocation of neutral molecules in a single CNT nano- 6 nm nanopore. A 48,500bp phage lambda DNA was successfully translocated channel with the resistive pulse technique. Furthermore, we employed finite on a 11 nm nanopore. Translocations were performed between potentials of element analysis to elucidate the relationship between CNT pore characteristics 200mV and 380mV in 1M KCl buffer, 10mM HEPES pH 8.0. (e.g., size, surface charge, slip length) and the resulting ionic transport and The versatility of the nanopore kit combined with the real-time analysis feature magnitude of the electro-osmotic component. We confirmed computationally allows for quick and easy characterizations of nanopores and their interaction that the unusually large conductance in our experimental platform results with analytes, for both educational and research purposes. from the presence of a strong electro-osmotic coupling between the CNT chan- nel and the SiNx nanopore in series with it. Finally, we apply first-principle sim- 756-Pos Board B521 ulations to show that cation-CNT interactions may help explain the origin of Ionic Transport Property under an Extremely High Electrical Field in a this electro-osmotic flow. Thin Pore Pinyao He, Kun Li, Kabin Lin, Zhongwu Li, Haojie Yang, Yunfei Chen. 759-Pos Board B524 Southeast University, Nanjing, China. Role of Solid State Nanopore Size and Charge on Molecular Transport Abnormal conductivity of electrolytic solutions is observed under an Kinetics extremely high electrical field, this phenomenon is called the Wien effect. Meni Wanunu. Generally, alternating voltage is applied on the electrodes because it is Physics, Northeastern University, Boston, MA, USA. intractable to produce a direct voltage as high as 100,000 volts. However, Nanopore-based detection of macromolecules has gained interest in recent extremely high electrical field which is akin to the high alternating electrical years, because of the enormous potential of single-molecule analysis at ultra- field could be generated by several-volt direct voltage if DC voltage is applied low analyte concentrations. In this work, we describe how nanopores can be on such pore whose thickness is approaching nanoscale. Through our exper- used to trap, manipulate, and study individual bimolecular complexes with con- iment increasing conductivity of electrolytic solutions is observed when trol over the applied force and other experimental parameters, such as temper- increasing the DC voltage over 2 volts. Additionally, we propose some hy- ature and pressure. Key to our results is the combination of ultrathin nanopores pothesis to account for the result. We speculate the mode of ionic transport and high-bandwidth electronics, which together enable high-resolution mea- will convert to a novel mode under very high electrical field. Our findings surements. In addition, characterizing the pore’s surface charge enables more demonstrate that DC voltage could produce similar phenomenon to the quantitative assessment of the role of electroosmosis on molecular capture Wien effect, and could help to explore the property of ionic transport under and transport. We will discuss specific examples of molecular transport on extreme conditions. well-characterized nanopores in different materials. Our results represent a key advance towards more quantitative macromolecular characterization at 757-Pos Board B522 the single-molecule level. Defect-Guided Transport of Biomacromolecules Manish Shankla1, Aleksei Aksimentiev2. 760-Pos Board B525 1Biophysics, University of Illinois at Urbana-Champaign, Champaign, IL, Structural Characterization of Vascular Endothelial Growth Factor by USA, 2Physics, University of Illinois at Urbana-Champaign, Champaign, Solid-State Nanopores IL, USA. Nitinun Varongchayakul1, Mark Grinstaff1, Amit Meller2. Defects in the structure of solid-state materials often deteriorate their functional 1Biomedical Engineering, Boston University, Boston, MA, USA, properties yet sometimes, they may grant the materials a new functionality. 2Biomedical Engineering, Technion Israel Institute of Technology, Haifa, Graphene, one of the most versatile 2D nanomaterials does not lack imperfec- Israel. tions. In fact, one of the most common graphene fabrication procedures - me- A novel solid-state nanopore sensing technique is described to study vascular chanical exfoliation - produces terraced islands of multi-layered carbon sheets. endothelial growth factor (VEGF). VEGF is a cytokine that stimulates vascu- Using all-atom molecular dynamics simulations, here, we show that such de- larization and is used as a cancer biomarker. Its biological activity depends fects in graphene can be used to direct the transport of adhered bio- on the isomerization state. Dimeric VEGF facilitates dimerization of the macromolecules. Our key observation is that, subject to an external force, a VEGF receptor which initiates the signal transduction pathway. Monomeric single-stranded DNA molecule moves much faster down a step-like defect VEGF consists of two domains; a VEGF receptor recognition domain and a on a graphene surface than against the defect, and even faster along the defect’s heparin binding domain, separated by a peptide sequence cleavable by plasmin. edge. We find the effect to be robust regardless of the nature of the force used to Translocation of VEGF through a 4 to 6 nm-diameter pore, fabricated via trans- drive the motion of the biomolecules (mechanical or electrokinetic), the height mission electron microscope sculpting on an thinned 10 nm free-standing SiN of the defects or its chemical termination. As a possible practical application of membrane, produces a structural-dependent electronic signature. By fine- the effect, we demonstrate a system for capturing and transporting a DNA tuning the pore’s geometry and experimental conditions, VEGF translocation molecule toward a graphene nanopore. Such defect-directed transport can be consistently produces multi-step patterns, which are used to differentiate mono- utilized for building two-dimensional nanoscale highways for precise delivery, meric and dimeric isoforms. The equilibrium dissociation constant depends on

BPJ 7746_7748 Sunday, February 12, 2017 155a concentration and pH. Further study with TCEP and plasmin show that the step synthetic versions are made from DNA which is ideal for predictable pattern corresponds to the translocation of individual protein domains. These de-novo design. The DNA channels are composed of six hexagonally results demonstrate the potential for nanopores to detect small proteins and arranged, interlinked duplexes that enclose a 2 nm-wide lumen. Lipid an- its capability to resolve the higher-order structure of proteins at the single- chors hold the negatively charged channels in the membrane[1–3] to form molecule level. an electrical seal required for electrical recordings[1, 4]. One DNA version mimics the function of biological ligand-gated ion channels. The gate of 761-Pos Board B526 the synthetic channel is a DNA strand that blocks the lumen, but a specific Solid-State Nanopore Detection of Hydrophobic Proteins DNA ligand can re-open the channel[1] (see figure). The pore can also distin- Adam R. Hall, Dhruba Jyoti Basu Roy. guish with high selectivity the transport of small-molecule cargo that differs Biomedical Engineering, Wake Forest School of Medicine, Winston-Salem, by the presence of a positive or negative charge. The synthetic analogue NC, USA. may be used for controlled drug release and the building of cell-like We report on a modified solid-state nanopore measurement scheme to probe networks. hydrophobic molecules that cannot be studied in conventional systems. Taking Related DNA channels show other hallmarks of the biological templates such advantage of the intrinsic alcohol solubility of LiCl as an electrolyte, we show as voltage-gating at high transmembrane potentials[1, 4, 5]. The artificial pores that the devices can be operated in alcohol-based azeotropic mixtures. We first can furthermore be programmed to function as cytotoxic agents by killing can- characterize nanopore conductivity as a function of ethanol content, nanopore cer cells via membrane-rupturing[7]. The synthetic pores expand the range of diameter, and salt concentration, showing predictable ionic response. Then, as a other DNA nanostructures that mimic biological functions of membrane pro- demonstration of resistive-pulse sensing, we measure and interpret electrical teins to control bilayer and cell shape[6]. translocations of zeins, a class of water-insoluble maize protein. References: 762-Pos Board B527 [1] J. R. Burns et al, Nat. Nanotechnol. 2016, 11, 152. The Nanometric Golden Ratio: The Relation between Gold Volume and [2] J. Burns et al, Nano Lett. 2013, 13, 2351. Nanopore Diameter [3] J. R. Burns et al, Angew. Chem. Int. Ed. 2013, 52, 12069. Lennart J. de Vreede. [4] A. Seifert et al, ACS Nano 2015, 9, 1117. Adolphe Merkle Institute, University of Fribourg, Fribourg, Switzerland. [5] V. Maingi et al, ACS Nano 2015, 9, 11209. In this work we build further on a recently discovered nanopore manufacturing [6] S. Howorka, Science 2016, 352, 890. scheme which is based on the heating of nanometric gold particles on a silicon [7] J. R. Burns et al, Angew. Chem. Int. Ed. 2014, 53, 12466. oxide surface. When heated to 1064C the lithographically patterned and sput- 765-Pos Board B530 tered gold particles dewet and create nanopores into the silicon oxide surface. Slowing Down DNA Translocation using Integrated Nanopore and Nano- The three leading phenomena which allow this nanopore creation are; a differ- pillars Precisely Deposited by Helium Ion Beam ence in the surface energies of the gold and the silicon oxide, mobility of the Yunsheng Deng, Qimeng Huang, Daming Zhou, Shuo Zhou, Liyuan Liang, silicon oxide and evaporation of the gold. [1] This approach promises to be Shaoxi Fang, Wanyi Xie, Shixuan He, Peng Tang, Deqiang Wang. relevant in the field of proteomics, since a transfer of this technique to silicon Chongqing Institute of Green and Intelligent Technology, Chinese Academy oxide or silicon nitride membranes allows the creation of nanopore through of Sciences, Chongqing, China. holes. [1] [2] Nanopores, either in singular or in array configuration, made in Slowing down DNA translocation speed in a nanopore is essential to real- silicon oxide or silicon nitride membranes could form interesting tools, for izing high resolution of individual bases. In this study, a uniform array of example, for the characterization of proteins. Platinum (Pt) and Silicon Oxide (SiO ) nanopillars created with Helium Currently however, it is not clear which volume of gold, combined with which x ion beam is introduced onto the trans side of Silicon Nitride layer to heat treatment, is needed to create which nanopore diameter. We conducted ex- slow down DNA translocation. Both of Pt and SiO nanopillars show a periments in which a silicon oxide surface is coated with a photosensitive layer x capability of great magnitude deceleration of dsDNA translocation. And and patterned by using deep ultraviolet (DUV) lithography and sputtered with a the periodic space and diameter of nanopillars are precisely controlled, layer of gold. The created nanometric gold volumes are measured and subse- enabling further investigation of the interaction between pillars and the quently heated to 1064 C. After cooling down the substrates are cleaved and dsDNA. Pt and SiO Plliars have different surface properties, hence we the nanopore diameters are measured. DUV lithography is used because it x could explore the different interaction mechanism between Pt/SiO Plliars has the ability to expose and develop features of 200 nm in diameter, thus al- x and dsDNA. lowing accurate volumetric variance when using different sputtering times. [1] Nanopore fabrication by Heating Au-Particles on Ceramic substrates, Nano 766-Pos Board B531 Lett. 15:727-731, 2015 [2] Nanopore fabrication in silicon oxynitride mem- Single-Channel Measurements of Conductance through Sub-Nanometer branes by heating Au-Particles, J. Micromech. Microeng. 26:037001, 2016. Carbon Nanotube Porins 763-Pos Board B528 Yun-Chiao Yao1,2, Robert Henley3, Ramya Tunuguntla2, Meni Wanunu3, Multiple-Nanopores Fabrication based on Dielectric Breakdown Aleksandr Noy1,2. 1 Yunlong Wang, Cuifeng Ying, Wenyuan Zhou, Zhibo Liu, Jianguo Tian. Chemistry and Chemical Biology, School of Natural Sciences, University of 2 NanKai University, TianJin, China. California, Merced, CA, USA, Biology and Biotechnology Division, Single molecule detection based on nanopore technology is becoming more and Physical and Life Sciences Directorate, Lawrence Livermore National 3 more established recently. Due to the Coulter counter and ion channels detec- Laboratory, Livermore, CA, USA, Department of Physics, Northeastern tion principle, single nanopore suffers from limitation of low throughput. University, Boston, MA, USA. Thus high-throughput nanopore detection based is a definite direction of devel- Carbon nanotubes (CNTs) have narrow hydrophobic inner pores, highly opment. Dielectric breakdown, a novel method reported by Tabard-Cossa reminiscent of protein channels, that enable fast transport of water and recently, shows advantages for low-cost and high fabrication efficiency ions. Their rigid structure and chemical robustness allow for diverse ex vivo compared to previous transmission electro microscopy (TEM) and focus ion applications. We have recently reported biomimetic membrane channels beam (FIB) drilling. Here we show that the by means of dielectric breakdown, cased on carbon nanotubes—carbon nanotube porins (CNTPs). We have several nanopores can be formed in one SiN membrane. By controlling the shown that CNTPs self-insert into lipid membranes and form stable pores. experiment conditions, we explore the number and diameters of nanopores In this study, we used electrophysiological single-channel measurements fabricated by our methods. Although it is still a challenge in making nanopore to quantify ion conductance and selectivity of 0.8 nm diameter CNTPs. We arrays due to the random formation, we show a simple and convenient method report the conductance of single CNTP channels, as well as the scaling to fabricate two or three nanopores at the same time. of the conductance with the ion concentration. We also used the reversal potential measurements to characterize the selectivity of the ion transport 764-Pos Board B529 and found that CNTPs are highly selective to cations over anions. We will Artificial DNA-Based Channels for Controlled Membrane Transport discuss the mechanism of this selectivity, and demonstrate ways to control Jonathan R. Burns1, Astrid Seifert2, Niels Fertig2, Stefan Howorka1. it. These findings are important and relevant for developing CNTPs into 1UCL Chemistry, London, United Kingdom, 2Nanion Technologies GmbH, a potential candidate nanopore for water treatment applications. Our Munich, Germany. studies on intrinsic characterization of CNTP transport properties will also We describe synthetic membrane channels that control transport of molecu- enable researchers to consider this new material for a variety of biophysical lar cargo across a lipid bilayer[1]. Unlike biological protein channels, the applications.

BPJ 7746_7748 156a Sunday, February 12, 2017

767-Pos Board B532 Nanotechnol. (2015), 11 (9), 1653–1661. [3] Bogdanovic, G., Djordjevic, A. Tunable Ion Selectivity in Sub-Nanometer Diameter Carbon Nanotube Srp. Arh. Celok. Lek. (2016), 144 (3–4), 222-231. Porins Robert Y. Henley1, Yun-Chiao Yao2, Ramya Tunuguntla2, 770-Pos Board B535 Pradeep Waduge1, Meni Wanunu1, Aleksandr Noy2. The Influence of Surfactant Structure and Geometry on Nanotoxicity and 1Physics, Northeastern University, Boston, MA, USA, 2Lawrence Livermore Dispersion of Carbon Nanotubes 1 _ 1 1 National Lab, Livermore, CA, USA. Jakub Zare˛ba , Justyna Izykowska , Michalina Skupin , Augustyn Molinski 1, Maria Dobies1,2, Stefan Jurga1,2, Maciej Kozak1,3. Carbon nanotube porins (CNTPs), ultra-short carbon nanotubes (5-20nm) that 1 can self-insert into lipid bilayers, allow single-channel planar lipid bilayer mea- Department of Macromolecular Physics, Adam Mickiewicz University, 2 surements of ionic transport through carbon nanotube (CNT) pores. CNTs with Poznan, Poland, NanoBioMedical Center, Adam Mickiewicz University, 3 sub-nanometer diameters have been predicted to display ultra-fast water trans- Poznan, Poland, Joint Laboratory for SAXS Studies, Adam Mickiewicz port and high levels of ionic exclusion resulting from one-dimensional water University, Poznan, Poland. wire transport through their hydrophobic cores. Here, we show that ultra-short Graphene, nanotubes and fullerenes, also known as carbon nanomaterials (CN), 0.8-nm-diameter CNTPs, can spontaneously insert into lipid bilayers to form because of the their unique properties have potential applications not only in highly cation selective channels that exhibit low ionic conductance. Using preparation of new composite materials or in electronics, but they can be also reversal potential in various asymmetric salt conditions, we observe that these components of drug delivery systems. However, the applications of CN in life CNTPs exhibit large permselectivity values, corresponding to a high selectivity sciences or biomedicine require stable dispersions of these materials in water. for both potassium and sodium over chloride. We also show evidence that The hydrophobic nature of CN causes serious problems with their separation neutralization of charges on the CNTs enhances ion selectivity. We also show in water solutions. Our study is aimed to find a way of dispersing such CN sys- that by using a solid-state nanopore to act as a support and form a completely tems in water, by the use of non-covalent surface modification via different sur- solvent-free bilayer system, we can observe extremely stable ionic currents factants. In this work we focused on the use of trimeric, dimeric and monomeric through these nanotubes, in contrast to previous reports with solvent containing surfactants fornon-covalent surface modification of selected carbon nanotubes. bilayers. These results establish CNTPs as a promising biomimetic platform for The toxicity of these systems (aqueous solutions of CN with surfactants and sur- developing cell interfaces, creating stochastic sensors, and water desalination. factants solutions) towards selected cell cultures (e. g. HeLa cells) was also tested. The surfactants studied can interact with carbon nanotubes in a number 768-Pos Board B533 of different modes: hydrophobic interactions (surfactant chains with CN side p p Sub-1-nm Carbon Nanotube Porins: Water Transport and Ion Selectivity walls) or - interactions (aromatic rings of surfactants with CN surface). in a Single-File Water Membrane Nanopore Infrared spectroscopy and atomic force microscopy were used for characteriza- Aleksandr Noy. tion of the systems studied. Finally, we focused on mechanical properties of cells Biology and Biotechnology Division, Lawrence Livermore National exposed to surfactants and CN. This study was supported by the Ministry of Laboratory, Livermore, CA, USA. Science and Higher Education (Poland), within the project ‘‘Najlepsi z najleps- Living systems control transport of ions or small molecules across biological zych!’’ (DEC - POWR.03.03.00-00-P001/15). membranes using ion channels that form pores in lipid bilayers. Membrane 771-Pos Board B536 pores formed by ultra-short carbon nanotubes (CNTs) assembled in the lipid Designer Peptides Self-Assemble on Graphene to Create Remarkably Sta- membranes have transport properties that come remarkably close to replicating ble, Precisely Organized Substrates the transport properties of biological channels. The defining features of these Gina-Mirela Mustata1, Meni Wanunu2, Gevorg Gregoryan3, nanostructures are their inner pores that have atomically smooth hydrophobic Yong Ho Kim4, Jian Zhang5, William F. DeGrado6. walls, which can confine water on a molecular level, and, in case of 0.8 nm 1Chemistry and Physics, Simmons College, Boston, MA, USA, 2Physics, diameter CNTPs, down to a single-file configuration. We present experimental Northeastern University, Boston, MA, USA, 3Dartmouth College, Hanover, results that demonstrate efficient water transport in CNTPs and explore its NH, USA, 4Sungkyunkwan University, Seoul, Korea, Republic of, physical origins. We also use single pore conductance measurements to demon- 5Chemistry, University of Nebraska-Lincoln, Lincoln, NE, USA, 6University strate ion selectivity in these pores and show that they can be configured into a of California, San Franciso, San Francisco, CA, USA. switchable nanofluidic diode. CNTPs represent a simplified biomimetic system We present a study of designed self-assembly of 2D peptide monolayer crystals that is ideal for studying fundamentals of nanofluidic transport and transport in on the surface of graphene and graphitic interfaces and their properties in various biological channels, and for building complex engineered mesoscale structures biologically significant conditions. Atomic force microscopy imaging of dried that could be the foundation of next-generation water treatment technologies. peptides adsorbed on graphitic surfaces reveals an amorphous monolayer struc- ture that contains voids due to drying. After rehydration, the peptide monolayer 769-Pos Board B534 reorganizes into highly ordered domains comprised by parallel arranged pep- Carbon Nanostructures of Different Spatial Geometry: Their Dispersion tides that are oriented on the graphitic structure with C3 symmetry, in close and Influence on Model Biological Systems agreement with computational predictions. The monolayers are remarkably sta- _ 1,2 1 1 Justyna Izykowska , Michalina Skupin , Weronika Andrzejewska , ble in a wide range of pH, ionic strengths, urea concentrations, and temperatures. Maria Dobies1,2, Stefan Jurga1,2, Maciej Kozak1,3. 1 Importantly, we find that alternating peptides that do not contain aromatic resi- Department of Macromolecular Physics, Adam Mickiewicz University, dues organize similarly, and conclude that aromatic residues are not essential for 2 Poznan, Poland, NanoBioMedical Center, Adam Mickiewicz University, this organization. The monolayers are highly stable to proteolytic digestion Poznan, Poland, 3Joint Laboratory for SAXS Studies, Adam Mickiewicz when full coverage is acquired, while voids in the layer become seeds to slow University, Poznan, Poland. degradation from the void inwards. A striking quality of these substrates is the The discovery of carbon nanostructures (CN) in the last century has revolution- preference to bind double stranded DNA imposing a preferred alignment to ized science, opening new research opportunities in biophysics, material match their own molecular arrangement on the graphene surface. This system sciences, biomedicine and pharmacology [1]. Unfortunately, carbon nanomate- of designed peptide-coated graphene surfaces, with its stability over a wide rials show a strong tendency towards aggregation and poor stability in solutions. range of situations, presents new opportunities for the design of structures and Such properties, especially the suspension stability, are critical for bio- systems that are significant in the study of various biological entities and pro- applications of carbon nanostructures [2]. Surface functionalization of nanocar- cesses, such as specific binding or designed catalysis. bons improves the dispersing properties in water solutions but it may also influence their physicochemical properties [3]. For preparation of biocompatible 772-Pos Board B537 CN samples we used novel dicationic surfactant (gemini) with imidazole head Modulation of Graphene Oxide Probiotic and Antibiotic Activity by Crit- groups. Nanocarbon suspensions were investigated in the context of their anti- ical Coagulation Concentration cancer activity, as well as their application as multimodal contrast/drug delivery Massimiliano Papi1, Valentina Palmieri2, Francesca Bugli3, agents. Different spatial geometries of CN may be of key importance for Maria Carmela Lauriola1, Margherita Cacaci3, Claudio Conti4, designing of the efficient anti-cancer system. Such systems were subjected to Maurizio Sanguinetti3, Marco De Spirito1. the cytotoxicity tests on HeLa cell cultures. The structural properties of selected, 1Physics, Universita` Cattolica del Sacro Cuore, Rome, Italy, 2Physics, the most effective systems based on CN suspensions were characterized by the Universita` Cattolica del Sacro Cuore, Roma, Italy, 3Microbiology Institute, use of SEM, AFM and NMR methods. This study was supported by the Ministry Universita` Cattolica del Sacro Cuore, Rome, Italy, 4Institute for Complex of Science and Higher Education (Poland), within the project ‘‘Najlepsi z naj- Systems, National Research Council (ISC-CNR), Rome, Italy. lepszych!’’ (DEC - POWR.03.03.00-00-P001/15). [1] Krokosz, A. et al., J. Ra- In recent years, significant research related to antibacterial properties and ef- diat. Phys. Chem. (2016), 128, 143–150. [2] Wang, L. et al., J. Biomed. fects on eukaryotic cells of Graphene Oxide (GO) and GO-based materials

BPJ 7746_7748 Sunday, February 12, 2017 157a has been conducted. Despite substantial efforts, results are controversial and a well-defined tail, thus offering us two positions to place a sensor: planar slide framework clarifying all effects reported in literature against bacteria still has and solution. We will first modify the device by introducing a clear fluorescent to be defined. In our work, we demonstrate that by modulating the stability of sensor for oxygen directly beneath the islets. We are currently exploring GO in solution, the antibacterial or growth enhancement effect can been RuII(bpy)3 [Tris(2,20-bipyridyl)dichlororuthenium(II) hexahydrate as an opti- controlled both on S. aureus and E. coli bacteria. We expose these two species cal sensor which has excitation and emission maxima at 450nm, and 600nm, to concentrations of GO ranging from 3 to 200 ug/ml, in different incubation respectively. The ruthenium compound will serve as a seed layer on the base conditions (ultrapure water, PBS, NaCl, MgCl2 and CaCl2). We analyze the of the device that will increase in fluorescence intensity when oxygen levels growth of microorganisms and characterize GO effects on cells with Atomic decrease. Furthermore, we are exploring putting the dye in solution and using Force Microscopy and Colony Forming Units Assay. Our data indicate that other sensors such as palladium and platinum compounds that react to oxygen. any buffer solution utilized during the GO-bacteria interaction alters specif- This device will enable toxicity screening of living pancreatic islets and other ically the GO surface zeta potential and the consequent GO clusters size and types of cells. structure. The GO stability influences antimicrobial activity and, while at low concentration, the sheets cut microorganisms membranes and, at high con- 775-Pos Board B540 centration, complexes between pathogens and aggregates inhibit or enhance Microcantilever Investigation of Nanoconfinement Effects on Water bacteria growth in a surface potential-dependent manner. The main conse- Transport quence of our results is the surprising possibility to finely modulate the GO ef- Michael DeLay. fects on bacteria and to produce versatile applications of GO materials in the Columbia University, Queens, NY, USA. environmental and medical sciences from treatments against multidrug resis- Nanoconfinement of water influences myriad applications including environ- tant bacteria to water remediation systems and probiotic therapies. mental engineering, desalination/filtration, as well as biological activities including membrane transport, signaling/turgor-sensing, and protein folding. 773-Pos Board B538 Understanding confinement effects in nature, outside of in situ SFM and Real-Time Flow Deformability Cytometry via Resistive Pulse Sensing AFM systems, requires consideration of heterogeneous, dynamic enclosures. Preston Hinkle. The nanoporous and water-responsive, peptidoglycan matrix atop the genomic Physics and Astronomy, UC Irvine, Irvine, CA, USA. core of Bacillus spores provides an opportunity to probe water confinement ef- We present resistive pulse and optical data of cancer cells deforming while fects when spores are layered atop an AFM cantilever. By measuring and inte- passing through microsized constrictions. Cell stiffness, the degree to which grating numerous transient nanomechanical responses as well as water mass cells deform under strain forces, is known to be an important physical param- displacements after brief evaporative photothermal pulses, we are able to eter that along with other physical and chemical biomarkers can be used to examine transport kinetics of confined water and observe that spores behave differentiate cell types and enable clinical detection of cancerous cells. Many as a poroelastic material and display an effective viscosity higher than bulk. desirable applications, such as detecting metastatic circulating tumor cells, We then investigate the temperature dependence of these kinetics and find rely on a high throughput of measurements, since the specimen of interest that the activation energy of spore water is higher than bulk and close to ice. may comprise only a total fraction of the total number of particles in a sample. We suggest two possible models for this phenomenon based on spore geome- Capable of measuring 1000s of cells/sec, real-time flow deformability cytom- try: high viscosity water due to total confinement, and decelerated transport due etry is a high throughput method for determining a cell’s stiffness that works by to frequent liquid-vapor-liquid transitions at negative pressure-driven nano- inducing cell deformation under strain forces in the pressure-driven laminar scale cavitations. We note that the slowed confinement kinetics observed could flow through a microfluidic channel. Currently, real-time flow deformability be involved in controlling reactions central to a range of biological processes cytometry relies on taking high-speed camera data of the sample, which is including spore germination. expensive to acquire and analyze. We propose to measure cell stiffness using resistive pulse sensing, whereby a particle’s physical properties are measured 776-Pos Board B541 from the change in the transchannel electrical current during the particle’s Sub-Diffraction STED Lithography using Orthogonally Functionalized transit. By using a microfluidic channel with regions differing in diameter, Resins 1 1 1 the particle is deformed to different degrees, distorting the resistive pulse signal Thomas A. Klar , Richard Wollhofen , Johannes Kreutzer , Bianca Buchegger1, Christine Eder2, Jaroslaw Jacak1,2. relative to the signal expected of a hard sphere. We show resistive pulse data 1 2 taken of hard spheres and cells with varying degrees of deformability, and using Johannes Kepler University Linz, Linz, Austria, Upper Austria University high speed video, show cells deforming in the channel. of Applied Sciences, Linz, Austria. Stimulated emission depletion (STED) is not only apt to provide diffraction- 774-Pos Board B539 unlimited imaging in fluorescence microscopy, but it also allows for sub- Quantifying Extracellular ROS Levels in Individual Pancreatic Islets using diffraction photo-polymerization. Nanoscale anchors to fix proteins on two an Optical Sensor and Microfluidic Device and three dimensional surfaces, even down to the single protein level, have Romario Regeenes1, Jonathan V. Rocheleau1,2. been developed in the past. However, the proteins were bound electrostatically. 1IBBME, University of Toronto, Toronto, ON, Canada, 2Physiology, Covalent bonds would be much stronger, insensitive to variations of buffer pH University of Toronto, Toronto, ON, Canada. and they would allow for orthogonal functionalization of a structure exhibiting The metabolism of pancreatic islet beta-cells is negatively regulated by reactive two different types of reactive groups. oxygen species (ROS), which is a toxic byproduct of the electron transport In this contribution, we present two acrylate-based photo-resins with chem- chain and excessive aerobic respiration. During oxidative stress it is evident ically reactive surfaces. Chemically functional monomers are copolymer- that ROS levels increase and result in apoptotic signaling. Chronic oxidative ized with a highly crosslinking triacrylate monomer to yield mechanically stress is associated with beta-cell failure and type 2 diabetes. Oxidative stress stable structures. Thereby, the polymer structure is equipped with chemical increases expression of antioxidant enzymes as a protective mechanism that functionality for post lithographical reactions. Two nanoscopic patterns, paradoxically reduces glucose stimulated insulin secretion. To remove ROS, one functionalized with mercapto- and the other one with carboxylate the cell uses superoxide dismutase (SOD) an enzyme that catalyzes the conver- groups, can be structured on top of each other with sub-diffraction sion of superoxide into hydrogen peroxide and oxygen. The hydrogen peroxide precision. In order to prove the surface reactivity, the bi-functionalized is then converted into water and oxygen via glutathione peroxidase or catalase. structure was labelled covalently with two different fluorophores. The Therefore, both hydrogen peroxide (H2O2) release and molecular oxygen (O2) resins show excellent performance in stimulated emission depletion lithog- consumption of islets can serve as proxies to measure conversion rates in the raphy and line widths below 60 nm can be achieved. Compound structures, tissue. We have designed a microfluidic device to hold individual pancreatic which are fabricated of both resins, will allow fabrication of two and islets separately in chambers optimized for live cell confocal imaging. This de- three dimensional scaffolds for proteins, for instance inside microfluidic vice puts islets very close to the planar slide and also collects islet effluent in a flow cells.

BPJ 7746_7748